Methylsulfonylmethane (msm) for treatment of drug resistant microorganisms
Abstract
A method to enhance the fermentation efficiency of a microorganism for the production of beer, cider, wine, a biofuel, bread, a dairy product or any combination thereof, the method comprising: provide a fermentation medium containing a carbon source and a microorganism capable of fermenting and contacting the fermentation medium with methylsulfonylmethane (MSM), wherein the MSM is provided at a concentration of 0.02% to 5% by weight of the fermentation medium or at a concentration of 0.02% to 5% by weight of the moisture content of the fermentation medium, wherein the MSM increases the fermentation efficiency of the microorganism compared to the fermentation efficiency in the absence of MSM.
Term
4.1 yearsto projected expiry
Projected expiry 29 October 2030, counted from filing; an application has no term until it is granted.
- Priority
- Filed
- Published
- Today
- Projected expiry
11 claims: 5 independent, 6 dependent
- 1ES 2 616 630 T3 REIVINDICACIONES 1. Un método para potenciar la eficacia de fermentación de un microorganismo para la producción de cerveza, sidra, vino, un biocombustible, pan, un producto lácteo o cualquier combinación de los mismos, comprendiendo el método:proporcionar un medio de fermentación que contiene una fuente de carbono y un microorganismo capaz de fermentar y poner en contacto el medio de fermentación con metilsulfonilmetano (MSM), en donde el MSM se proporciona a una concentración del 0,02 % al 5 % en peso del medio de fermentación o a una concentración del 0,02 % al 5 % en peso del contenido de humedad del medio de fermentación, en donde el MSM aumenta la eficacia de fermentación del microorganismo en comparación con la eficacia de fermentación en ausencia de MSM.
- 2El método de la reivindicación 1, en el que la potenciación de la eficacia de fermentación comprende un aumento de al menos el 50 % en la producción de alcohol, dióxido de carbono o ácido por el microorganismo en presencia de MSM en comparación con la producción de alcohol, dióxido de carbono o ácido en ausencia de MSM.
- 3El método de la reivindicación 2, en el que la potenciación de la eficacia de fermentación comprende un aumento de al menos el 50 % en la producción de etanol, metanol o una combinación de los mismos en comparación con la producción de etanol, metanol o una combinación de los mismos en ausencia de MSM.
- 4El método de la reivindicación 2, en el que la potenciación de la eficacia de fermentación comprende un aumento de al menos el 50 % en la producción de dióxido de carbono por el microorganismo en presencia de MSM en comparación con la producción de dióxido de carbono en ausencia de MSM, el microorganismo es levadura y el método para potenciar la fermentación es para la producción de pan.
- 5El método de la reivindicación 2, en el que la potenciación de la eficacia de fermentación comprende un aumento de al menos el 50 % en la producción de ácido láctico por el microorganismo en presencia de MSM en comparación con la producción de ácido láctico en ausencia de MSM y el método para potenciar la fermentación es para la producción de un producto lácteo.
- 6El método de una cualquiera de las reivindicaciones 1-5, en el que la concentración de MSM es del 0,5 %.
- 7El método de una cualquiera de las reivindicaciones 1-6, en el que el medio de fermentación comprende una concentración de cloruro de sodio de menos del 5 % del contenido de humedad total del medio de fermentación.
- 8Un método in vitro para potenciar el crecimiento de uno o más microorganismos probióticos, comprendiendo el método:poner en contacto uno o más microorganismos probióticos con un medio capaz de soportar el crecimiento de uno o más microorganismos probióticos;y proporcionar metilsulfonilmetano (MSM) al medio a razón de un 0,02 % a un 5 % en peso del medio o en peso de un contenido de humedad del medio, potenciando de este modo el crecimiento in vitro de los uno o más microorganismos en comparación con el crecimiento in vitro de los uno o más microorganismos en ausencia de MSM.
- 9El método de la reivindicación 8, en el que la concentración de MSM es del 1 % al 3 % del peso del medio o del contenido de humedad del medio.
- 10El método de las reivindicaciones 8 o 9, en el que los uno o más microorganismos probióticos comprenden Lactobacillus acidophilus, Lactobacillus delbrueckii, Bacillus coagulans, Lactobacillus rhamnosus, Bifidobacterium bifidum o cualquier combinación de los mismos.
- 11Un método para potenciar el crecimiento de un microorganismo en una muestra de ensayo diagnóstica, comprendiendo el método:poner en contacto la muestra de ensayo diagnóstica que comprende uno o más microorganismos con un medio capaz de soportar el crecimiento de los uno o más microorganimos;proporcionar metilsulfonilmetano (MSM) al medio a una concentración del 0,02 % al 5 % en peso del medio o en peso de un contenido de humedad del medio, potenciando de este modo el crecimiento de los uno o más microorganismos en la muestra de ensayo diagnóstica en comparación con el crecimiento de los uno o más microorganismos en ausencia de MSM. 122
Independent claims11
2,446 paragraphs in 102 sections, as filed
ES 2 616 630 T3
DESCRIPTION
Use of methylsulfonylmethane (MSM) to modulate microbial activity
Disclosure field
The present disclosure relates to the field of methylsulfonylmethane (MSM), specifically, to methods of using MSM to modify biological activity, such as to enhance or inhibit microbial activity, including bacterial growth.
Background
Microorganisms (or microbes) are microscopic organisms that include bacteria, fungi, archaea, protists, plants (eg, green algae), viruses, prions, parasites, and animals, such as amoebae and planktonic microorganisms. Depending on the context, microorganisms can be viewed as either harmful or beneficial. In some cases, microorganisms can be harmful and lead to disease and illness in plants, animals, or humans. Furthermore, in addition to causing infection or disease, unwanted microbial growth can also occur in consumer products, such as food contamination. In other cases, the growth of microorganisms is beneficial and is routinely exploited in biotechnology, in modern development technologies, in chemical processes (e.g. fermentation), in food and beverage preparation, in environmental and industrial applications, and in the maintenance and promotion of human health.
Summary
Methods for modulating the activity of microorganisms with MSM are disclosed herein. MSM is an organosulfur compound of formula (CH<sub>3</sub>)<sub>2</sub>SW<sub>2</sub>. In particular, herein is disclosed the surprising ability of MSM to enhance or inhibit the activity of microorganisms, such as the growth or survival of microorganisms, depending on the concentration of MSM provided to the microorganism (for example, in the medium in which the microorganism is cultured). MSM at a concentration of about 0.5% to about 5% by weight of medium or by weight of moisture content of the medium enhances microbial activity, while MSM at a concentration of about 6% to about 17 % by weight of medium or by weight of moisture content of the medium inhibits microbial activity.
Disclosed herein is the surprising discovery that MSM can both inhibit and enhance microbial activity, depending on the concentration of MSM. For example, MSM concentrations of between about 6 and about 17 percent by weight of medium (or moisture content of the medium) inhibit microbial activity by reducing or otherwise impacting growth, survival rate ( for example, causing or causing cell damage or death, such as programmed cell death), metabolism, reproduction (for example, gene expression, protein expression, signal transduction, transcription, translation, protein folding, etc.), proliferation, vitality, robustness, action, and / or function of the microorganism. In contrast, MSM concentrations of between about 0.04% to about 5% by weight enhance microbial activity, including enhancing microbial fermentation efficiency, microbial growth, and / or culture efficiency.
As such, methods of using MSM to modulate microbial activity, such as to enhance or inhibit the activity of microorganisms, are disclosed herein.
In some embodiments, a method of enhancing the fermentation efficiency of a microorganism is disclosed. For example, the method includes contacting MSM with the medium containing a microorganism capable of fermenting, wherein the MSM is provided at a concentration of from about 0.5% to about 5% by weight of the medium or at a concentration from about 0.5% to about 5% by weight of the moisture content of the medium, wherein MSM increases the fermentation efficiency of the microorganism compared to the fermentation efficiency in the absence of MSM.
In some embodiments, in vitro methods are disclosed for enhancing the growth of one or more probiotic organisms. In some examples, the method comprises contacting one or more probiotic microorganisms with a medium capable of supporting the growth of one or more probiotic microorganisms; and providing MSM to the medium at from about 0.4% to about 5% by weight of the medium or by weight of a moisture content of the medium, thereby enhancing the growth of the one or more microorganisms in vitro compared to the growth of the one or more microorganisms in vitro in the absence of MSM.
Methods for enhancing the growth of a microorganism in a diagnostic test sample are also provided. In some examples, the method comprises contacting the diagnostic test sample comprising one or more microorganisms with a medium capable of supporting the growth of the one or more microorganisms; provide MSM to the medium at a concentration from about 0.4% to about
ES 2 616 630 T3 5% by weight of the medium or by weight of a moisture content of the medium, thereby enhancing the growth of the one or more microorganisms in the diagnostic test sample compared to the growth of the one or more more microorganisms in the absence of MSM.
Furthermore, methods for inhibiting microbial activity are disclosed. In some examples, the method comprises selecting a medium that is susceptible to H1N1 influenza contamination; and contacting the medium with MSM at a concentration of from about 10% to about 16% by weight by volume, thereby inhibiting the microbial activity of the H1N1 flu.
The above disclosure and other features will be apparent from the following detailed description of various embodiments.
Detailed description
I. Overview of various achievements
It is disclosed herein a surprising discovery that MSM can both inhibit and enhance microbial activity, depending on the concentration of MSM. For example, MSM concentrations of between about 6 and about 17 percent by weight of medium (or moisture content of the medium) inhibit microbial activity by reducing or otherwise impacting growth, survival rate ( for example, causing or causing cell damage or death, such as programmed cell death), metabolism, reproduction (for example, gene expression, protein expression, signal transduction, transcription, translation, protein folding, etc.), proliferation, vitality, robustness, action, and / or function of the microorganism. In contrast, MSM concentrations of between about 0.04% to about 5% by weight enhance microbial activity, including enhancing microbial fermentation efficiency, microbial growth, and / or culture efficiency.
As such, methods of using MSM to modulate microbial activity, such as to enhance or inhibit the activity of microorganisms, are disclosed herein.
In some embodiments, a method of enhancing the fermentation efficiency of a microorganism is disclosed. For example, the method includes contacting MSM with the medium containing a microorganism capable of fermenting, wherein the MSM is provided at a concentration of from about 0.5% to about 5% by weight of the medium or at a concentration from about 0.5% to about 5% by weight of the moisture content of the medium, wherein MSM increases the fermentation efficiency of the microorganism compared to the fermentation efficiency in the absence of MSM. In some examples, increasing the efficiency of the fermentation comprises an increase of at least 50% in the production of alcohol, carbon dioxide or acid by the microorganism in the presence of MSM compared to the production of alcohol or acid in the absence. by MSM. For example, increasing fermentation efficiency comprises an increase of at least 50% in the production of ethanol, methanol, or a combination thereof compared to the production of ethanol, methanol, or a combination thereof in the absence of MSM.
In some examples, the increase in fermentation efficiency comprises an increase of at least 50% in the production of carbon dioxide by the microorganism in the presence of MSM compared to the production of carbon dioxide in the absence of MSM, the microorganism it is yeast and the method to enhance fermentation is for the production of bread.
In some examples, the increase in fermentation efficiency comprises an increase of at least 50% in the production of lactic acid by the microorganism in the presence of MSM compared to the production of lactic acid in the absence of MSM and the method for enhancing fermentation is for the production of a dairy product.
In some embodiments, the method of enhancing fermentation efficiency is for the production of beer, cider, wine, a biofuel, bread, a dairy product, or any combination thereof. In some examples, the microorganism is yeast and the method to enhance fermentation is for beer production. In some examples, the microorganism is an algae and the method to enhance fermentation is for biofuel production.
In some embodiments, the MSM concentration is about 0.5%. In some examples, the medium comprises a sodium chloride concentration of less than 5% of the total moisture content.
In vitro methods for enhancing the growth of one or more probiotic microorganisms are also disclosed. In some embodiments, the method comprises contacting one or more probiotic microorganisms with a medium capable of supporting the growth of one or more probiotic microorganisms; and providing MSM to the medium at from about 0.4% to about 5% by weight of the medium or by weight of a moisture content of the medium, thereby enhancing the growth of the one or more microorganisms in vitro compared to the growth of the one or more microorganisms in vitro in the absence of MSM.
ES 2 616 630 T3
In some examples, the concentration of MSM is from about 1% to about 3% of the weight of the medium or the moisture content of the medium.
In some examples, the one or more probiotic microorganisms comprise Lactobacillus acidophilus, Lactobacillus delbrueckii, Bacillus coagulanss, Lactobacillus rhramnosus, Bifidobacterium bifidum, or any combination thereof.
In some examples, the medium comprises a probiotic-containing product, such as milk, yogurt, rice yogurt, frozen yogurt, chocolate, cheese, beer, wine, vinegar, sauerkraut, or any combination thereof.
Methods for enhancing the growth of a microorganism in a diagnostic test sample are also disclosed. In some examples, the method comprises contacting the diagnostic test sample comprising one or more microorganisms with a medium capable of supporting the growth of the one or more microorganisms; providing MSM to the medium at a concentration of from about 0.4% to about 5% by weight of the medium or by weight of a moisture content of the medium, thereby enhancing the growth of the one or more microorganisms in the sample of diagnostic test compared to the growth of the one or more microorganisms in the absence of MSM.
Furthermore, methods for inhibiting microbial activity are disclosed. In some examples, the method comprises selecting a medium that is susceptible to H1N1 influenza contamination; and contacting the medium with MSM at a concentration of from about 10% to about 16% by weight by volume, thereby inhibiting the microbial activity of the H1N1 flu. In some examples, the medium comprises a body fluid, a body tissue, or a surface. In some examples, contacting the medium comprises spraying or adding MSM to the medium susceptible to microbial contamination. In some examples, the surface is a household surface, bedding, coatings, equipment or industrial surfaces, blood, skin, or a combination thereof. In some examples, MSM is provided in a composition, wherein said composition does not contain bleach or alcohol or consists essentially of water. In some examples, the method further comprises sterilizing the medium after adding said MSM. In some examples, the medium has no preservatives. In some examples, MSM inhibits microbial activity by reducing the growth rate of H1N1 influenza by at least 50% compared to the growth rate of H1N1 influenza in the absence of MSM.
II. Abbreviations and terms
DMEM: Dulbecco's Modified Eagle's Medium
DMSO: Dimethylsulfoxide
DNA: Deoxyribonucleic acid
ELISA: Enzyme Linked Immunosorbent Assay
IC50: Inhibitory concentration at 50
LAB: Lactic acid bacteria
MIC: Minimum inhibitory concentration
MSM: Methylsulfonylmethane
PAGE: Polyacrylamide Gel Electrophoresis
PBS: Phosphate Buffered Saline
PDA: Potato Dextrose Agar
SDS: Sodium dodecyl sulfate
TNTC: Too Many to Count
TSB: Tryptic Soy Broth
The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in practicing the present disclosure. The singular forms a, an, and the or the refer to one or more than one, unless the context clearly indicates otherwise. For example, the term comprising a bacterial cell includes a single or several bacterial cells and is considered equivalent to the term comprising at least one bacterial cell. The term "o" refers to a single item of indicated alternative items or a combination of two or more items, unless the context clearly indicates otherwise. As used herein, "comprise" means "includes." Therefore, "comprising A or B" means that it includes A, B, or A and B, without excluding additional elements.
Unless otherwise explained, all technical and scientific terms used in this document have the same meaning as that normally understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting. For example, conventional methods well known in the art to which the disclosed invention pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular
ES 2 616 630 T3
Cloning: A Laboratory Manual, 2<sup>to</sup> ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Clowning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999; Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith and March, March's Advances Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analyzes, Fourth Edition, New York: Longman, 1978.
Additional terms used in molecular genetics can be found in Benjamin Lewin, Genres V published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
Additional terms used in chemistry can be found in Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978.
Administration: Providing or administering to a subject a compound, such as MSM, by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal (such as topical), intranasal, vaginal, and inhalation. A typical type of administration is topical.
Bacterial pathogen: A bacterium that causes disease (pathogenic bacteria). Examples of pathogenic bacteria for which MSM can be used for modification include, without limitation, any one or more of (or any combination of) Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonella bacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetella sp. (such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi), Brucella sp. Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis), Burkholderia sp. (Such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium), Clostridium sp. (such as Clostridium perfrigens, Clostridium difficile, Clostridium botulinum and Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae, and Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli, and E. coli uropathogenic) Enterococcus sp. (such as Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus, and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, Helicobacter cinaedi, and Helicobacter fennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiella pneumoniae, Klebsiella granulomatis and Klebsiella oxytoca), Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Mobilcouncus sp., Mobilcouncus sp., Mobilcouncus sp., Morganella sp. . (such as Mycobacteriu leprae, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum), Mycoplasma sp. (such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (such as Nocardia asteroides, Nocardia cyriacigeorgica, and Nocardia brasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides. Prevotella sp., Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri, and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari, and Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi), and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium), Serratia sp. (such as Serratia marcesans and Serratia liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei), Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcus
ES 2 616 630 T3 pneumoniae (for example, Streptococcus pneumoniae serotype 4 resistant to chloramphenicol, Streptococcus pneumoniae serotype 6B resistant to spectinomycin, Streptococcus pneumoniae serotype 9V resistant to streptreomycin, Streptococcus pneumoniae resistant to serotype 6B of spectinomycin optokine-resistant serotype 14, Rifampin-resistant serotype 18C Streptococcus pneumoniae, Streptococcus pneumoniae serotype 19F resistant to tetracycline, Streptococcus pneumoniae serotype 19F resistant to penicillin, and Streptococcus pneumoniae serotype 23F resistant to trimethoprim, Streptococcus pneumoniae resistant to chloramphenicol-resistant serotype 4, Streptococcus pneumoniae resistant to chloramphenicol serotype 6, Streptococcus pneumoniae resistant to chloramphenicol serotype 6 Streptococcus pneumoniae serotype 9V resistant to streptomycin, Streptococcus pneumoniae serotype 14 resistant to optokine, Rifampin-resistant Streptococcus pneumoniae serotype 18C, Streptococcus pneumoniae serotype 19F penicillin-resistant, or Streptococcus pneumoniae serotype 23F resistant to trimethoprim), Streptococcus agalactiae, Streptococcus Agalactiae, Streptococcus mutans, Streptococcus streptocogenesis, Streptococcus mutans, group B, Streptococcus agalactiae, group C streptococci, Streptococcus anginosus, Streptococcus equismilis, Group D streptococci, Streptococcus bovis, group F streptococci, and Streptococcus anginosus, group G streptococci), Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela, and Vibrio furnisii), Yersinia sp. (such as Yersinia enterocolitica, and Yersinia pestis) and Xanthomonas maltophilia, among others.
In some embodiments, MSM is used to modify, such as to increase or decrease the biological activity of one or more of the organisms listed above.
Beta-lactam antibiotics: A class of antibiotic agents that contain a β-lactam core in their molecular structure. Examples include the penicillin, cephalosporin, monobactam, and carbapenem families of antibiotics. Methicillin and oxacillin are beta-lactam antibiotics.
Biological activity: An expression that describes the beneficial or adverse effects of a substance on living matter. When the agent is a complex chemical mixture, this activity is exerted by the active principle of the substance or pharmacophore, but it can be modified by the other constituents. Activity is generally dose dependent and it is not uncommon to have effects ranging from beneficial to adverse for a substance when going from low to high doses. In one example, MSM alters, such as increases or decreases the biological activity of a microorganism, such as a bacterium.
Biofuel: A fuel derived from a metabolic product of a living organism. It is a renewable energy source, unlike other natural resources, such as oil, coal, and nuclear fuels. A biodiesel fuel is a processed fuel equivalent to diesel from biological sources that can be used in unmodified diesel powered vehicles. Biodiesel is attractive for fuels, and for other different uses, because they have a low vapor pressure, are not toxic, are stable and do not deteriorate or detonate after heating them slightly. Chemically, biodiesel is generally defined as the monoalkyl esters of long-chain fatty acids from renewable lipid sources.
Bleach: A solution of approximately 3-6% sodium hypochlorite (NaClO), and oxygen bleach, that contains hydrogen peroxide or a compound that releases peroxide, such as sodium perborate, sodium percarbonate, sodium persulfate, tetrasodium pyrophosphate, or urea peroxide together with catalysts and activators, for example, sodium tetraacetylethylenediamine and / or nonanoyloxybenzenesulfonate. The bleaching powder is sodium hypochlorite. Many bleaches have strong bactericidal properties, and are used to disinfect and sterilize. Conditions that allow production: Any fermentation or culture conditions that allow a microorganism to grow and / or produce a desired product, such as alcohols and carbon dioxide or organic acids. Such conditions typically include temperature ranges, aeration levels, and selection of media that, when combined, allow the microorganism to grow. Illustrative media include broths or gels. To determine if culture conditions allow product production, the microorganism can be cultured for 2, 4, 6, 8, 12, 24, 36, 48, or 72 and a sample can be obtained and analyzed. For example, cells in the sample or the medium in which the cells were grown can be tested for the presence of the desired product. When checking for the presence of a product, assays, such as those provided herein, including those presented in the examples below, can be used.
Putting in contact: Putting in direct physical association; including in solid, liquid and gas forms. Contacting includes contact between one molecule and another molecule. Contact can occur in vitro with isolated cells, tissues, or a solid surface (such as a domestic or industrial surface) or in vivo, by administration to a subject.
Control: Samples that are considered normal (for example, representative of the activity or function in the absence of the variable being tested) as well as laboratory values, although they may have been established arbitrarily, taking into consideration that these values may vary from one laboratory to another. A control group is practically identical to the treatment group, except for the individual variable of interest whose effects are being tested, which only applies to the treatment group.
Culture: Keeping a cell in a medium that allows the organism to continue living. For example, cultivation
ES 2 616 630 T3 includes Incubate a microorganism in a fermentation medium, such as a fermentation broth or a fermentation gel. One of ordinary skill in the art will appreciate that the time, temperature, and other physical conditions associated with the culture will depend on the organism being cultivated and the desired culture result. For example, a microorganism that is grown to produce ethanol can be placed in a fermentation broth containing a carbohydrate source, various minerals and trace elements, as well as MSM and compounds useful to induce production, including less than 5% NaCl.
Reduction: Reducing the quality, quantity, or strength of something. In one example, the administration of MSM decreases or reduces one or more biological activities, such as growth, reproduction, proliferation, survival rate, metabolism, vitality, robustness, action, and / or function of microorganisms by at least 10%, at least 20%, at least 50%, or even at least 90%, including between 10% to 95%, 20% to 80%, 30% to 70%, 40% to 50%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 85%, 90%, 95%, 98%, or 100%. For example, MSM administration reduces or inhibits bacterial growth, for example, by at least a factor of 2, for example, at least a factor of 3 or at least a factor of 4, compared to a control (such as bacterial growth in the absence of MSM or a reference value known to be representative of bacterial growth in a subject affected by a bacterial infection). Such reductions can be measured using the methods disclosed herein, as well as those known to one of ordinary skill in the art. In some embodiments, MSM is used to inhibit the growth of specific microorganisms. In other embodiments, MSM is used to inhibit the growth of a wide variety of microorganisms in certain media or products. In some embodiments, large-scale reductions are made after the first 24 hours.
Dimethylsulfoxide (DMSO): Dimethylsulfoxide (DMSO), also known as methylsulfinylmethane or methyl sulfoxide, is an organosulfur compound of formula (CH3) 2SO. This colorless liquid is a polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible in a wide variety of organic solvents, as well as in water. It has the distinctive property of penetrating the skin very quickly, so its taste can be perceived shortly after it comes into contact with the skin. DMSO is widely known as a nutritional supplement and as a pharmaceutical agent. One skilled in the art will be familiar with these uses. Various grades of DMSO are commercially available (eg, Sigma-Aldrich, Corp., St. Louis, MO. Product # 472301) and one of skill in the art will be familiar with a source of DMSO. Empowerment or augmentation: Increasing the quality, quantity, or strength of something. In one example, MSM increases or enhances the activity of a microorganism, for example, relative to activity in the absence of MSM. In a particular example, MSM increases the activity of a microorganism, such as enhancing growth, reproduction, proliferation, survival rate, metabolism, vitality, robustness, action, and / or function of a microorganism in at least 10%, at least 20%, at least 50%, or even at least 90%, including between 10% to 95%, 20% to 80%, 30% to 70 %, 40% to 50%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85 %, 90%, 95%, 98%, or 100%. The terms activity and growth are used interchangeably in some contexts. In some examples, MSM is used to enhance the growth of specific microorganisms. In other examples, MSM is used to enhance the growth of a wide variety of microorganisms in certain media or products. In some examples, enhancement of microbial activity includes enhancement of microbial products or microbial metabolites. For example, MSM increases or enhances the efficiency of the fermentation or the efficiency of the culture such as by at least 10%, at least 20%, at least 50%, or even at least 90%, including between a 10% to 95%, 20% to 80%, 30% to 70%, 40% to 50%, such as 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 85%, 90%, 95%, 98%, or 100%. Such increases can be measured using the methods disclosed herein.
Fermentation: A process for deriving energy from the oxidation of organic compounds, such as carbohydrates, and using an endogenous electron acceptor, which is normally an organic compound. During fermentation, pyruvate is metabolized into a number of different compounds. Homolactic fermentation is the production of lactic acid from pyruvate; alcoholic fermentation is the conversion of pyrovate into ethanol and carbon dioxide; a heterolactic fermentation is the production of lactic acid as well as other acids and alcohols. Fermentation does not necessarily have to take place in an anaerobic environment. For example, even in the presence of abundant oxygen, yeast cells prefer fermentation to oxidative phosphorylation, as long as sugars are readily available for consumption. Sugars are a common substrate for fermentation, and typical examples of fermentation products are ethanol, lactic acid, and hydrogen. However, more exotic compounds can be produced by fermentation, such as butyric acid and acetone. Yeast carries out fermentation in the production of ethanol in beers, wines, and other alcoholic beverages, along with the production of large amounts of carbon dioxide.
Fermentation broth: Any medium that supports the life of microorganisms (for example, a microorganism that is actively metabolizing carbon). A fermentation medium normally contains a carbon source. The carbon source can be anything that can be used, with or without additional enzymes, by the microorganism for energy.
Fermentation efficiency: An expression of how much of the fermentation product, such as alcohol, lactic acid, microorganisms, or other desired fermentation product has been produced relative to a control (such as in the absence of MSM) or to an amount that could have been produced theoretically.
Fermentation medium: Any substance used to grow cells, such as mammalian cells and microorganisms. The fermentation medium includes any growth medium (eg, broth or gel) that supports the life of microorganisms (eg, a microorganism that is metabolizing carbon in a
ES 2 616 630 T3 active). A fermentation medium typically contains a carbon source, such as glucose, xylose, cellulosic material, and the like. The carbon source can be anything that can be used, with or without additional enzymes, by the microorganism for energy.
Fungal pathogen: A fungus that causes disease. Examples of fungal pathogens for which MSM can be used for modification include, without limitation, any one or more of (or any combination of) Trichophyton rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, Pityrosporum orbiculare (Malassezia furfur), Candida sp. (such as Candida albicans), Aspergillus, sp. (such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus, glaucus, Aspergillus, nidulans, Aspergillus, oryzae, Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor, and Aspergillus clavatus), Cryptococvatcus sp. (such as Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii, and Cryptococcus albidus), Coccidioides sp., Histoplasma sp. (such as Histoplasma capsulatum), Pneumocystis sp. (such as Pneumocystis jirovecii), Stachybotrys sp. (such as Stachybotrys chartarum), Paracoccidioides, Blastomyce, Fusarium, Sporothrix, Trichosporon, Rhizopus, Pseudallescheria, Paecilomyces, Alternaria, Curvularia, Exophiala, Wangiella, Penicillium, and Cephalosphorium. In some embodiments, MSM is administered to inhibit or prevent an infection or disorder associated with one or more of the aforementioned fungal pathogens.
Incubation: A period that includes a sufficient amount of time for an agent, such as MSM, to interact with a cell or tissue.
Inhalation Device: A device capable of delivering a composition to a subject, eg, to the lung tissue of a subject. For example, an inhalation device can be an inhaler, a nebulizer, or a ventilator. The inhalation devices described herein are constructed of a material adapted to come into contact with DMSO and / or MSM. In some embodiments, an inhalation device is disposable or replaceable. The inhalation devices described herein are configured to deliver a composition containing DMSO or MSM for direct contact with bacterial pathogens in the lung tissue of a subject. Inhalation devices are configured to generate particles of composition that vary in size. In some embodiments, an inhalation device is configured to generate particles of a composition that have a size in the range of about 0.1 pm to about 10 pm or from about 0.5 pm to about 5 pm.
Inhibition of microbial activity or inhibition of an infection or disease: The phrase inhibit microbial activity refers to reducing growth, reproduction, proliferation, survival rate, metabolism, vitality, robustness, action, and / or the function of microorganisms. The term "inhibiting or treating an infection, disease, or condition" refers to preventing or reducing the full development of an infection, disease, or condition, for example, in a subject who is at risk of developing an infection, such as an infection. bacterial. Treatment refers to a therapeutic intervention that relieves a sign or symptom of a disease or pathology after it has begun to develop. As used herein, the term "ameliorate" in reference to a disease, pathology, or symptom, refers to any observable beneficial effect of the treatment. The beneficial effect may be evidenced, for example, by a delayed onset of the clinical symptoms of the infection / disease in a susceptible subject, a reduction in the severity of some or all of the clinical symptoms of the infection / disease, a slower progression of infection / disease, a reduction in the number of relapses of the infection / disease, an improvement in the general health or well-being of the subject, or by any other parameters well known in the art that are specific for the particular infection / disease, such as a specific bacterial infection.
Medium (s): An environment that contains or is suitable to support microorganisms, including, but not limited to, broths, agar, cultures, foods, beverages, cell suspensions, biological tissue, biological fluids, inorganic surfaces, organic surfaces, substrates, cells live, host cells, diagnostic assays, and other solid, liquid, matrix, gelatinous, or gaseous environments.
Methylsulfonylmethane (MSM): An organosulfur compound of formula (CH3) 2SO2. MSM has been marketed and sold largely as a dietary supplement. MSM is also known as DMSO2, dimethyl sulfone, and methyl sulfone.
MSM is structurally related to dimethyl sulfoxide (DMSO), but the behavior of these two is different. DMSO is a highly polar solvent and an excellent ligand, with dissolution properties similar to water, while MSM is less polar and less reactive. MSM is also a metabolite of DMSO. The MSM has the following structure:
<img file="ES2616630T3_D0001.tif" />
Microorganism: A member of the prokaryotic or eukaryotic microbial species of the Archaea, Bacteria, and Eucarya domains, the latter including yeast and filamentous fungi, protozoa, algae, or higher protists. The terms microbial cells and microbes are used interchangeably with the term microorganism. Microbes can include wild-type organisms or engineered or modified organisms.
ES 2 616 630 T3 by genetic engineering. Microorganisms include viruses, prlones, parasites, fungi, molds, yeasts, and bacteria.
In some embodiments, MSM is used to enhance the activity of a broad spectrum of microorganisms, including, but not limited to, viruses, prions, parasites, fungi, molds, yeasts, algae, and bacteria. In other embodiments, MSM is used to inhibit the activity of microorganisms, including, but not limited to, fungi, molds, yeasts, bacteria, and viruses. Modulate or modulation: Adjust, alter, regulate an activity, a degree or a speed, including an increase or a reduction in the biological activity of a molecule. In one example, MSM is administered to modulate, either increase or decrease, microbial activity, such as bacterial growth.
Parasite: An organism that lives inside humans or other organisms that act as hosts (for the parasite). Parasites depend on their hosts for at least part of their life cycle. Parasites are harmful to humans, because they consume the necessary food, feed on tissues and cells of the body, and eliminate toxic waste, which makes people sick. Examples of fungal pathogens for use in accordance with the disclosed methods and compositions include, without limitation, any one or more (or any combination of) Malaria (Plasmodium falciparum, P. vivax, P. malariae), schistosomes, trypanosomes, Leishmania , filarial nematodes, trichomoniasis, sarcosporidiasis, tapeworm (T. saginata, T. sodium),
Leishmania, Toxoplasma gondii, trichinosis (Trichinella spiralis) or coccidiosis (Eimeria sp.). MSM can be used to inhibit or prevent the activity of one or more of the organisms listed above.
Pharmaceutical Composition: A compound or chemical composition capable of inducing a desired therapeutic or prophylactic effect when appropriately administered to a subject. A pharmaceutical composition can include a therapeutic agent, a diagnostic agent, or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or in conjunction with an additional compound induces the desired response (such as the induction of a therapeutic or prophylactic effect when administered to a subject). In a particular example, a pharmaceutical agent is an agent that significantly reduces one or more symptoms associated with an infection, such as a bacterial or viral infection. In some embodiments, a therapeutic agent is an antibiotic agent, such as methicillin or oxacillin.
Pharmaceutically acceptable carriers or vehicles: The pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 19<sup>to</sup> Edition (1995), describes compounds and compositions suitable for the pharmaceutical delivery of one or more therapeutic compounds or molecules, such as one or more peptides provided herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral compositions typically comprise injectable fluids including pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a carrier. In a particular embodiment, the carrier is one that allows the therapeutic compound to cross the blood-brain barrier. For solid compositions (eg, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions for administration may contain minor amounts of non-toxic adjuvant substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.
Prebiotic: An indigestible food ingredient that stimulates the growth and / or activity of bacteria in the digestive tract that are beneficial to the health of the body. Typically, prebiotics are carbohydrates (such as oligosaccharides); however, non-carbohydrates are also sources of these ingredients. Prebiotics can be short chain, long chain, and / or full spectrum prebiotics. Short-chain prebiotics (such as oligofructose), contain 2-8 bonds per polysaccharide molecule, and normally ferment more rapidly on the right side of the colon, providing nutrition to bacteria in that area. Longer chain prebiotics (such as inulin) contain 9-64 bonds per saccharide molecule, and tend to ferment more slowly, feeding bacteria predominantly in the left colon. Full-spectrum prebiotics provide the full molecular range of bond lengths of 2-64 bonds per molecule, and nourish bacteria throughout the entire colon (such as oligofructose-enriched inulin, OEI). In some examples, a perbiotic increases the number and / or activity of bifidobacteria and lactic acid bacteria. Bifidobacteria and lactic acid bacteria (Lactobacillus or LAB) are bacteria that improve digestion (including enhancing the absorption of minerals) and the effectiveness and intrinsic strength of the immune system. A product that stimulates bifidobacteria, such as MSM, is considered a bifidogenic factor. Traditional dietary sources of probiotics include soybeans, inulin sources (such as Jerusalem artichoke, jicama, and chicory root), raw oats, unrefined wheat, unrefined barley, garlic, leek, asparagus onion, banana, and yacon. Prebiotic oligosaccharides are increasingly being added to foods due to their health benefits. Some oligosaccharides used in this way are fructooligosaccharides (FOS), xylooligosaccharides (XOS), polydextrose, and galactooligosaccharides (GOS). Some monosaccharides, such as tagatose, are also sometimes used as prebiotics. As used herein, MSM is a prebiotic. Probiotic: A microorganism that confers a health benefit on the host, including, but not limited to, conferring protection against or treating disease or its unwanted effects. Probiotics can confer health benefits on a product, such as increasing the nutritional quality of edible products. Probiotics include beneficial bacteria, such as
ES 2 616 630 T3 such as lactic acid bacteria (such as Lactobacillus bulgaricus, Lactobacillus rhamnosus, Lactobacillus casei and Lactobacillus johnsonii) and bifidobacteria (such as Lactobacillus bifidus) which are the most common types of microbes used as probiotics; although certain yeasts and bacilli can also be probiotics. Probiotics are normally consumed as part of fermented foods; such as in yogurt, soy products, or as dietary supplements. Live probiotic cultures are available in fermented dairy products and probiotic fortified foods. However, tablets, capsules, powders and sachets containing the bacteria in freeze-dried form are also available. Illustrative probiotic strains include, but are not limited to, Bacillus coagulans GBI-30, 6086 (Ganeden Biotech), Bifidobacterium LAFTI® B94 (Institut-Rosell-Lallemand), Lactobacillus acidophilus LAFTI® L10 (Institut-Rosell-Lallemandus), casei Lactobacillus LAFTI® L26 (Institut-Rosell-Lallemand), Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium breve (Yakult), Bifidobacterium infantis 35624 (Procter & Gamble), Bifidobacterium animalis subsp. lactis HN019 (Danisco), Bifidobacterium longum BB536 (Morinaga Milk Industry), Lactobacillus acidophilus DDS-1 (Nebraska Cultures), Lactobacillus acidophilus LA-5, Lactobacillus acidophilus NCFM (Danisco), Lactobacillus casei 43001, Lactobacillus 43001, Lactobacillus casei DN114-001 , Lactobacillus casei F19 (Arla Foods), Lactobacillus casei (Yakult), Lactobacillus paracasei St11 (or NCC2461. Nestle), Lactobacillus johnsonii La1 (Lactobacillus LC1, Lactobacillus johnsonii NCC533, Nestle), Lactococcus lactis L1A (Norrmejerier), Lactobacillus plantarum 299v (Probi), Lactobacillus reuteri giovani ATTC13730, Lactobacillus reuteri Gilliactobacillnos 5, Lactobacillnos 5 Ccillus reuterhami ATTC21730, Lactobacillus reuterhami ATTC 5512730 (BioCCillus reuterhami SDTC132123) ), Lactobacillus rhamnosus LB21 (Norrmejerier), Bifidobacterium bifidum, Lactobacillus gasserí PA16 / 8, Bifidobacterium bifidum MF20 / 5, Bifidobacterium longum SP07 / 3, Streptococcus thermophilus, Lactobacillus salivarius, Bifidobacterium longum Resell-175, Lactococcus lactis Rosell-1058, Bifidobacterium breve Rosell-70, Lactobacillus rhamnosus Rosell-11, Lactobacillus acidophilus Rosell-52, Bifidobacterium bifidum resell-71, Lactobacillus factobacillus, Lactobacillus varasecoustocillus subtilis Bifidobacterium animalis, Lactobacillus delbrueckii, and Saccharomyces cerevisiae.
Quantification: Determining or measuring an amount (such as a relative amount) of a molecule or the activity of a molecule, such as the amount of analyte present in a sample.
Stem cell: A cell that has the ability to self-replicate indefinitely and that, under the right conditions, or by providing it with the right signals, can differentiate into some or all of the different cell types that make up an organism. Stem cells have the potential to develop into differentiated mature cells, such as heart cells, skin cells, or nerve cells. The fertilized egg is a stem cell since it has the potential to generate all the cells and tissues that make up an embryo and that support its development in the uterus. Adult mammals have more than 200 cell types, eg, neurons, myocytes, epithelial cells, erythrocytes, monocytes, lymphocytes, osteocytes, and chondrocytes. Other cells that are essential for embryonic development but are not incorporated into the embryo include extra-embryonic tissues, the placenta, and the umbilical cord. All of these cells are generated from a single fertilized egg.
Pluripotent cells can give rise to cells from the three embryonic germ layers, mesoderm, endoderm, and ectoderm. Therefore, pluripotent cells have the potential to give rise to any type of cell. Unipotent stem cells are capable of differentiating only into one lineage. Embryonic stem cells are pluripotent cells derived from the blastocyst. Adult stem cells are undifferentiated cells found in differentiated tissue that can replicate and specialize to provide all of the specialized cell types for the tissue from which they originate. Adult stem cells are capable of self-renewal throughout the life of the body. Sources of adult stem cells have been found in the bone marrow, bloodstream, cornea, retina, dental pulp, liver, skin, gastrointestinal tract, and pancreas. Herein, MSM is used to increase stem cell culture efficiency, stability, and / or viability.
Sterilization: A process that removes (removes) or kills all life forms, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound, such as a biological culture medium. Sterilization can be accomplished by methods known to one of ordinary skill in the art, including the application of suitable combinations of heat, chemicals, irradiation, high pressure, and filtration.
Subject: Living vertebrate multicellular organisms, a category that includes human and non-human mammals.
Symptom and sign: Any subjective evidence of disease or a condition of a subject, for example, evidences, such as those perceived by the subject; a perceptible change in a patient's condition indicative of some bodily or mental state. A sign is any abnormality indicative of disease, which can be discovered upon examination or evaluation of a subject. In general, a sign is an objective indication of disease. Signs include, but are not limited to, any measured parameter, such as tests to detect a disorder or disease, such as a bacterial or viral infection. In one example, reducing or inhibiting one or more symptoms or signs associated with a bacterial or viral infection includes reducing or inhibiting bacterial growth or viral infection by a desired amount, for example, by at least 20%, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, in comparison with bacterial growth or viral infectivity in the absence of MSM.
Therapeutically effective amount or concentration: An amount of a composition that by itself, or together
ES 2,616,630 T3 with one or more additional therapeutic agents is sufficient to achieve a desired effect in a subject, or in a cell, being treated with the agent. The effective amount of the agent will depend on a number of factors, including, but not limited to, the subject or cells being treated, and the mode of administration or the therapeutic composition. In one example, a therapeutically effective amount or concentration is one that is sufficient to prevent progression, delay progression, or cause regression of a disease, or that is capable of reducing symptoms caused by a condition or disease.
In one example, a desired effect is to reduce or inhibit one or more symptoms associated with the disease. The one or more symptoms need not be completely eliminated for the composition to be effective. For example, a composition can reduce the sign or symptom by a desired amount, for example, by at least 20%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, compared to the sign or symptom in the absence of MSM. In a particular example, a desired response is to reduce or inhibit the activity of microorganisms (such as bacterial growth) by a desired amount, for example, by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, compared to the activity of the microorganism in the absence of MSM.
A therapeutically effective amount of a disclosed pharmaceutical composition can be administered in a single dose, or in several doses, eg, daily, during a treatment cycle. However, the therapeutically effective amount may depend on the subject being treated, the severity and type of the condition being treated, and the mode of administration. A therapeutically effective amount of an agent can be measured as the concentration (moles per liter or molar, M) of the agent in blood (in vivo) or in a buffer (in vitro) that produces the desired effect (s). ). Alternatively, a therapeutically effective amount of an agent can be measured as the amount administered to a subject per body weight of the subject, eg, mg agent / kg body weight.
Untreated cell: A cell that has not been contacted with a desired agent, such as MSM. In one example, an untreated cell is a cell that receives the vehicle in which the MSM was delivered.
Virus: A microscopic infectious organism that reproduces within cells. A virus consists essentially of a nucleic acid nucleus surrounded by a protein envelope, and has the ability to replicate only within a living cell. Viral replication is the production of additional viruses through the appearance of at least one viral life cycle. A virus can alter the normal functions of a host cell, causing the cell to behave in a way determined by the virus. For example, a viral infection can result in the cell producing a cytokine, or responding to a cytokine, when the uninfected cell normally does not. In some examples, a virus is a pathogen.
Specific examples of viral pathogens that can be treated in accordance with the disclosed methods and compositions include, without limitation, any one or more of (or any combination of): arenaviruses (such as Guanarito virus, Lassa virus, Junin virus, Machupo and Sabia virus), arterivirus, ronivirus, astrovirus, buniavirus (such as Crimean-Congo hemorrhagic fever virus and hantavirus), barnavirus, birnavirus , bornavirus (such as Borna disease virus), bromovirus, calcivirus, chrysovirus, coronavirus (such as coronavirus and SARS), cystovirus, closterovirus, comovirus, dicistrovirus, flaviviruses (such as yellow fever virus, west nile virus, hepatitis C virus, and dengue fever virus), filovirus (such as Ebola virus and Marburg virus), flexivirus , herpesviruses (such as hepatitis E virus), human adenoviruses (such as human adenovirus AF), human astroviruses, human BK polyomaviruses, human bocaviruses, human coronaviruses (such as human coronavirus HKU1, nL63, and OC43), human enteroviruses (such as human AD enteroviruses), human erythrovirus V9, human foamy viruses, human herpesviruses (such as human herpesvirus 1 (herpes simplex virus type 1), human herpesvirus 2 (herpes simplex virus type 2) , human herpesvirus 3 (varicella zoster virus), human herpesvirus 4 type 1 (Epstein-Barr virus type 1), human herpesvirus 4 type 2 (Epstein-Barr virus type 2), human herpesvirus 5, strain AD169 , human herpesvirus, Merlin Strain strain, human herpesvirus 6A, human herpesvirus 6B, human herpesvirus 7, human herpesvirus 8 type M, human herpesvirus 8 type P, and human cytomegalovirus), human immunodeficiency virus (HIV) (such as HIV 1 and HIV 2 ), human metapneumovirus, human papillomavirus (such as human papillomavirus-1, human papillomavirus-18, human papillomavirus-2, human papillomavirus-54, human papillomavirus-61, human papillomavirus-cand90, human papillomavirus RTRX7, human papillomavirus type 10, human papillomavirus type 101, human papillomavirus type 103, human papillomavirus type 107, human papillomavirus type 16, human papillomavirus type 24, human papillomavirus type 26, human papillomavirus type 32, human papillomavirus type 34, human papillomavirus type 4, human papillomavirus type 41, human papillomavirus type 48, human papillomavirus type 49, human papillomavirus type 5, human papillomavirus type 50, human papillomavirus type 53, human papillomavirus type 60, human papillomavirus type 63, human papillomavirus type 6b, human papillomavirus type 7, human papillomavirus type 71, human papillomavirus type 9, human papillomavirus type 92, and papillomavirus human type 96), human parainfluenza viruses (such as human parainfluenza viruses 1-3), human parecoviruses, human parvoviruses (such as human parvovirus 4 and human parvovirus B19), Human respiratory syncytial virus, human rhinovirus (such as human rhinovirus A and human rhinovirus B), human foam retrovirus, human T lymphotrophic virus (such as human T lymphotrophic virus 1 and human T lymphotrophic virus 2), human polyoma virus, hypovirus, leviviruses, luteovirus, lymphocytic choriomeningitis virus (LCM), marnavirus, narnavirus, nidivirus, nodavirus, orthomyxovirus (such as
ES 2 616 630 T3 influenza virus), partitivirus, paramlxovlrus (such as measles virus and mumps virus), plcornavlrus (such as poliovirus, common cold virus, and hepatitis A virus), potivirus, poxivurs (such as chickenpox and vaccinia virus), sequivirus, reovirus (such as rotavirus), rhabdovirus (such as rabies virus), rhabdovirus (such as vesicular stomatitis virus, tetravirus , togaviruses (such as rubella virus and Ross River virus), tombusvirus, totivirus, thymovirus, and norovirus, among others.
In some embodiments, MSM is used to inhibit a biological activity of one or more of the viruses listed above.
Yeast: A eukaryotic microorganism classified in the kingdom of fungi, with approximately 1,500 described species. Most reproduce asexually by budding, although some reproduce by bipartition. Yeasts are generally unicellular, although some species can become multicellular by forming a strand of connected budding cells, known as a pseudohypha, or false hypha. Illustrative yeasts that can be used in the disclosed methods and compositions include, but are not limited to, Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Pichia, Cryptococcus, Zygosaccharomyces, Torulopsis, Hansenula, and Debaryomyces.
III. MSM Compositions
Disclosed herein are MSM compositions for use in modulating microbial activity, such as enhancing or reducing microbial activity. In some embodiments, an MSM composition for use in enhancing microbial activity includes from about 0.02% to about 5% MSM by weight of the medium (such as culture medium) or by weight of the wet content. of the medium (such as culture medium), such as from about 0.04% to about 4%, from about 1% to about 3%, including about 0.02%, about 0.03% , approximately 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 3%, about 0 , 5%, about 1%, about 2%, about 2.5%, about 3%, about 4%, or about 5% of the weight of the medium or the moisture content of the medium. In some examples, the percentages of MSM provided herein are calculated from the amount of a polar solvent, eg, water, in a product. As an example, a composition with 5% MSM by weight of the medium could contain 5 grams of MSM per 100 grams of medium or a composition with 5% MSM by weight of the moisture content of the medium could contain 5 grams of MSM per 100 grams of the polar solvent in the medium, excluding solids.
In some embodiments, the disclosed compositions include a medium capable of supporting the growth of a microorganism, a microorganism, and MSM. In some examples, a medium includes one or more of the following: probiotic-containing products, dairy products, milk, yogurt, rice yogurt, frozen yogurt, chocolate, cheese, fermented beverages (such as beer, cider, wine), and Water. In some examples, the medium also includes other products, whether edible or not, that benefit from the enhanced microbial activity.
In some embodiments, enhancing microbial activity includes enhancing the fermentation of a microorganism. Thus, in particular examples, a composition includes a medium capable of supporting the growth of fermenting microorganisms, a fermenting microorganism, and MSM. In some examples, MSM is provided at a concentration of from about 0.04% to about 5%, such as from about 0.1% to about 4%, from 0.5% to about 3%, from about 1% to about 2%, including about 0.04%, up to about 0.05%, about 0.06%, about 0.07%, about 0.08%, about a 0.09%, about 0.1%, about 0.3%, about 0.5%, about 0.7%, about 1%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 4 %, or about 4.5% MSM by weight of the medium or by weight of the moisture content of the medium, wherein the concentration of MSM is effective to enhance the fermentation of the microorganisms. In some embodiments, the disclosed compositions are used to produce a fermented beverage, such as beer, cider, and / or wine. In some embodiments, a composition for enhancing fermentation efficiency includes MSM added to yeast packets to generate a fast or fast-activating yeast for home or commercial use.
In some embodiments, enhancing microbial activity includes enhancing probiotic growth. Thus, in some examples, a probiotic growth enhancing composition includes a medium capable of supporting the growth of probiotics and MSM at a concentration of from about 0.04% to about 5% by weight of the medium or by weight of the wet content of the medium, in which the concentration of MSM is effective in enhancing the activity (eg, growth) of the probiotics. In addition, a composition for enhancing the growth of probiotics includes from about 0.04% to about 5% MSM, such as from about 0.1% to about 4%, from about 0.5% to about 3%, from about 1% to about 2%, including about 0.04%, to about 0.05%, about 0.06%, about 0.07%, about 0.08
ES 2 616 630 T3%, about 0.09%, about 0.1%, about 0.3%, about 0.5%, about 0.7%, about 1%, about 1 , 5%, about 2.0%, about 2.5%, about 3.0%, about 4%, or about 4.5% MSM by weight of medium or by weight of moisture content medium.
In some embodiments, enhancing micro-activity includes enhancing microbial production of biofuel. Thus, in some examples, a composition for enhancing microbial biofuel production includes a medium capable of supporting algae growth, algae capable of producing a biofuel, and MSM at a concentration of about 0.04% to about 5%. %, such as from about 0.1% to about 4%, from 0.5% to about 3%, from about 1% to about 2%, including about 0.04%, up to about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.3%, about 0.5%, about 0.7%, about 1%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 4 %, or about 4.5% MSM by weight of the medium or by weight of the moisture content of the medium, in which the concentration of MSM is effective to enhance biofuel production from algae. In other examples, a composition includes algae and MSM, and optionally other ingredients to enhance the growth of the algae. In various embodiments, the composition is useful for enhancing the activity of algae for biofuel, algae cultivation, aquaculture, medicine, etc. In one embodiment, the method comprises exposing the algae to MSM at, for example, a concentration of from about 0.04% to about 5% by weight of the medium or by weight of the moisture content of the medium.
MSM compositions are disclosed to inhibit microbial activity. In some cases, an MSM composition for inhibiting microbial activity includes from about 6% to about 17%, such as from about 7% to about 15%, from about 10% to about 12%, such as like about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 13%, about 14%, about 15%, or about 16% MSM by weight of the medium or by weight of the moisture content of the medium, wherein the concentration of MSM is effective to inhibit microbial activity, including, but not limited to, microbial growth, infection rate , or a combination thereof.
It is contemplated that any of the disclosed compositions that include MSM to modulate microbial activity have a sodium chloride concentration of less than 5% of the total moisture content of the medium, such as from about 1% to about 3% of sodium chloride. sodium, including 0%. 0.1%, 0.3%, 0.5%, 0.75%, 1%, 2%, 2.5%, 3%, or 4%. In some examples, a disclosed MSM composition has no preservatives. For example, MSM is added to a food, cosmetic, or beverage that requires or requires an all-natural ingredient list. In some embodiments, an MSM composition consists or consists essentially of MSM and non-toxic all-natural ingredients. In other examples, a disclosed MSM composition includes one or more additional preservatives. Preservatives include, but are not limited to, one or more or a combination of the following: formaldehyde, potassium sorbate, methyl paraben, methylchloroisothiazolinone, phthalates, cocamidopropyl betaine, parabens, decyl polyglucose, polyaminopropyl biguanide, phenoxyethanol, sodium lauryl tetrasodium ether sulfate ED , decyl glucoside, polyethylene glycol, and propylene glycol.
In several examples, MSM is used to extend the shelf life of products and is capable of reducing microbial activity by at least a factor of 2, 3, 4, 5, 10, 25, 50, 100, or 1000 compared to products without MSM or compared to products with less effective antimicrobial agents. In other examples, MSM is capable of achieving comparable levels of antimicrobial activity compared to agents that produce unwanted side effects. Thus, in one example, MSM can be used in place of an unwanted preservative. In several examples, the compositions include preservative-free or preservative-reduced formulations comprising MSM.
In various embodiments, MSM is used in products (eg, cosmetics) that have an acidic, basic, or neutral pH. Because MSM can inhibit microbial activity, cosmetics and other products may have more flexibility in pH selection. Therefore, a pH can be selected that is optimal for the product. In various embodiments, the compounds comprising MSM do not require refrigeration and can be stored at room temperature. In other embodiments, products comprising MSM do not require sterilization, including, but not limited to, sterilization by chemicals, heating, radiation, filtration, or ultraviolet light.
In various embodiments, the disclosed compositions include MSM in addition to one or more thickening agents, emollients, and / or aromatic agents. In some embodiments, a product or other medium is supplemented with continuous or periodic additions of MSM to, for example, extend the inhibitory or stimulating actions of MSM.
In certain examples, the addition of MSM is an effective antimicrobial agent. For example, in one example, a composition comprising MSM has the same or enhanced antimicrobial effect compared to a formulation without MSM. In other examples, MSM serves as an antibacterial agent. In certain examples, MSM is used as a substitute for a chemical food preservative. In still other examples, MSM can be used in
ES 2 616 630 T3 in combination with a preservative. In some of these examples, the use of MSM reduces the amount of, or completely replaces, traditional preservatives. In some examples, MSM can increase the shelf life of a product, including products that traditionally would not have a preservative. In still other examples, MSM serves as a viricide, fungicide, and / or bactericide. In additional examples, MSM is a bacteriostatic. In some examples, MSM is a broad spectrum inhibitor of microbial activity. In other examples, MSM selectively kills a particular kingdom, genus, or species. In some examples, MSM selectively inhibits aerobic bacteria. In other examples, MSM selectively inhibits anaerobic bacteria. In some examples, MSM selectively inhibits gram-positive bacteria. In other examples, MSM selectively inhibits gram-negative bacteria.
In various examples, MSM is used to inhibit the growth of microorganisms, including those used in cosmetics, health and beauty supplements, parenterals, products used topically, and oral products. In several examples, MSM is used to inhibit the growth of microorganisms in products packaged in single or multi-dose containers. MSM formulations according to various of the examples described herein are in any suitable form, including, but not limited to, powder, cream, liquid, paste, solid, or gel.
In several examples, MSM is added to cosmetic products susceptible to microbial contamination. Cosmetics may include, but are not limited to, lipstick, lip gloss, lip liner, lip volumizer, lip balm, lip conditioner and lip volumizers, foundations, powder, blush, glosses, bronzer, mascara, eyeliner , eye shadow, eye reflection, eye glitter pencils, eyebrow pencils, nail varnish, concealer, skin care products, creams, lotions, serums, moisturizer, sunscreens, skin repair products (eg, for acne, sunburn, wrinkles, blemishes), and sunscreen.
In several examples, MSM is added to a cosmetic cream matrix susceptible to microbial contamination. In some similar examples, the cream includes jojoba, aloe vera, cocoa butter, shea butter, coconut oil, or combinations thereof.
In several examples, MSM is added to personal care products susceptible to microbial contamination. Such products include products for daily hydration needs, products to treat psoriasis or eczema, products to treat dry or itchy skin, products to treat sun and wind burns, before and after shave products. , massage oils or creams, personal lubricants, acne treatment products, and exfoliants or emollients. According to other examples, MSM is added to products to soften the skin, hands or feet (such as calluses), skin care products after swimming, makeup removers, children's skin lotions, and cream for the skin. diaper eczema. In some examples, MSM inhibits contamination while simultaneously conferring a beneficial cosmetic effect or health benefit.
In several examples, MSM is added to medicinal products or equipment susceptible to microbial contamination. Medicinal products include, but are not limited to, cold or flu prevention agents and treatment, allergy prevention agents and treatment, nasal irrigations, medicated drops, eye drops, inhalers, foot treatments athlete, medications for herpes and cold sores rashes, creams for burns, ointments for cuts and infections, and bactericidal, fungicidal and virucidal sprays or lotions. In some examples, MSM is used to inhibit microbial activity in inhalers, nebulizers, ventilators, catheters, syringes, intubation tubes, room equipment, hospital furniture and surfaces, diagnostic equipment, fabrics, bedding, and patient bedspreads. . In several examples, MSM is used to disinfect tissues and body fluids. For example, MSM can be used as part of a dialysis system to inhibit microbial activity in the blood, which can be particularly useful for patients with sepsis. In other examples, MSM is injected into a patient to inhibit microbial activity locally or systemically. In other embodiments, a composition including MSM is applied topically to a microbial target displayed on a dermal surface.
In some examples, MSM products are used nasally. In other embodiments, such products are used orally and / or in vapor form. In still other embodiments, the product is an eye drop or other re-use medicated eye product.
In several examples, MSM is added to medicinal products used to prevent or treat fungal infections. In some similar examples, the product is used to prevent or treat athlete's foot. In some examples, the product is used topically. In some similar examples, the product is a cream, ointment, spray, gel, or powder. In other examples, the product is used orally.
In several examples, MSM inhibits the activity of mycotoxins, toxic metabolites produced by an organism from the fungal kingdom, including mushrooms, molds, and yeasts. Products comprising MSM are also useful for decontaminating surfaces and equipment that are susceptible to contamination by such organisms and / or metabolites. In some examples, MSM inhibits the activity of organisms from the fungal kingdom (eg, mushrooms, molds, and yeasts). In still other examples, MSM directly and / or indirectly inhibits microbial toxins by inhibiting the activity of the microbes. In one example, MSM inhibits the formation and / or release of microbial metabolites.
ES 2 616 630 T3
In several examples, MSM is added to products used to prevent or treat viral infections. In one example, antiviral nasal sprays comprising MSM are provided. Products comprising MSM are also useful for decontaminating surfaces and equipment that are susceptible to virus contamination. In one example, MSM is used to inhibit influenza virus, including H1N1, either in biological tissue or on an external surface. In some examples, MSM is used to inhibit human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza virus, hepatitis virus, and others.
In some examples, MSM inhibits algae. In one example, MSM inhibits algal blooms. In some examples, MSM inhibits the unwanted activity of phytoplankton. In other examples, MSM inhibits macroalgal species. In another example, MSM inhibits dinoflagellates of the genera Alexandrium and Karenia. In several examples, MSM inhibits the toxic metabolites (including by-products) of algae.
In some examples, MSM is added to medicinal products used to treat a burn, cut, or wound. Wounds may include, but are not limited to, lacerations, open lacerations, overstretches, grinding compression, cut lacerations, tears, incisions, incisive wounds, abrasions, puncture wounds, penetrating wounds. In some examples, MSM is incorporated into a bandage used to cover a wound. In other examples, MSM is added to a cream or ointment. In some similar examples, the product formulated with MSM acts as an antiseptic.
The microflora of the skin (bacteria, fungi, viruses, phage, archaea) play a significant role in common dermatological conditions, such as atopic dermatitis (a common form of eczema). Normally, a specific microbe colonizes the skin to alter the balance of the commensal microflora, or the microbes release toxic substances or invade cells to directly induce an inflammatory response. Therefore, in some examples, MSM is incorporated into a topical product that inhibits the growth of said microflora. Subcutaneous administration of MSM is provided in other examples.
In other examples, MSM is added to optics susceptible to microbial contamination and / or optics to enhance their antimicrobial activity. Optical products may include solutions to clean or disinfect contact lenses. In some similar examples, MSM is incorporated into various products applied to contact lenses, such as a contact lens preservation solution. In some examples, MSM is added to eye drops used in conjunction with contact lenses. In other examples, MSM is added to chemical solutions used in ocular diagnostic procedures, such as multipurpose pupil dilation solution.
In several examples, MSM is added to oral products susceptible to microbial contamination and / or oral products to enhance their antimicrobial activity. In some examples, such products are used for dental hygiene. In some examples, MSM is incorporated into toothpaste or gel. In some examples, MSM is incorporated into or coated onto the bristles of a toothbrush. In other examples, MSM formulations are incorporated into or used to coat dental floss. In other examples, the product is used to clean the tongue. In other examples, the product is a mouthwash, mouthwash or mouth irrigation for home or professional dental use. In still other examples, the product is a chewing gum or confectionery. In some examples, MSM is added to storage or cleaning solutions for dental implants, dentures, and the like.
In some embodiments, MSM is added to foods containing probiotic organisms, such as milk, yogurt, rice yogurt, frozen yogurt, kefir, juice, pickled vegetables, sauerkraut, fermented bean paste, olives in brine, chocolate, cheeses. and other dairy products, and certain cereals. In some embodiments, MSM is added to products that are dietary supplements, including, but not limited to, probiotic pills, capsules, and liquids. In some such embodiments, MSM is added to a supplement for human consumption. In other embodiments, MSM is added to an animal supplement. In some embodiments, MSM is added to a product that is formulated as a capsule or tablet. In some embodiments, MSM is added to a product that is formulated as a solid or a liquid. In other embodiments, MSM is added to a food during the production process, while, in other embodiments, MSM is added to finished food products.
In several examples, MSM is added to a food product that is susceptible to microbial infection. In some examples, MSM can be mixed, premixed, compounded, or otherwise incorporated into the food product. In other examples, MSM is applied to the surface of a food product. For example, in some examples, MSM can be sprayed on a food product. Such food products may include, but are not limited to, fruits, vegetables, fish, and meat products. In some examples, MSM is used in processing or packaging facilities to extend the shelf life of food products. In several examples, the addition of MSM (eg, from about 5% to about 25%) increases the time to spoilage of the ingestible products. For example, MSM can be baked into or added to breads, pastas, or doughs to increase the shelf life of edible products by approximately 10% to 100% (e.g. 20%, 30%, 40%, 50%). , 75%, 150%, 200% or more). For example, in one example, in case the shelf life of an edible product is 10 days, the addition of MSM will increase the shelf-life to at least 11 days in some examples (e.g. 11 days, 14 days, 15 days, 20 days, or 25 days). As a further example, if an edible product has a shelf life of 14 days at room temperature and / or 30 days in the fridge and / or 3 months in the freezer, the addition
ES 2 616 630 T3 from MSM will increase shelf life to 30 days at room temperature and / or 60 days in the refrigerator and / or 6 months in the freezer. In additional examples, the use of MSM will allow the shipment and / or storage of an edible product at room temperature, where otherwise the product would have to be shipped and / or stored at cooler temperatures. In still other examples, the use of MSM will obviate the need for sterilization of edible products.
In some examples, any of the disclosed MSM compositions consist essentially of water. For example, MSM is particularly useful when combined with water or other liquid components. In some examples, a disclosed MSM composition does not contain bleaches, does not contain alcohols, or a combination thereof. In various embodiments, a composition for modulating microbial activity includes an MSM-related compound in place of or in addition to MSM. Related compounds include, but are not limited to, DMSO and dimethylsulfide (DMS).
MSM used in accordance with any of the embodiments provided herein can be isolated, purified, or processed. MSM which is designated GRAS (Generally Recognized As Safe) is used for various embodiments described herein. In accordance with various embodiments, formulations for consumption by humans, domesticated animals, and farm animals are provided herein.
In some embodiments, MSM is combined with one or more of the following ingredients (or derivatives, metabolites, precursors, oils, extracts, esters, acids, salts, and related compounds thereof): abietic acid, gum arabic, gum arabic from Senegal, acai extract, acetic acid, acetone, acetylglucosamine, Acmella olerácea extract, Adenophora stricta, Alaria marginata (marine vegetable), albumin, alcohol, aldenine, alfalfa, algae extract, alkyl guanine transferase, alkyl amides, allantoin, aluminum hydroxide, almond, aloe vera, alpha-lipoic acid, aluminum benzoate, aluminum chloride, amino acids, aminopropane sulfonic acid 3, ammonium glycolate, ammonium lauryl sulfate, Anemarrhenae asphodeloides root extract, anise oil, antioxidants, apigenin, apricot, apricot kernel, arachidonic acid, arbutin, argan oil, Argania spinosa leaf extract, arginine, argireline, arnica extract, Artemisia dracunculus (tarragon) oil, ascorbic acid, ascorbyl palmitate, ascorbyl tetraisopalmitate, Aspergillus ferment, Aspidosperma quebracho, astaxanthin, atelocollagen, Avena sativa (oat) germ extract, avobenzone, azelic acid, adzuki beans, peppermint balsamic extract, peru balsam, bamboo stem extract, barley extract (Hordeum vulgare), barium sulfate, barley, basil, pollen of bee, beeswax, bentonite, benzoyl peroxide, Beta vulgaris (beet) root extract, beta carotene, bilberry, biotin, bismuth oxychloride, vesicular sargassum extract, borage oil, boric acid, boric oxide, bovine placental fluid, brewer's yeast, bronopol, butyl acetate, butyl stearate, butylated hydroxyanisole, butylated hydroxytoluene, butylene glycol, butyl paraben, Butyrospermum parkii, C18-36 triglyceric acid, caffeine, calamine, calcium, calendula extract, wax carnauba, Camellia oleifera leaf extract, Camellia sinenis leaf extract, camphor, Canaga odorata flower oil (ylang ylang), candelilla wax, candelilla fence, rapeseed sterols, caprylic acid, Caprylic / Capric Triglyceride, Caprylyl Glycol, Caprylyl Glycol, Capsicum Oleoresin, Caramel, Carmine, Carotenoids, Carrageenan, Carrot Oil, Carrot Seed Oil, Carthamus tinctorius (Safflower) Seed Oil, Castor Oil, Cellulose, Calendula asiatica officinalis, cera alba, carnauba wax, ceramide, cerebrosides, ceric ammonium ferrocyanide, ceteareth-3, cetearyl alcohol, cetearyl glucoside, cetearyl olivate, cetyl alcohol, cetyl alcohol, Chamomile oil, Chamomilla recutita flower extract (feverfew), chestnut, chestnut extract, chloroxylenol, chlorphenesin, cholesterol, choline, Chondrus crispus (Irish moss), chromium hydroxide green, chromium oxide greens, cinnamyl alcohol, citric acid, citronellol, citrus fruits, Citrus nobilis (green tangerine) oil, clove powder, clove flower extract, glyceryl coconate, cocamidopropyl betaine, cocoa, cocoa butter, caprylate / caprate, coconut oil, coconut wax, cod liver oil, coenzyme q10, collagen, comfrey extract, echinacea extract, Copernica cerifera (carnauba) wax, copper, coriander, Coriandrum sativum (coriander) oil, corn starch, flower extract from corn, creatine, Crithmum maritimum extract, cucumber, cyclomethicone, cyclopentasiloxane, dantoin 685, decyl glucoside, deionized water, diazolidinyl urea, dehydrated dicalcium phosphate, dicaprylyl carbonate, diethanolamine, dilaurate, dimethicone, dimethylaminoethanol, Dioscorea villosa (wild sweet potato) root extract, dipotassium glycyrrhizinate, disodium diestyrylbiphenyl disulfonate, disodium edta, dmdm hydantoin, Echinace angustifolia (echinacea) extract, edta, rose sausage, oleander, florida emollients, enzymes, Epilobium fleischeri (gravel willow) extract, Equisetum hiemale (horsetail) leaf extract, erucate, essential fatty acids, essential oils, ethanol, ethoxydiglycol, ethyl acetate, ethylene / acrylic acid copolymer, ethylhexyl pamitate, ethylhexylglycerin, ethylparaben, eucalyptus extract, eukarion, Euterpe oleracea fruit extract, evening primrose oil, scrubs, fatty acids, fatty alcohols, fennel oil, ferric oxide, flavanoids, flavonolignan, fish oils, flax, floralozone, fluoride, formaldehyde, fruit acids, fruit extract, fruit extracts, gaba, gamma linolenic acid, gelatin, geraniol, Geranium oil, Gigartina papillata (wild algae), ginger, ginger oil, Ginko biloba oil, ginseng, glucosamine, glucose oxidase, glucose sugar, glicereth, glicereth-26, glycerin, glycerol, glycerol stearate, hydrogenated rosinate glyceryl, glyceryl oleate, glyceryl stearate, glycol distearate, glycolic acid, gold, hydrastis extract, grape seed, grape seed oil, grapefruit, grapefruit oil, grapefruit seed extract, green tea, gums, hazelnut oil, hdi / trimethylolhexillactone crospolymer, hemp seed oil, hexamidine, hexylene glycol, homosalate, honey, Hordeum distychum extract, hordihydroguaiaretic acid, hormones, humectant, Humulus lupulus extract, hyaluronic acid, Hydisradens cana extract (hydrastis), hydrocortisone, hydrocotyl extract, hydrogenated rapeseed oil laurate, hydrogenated polyisobutene, hydrogenated polyisobulene, hydrolyzed animal protein, keratin
ES 2 616 630 T3 hydrolyzed, hydrolyzed rhizobian gum, hydrolyzed soy protein, hydrolyzed wheat protein, hydroxy acids, hydroxyethylcellulose, hydroxyethylcellulose, hydroxyisohexyl 3-cyclohexene carboxaldehyde, hydroxypropylcellulose, hydroxypropylbentrimonium extract, hydroxypropylbentrimonium extract, hydroquinone hydrochloride, hydroquinone extract, hydroxypropylbentrimonium imidazolidinyl urea, iodine, Irish moss, iron oxides, isobutlparaben, isododecane, isohexadecane, isononyl isononanoate, isopentyldiol, isopropyl alcohol, isopropyl lanolate, isopropyl linoleate, isopropyl myristate, isostearate, isostearic acid, ivy extract, jasmine oil, jojoba butter, jojoba oil, juniper extract, juniper oil, kaolin, keratin, ketones, kinerase, kinetin, kojic acid, kukui nut oil, lactic acid, lactoperoxidase, lady's mantle leaf extract, Laminaria digitata (kelp) extract, lanolin, Larix sibirica wood extract, lauramide, laurate, laureth, lauryl alcohol, lauryl glycoside, lavender, lavender oil, lecithin, lemon oil, licorice, lime oil, limonene, linden extract, linoleic acid, linolenic acid, liposomes, carob, Lycium barbarum fruit extract, Lycium barbarum fruit extract (goji berries), lycopene, macadamia nut oil, matcha, magnesium aluminum silicate, magnesium ascorbyl phosphate, magnesium myristate, magnesium stearate, magnesium sulfate (Epsom salts), Malpighia punicifolia (Acerola) fruit extract, manganese violet, mango butter, calendula, marshmallow extract, matcha tea powder, feverfew oil, mea, meadowsweet, melaleuca oil, cantaloupe extract, mentha extract piperite (organic peppermint), menthol, methyl acetate, methyl ethyl ketone, methyl dihydrojasmonate, methyl paraben, mica, microdermabrasion compounds, lactic protein, minerals, mineral oil, mipa, monoethanolamine, monostearate, Montmorillonite (green clay), mugwort extract (Artemisa vulgaris), blackberry, blackberry root extract (Morus nigra), murumuru, mushrooms, myristate, myristate, myristic acid, myristyl myristate, Myrtus communis (green myrtle) oil, n-acetyl glucosamine, nephrite powder, neroli oil, nettle leaf, neuropeptides, niacin, nitrosamines, nonyl nonoxynol-150, nucleic acids, nutmeg powder, walnut, oats, oatmeal, oats, Ocimum basilicum linalool oil (basil linalool), octinoxate, octsalate, oleate, oleic acid, oleyl alcohol, oligopeptides, oligosaccharides, olive fruit extract, olive oil, omega-3, orange peel oil, acid orthoboric, oxybenzone, ozokerite, Padina pavonica thallus extract, palm oil, palmitate, palmitic acid, palmitoyl, pantethine, panthenol, para-aminobenzoic acid, paraben, paraffin, Passiflora incarnata fruit extract, passion fruit, patchouli, peach pit, peat extract, pectin, peg, peppermint, peppermint oil, peptides, petroleum jelly, Phellodendron amurense bark extract, phenoxyethanol, phenyl trimethicone, phenylethyl resorcinol, phosphoric acid phytochemicals, derived from Pine extract, Pineapple extract, Plantago lanceolata leaf extract, Banana leaf extract, Pollen extract, Polygonum cuspidatum root extract, Polypeptides, Polysaccharides, Polysilicone, Polysorbate, polysorbate, polyvinylpyrrolidone, progesterone, propylene glycol, propylheptyl caprylate, propylparaben, pumpkin seed extract, Punica granatum (pomegranate) extract, Punica granatum / Punica granatum extract, pynogenol, quaternium-15, Quillaja saponaria soap bark extract ), quillia extract, reserveratol, retinoic acid, retinoids, retinol, retinyl palmate, Ribes rubrum fruit extract, rice, rice bran wax, ricinoleate, rose oil, rose hips, rosemary, rosemary oil, rose water, royal jelly, Rubus villosus fruit extract, Saccharum officinarum (sugar cane), salicylic acid, sage, sandalwood oil, saponins, sassafras, sawwood, Saxiphage extract sarmentose, clarareolida, Scutellaria baicalensis extract, seaweed, Secale cereale (rye) seed extract, Selaginella tamariscina (moss spike) extract, selenium, sesame oil, sesquioleate, razor grass, shea butter, silibinin, silica, silicone, sirtuins, sodium alginate, sodium ascorbate, sodium bisulfate, sodium borate, sodium carbonate, sodium chloride, sodium citrate, sodium dehydroacetate, sodium ethyl paraben, sodium glycyrrhinate, hyaluronate sodium, sodium lactobionate, sodium lauryl sulfate, sodium methyl paraben, sodium polystyrene sulfonate, sodium propyl paraben, sodium stearate, sodium thioglycolate, sodium acrylodimethyl laurate, sorbitan isostearate, Sorbitan olivate, sorbitan sesquioleate, sorbitan stearate, sorbitol, sorbitol, soybeans, soy wax, soybean oil, peppermint oil, squalane, St. Paul's / St. John's wort, stearate, stem cells, sucrose stearate, extract from sugar cane, sulfate, sunflower seed oil, sweet almond oil, Symphytum officinale (comfrey) leaf extract, Symphytum officinale leaf extract, synthetic fluorflolopite, Tamarindus indica seed extract, tea tree oil, thyme extract, tin oxide, titanium dioxide, titanium dioxide, tocopherol, tocopheryl acetate, tocopheryl acetate, toluene, tomato, tragacanth, tretinoin, tribehenin, triclosan, tridecyl trimellitate, triethanolamine, trihydroxystearin, tri-stearyl citrate, trimethylolpropane tri-stearate, trimethylsilyloxysilicate, trimyristate, tripeptide, turmeric, tyrosine, ubiquinone, ultramarines, undecylenoyl phenylalanine, urea, uridine, Vaccinium macrocarpon fruit extract, vegetable glycerin, vetiver oil, vitamin A, vitamin B1-B12, vitamin C, vitamin C ester, vitamin D, vitamin E, vitamin K, vitamins, walnut shell powder, water, oil wheat germ, whey protein (lactic protein), white birch bark extract, willow bark, wintergreen oil, witch hazel, xanthan gum, xanthan gum, yarrow extract, yeast, yerba mate, yucca , zinc oxide, zinc stearate.
In some embodiments, the composition comprises, consists or consists essentially of MSM in combination with one, two, three, four, five, or more of the previously identified ingredients. In several examples, MSM inhibited microbial activity in the formulation. In other examples, MSM offers the same or better antimicrobial effect when used to replace a preservative in the formulation (some of which have been identified above). In certain examples, MSM offers the same or better antimicrobial effect when used with a reduced amount of preservative. In still other examples, the addition of MSM to a formulation having a preservative enhances the effects of the preservative. The ingredients identified herein can be used with MSM in a cosmetic formulation (eg, oral, injectable, or topical), or in other types of formulations (eg, oral, injectable, or topical medical formulations).
ES 2 616 630 T3
In some embodiments, the composition includes MSM, but lacks one or more of the following compounds: sulfates, GMOs, synthetic fragrances, synthetic colorants, formaldehyde, potassium sorbate, methyl paraben, methylchloroisothiazolinone, cocamidopropyl betaine, parabens, decyl polyglucose, polyamidaminopropyl biguanidamine. phenoxyethanol, sodium lauryl ether sulfate, tetrasodium EDTA, decyl glucoside, polyethylene glycol, propylene glycol, phthalates, and triclosan. In some embodiments, the use of MSM allows the production of a formulation that is devoid of any synthetic ingredients. In still other embodiments, the use of MSM allows the production of a formulation that is devoid of any allergy-causing, immunosuppressive and / or inflammatory ingredients.
In several examples, the antimicrobial properties of MSM reduce or eliminate the need for sterilization, reduced temperatures, sterile environments, special closures, and / or special packaging, etc. MSM has a dual or multipurpose function according to some embodiments. For example, MSM not only inhibits the growth of unwanted microorganisms, MSM also beneficially affects the product where it is added in various examples (for example, MSM serves as an antioxidant, regenerating compound, anti-wrinkle compound, moisturizer, skin brightener skin, softener, circulation stimulant, neutralizer, repairer, hair / nail strengthener, healing catalyst, rejuvenating agent, etc., or combinations of two or more thereof). In several examples, the antimicrobial properties of MSM increase the shelf life, half-life, efficacy, and / or stability of the formulation (or a specific ingredient identified herein). The use of MSM may be particularly beneficial in some examples for cosmetic or other formulations that are shared by more than one person (eg, cosmetics used by makeup artists or on cosmetic counters).
Cosmetics may include, but are not limited to, lipstick, lip gloss, lip liner, lip volumizer, lip balm, lip conditioner and lip volumizers, foundations, powder, blush, glosses, bronzer, mascara, eyeliner , eye shadow, eye reflection, eye gloss pencils, eyebrow pencils, nail varnish, concealer, skin care products (e.g. microdermabrasion products, soothing gels), creams, lotions, serums, moisturizer, sunscreen, skin repair products (eg, for acne, sunburn, wrinkles, blemishes), and brushes. In various embodiments, formulations are provided for the face, hair, and body (eg, shampoo, soaps, conditioners, sprays, gels, serums, restorative treatments, deodorants, etc.). In various embodiments of the invention, cosmetics are provided, such as cosmeceuticals and nutraceuticals. In various embodiments, dermal filters and other dermatological products are provided (such as hyaluronic acid, waglerin 1, acetyl hexapeptide-8, palmitoyl tetrapeptide-7, palmitoyl oligopeptide, liposomes, collagen, calcium hydroxylapatite, polylactic acid, and botulinum toxin. ). In various embodiments, anti-wrinkle, anti-acne, anti-aging, exfoliating, moisturizing, and anti-stretch mark formulations, fragrances, mineral makeup, and foundations are provided. In some embodiments dermal gels are provided, for cosmetic and medical use (eg, inhibiting or preventing microbial infection and / or wound healing).
In several examples, MSM-containing products can be shipped and / or stored under high temperature and high humidity conditions that would otherwise be favorable for microbial activity.
In some examples, products that include MSM are packaged in containers adapted for multipurpose applications and exposure to external microorganisms, such as from air or contact with a part of the body (eg, fingers). The use of MSM is particularly beneficial in several examples, because it increases the shelf life of such products. In some examples, products comprising MSM are also packaged in sealed single-use containers. In one example, a single-use product (such as a condiment pack, seasoning pack, or travel cosmetic pack, etc.) will have a longer shelf life and / or will no longer need refrigeration if it is use MSM in conjunction with the product and / or container.
In some examples, MSM is incorporated directly into packaging materials to, for example, improve shelf life. For example, MSM can be incorporated into food storage bags to inhibit bacterial growth. In other examples, MSM can be incorporated into containers and / or lids to enhance the shelf life of foods, cosmetics, or other products by inhibiting unwanted bacterial growth. In still other examples, MSM can be incorporated into coating products, such as plastic wrap and adhesives.
In various examples, a composition for inhibiting microbial activity in a topical cream or ointment includes MSM, where the MSM is configured to affect microbial contamination by inhibiting microbial activity. MSM is provided at a concentration of at least the MSM according to one example (e.g. 5-10%, 10-16%, 16-20%, 20-30%, 30-40%, 40-50% , 50-75% or greater, and the intermediate ranges thereof). In some examples, the composition is a preservative-free cream. In one example, MSM inhibits microbial activity by at least 50% in cream at room temperature.
In some examples, the pharmaceutical compositions include MSM, DMSO, and / or antimicrobial agents, or combinations thereof, that are formulated for use in human or veterinary medicine.
For example, the pharmaceutical compositions provided include from about 0.01% MSM by weight to about 20% MSM by weight. In some examples, a pharmaceutical composition contains between about 0.01% to about 5% mSm by weight. Other examples contain between
ES 2 616 630 T3 about 5% to about 10% MSM, about 10% to about 15% MSM, or about 15% to about 20% MSM, such as about 5% , about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15% , about 16%, about 17%, about 18%, about 19%, or about 20% MSM. Some examples include about 10-16% MSM, about 10-14% MSM, or about 10-12% MSM.
Additional exemplary antimicrobial agents that may be included in a disclosed composition include, but are not limited to, penicillin derivatives, cephalosporins, penems, monobactams, carbapenems, beta-lactamase inhibitors, and combinations thereof. Examples of derivatives of penicillin include, but are not limited to, aminopenicillins (eg, amoxacillin, ampicillin, and epicillin); carboxypenicillins (eg, carbenicillin, ticarcillin, and temocillin); ureidopenicillins (eg, azlocillin, piperacillin, and mezlocillin); mecillinam, sulbenicillin, benzathine penicillin, penicillin G (benzylpenicillin), penicillin V (phenoxymethylpenicillin), penicillin O (allylmercaptomethylpenicillin), procaine penicillin, oxacillin, methicillin, nafcillin, cloxacillin, fluoampicillin, nafcillin, cloxacillin, fluxillin co-amoxiclav (amoxicillin plus clavulanic acid), and piperacillion. Examples of cephalosporins include, but are not limited to, cephalexin, cephalothin, cefazolin, cefaclor, cefuroxin, cefamandeol, cefotetan, cefoxitin, ceforanide, ceftriaxone, cefotaxin, cefpodoxime proxetil, ceftazidime, cefepime, ceforazidime, cefeponafix, and ceforazidime, cefeponafix, and ceforazidime. Examples of penems include, without limitation, faropenem. Examples of monobactams include, without limitation, aztreonam and tigemonam. Examples of carbapenems include, but are not limited to, biapenenvdoripenem, ertapenem, imipenem, meropenem, and panipenem. Examples of beta-lactamase inhibitors include, but are not limited to, tazobactam (sodium salt of 4,4-acid dioxide
[2S- (2alpha, 3beta, 5alpha)] - 3-methyl-7-oxo-3- (1H-1,2,3-triazol-1-ylmethyl) -4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid), sulbactam acid 4,4-dioxide sodium salt (2S, 5R) -3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptan-2 -carboxylic), and clavulanic acid ((2R, 5R, Z) -3- (2-hydroxyethylidene) -7-oxo-4-oxa-1-aza-bicyclo [3.2.0] heptane-2-carboxylic acid), or another beta-lactam antibiotic.
Many antibiotics have a set minimum inhibitory concentration (MIC) at which they are effective in reducing or killing certain bacteria. In some examples, a disclosed pharmaceutical composition includes an amount of beta-lactam antibiotic equal to about 0.001 to 100 MIC for the particular bacterial pathogens disclosed herein. In some examples, the pharmaceutical composition comprises about 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or about 90-100 MIC of a beta-lactam antibiotic. In some examples, the pharmaceutical composition comprises about 0.001, 0.01, 0.1. 0.5 or 1 MIC of a beta-lactam antibiotic.
The pharmaceutical compositions provided herein also include combinations of MSM and antimicrobial compounds, for example, a combination of MSM and a beta-lactam antibiotic. In some examples, the pharmaceutical compositions provided herein include 10-16% of: S; a beta-lactam antibiotic quantity equal to 1 MIC for a bacterial pathogen with which the composition will come into contact.
One of skill in the art will know the MIC of an antibiotic for a particular bacterial pathogen, or one of skill in the art will know how to determine the MIC of an antibiotic for a particular bacterial pathogen. Methods for determining a MIC of a particular antibiotic for a particular pathogen are described herein, for example, using the Etest® Antibiotic Test System (bioMérieux, Durham, NC).
The dosage form of the pharmaceutical composition will be influenced by the selected mode of administration. For example, in addition to injectable fluids, inhalation, topical, ophthalmic, peritoneal, and oral formulations may be employed. Inhalation preparations can include aerosols, particles, and the like. In general, the target particle size for inhalation is about 1 pm or less for the pharmaceutical agent to reach the alveolar region of the lung for absorption.
Pharmaceutical compositions that include MSM, DMSO, an antimicrobial therapeutic agent or compound as described herein, such as an active ingredient, or that include a mixture of two or more thereof, with or without additional agents as ingredients. Active ingredients can be formulated with a suitable solid or liquid carrier, depending on the particular mode of administration chosen. Oral formulations can be liquid (eg, syrups, solutions, or suspensions), or solid (eg, powders, pills, tablets, or capsules). For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods for preparing such dosage forms are known or will be apparent to those of ordinary skill in the art.
For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients, such as agents.
ES 2 616 630 T3 binders (eg, pregelatinized cornstarch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (eg, lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (eg, magnesium stearate, talc, or silica); disintegrants (eg potato starch or sodium starch glycolate); or wetting agents (eg, sodium lauryl sulfate). Tablets can be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (eg, lecithin or acacia); non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (eg, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents, as appropriate.
For administration by inhalation, the compounds for use in accordance with the present disclosure are conveniently delivered in the form of an aerosol spray presentation from pressure packs or from a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorotrufluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges can be formulated for use in an inhaler or insufflator containing a powder mixture of the compound and a suitable powder base, such as lactose or starch.
For topical administration, the compounds can, for example, be mixed with a liquid delivery agent for local administration. Agents used therapeutically (such as DMSO, MSM, and / or other therapeutic compounds as described herein) are readily soluble or suspended in water, and as such this could be useful for their delivery, as water does not cause adverse effects on biological tissues. This allows the administration of sufficiently high doses locally or systemically, without secondary toxicity from the delivery vehicle.
Pharmaceutical compositions that include a therapeutic amount of MSM as described herein as an active ingredient will normally be formulated with a suitable solid or liquid carrier, depending on the particular mode of administration chosen. The pharmaceutically acceptable carriers and excipients useful in the present disclosure are conventional. For example, parenteral formulations typically comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid carriers, such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol, or the like. Excipients that can be included are, for example, proteins, such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate. . Actual methods for preparing such dosage forms are known or will be apparent to those of skill in the art.
Pharmaceutical compositions that include a therapeutically effective amount of MSM will, in some embodiments, be formulated in unit dose form, suitable for individual administration of precise doses. The amount of MSM administered will depend on the subject being treated, the severity of the condition, and the mode of administration, and is preferably at the discretion of the prescribing physician. Within these limits, the formulation to be administered will contain an amount of the active components in amounts effective to achieve the desired effect in the subject being treated.
Preparations for administration may be suitably formulated to provide controlled release of the therapeutic agent (s) (eg, DMSO, MSM, beta-lactam antibiotic, and so on). For example, pharmaceutical compositions can be in the form of particles comprising a biodegradable polymer and / or a polysaccharide gelling agent and / or a bioadhesive polymer, an amphiphilic polymer, an agent that modifies the interfacial properties of the particles, and a pharmacologically active substance. . These compositions show certain biocompatibility characteristics that allow a controlled release of the active substance. See, for example, US Patent No. 5,700,486.
Polymers can be used for controlled release. Various degradable and non-degradable polymeric matrices are known in the art for use in drug delivery (Langer, Account Client. Res. 26: 537, 1993). For example, the block copolymer, poloxamer 407, exists as a viscous liquid although mobile at low temperatures but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained release of interleukin-2 and urease (Johnston et al., Pharm. Res. 9: 425, 1992; Pec, J. Parent. Sci. Tench. 44 (2 ): 58, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled protein release (Ijntema et al., Int. J. Pharm. 112: 215, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of lipid encapsulated compounds (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA, 1993). Numerous additional systems are known for the controlled delivery of therapeutic proteins (for example,
ES 2 616 630 T3
US Patent No. 5,055,303; US Patent No. 5,188,837; US Patent No. 4,235,871; US Patent No. 4,501,728; US Patent No. 4,837,028; States Patent
States No. 4,957,735; and US Patent No. 5,019,369; US Patent No. 5,055,303;
US Patent No. 5,514,670; US Patent No. 5,413,797; US Patent No.
5,268,164; US Patent No. 5,004,697; US Patent No. 4,902,505; States Patent
States No. 5,506,206; US Patent No. 5,271,961; US Patent No. 5,254,342; and US Patent No. 5,534,496).
In various examples, the pharmaceutical compositions include DMSO and / or MSM, and a therapeutic agent to treat an infectious disease, such as H1N1, the herpes simplex virus, or HIV. In some examples, compositions are provided that include DMSO and / or MSM in the form of an inhalant to treat an infectious disease. In some examples, pharmaceutical compositions for treating infectious disease include DMSO and / or MSM formulated as solids, while in various other embodiments, compositions including DMSO and MSM are formulated as liquids. In some examples, the compositions are consumed orally to treat infectious disease, while in other embodiments, the compositions are applied topically. In a particular example, the compositions are delivered in an inhalation device that is configured to generate particles of the formulation that have a size in the range of about 0.5 pm to about 5 pm.
In some examples, pharmaceutical compositions that include DMSO and / or MSM allow antibiotics (or other therapeutic agents) to penetrate lung tissue infected by an infectious disease. In one embodiment, such compositions that include DMSO and / or MSM: (i) allow antibiotics to reach deeper levels of infected tissue; (ii) allow direct contact with infected tissue; (iii) prolong the exposure time of the antibiotic in the infected tissue; and / or (iv) reduce the time to achieve a desired antibiotic effect. In one example, DMSO and / or MSM achieve one or more of these desired effects through use as an inhalant, wherein the inhalant further comprises one or more antibiotics or other therapeutic agents.
In some examples, pharmaceutical compositions that include DMSO and / or MSM formulations further include anti-parasitic agents that are effective in treating infections caused by parasites, such as nematodes, cestodes, trematodes, protozoa, or amoebae.
In some examples, pharmaceutical compositions that include DMSO and / or MSM formulations further include antifungal agents that are effective in treating fungal infections, such as those caused by ringworm, candidiasis, and Cryptococcus (cryptococcal meningitis, for example).
In some examples, pharmaceutical compositions that include DMSO and / or MSM formulations further include antiviral agents that are effective to treat viral infections. In some examples, specific classes of antiviral agents are used to treat infections caused by particular virus types. In some examples, agents are used that target HIV, herpes viruses, hepatitis B or C viruses, and influenza viruses, such as H1N1.
In various examples, the DMSO and / or MSM compositions include antibiotics that are effective in treating bacterial infections by, for example, inhibiting bacterial growth, metabolism, proliferation, activity, and / or function. In some examples, bacteriostatic antibiotics are used, while in other embodiments, bactericidal antibiotics are used. In still other examples, both bacteriostatic and bactericidal antibiotics are incorporated into a single formulation comprising DMSO and / or MSM. In some examples, antibiotics of one or more classes are incorporated into a composition that includes DMSO and / or MSM. In certain examples, a composition includes one or more than one: aminoglycoside, ansamycin, carbacephem, carbapenem, cephalosporin (1<sup>to</sup>, 2<sup>to</sup>, 3<sup>to</sup>, 4<sup>to</sup>, or 5<sup>to</sup> generation), glycopeptides, macrolides, monobactam, penicillin, polypeptide, quinolone, sulfonamide, tetracycline, and the like.
In some examples, specific diseases are targeted by incorporating specific antibiotics into a disclosed composition that includes DMSO and / or MSM. For example, macrolides, such as azithromycin or erythromycin are incorporated into formulations used to treat respiratory or mycoplasmic infections. Likewise, penicillins, such as amoxicillin or oxacillin are incorporated into formulations used to treat a wide variety of streptococcal infections.
In still other examples, specific disease-causing microorganisms are targeted by specific antibiotics incorporated in a formulation comprising DMSO and / or MSM. For example, aminoglycosides, such as neomycin, are incorporated into formulations used to treat Escherichia coli infections. In various examples, antibiotics commonly used to combat microbial infections are used. In certain examples, antibiotics including, but not limited to, isoniazid, rifampin, pyrazinamide, and ethanol are incorporated into formulations comprising one or more of DMSO and MSM, and used to treat an infectious disease, including a drug-resistant infectious disease.
In various examples, compositions are provided that include DMSO, MSM, and one or more of the following agents
Therapeutic ES 2 616 630 T3: rifampicin, isoniazid, pyrazinamide and ethambutol. In other examples, compositions are provided that include DMSO and at least one of rifampin, isoniazid, pyrazinamide, and ethambutol. In further examples, compositions are provided that include MSM and at least one of rifampin, isoniazid, pyrazinamide, and ethambutol. In various examples, compositions are provided that include DMSO and / or MSM in combination with rifampin, isoniazid, pyrazinamide, and ethambutol to treat an infectious disease, including a drug-resistant infectious disease.
In some examples, rifampicin is provided in a total daily dose of from about 400 mg to about 800 mg per day. In some examples, rifampicin is provided in a total daily dose of about 500 mg to about 700 mg per day, while in other examples, it is provided in a total daily dose in the range of about 550 to about 650 mg per day, including 560, 570, 580, 590, 600, 610, 620, 630, and 640 mg daily.
In some examples, isoniazid is provided in a total daily dose of from about 100 mg to about 500 mg per day. In some examples, isoniazid is provided in a total daily dose of about 200 mg to about 400 mg per day, while in other examples, it is provided in a total daily dose of about 250 mg to about 350 mg per day, including 260, 270, 280, 290, 300, 310, 320, 330, and 340 mg daily.
In some examples, pyrazinamide is provided in a total daily dose in the range of about 1.0 to about 4.0 g per day. In some examples, pyrazinamide is provided in a total daily dose in the range of about 2.0 to about 3.0 g per day, while in other examples, it is provided in a total daily dose in the range of about 2.0. at about 2.5 g per day, including 2.1,2,2, 2.3, and 2.4 g.
In some examples, ethambutol is provided in a total daily dose in the range of about 0.5 to about 2.5 g per day. In some examples, ethambutol is provided in a total daily dose in the range of about 1.0 to 2.0 g per day, while in other embodiments, it is provided in a total daily dose in the range of about 1.0 to approximately 1.5 g per day, including 1.1, 1.2, 1.3, and 1.4 g.
In some examples, pharmaceutical compositions including DMSO and / or MSM are used to pretreat a patient suffering from an infectious disease, such as H1N1. In some examples, the dose of DmsO and / or MSM used to pretreat patients ranges from about 10% to 50% weight to volume. In some examples, the pretreatment dose of DMSO and / or MSM ranges from about 20% to about 40%, from about 25% to 35%, including 26, 27, 28, 29, 30, 31,32 , 33, and 34%. In some examples, DMSO and / or MSM is used from about 50% to about 100%. In several examples, pretreatment with DMSO and / or MSM enhances the ability of an antibiotic to inhibit bacterial activity and / or sensitizes a drug resistant strain to a drug that was previously ineffective.
In some examples, a pharmaceutical composition is prepared in which antimicrobial agents are dissolved in DMSO and / or MSM prior to administration. This is particularly advantageous in certain examples, because the antimicrobial and DMSO (and optionally MSM) can be administered to a subject by inhalation. Inhalers, according to some examples, provide direct access of DMSO and / or MSM to infected lung tissue to sensitize bacterial cells to the antibiotic.
In one example, an inhaler is provided to target the site of infection (eg, the lungs) of various infectious diseases. In some similar examples, the inhalation device comprises a nebulizer. In other examples, an inhaler is used. In some examples, a metered dose pressure inhaler is used, and the formulation is inhaled as a liquid aerosol. In other examples, dry powder inhalers are used, and the formulation is inhaled in an aerosol powder form. In various embodiments, oral, intravenous, intramuscular, or subcutaneous administration is used in addition to or in place of inhalation therapy.
The ability to deliver antimicrobial agents in the form of an inhalation (for example, in a powdered aerosol form) with DMSO and / or MSM is especially advantageous in some examples because it allows greater stability during storage and prepackaged doses. . This is particularly useful for individuals in underdeveloped or developing countries who do not have regular access to sanitary facilities. Full cycles of treatment can be administered to an affected subject in a single visit to a healthcare professional without the need for a hospital stay or repeat visits. In various examples, the formulations disclosed herein are suitable for self-administration (eg, by inhalation devices) and therefore are especially suitable for patients with limited access to healthcare.
In certain examples, the total volume of inhaled DMSO and / or MSM is approximately 2-8 ml. In some examples, the total volume of inhaled DMSO and / or MSM is from about 2 ml to about 4 ml. In some examples, the total volume of inhaled DMSO and / or MSM is from about 6 ml to about 8 ml. In still other examples, the total volume of inhaled DMSO and / or MSM is from about 3ml to about 7ml. including 4, 5, and 6 ml. Therefore, in some examples, the DMSO concentration
ES 2,616,630 T3 administered by inhalation ranges from about 65% to about 95%, including 70, 75, 80, 85, 86, 87, 88, 89, 90, 91.92, 93, and 94%.
In several examples, MSM is included with DMSO and antimicrobial compounds. In certain examples, the amount of mSm inhaled ranges from about 0.01% by weight to about 70% by weight of the inhalant. In other examples, the inhaled formulation contains between about 0.01% and 10% MSM by weight. Other examples contain between about 10-20% MSM, about 20-30% MSM, about 30-40% MSM, about 40-50% MSM, about 50-60% MSM, or about 60-70% mSm, including 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70% MSM. Still other examples comprise a formulation containing about 7 and 15% MSM, about 15-25% MSM, about 25-35% MSM, about 35-45% MSM, about 55-60. % MSM, about 60-65% MSM, or about 65-70% MSM. Therefore, in some examples of the inhaled formulation containing MSM, the administered DMSO concentration ranges from about 50% to about 95%, including 55, 60, 65, 70, 75, 80, 85, 86, 87 , 88, 89, 90, 91, 92, 93, and 94%.
In several examples, the use of MSM reduces the amount of DMSO needed to achieve a comparable effect and / or improves the effectiveness of DMSO by at least 10%, 25%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 50 times, or 100 times. In other examples, the use of MSM reduces the amount of a therapeutic agent necessary to achieve a comparable effect and / or improves the efficacy of the therapeutic agent by at least 10%, 25%, 50%, 100%, 2 times, 3 times, 5 times, 10fold, 50 times, or 100 times. In additional examples, the use of DMSO reduces the amount of a therapeutic agent necessary to achieve a comparable effect and / or improves the efficacy of the therapeutic agent by at least 10%, 25%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 50 times, or 100 times. In still other examples, the use of DMSO and MSM reduces the amount of a therapeutic agent necessary to achieve a comparable effect and / or improves the efficacy of the therapeutic agent by at least 10%, 25%, 50%, 100%, 2 times, 3 times, 5 times, 10 times, 50 times, or 100 times compared to DMSO or MSM alone and / or the therapeutic agent alone.
In various examples, a pretreatment formulation that includes DMSO alone or in combination with MSM is administered intravenously, intramuscularly, topically, or orally to a subject to enhance the effects of an inhalation therapy comprising DMSO and / or MSM with therapeutic agents. , such as antibiotics. Pretreatment with DMSO, alone or in combination with MSM, improves the therapeutic effects of the inhalant by at least 10%, 25%, 50%, 100%, 2 times, 3 times, 5fold, 10 times, 50 times, or 100 times.
In various examples, subjects having an infectious disease are re-treated with a formulation comprising, consisting or consisting essentially of DMSO, alone or in combination with MSM, and one or more therapeutic agents, such as antibiotics. In some examples, the formulation further includes other therapeutic agents, carriers, or excipients. In one example, the formulation further includes arginine, vitamin D, antioxidants, macrolides, linezolid, thioacetazone, thioridazine, or combinations thereof.
DMSO easily alters the integrity of many materials (particularly plastics and polymers used in the manufacture of disposable medical equipment). Accordingly, several examples comprise devices to facilitate the storage and administration of DMSO. In some examples, DMSO is stored in glass bottles and administered through non-reactive lines. In other examples, the inhaler devices are specifically designed to be resistant to DMSO. In some examples, the parts of the inhaler devices are disposable or replaceable. According to various examples, formulations comprising DMSO are manufactured, stored and / or administered using materials and devices disclosed in United States Patent Application No. 12 / 066,480, which is the national phase entry of the International Application No.: PCT / US06 / 35499, filed September 11, 2006.
In certain examples, the delivery device delivers drops or particles of the inhaled formulation of a size capable of reaching the bronchioles in the lungs of patients. In some examples, the delivery device is synchronized with the patient's respiratory rate to deliver the formulation to the bronchioles. Inhalation therapy, according to one example, allows more direct administration of the inhaled formulation to target lung tissues. Direct targeting is advantageous in some examples because it allows reduction of the amount of antimicrobial compounds incorporated into the formulation while maintaining or improving the efficacy of the formulation against infectious microorganisms. In other examples, direct administration increases the efficacy of a given antimicrobial regimen against one or more drug resistant strains of the microorganism. Direct targeting, according to other examples, minimizes side effects by minimizing contact with non-target tissue.
The small droplet or particle size provided according to some examples reduces the volume of DMSO and / or MSM delivered compared to traditional ventilatory therapy. For example, in one example, use of an inhalation device (eg, nebulizer) will be effective with from about 6 mg to about 25 mg of DMSO and / or MSM per day, compared to 50-100 mg per day when it is administered through other specific routes. Reducing DMSO is beneficial in some examples because it reduces unwanted side effects and odor. In other examples, larger amounts of DMSO are used and tolerated.
ES 2 616 630 T3
In several examples, the addition of MSM unexpectedly reduces the unpleasant odor normally experienced with the use of DMSO. For example, in certain examples, the DMSO and MSM formulations do not produce a noticeable odor after use. In some other examples that have DMSO concentrations approaching or exceeding 50%, the combination with MSM in the formulation reduces or eliminates the odor caused by the DMSO. Such a result is unexpected, since the use of DMSO is normally associated with a strong unpleasant odor.
In some examples, the use of DMSO and / or MSM with therapeutic agents (such as antibiotics) allows the manufacture and / or administration of small droplets or particle sizes, thereby reducing irritation of the mucosa of the mouth and the mouth. throat, and the droplets or particles are transported deeper into the patient's lungs. In some examples, the depth of transport of the droplets or particles increases the concentration of the dissolved antibiotics in the lungs of the patient.
In various examples, the DMSO and / or MSM compositions are combined with therapeutic agents (such as antibiotics) and provided in the form of an aerosol to deliver locally active drugs to the respiratory system to treat respiratory disease. In one example, the lower respiratory tract is contacted (or exclusively contacted) with the composition. In other examples, the composition is used to treat diseases systemically. For systemically active drugs, aerosol particles are given a size such that they reach the alveolar surface in the peripheral areas of the lung.
In some examples, the use of DMSO and / or MSM compositions comprising a therapeutic agent (such as an antibiotic) is particularly advantageous because it provides rapid onset of action. In one example, delivery by inhalation provides a large lung absorption zone. For locally acting drugs, in some examples, the onset of action is immediate. Systemically active inhaled formulations, according to some embodiments, quickly reach the bloodstream. Inhalation therapy provides, in some examples, a therapeutic effect in about 1-90 minutes. In one example, DMSO and / or MSM enhance the bioavailability of the therapeutic agent. In a further example, DMSO and / or MSM reduce the degradation of the therapeutic agent. In another example, the aerosol formulations disclosed herein reduce gastrointestinal side effects or skin irritation that can occur with oral or topical treatment.
In several examples, the inhalant particles have a size that minimizes the deposition of these particles by inertial impact in the upper respiratory tract without reaching the site of action. In several examples, the particles are sized to minimize deposit in the mouth and throat, thereby minimizing ingestion and unwanted local or systemic side effects. In several examples, the particles are smaller than 2, 5 or 10 pm. In one example, the particles are about 3-5 pm and are transported into the bifurcations and the smaller airways of the bronchi and bronchioles. In another example, the particles are smaller than 3 pm and follow the airflow to the alveoli. In various examples, the use of DMSO and / or MSM allows for the optimization of the particle size of the therapeutic agent. Therefore, diseases, such as an infectious disease, can be more effectively treated. Furthermore, in various examples, the use of DMSO and / or MSM sensitizes drug resistant microorganisms to antibiotics.
In various examples, DMSO and / or MSM form a solution, mixture, emulsion, suspension, or other suitable combination with the therapeutic agent. In one example, homogenization, sonication, high shear fluid processing, or other mechanical methods are used to combine the therapeutic agent with the DMSO and / or MSM. In other examples, the therapeutic agent readily dissolves in DMSO. Unlike other strong solvents, DMSO is not harmful to lung tissue. Therefore, DMSO is especially advantageous in some examples because it can both dissolve the therapeutic agent and deliver said agent without damaging lung tissue. In some examples, DMSO dissolves at least 50%, 75%, 90%, 95%, or 99% of the therapeutic agent, and in one example, it is capable of preventing unwanted precipitation of the therapeutic agent.
In some examples, sprays, gels, or wet wipes comprising DMSO alone or in combination with MSM and antibacterial agents are provided to sanitize medical equipment, surfaces, and the body to minimize the spread of infectious diseases.
In various examples, a pharmaceutical composition comprising DMSO and / or MSM and antimicrobial agents is used as a treatment for an infectious disease.
In certain examples, the compositions disclosed herein are effective in treating various infectious diseases including, but not limited to, Acinetobacter infection, actinomycosis, adenovirus infection, African sleeping sickness (African trypanosomiasis), AIDS, amebiasis, anaplasmosis, anthrax. , Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis (BV), Bacterioid infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black stone, Blastocystis hominis infection, Blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, Botulism, hemorrhagic fever
Brazilian ES 2 616 630 T3, brucellosis, Burkholderia infection, calicivirus infection, campylobacteriosis, candidiasis (moniliasis; thrush), cat scratch disease, cellulitis, Chagas disease, chancroid, chickenpox, chlamydia, Chlamydophila pneumoniae infection, cholera, chromoblastomycosis, clonorchiasis, Clostridium difficile infection, coccidioidomycosis, Colorado fever, common cold, Creutzfeldt disease Jacob, Crimean-Congo hemorrhagic fever, cryptococcosis, cryptosporidiosis, cutaneous larva migrant (CLM), cycloesporiasis, cystocerciasis, cytomegalovirus infection, dengue fever, Dientamoebiasis, diphtheria, diphyllobothriasis, dracunculiasis, Ebola hemorrhagic fever, echinococcosis, ehrlichiosis, enterobiasis (roundworm infection) Enterococcus infection, enterovirus infection, epidemic phytus, infectious erythema, sudden fasciolitis, fossil fungus ), filariasis, food poisoning, free living amoeba infection, Fusobacterium infection, gas gangrene (clostridial myonecrosis), geotrichosis, Gerstmann-Straussler-Scheinker syndrome (GSS), giardiasis, glanders, gnatostomiasis, gonorrhea, inguinal granuloma (donovanosis), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand disease, foot and mouth (HFMD), Hantavirus, Helicobacter pylori infection, Hemolytic uremic syndrome (HUS), Hemorrhagic fever with renal syndrome (HFRS), Hepatisis A, B, C, D, or E, Herpes Simplex, Histoplasmosis, Hookworm infection, human bocavirus infection, human ewingii ehrlichiosis, human granulocytic anaplasmosis (HGA), human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus (HPV) infection, human parainfluenza virus infection, and hymenolepiasis.
In certain examples, the formulations disclosed herein are also effective for treating one or more of the following infectious diseases, Epstein-Barr virus infectious mononucleosis (mononucleosis), influenza, isporiasis, Kawasaki disease, keratitis, Kingella infection kingae, Kuru, Lassa fever, legionellosis, leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease, lymphatic filariasis, lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever (MHF), measles, melioidosis (Whitmore disease), meningitis, meningococcal disease, metagonimiasis, Microsporidiosis Microsporidia, Molluscum contagiosum (MC), mumps, murine typhus, Mycoplasma pneumonia, mycetoma, myiasis, neuropathic conjunctivitis River blindness), paracoccidioidomycosis (South American blastomycosis), paragonimiasis, pasteurellosis, capillary head lice (lice), body pediculosis (body lice), pubic pediculosis (pubic lice, crabs), pelvic inflammatory disease (PID), whooping cough, plague, pneumococcal infection, Pneumocystis pneumonia (PCP), pneumonia, polio, poliovirus, primary amoebic meningoencephalitis (PAM), multifocal leukoencephalopathy, progressive fever, Q, rabies, rat bite fever, respiratory syncytial virus, rhinosporidiosis, rhinovirus infection, rickettsial infection, Rickettsialpox, Rift Valley fever (RVF), Rocky Mountain Spotted Fever (RMSF), rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, schistosomiasis, septicemia, shigellosis, herpes (shingles), smallpox, sporotrichosis, staph food contamination, infection Staphylococcal, strongyloidiasis, syphilis, taeniasis, tetanus (trismus), Tinea barbae (barber's itch), Tinea capitis (ringworm of the scalp), Tinea corporis (ringworm of the body), Tinea cruris (jock itch), Tinea manuum (ringworm of the hand), Tinea nigra, Tinea pedis (athlete's foot), Tinea unguium (onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis (Ocular Larva Migrans (OLM)), Toxocariasis (Visceral Larva Migrans (VLM)), toxoplasmosis, trichinellosis, trichomoniasis, trichuriasis (whipworm infection), tularemia, Ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, white stone, yersiniosis, yellow fever, and zygomycosis.
In various examples, the compositions disclosed herein are particularly effective in treating one or more infections that are resistant to drug therapies. In addition to those infectious diseases listed above, which may become or become drug resistant in the future, certain examples are effective in treating, among others, the following drug resistant diseases: measles, tetanus, malaria, upper and lower respiratory infections, hepatitis, typhoid fever, vancomycin-resistant Staphylococcus auerus infection / glycopeptide intermediate, vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus pneumoniae.
In some examples, treatment of an infectious disease comprises pretreatment of a patient with DMSO, followed by administration of a pharmaceutical composition comprising DMSO and antimicrobial agents. In other examples, treatment of an infectious disease comprises pretreatment of a patient with DMSO, followed by administration of a formulation comprising DMSO, MSM, and antimicrobial agents. In some examples, DMSO pretreatment is administered intravenously through a rapid IV drip catheter. In other examples, DMSO is given as an IV bolus injection. In still other examples, pretreatment with DMSO is not performed. Pretreatment compositions further include MSM, a therapeutic agent, or a combination thereof in some examples.
In various examples, compositions including DMSO and antimicrobial agents, or DMSO, MSM, and antimicrobial agents are administered orally, intravenously, intramuscularly, or subcutaneously. However, because the site of infection for various infectious diseases is the lungs, in some examples, the formulations are administered by inhalation. In some similar examples, the inhalant comprises a nebulizer. In another example, an inhaler is used.
In various examples, subjects are pretreated with DMSO using intravenous DMSO via rapid dropper over, for example, a ten minute period. In one example, DMSO will be provided in glass bottles with tubes not
ES 2 616 630 T3 own reagents. The subjects will then receive the antibiotics dissolved in DMSO in 3 ml doses via inhaler or a mouth spray three times a day with meals. In one example, DMSO pretreatment is provided in the range of about 25 mg to about 75 mg (eg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg) in 200 ml of 5% dextrose and water. . In one example, 56 mg of DMSO is provided in 200 ml of 5% dextrose and water. In one example, the following antibodies are provided: rifampin, isoniazid, pyrazinamide, and ethambutol. In one embodiment, approximately 600 mg of rifampin, 300 mg of isoniazid, 2.4 g of pyrazinamide, and 1.2 g of ethambutol are administered per day, via an inhaler / nebulizer or mouth spray delivered in 3 ml doses. three times a day. In one example, antibiotics are combined with DMSO for delivery by inhalation, with or without pretreatment with DMSO. In several examples pretreatment with MSM is also provided. Pretreatment with DMSO, MDM, or the combination of the two is provided in some examples. In some examples, the pretreatment formulations include therapeutic agents.
In various examples, therapeutic effects are obtained at two weeks of treatment, two weeks of treatment, and / or six months of treatment. Other therapeutic windows are also provided.
In some examples, patients pretreated with DMSO show greater improvement than those treated with inhaled DMSO and antibiotics without intravenous DMSO pretreatment. In some examples, patients treated with DMSO with inhaled DMSO and antibiotics show greater improvement than those treated with antibiotics alone. In various examples, the addition of MSM to the formulation improves therapeutic effects or reduces side effects. In one example, only MSM is used as a pretreatment.
In various examples, the compositions disclosed herein are used not only to treat unwanted symptoms and illnesses, but can also act as preventive agents. For example, the formulation can be taken on a regular basis to prevent disease occurrence. In one example, subjects at risk (eg, relatives or subjects who are exposed to patients who have an infectious disease) are given lower doses of DMSO and / or MSM and antibiotics to prevent the onset of infection.
IV. MSM usage methods
Disclosed herein are methods for using any of the disclosed MSM compositions (as described in section III) to modulate microbial activity, such as to enhance or inhibit the activity of microorganisms. For example, methods for enhancing microbial activity are disclosed including methods for enhancing microbial growth, fermentation efficiency, culture efficiency, microbial survival, or any combination thereof. Methods for inhibiting microbial activity including methods for inhibiting microbial growth or infection (such as bacterial growth) are also disclosed. In some embodiments, MSM selectively enhances the activity (eg, growth) of a microorganism (such as a probiotic microorganism) and inhibits the activity of unwanted microbes (such as unwanted bacterial or fungal activity).
A. Methods to enhance microbial activity
Methods for enhancing microbial activity are disclosed. In one embodiment, a method of enhancing the activity of a microorganism includes providing microorganisms, a medium capable of supporting the growth of the microorganisms, and MSM in an amount sufficient to enhance the activity (e.g., fermentation efficiency, growth , culture efficiency, and / or microbial survival) of the microorganisms and contacting the MSM with the medium, thus enhancing the growth of microorganisms in the medium. It is contemplated that MSM may be added to the medium prior to, concurrently with, or after the medium has been contacted with the microorganisms. In a particular embodiment, MSM is provided at a concentration of from about 0.04% to about 5% by weight of the medium or by weight of the moisture content of the medium. As such, in some examples, MSM (such as a composition that includes from about 0.5% to about 5% MSM) is used to enhance microbial growth. For example, MSM is used to enhance the efficiency of fermentation, such as to enhance the efficiency of fermentation associated with the production of beer, cider, wine, a biofuel, a dairy product, or any combination thereof. In several examples, MSM enhances the production of certain food or beverage manufacturing processes that depend on microorganisms, such as beer production, wine production, bakery, pickling, dairy production, and the like. In additional examples, MSM is used to enhance the growth of one or more probiotic microorganisms or of a microorganism in a diagnostic test sample. In still other examples, MSM is used to enhance culture efficiency and / or survival of microorganisms.
i. Methods to enhance the efficiency of fermentation of microorganisms with MSM
In various embodiments, MSM is used to facilitate energy production. Therefore, methods for enhancing energy production are disclosed herein, including methods for enhancing the efficiency of the fermentation of microorganisms. For example, microorganisms can be used in a fermentation process to produce ethanol, and in biogas reactors to produce methane. Fermentation is an energy-producing process
ES 2 616 630 T3 by which organic or synthetic molecules are degraded by the metabolism of microorganisms. Some forms of microorganisms, such as bacteria or yeasts, can be used to convert various forms of agricultural and municipal waste into usable fuels. Microorganisms can be used as living microbial fuel cells. In some embodiments, MSM enhances bacterial growth and metabolism. In some embodiments, MSM enhances bacterial energy production. In some embodiments, MSM enhances yeast growth and metabolism. In some embodiments, MSM enhances yeast energy production.
In various embodiments, MSM is used to activate or enhance one or more of the following: (i) ethanol fermentation or other anaerobic respiration used primarily by yeast when oxygen is not present in an amount sufficient for normal cellular respiration; (ii) fermentative hydrogen production; (iii) industrial fermentation and other degradation and reconfiguration of biochemical agents for industries; (iv) the conversion of carbohydrates to alcohols or acids under anaerobic conditions used for food preparation (eg, breads, dairy products, beans, vinegar, sauerkraut, kimchi, fish, and tofu); (v) fermentation to produce brandy, whiskey, vodka, beer, wine or cider, (vi) fermentation to produce glucosamine; and (vii) fermentation for the aerobic treatment of tea leaves to break down unwanted chemical agents and produce others that impact, for example, the aroma, and / or nutrients of the tea.
In one embodiment, a method of enhancing the fermentation of a microorganism includes contacting a medium containing a microorganism capable of fermentation with MSM, wherein the MSM is provided at a concentration of from about 0.04% to about 5%. % by weight of the medium or at a concentration of from about 0.04% to about 5% by weight of the moisture content of the medium, wherein the concentration of MSM increases the fermentation efficiency of the microorganism compared to the fermentation efficiency in the absence of MSM.
In one embodiment, the enhanced fermentation efficiency is indicated by an increase of at least 10%, such as from about 20% to about 80%, an increase from about 30% to about 50%, including a increase of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% in the production of alcohol, carbon dioxide or acid in the presence of MSM by microorganisms compared to the production of alcohol, carbon dioxide or acid in the absence of MSM. For example, the method of enhancing the efficiency of fermentation is for the production of beer, cider, wine, a biofuel, bread, a dairy product, or any combination thereof. In some examples, enhancing fermentation efficiency includes an increase of at least 10%, such as from about 20% to about 80%, an increase from about 30% to about 50%, including an increase in about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% in the production of ethanol, methanol, or a combination thereof compared to the production of ethanol, methanol, or a combination thereof in the absence of MSM. In a particular example, the microorganism is yeast and the method to enhance fermentation is for the production of beer. In another example, the microorganism is an algae and the method to enhance fermentation is for biofuel production.
In some embodiments, enhancing fermentation efficiency includes an increase of at least 10%, such as from about 20% to about 80%, an increase from about 30% to about 50%, including an increase in about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% in the production of carbon dioxide in the presence of MSM by the microorganisms compared to the production of carbon dioxide in the absence of MSM. In a particular example, the microorganism is yeast and the method to enhance fermentation is for the production of bread.
In additional embodiments, MSM is used to control the fermentation process in the production of cultured dairy products, such as yogurt, milk, cheese, and the like. For example, methods for enhancing fermentation efficiency include an increase of at least 10%, such as from about 20% to about 80%, an increase from about 30% to about 50%, including a increase of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% in the production of lactic acid in the presence of MSM by the microorganisms compared to the production of lactic acid in the absence of MSM.
In some embodiments, the concentration of MSM that is effective in enhancing fermentation efficiency is from about 0.04% to about 5%, such as from about 0.1% to about 0.5%.
ES 2 616 630 T3%, from 0.5% to about 3%, from about 1% to about 2%, including about 0.04%, to about 0.05%, about 0, 06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.3%, about 0.5%, about 0.7 %, about 1%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 4%, or about 4.5% by weight of medium or moisture content of the medium. In some embodiments, MSM is added to yeast packages to generate rapid or rapid activation yeast for home or commercial use.
In some examples, the medium for the method of enhancing the efficacy of a microorganism includes a sodium chloride concentration of less than 5% of the total moisture content of the medium, such as from about 1% to about 3% chloride. sodium, including 0%. 0.1%, 0.3%, 0.5%, 0.75%, 1%, 2%, 2.5%, 3%, or 4%.
In a certain embodiment, MSM is used to produce beer. Yeast cultures are involved in the production of beer during the fermentation process to produce ethanol and carbon dioxide. In some examples, MSM is used to accelerate or facilitate yeast culture activation, enhance fermentation, reduce potential environmental contamination (such as by unwanted airborne microorganisms), or a combination thereof. For example, an increase in the efficiency of activating yeast (such as an increase in the efficiency of the starter process), an increase in the efficiency of the fermentation process, or a combination thereof is indicated by an increase of at least 10 %, such as from at least 20% to 80%, an increase from about 30% to about 50%, including an increase from about 10%, about 20%, about 30%, about 40 %, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% compared to a control ( such as the efficiency of these processes in the absence of MSM).
In various embodiments, MSM is used to enhance the activity of algae, including the fermentation process associated with the generation of biofuel through the use of algae. In one embodiment, this is particularly beneficial for algae cultivation (algae farms) to produce vegetable oil, biodiesel, bioethanol, biogasoline, biomethanol, biobutanol, and / or other biofuels. In one embodiment, the addition of MSM increases the growth rate of algae by about 25%, about 30%, about 40%, about 50%, about 100%, about 200%, about 300%. , about 400%, about 500% or more. MSM can be particularly advantageous because, by enhancing algae activity (such as algae growth), biofuel production can be made scalable, economically competitive, and / or commercially viable. In one embodiment, MSM enhances the process by which the product is harvested from the algae and converted to biodiesel. In other embodiments, MSM enhances the process by which the carbohydrate content of algae is fermented into bioethanol and biobutanol. In some embodiments, MSM enhances the algae process by (i) increasing the yield of the algae, (ii) forming more robust algal colonies, (iii) shortening the time to harvest, (iv) shortening the fermentation time , (v) enhancing fermentation, and / or otherwise supporting or enhancing growth, reproduction, proliferation, survival rate, metabolism, vitality, robustness, action, and / or function of the algae . Algae, including, but not limited to, Botryococcus braunii, Chlorella, Dunaliella tertiolecta, Gracilaria, Pleurochrysis portfolioe, and Sargassum are enhanced by MSM in accordance with various embodiments.
ii. Methods to enhance microbial growth with MSM
In some embodiments, the addition of MSM is particularly advantageous because MSM promotes the growth of certain microorganisms (eg, probiotics). In some embodiments, microorganisms grown with a medium composition comprising MSM have a higher growth rate curve compared to a comparable composition without MSM. In some embodiments, microorganisms grown with a composition comprising MSM have an increased general population density compared to a comparable composition without MSM. In certain embodiments, MSM significantly enhances the simultaneous growth of one or more microorganisms. In some embodiments, a medium supplemented with an MSM composition to enhance microbial activity (such as a concentration range from about 0.4% to about 5% or any of the MSM compositions to enhance microbial growth provided in section III) enhances the growth of microorganisms.
Some microorganisms are anaerobic (anaerobic) organisms. Anaerobic organisms do not need oxygen for their growth. Anaerobic organisms can be used for fermentation and / or cultivation. In some embodiments, MSM has a positive impact on anaerobic organisms, such as Bifidobacterium, among others. In some of these embodiments, MSM has a greater positive impact on the growth of anaerobic organisms than on other microorganisms. In other embodiments, MSM has a greater positive impact on the growth of aerobic bacteria compared to other microorganisms. In still other embodiments, aerobic and anaerobic organisms are positively affected by the presence of MSM.
ES 2 616 630 T3
Bacteria can be generally classified as gram-positive or gram-negative, depending on the structure of their cell wall. Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas, Salmonella, Shigella, Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella, alpha-proteobacteria, cyanobacteria, spirochetes, and green sulfur bacteria. greens not sulfur. Enteric bacteria are rod-shaped gram-negative bacteria; most appear normally or pathogenically in the intestines of humans and other animals. In some embodiments, MSM has a positive impact on the growth of gram-positive bacteria. In other embodiments, MSM has a positive impact on the growth of gram-negative bacteria. In some of these embodiments, MSM has a greater positive impact on gram-negative bacteria than on gram-positive bacteria. In other embodiments, MSM has a greater impact on gram-positive bacteria than on gram-negative bacteria. In still other embodiments, MSM has a positive impact on both gram-negative and gram-positive bacteria.
Probiotics include live microorganisms that are considered to be healthy for the host organism. Lactic acid bacteria (LAB) and bifidobacteria are common types of microbes used as probiotics. Certain yeasts and bacilli are also used. In various embodiments, MSM is used to enhance the survival or growth of at least one probiotic. The effect on survival of probiotic organisms can be measured at three points according to some embodiments: survivability, colonization, and lactic acid production. To be effective in maintaining the health of the gastrointestinal tract, probiotic bacteria must be able to survive. Bacteria that are dead on arrival, in most cases, provide no benefit. Therefore, in some embodiments, MSM positively affects the survival of probiotics. In certain embodiments, MSM improves initial survival after exposure of the bacteria to a new environment. Therefore, in such embodiments, a product comprising a probiotic and MSM establishes a larger or healthier population (or both) of probiotic bacteria in the gut compared to probiotic products alone. In certain embodiments, MSM improves the long-term survival of probiotics. Therefore, in such embodiments, a product comprising a probiotic and MSM establishes a longer-lasting, and growth-based, population of probiotic bacteria in the gut compared to probiotic products alone. Of those probiotic bacteria that reach the intestine alive, those that colonize (multiply in) the intestine provide benefits. Therefore, in various embodiments, MSM improves the speed and frequency of probiotic multiplication. In still other embodiments, MSM increases lactic acid production.
In some embodiments, MSM has a positive impact on probiotic growth. In some embodiments, MSM has a positive impact on the microbial flora of the gastrointestinal tract. In some of these embodiments, MSM has a positive impact on intestinal health. In some embodiments, the probiotic-containing antibodies are supplemented with MSM, and the resulting probiotic levels achieved in the intestinal tract are higher than after ingestion of the probiotic-containing food alone. In some such embodiments, the addition of MSM results in a higher level of probiotic organisms in a shorter period of time compared to ingesting only a probiotic-containing food. In some embodiments, probiotics need 24 to 48 hours before effects are seen and become more effective because MSM increases their lifespan.
Bacterial growth normally has an initial lag phase where bacteria adjust to the environment, before moving to the logarithmic phase, where cells duplicate. After the logarithmic phase, a stationary phase occurs. During the stationary phase, growth rate is reduced as a result of nutrient depletion and accumulation of metabolic by-products. This phase is reached as the microbes begin to deplete the resources that are available to them. This phase is at a relatively constant value since the rate of microbial growth is equal to the rate of microbial death. In the death phase, bacteria have normally depleted nutrients and population numbers are greatly reduced.
In some embodiments, MSM impacts lag phase, log phase, stationary phase, death phase, or any combination thereof. In certain embodiments, MSM shortens the lag phase such that bacteria, such as probiotic bacteria, begin the log phase earlier. In various embodiments, MSM extends the stationary phase. In certain embodiments, the death rate is reduced in the presence of MSM. Certain embodiments of the disclosure as described herein affect one or more, and in certain embodiments all, stages of probiotic bacteria growth.
In some embodiments, MSM impacts the metabolism of microbes (eg, probiotics), in the lag phase. During the lag phase, the microbes are maturing (increasing in size) and are not yet able to divide (therefore, they do not increase in number). During the logarithmic phase of the microbial growth cycle, the synthesis of RNA, enzymes, and other molecules occurs. In some embodiments, MSM reduces the duration of the lag phase by accelerating the maturation (and adaptation of the microorganisms to environmental stressors) of the microorganisms, thereby allowing microbial division earlier than in medium without MSM.
In some embodiments, MSM supplementation results in an increase in the log phase of
ES 2 616 630 T3 growth of microbes (eg probiotics). The exponential phase (sometimes called the logarithmic phase) of growth is a period characterized by cell duplication. The number of new microbes that appear per unit of time is proportional to the population present. In case of not limiting growth, doubling will continue at a constant rate, whereby the number of cells and the rate of population increase doubles with each consecutive period of time. However, exponential growth cannot continue indefinitely, as the medium is depleted of nutrients and rapidly enriched with waste. In some embodiments, MSM increases the overall duration of the exponential phase. In other embodiments, the presence of MSM in the growth medium promotes microbial entry into the exponential phase more rapidly than in microorganisms in medium without MSM. The initial growth environment with medium supplemented with MSM can lead to cell multiplication and survival.
In various embodiments, MSM affects the stationary phase of microbial (eg, probiotic) growth. In one example, supplementation of the medium with MSM prolongs the stationary phase for the microbes compared to the medium without MSM.
In some embodiments, MSM enhances probiotic growth, which in turn saturates and deprives unwanted microbes of nutrients. In other embodiments, MSM enhances probiotic activity, which in turn enhances lactic and acetic acid production to lower ambient pH and inhibit the activity of unwanted bacteria. In additional embodiments, MSM enhances probiotic activity, which in turn stimulates the production of immunomodulatory agents (eg, cytokines), thereby enhancing the immune response. In certain embodiments, MSM enhances probiotic activity, which in turn enhances bactericidal activity against unwanted microbial contamination. In one embodiment, MSM enhances probiotic growth at a higher rate than that of unwanted microbes, thereby allowing the probiotics to preferentially colonize an environment (eg, edible products, intestinal tract).
Without being bound by theory, in various embodiments, MSM has a biochemical effect on microbial metabolism. For example, in some embodiments, the addition of MSM has a positive effect on the metabolism of certain microorganisms, such that certain microorganisms are able to better adapt and / or recover from environmental changes. In some embodiments, MSM serves as a substrate or cofactor for microbial metabolism and / or anapleurotic biochemical pathways. In some embodiments, MSM has a positive impact on the delayed growth phase. In some embodiments, MSM increases the log phase of microbial growth. In still other embodiments, MSM increases the duration of the stationary phase of microbial growth. In some embodiments, MSM slows down the population reduction of certain microbes. In certain embodiments, MSM provides a selective or semi-selective growth environment, such that certain microbial species grow faster (or reach a larger population size, or both) compared to other microbial species. In certain embodiments, MSM impacts the metabolic activity of microorganisms, while in other embodiments, MSM creates an environment more conducive to microbial growth.
As such, methods are provided to enhance microbial growth. In some embodiments, methods for enhancing microbial growth include in vitro methods for enhancing the growth of one or more microorganisms. In one example, in vitro methods for enhancing the growth of one or more microorganisms include contacting one or more microorganisms with a medium capable of supporting the growth of one or more microorganisms; and providing MSM to the medium at from about 0.4% to about 5% by weight of the medium or by weight of moisture content of the medium, thereby enhancing the growth of the one or more microorganisms in vitro compared to the growth of the one or more microorganisms in vitro in the absence of MSM. It is contemplated that similar methods may be used to enhance the growth of desired microorganisms (such as probiotics) in vivo. For example, an increase in microbial growth is indicated by an increase in microorganism weight or cell number such as an increase of at least 10%, such as from about 20% to 80%, an increase of about 30% to about 50%, including an increase of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% compared to a control (such as the weight of the microorganism or the number of cells in the absence of MSM ). Increases in the growth of microorganisms can be detected by methods known to those of skill in the art, including those described in the examples.
to. Methods to enhance the growth of a probiotic microorganism
Methods for enhancing the growth of one or more probiotic microorganisms are disclosed. For example, methods of enhancing the growth of one or more probiotic microorganisms include contacting one or more probiotic microorganisms with a medium capable of supporting the growth of one or more probiotic microorganisms; and providing MSM to the medium at from about 0.4% to about 5% by weight of the medium or by weight of moisture content of the medium, thereby enhancing the growth of the one or more microorganisms as compared to the growth of the one or more microorganisms in the absence of MSM. In a
For example, the concentration of MSM is from about 1% to about 3% of the weight of the medium or by weight of the moisture content of the medium. An increase in probiotic growth is indicated by an increase of at least 10%, such as from approximately 20% to 80%, an increase from approximately 30% to approximately 50%, including an increase of approximately 10%. , about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% in cell growth compared to a control (such as cell growth in the absence of MSM).
In some examples, the medium for enhancing microbial growth, such as probiotic growth, includes probiotic-containing products, such as milk, yogurt, rice yogurt, frozen yogurt, chocolate, cheese, beer, wine, vinegar, sauerkraut or any combination thereof.
It is contemplated that the method may be used to enhance the growth of any probiotic microorganism, including, but not limited to, Lactobacillus acidophilus, Lactobacillus delbrueckii, Bacillus coagulans, Lactobacillus rhamnosus, Bifidobacterium bifidum, or any combination thereof. In one embodiment, the disclosed methods are used to enhance the activity of the bacterium Lactobacillus rhamnosus. In other embodiments, the disclosed methods are used to enhance the activity of species within the genus Lactobacillus. For example, a method of enhancing the activity (eg, growth) of Lactobacillus acidophilus includes contacting Lactobacillus acidophilus with a medium capable of supporting the growth of Lactobacillus acidophilus; and providing MSM to the medium at about less than about 1% (such as at about 0.04%, 0.05%. 0.1%, 0.2%, 0.3%, 0.4%, 0.5%. 0.6%, 0.75, 0.8% or 0.9%) by weight of the medium or by weight of a moisture content of the medium, thereby enhancing the growth of Lactobacillus acidophilus compared to the growth of Lactobacillus acidophilus in the absence of MSM.
In other embodiments the disclosed methods are used to enhance the activity of Bifidobacterium bifidum. For example, a method of enhancing the activity (eg, growth) of Bifidobacterium bifidum includes contacting Bifidobacterium bifidum with a medium capable of supporting the growth of Bifidobacterium bifidum; and providing MSM to the medium at about less than about 1% (such as at about 0.04%, 0.05%. 0.1%, 0.2%, 0.3%, 0.4%, 0.5%. 0.6%, 0.75, 0.8% or 0.9%) by weight of the medium or a moisture content of the medium, thereby enhancing the growth of Bifidobacterium bifidum compared to the growth of Bifidobacterium bifidum in absence of MSM.
An increase in probiotic growth is indicated by an increase in the weight of the probiotic microorganism or the number of cells thereof, including an increase of at least 10%, such as from approximately 20% to 80%, an increase of about 30% to about 50%, including an increase of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% compared to a control (such as the weight of the probiotic microorganism or the number of cells in the absence of MSM). Increases in the growth of probiotic microorganisms can be detected by methods known to those of skill in the art, including those described in the examples.
b. Methods to enhance the growth of a microorganism in a diagnostic test sample or an industrial test sample
Methods for enhancing the growth of a microorganism in a diagnostic test sample or an industrial test sample are disclosed. In one embodiment, a method is provided for enhancing the growth of a microorganism in a diagnostic test sample. In one example, the method includes contacting a diagnostic test sample (eg, blood, tissue, scrapings, body fluids and metabolic products, and the like) comprising one or more microorganisms with a medium capable of supporting the growth of the microorganisms. one or more microorganisms; and providing MSM to the medium at a concentration sufficient to enhance microbial growth, thereby enhancing the growth of the one or more microorganisms in the diagnostic test sample compared to the growth of the one or more microorganisms in the absence of MSM.
In some embodiments, a method is provided for enhancing the growth of a microorganism in an industrial test sample. In one example, the method includes contacting an industrial test sample (e.g., water sample, household mold, or sample of bacteria and other similar samples) comprising one or more microorganisms with a medium capable of supporting the growth of the bacteria. one or more microorganisms; and providing MSM to the medium at a concentration sufficient to enhance microbial growth, thereby enhancing the growth of the one or more microorganisms in the industrial test sample compared to the growth of the one or more microorganisms in the absence of MSM.
In various embodiments, MSM is provided in a composition to facilitate diagnostic testing or testing of industrial samples, such as at a concentration of from about 0.04% to about 5% by weight of the sample or by weight of the moisture content of the sample. In some embodiments, MSM is provided
ES 2 616 630 T3 in a composition to facilitate diagnostic tests or trials of industrial test samples, such as any of the MSM compositions capable of enhancing microbial activity that are described in section
III. In certain embodiments, MSM is added directly to the diagnostic or industrial test sample comprising microorganisms.
According to various embodiments described herein, MSM can shorten detection and / or analysis time by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. %. According to various embodiments described herein, MSM can enhance microbial activity (such as growth) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 90%, 100%, 2 times, 5 times, 100 times, 500 times, or 1000 times. For example, an increase in microbial growth is indicated by an increase in the weight of the microorganism or the number of cells in the microorganism, including an increase of at least 10%, such as from about 20% to 80%, an increase from about 30% to about 50%, including an increase of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% compared to a control (such as the weight of the microorganism or the number of cells in the absence of MSM ). Increases in the growth of microorganisms can be detected by methods known to those of skill in the art, including those described in the examples.
In various embodiments, MSM is used in conjunction with exploratory medical tests and rapid diagnostic tests, such as in urine or blood samples. In many cases, diagnostic tests are done to identify possible microbial infections. Various groups of microorganisms, including bacteria, viruses, molds, and yeasts, can cause infections. If a microorganism is found, further tests are done to determine which antibiotics might be effective in treating the infection. To diagnose such infections as early as possible, in some embodiments, MSM is used to supplement the growth medium used in diagnostic tests to increase the growth rate of microorganisms in the patient sample, thereby improving the detection time of the proof. In some embodiments, MSM can enhance the detection sensitivity of a diagnostic test. In some embodiments, the diagnostic test is a urine test. In some embodiments, the diagnostic test is a blood test. In other embodiments, other patient samples may be cultured for diagnostic purposes, such as sputum, saliva, skin swabs, dental swabs, cervical or vaginal swabs, and the like. In one embodiment, the MSM is used to provide a rapid test strip. For example, a body fluid sample (the diagnostic test sample) is added to a test tube or culture dish (the medium). The medium supports the growth of any microbes that may exist in the body fluid. By providing a medium pre-dosed with MSM or adding MSM to it before or after adding the body fluid to the test tube or culture dish, microbes in the body fluid (or its analyzable products or metabolites) could increase , it might be easier to rehearse. Therefore, the diagnosis is facilitated.
In various embodiments, the use of MSM facilitates the medical diagnosis of viral infections by supporting virus growth for diagnostic tests. Viruses include, but are not limited to, human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza, hepatitis, and other viruses. Similarly, the medical diagnosis of other infections, such as those caused by bacteria, fungi, yeasts and parasites is also facilitated by MSM according to various embodiments. In one embodiment, the use of MSM facilitates vaccine development.
In various embodiments, MSM is used to enhance microbial detection in a commercial or industrial test. Microorganisms are a common water pollutant. Many water safety test kits evaluate the quality of drinking water using Environmental Protection Agency (EPA) test methods for the presence of bacteria, among others. Molds found in home, office and school environments have been linked to lung disorders and allergic symptoms. However, some tests used to detect bacteria or mold can take a long time to analyze, while some tests also detect only viable (live) organisms. Therefore, in various embodiments, MSM is used to supplement the growth medium used in commercial screening tests. In some embodiments, the MSM supplemented medium improves the detection time of the tests. In some embodiments, the medium supplemented with MSM improves the detection sensitivity of such tests. In certain embodiments, MSM restores environmentally stressed bacteria that were previously not viable. In still other embodiments, diagnostic test kits comprising medium supplemented with MSM specific for microorganisms are used to enhance the detection time or sensitivity of a test designed to detect a particular microorganism. In other embodiments, MSM is used to supplement a wide variety of culture media, such that various microorganisms are detected more quickly or with greater sensitivity.
iii. Methods to enhance the survival capacity of microorganisms and cells with MSM
Methods for enhancing the survivability of microorganisms (including, but not limited to, probiotic microorganisms) or cells (such as stem cells or recombinant cells) are disclosed. For example,
ES 2 616 630 T3 Methods for enhancing the survivability of microorganisms or cells, such as in cell culture, include contacting one or more microorganisms or selected cells with MSM at from about 0.4% to about 5%. % by weight of the medium or by weight of a moisture content of the medium, thereby enhancing the survivability of the one or more microorganisms or the collection of cells compared to the survivability of the one or more microorganisms or the collection of cells in the absence of MSM. In one example, the concentration of MSM is from about 1% to about 3% of the weight of the medium or the moisture content of the medium. An increase in survivability is indicated by an increase of at least 10%, such as from approximately 20% to 80%, an increase from approximately 30% to approximately 50%, including an increase of approximately 10%. %, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% in the number of colonies or cells compared to a control (such as the number of colonies or cells in the absence of MSM).
According to various embodiments, MSM improves the initial survival of microorganisms (including, but not limited to, probiotic microorganisms). In one embodiment, MSM improves the long-term survival of the microorganisms. In one embodiment, MSM prolongs the stationary phase of a microorganism growth curve.
In various embodiments, MSM lengthens the shelf life of a product by extending the life span of beneficial bacteria compared to products without MSM. For example, a product containing probiotics may have a shelf life of several weeks, after which the probiotic organisms begin to lose their health and / or population. However, in some embodiments, the addition of MSM to a probiotic-containing product increases the time span from product packaging to reduction in the health and / or population of the probiotic. In such embodiments, the probiotic product is functional (in terms of delivering a population of healthy and active probiotics to the consumer's GI tract) for a longer period of time after packaging.
In various embodiments, the addition of MSM increases the time to spoilage of ingestible products by supporting or enhancing the activity of beneficial microbes, with a resulting reduction in the activity of unwanted microbes. For example, MSM can increase the shelf life of edible products, such as a probiotic product, by approximately 10% to 100% (e.g., 20%, 30%, 40%, 50%, 75%, 150%, 200% or more). For example, in one embodiment, in case the shelf life of an edible product is 10 days, the addition of MSM will increase the shelf life to at least 11 days in some embodiments (e.g. 11 days, 14 days, 15 days, 20 days, or 25 days). By way of further example, in another embodiment, in case an edible product has a shelf life of 14 days at room temperature and / or 30 days in the fridge and / or 3 months in the freezer, the addition of MSM will increase shelf life to 30 days at room temperature and / or 60 days in the refrigerator and / or 6 months in the freezer. In some embodiments, the use of MSM unexpectedly enhances the activity of beneficial microbes and inhibits (either directly or indirectly) the activity of unwanted bacteria, thereby reducing or eliminating the need for sterilization (e.g., by irradiation, filtration, heat, chemical agents, etc.).
In some embodiments, MSM is provided to enhance the activity of genetic vectors, such as recombinant viral vectors in recombinant cells. This can be beneficial for diagnostic as well as therapeutic agents, such as gene therapy. In some embodiments, MSM is used to enhance the activity ( eg, growth, culture, or viability) of one or more plasmid vectors, binary vectors, cloning vectors, expression vectors, shuttle vectors, and viral vectors. As such, methods for enhancing gene therapy are disclosed in which one or more processes associated with gene therapy are enhanced or augmented by treating the recombinant cell or organism with a concentration of MSM (such as a concentration of about 0 , 04% to approximately 5% MSM) capable of enhancing one or more gene therapy processes (such as expression, growth or survivability of recombinant cells or microorganisms), thereby increasing the efficacy of gene therapy.
iv. Methods to enhance the efficiency of culture with MSM
Methods for enhancing the efficiency of culture with MSM are disclosed herein. In one embodiment, methods are provided for enhancing various types of culture, including, but not limited to, enhancing the efficacy of culturing antibiotics, steroids, cells (eg, recombinant and wild-type), microorganisms, and fertilizers. For example, in various embodiments, MSM is used to supplement culture media used for the growth or propagation of microbial organisms. In various embodiments, MSM supplemented media enhances culture efficiency by enhancing cell growth.
In some embodiments, methods for enhancing culture efficiency include enhancing / promoting microbial activity in the environmental and industrial fields. Microorganisms participate in the cycles of elements such as the carbon cycle and the nitrogen cycle, as well as in the performance of other vital roles in practically all ecosystems, such as the recycling of waste products and / or the remains of other organisms through decomposition. Therefore, in some embodiments, the use of MSM can enhance decomposition
ES 2 616 630 T3 waste and waste management. Many biological oxidation processes for treating industrial wastewater have the use of oxygen (or air) and microbial action in common. Specifically grown microbes are used in the biological treatment of wastewater and industrial waste effluents, a process known as bioaugmentation. Bioaugmentation is used to ensure that microorganisms in situ can degrade contaminants. In some embodiments, MSM enhances degradation of contaminants by certain microorganisms. In some embodiments, MSM is added to garden products, such as substrates, fertilizers, and compost bins, to enhance the activity of beneficial microorganisms. As such, MSM is used to increase the efficiency of fertilizers and composting reactions.
In one embodiment, a method of enhancing the effectiveness of a fertilizer includes applying MSM to the medium in an amount sufficient to enhance the activity of a fertilizer, thereby enhancing the activity of the fertilizer. In a particular embodiment, MSM is dissolved in a solution at a final concentration of approximately 0.04% to approximately 5%. This solution is then sprayed onto a vegetable surface before, after or simultaneously with the fertilizer. An increase in fertilizer efficiency is indicated by an increase of at least 10%, such as from about 20% to 80%, an increase from about 30% to about 50%, including an increase of about 10%. %, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% in plant growth compared to a control (such as plant growth in the absence of MSM).
In another embodiment, a method of enhancing composting efficiency is disclosed. This method includes applying MSM to the compost in an amount sufficient to enhance the activity of one or more microorganisms or substances present in the compost. In a particular embodiment, MSM is dissolved in a solution at a final concentration of approximately 0.04% to approximately 5%. This solution is then applied to the compost (such as by pouring or spraying the solution) and sufficient time is allowed to elapse to enhance the effectiveness of composting. An increase in composting efficiency is indicated by an increase of at least 10%, such as from about 20% to 80%, an increase from about 30% to about 50%, including an increase of about 10%. %, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% nitrate levels compared to a control (such as nitrate levels in the absence of MSM). In other examples, an increase in composting efficiency is indicated by an increase of at least 10%, such as from about 20% to 80%, an increase from about 30% to about 50%, including a reduction. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% , about 150%, about 200%, about 300% in the amount of time it takes for organic matter decomposition to occur compared to a control (such as decomposition rate in the absence of MSM).
B. Methods to inhibit microbial activity
Methods for inhibiting microbial activity are disclosed. In one example, a method of inhibiting microbial activity includes selecting a medium that is susceptible to contamination; and contacting the medium with MSM at a concentration of from about 6% to about 16% by weight per volume, thereby inhibiting microbial activity compared to microbial activity in a control (such as microbial activity in the absence MSM), by at least 10%, such as a reduction of about 20% to 80%, a reduction of about 30% to 50%, including a reduction of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% compared to a control (such as microbial activity in the absence of MSM).
In some examples, a method of inhibiting microbial activity includes selecting a medium that is susceptible to bacterial contamination; and contacting the medium with MSM at a concentration of from about 6% to about 16% by weight by volume, thereby inhibiting bacterial activity. In some examples, a method of inhibiting microbial activity includes selecting a medium that is susceptible to viral contamination (such as contamination by human immunodeficiency virus, H1N1, herpes simplex virus, papilloma virus, virus parainfluenza, flu, hepatitis, or other similar viruses); and contacting the medium with MSM at a concentration of from about 6% to about 16% by weight by volume, thereby inhibiting viral activity.
In a particular example, a method of inhibiting microbial activity includes selecting a medium that is susceptible to contamination by H1N1 influenza; and contacting the medium with MSM at a concentration of from about 10% to about 16% by weight by volume, thereby inhibiting the activity.
ES 2 616 630 T3 microbial of the H1N1 flu. In some examples, MSM inhibits microbial activity by reducing the growth rate of the H1N1 flu by at least 10%, such as a reduction of about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300% growth or infectivity of H1N1 flu compared to a control (such as activity or infectivity of H1N1 flu in the absence of MSM).
In various embodiments, the methods include MSM at about 8% (by weight) or more, of the total product weight or moisture content. In certain examples, MSM is an effective antimicrobial agent when used at concentrations of between about 5% and about 16%. In certain examples, MSM is an effective antimicrobial agent when used at concentrations (based on the total weight of a product or its moisture content) of between about 9% and about 16%, between about 10% and about 16%, between about 12% and about 16%, between about 9% and about 13%, and between about 10% and about 12%. In certain examples, MSM is an effective antimicrobial agent when used at concentrations of between about 5% and about 16%, including 6%. 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14, and 15%. In various embodiments disclosed herein, the percentages of MSM are based on the moisture content of a product. In some embodiments, MSM is particularly useful when combined with water or other liquid components. In various embodiments, the percentages of MSM provided herein are based on the amount of a polar solvent in a product or other medium.
In some examples, the disclosed methods of inhibiting microbial activity include inhibiting the growth of specific microorganisms. In some examples, the methods include inhibiting the growth of a wide variety of microorganisms in certain media or products. In some examples, large-scale reductions are made after the first 24 hours. In other different examples, significant logarithmic scale reductions are evidenced at 24-48 hours. In some examples, the disclosed methods include MSM formulations that provide a reduction in microbial (eg, bacterial) levels ranging from about 0.5 log to about 5 log or more in two weeks. In some examples, the disclosed methods for inhibiting microbial activity result in a log reduction between a reduction of about 1 log and reductions of about 3 log or more. In other examples, the disclosed methods for inhibiting microbial activity lethally inhibit the growth of certain microorganisms. In one example, a method using an MSM formulation at between about 12% and about 16% lethally kills certain microbes (eg, bacteria) in about 48 hours. In another example, a formulation comprising MSM at between about 8% and about 12% lethally kills certain microbes (eg, bacteria) in about three to seven days. In other examples, the methods employ a formulation of MSM at between about 5% and about 8%, combined with a reduced amount of conventional preservative, which lethally kills certain microbes (eg, bacteria) in about 48 hours. With higher concentrations of preservative, MSM levels can be further reduced.
In some examples, the disclosed methods of inhibiting bacterial activity with MSM (such as with about 6% to about 16% MSM) impact the metabolism of microbes in the lag phase. For example, the disclosed method increases the duration of the delay phase. An alteration, such as an increase in the lag phase, can be detected by methods known to those of skill in the art, including those described in the examples.
In some embodiments, MSM supplementation results in a reduction in the logarithmic phase of growth of the microbes. The exponential phase (sometimes called the logarithmic phase) of growth is a period characterized by cell duplication. The number of new microbes that appear per unit of time is proportional to the population present. In case of not limiting growth, doubling will continue at a constant rate, whereby the number of cells and the rate of population increase doubles with each consecutive period of time. However, exponential growth cannot continue indefinitely, as the medium is depleted of nutrients and rapidly enriched with waste. In some embodiments, MSM reduces the overall duration of the exponential phase. In other embodiments, the presence of MSM in the culture medium inhibits microbial entry into the exponential phase.
In various examples, disclosed methods for inhibiting microbial activity include modulating the stationary phase of microbial growth. During the stationary phase, growth rate is reduced as a result of nutrient depletion and accumulation of metabolic by-products. This phase is reached as the microbes begin to deplete the resources that are available to them. This phase is at a relatively constant value since the rate of microbial growth is equal to the rate of microbial death. In one example, supplementation of the medium with MSM at certain concentrations shortens the stationary phase for microbes.
A medium is contemplated to include any medium or environment that contains or is suitable to withstand contamination, including, but not limited to, cosmetics, broths, agar, cultures, foods, beverages, suspensions.
ES 2 616 630 T3 cellular, biological tissue, biological fluids, inorganic surfaces, organic surfaces, substrates, living cells, host cells, diagnostic assays, and other solid, liquid, matrix, gelatinous or gaseous environments. In some examples, the medium is a body fluid, a body tissue, or a surface.
In some embodiments, contacting the medium includes topical, oral, intravenous, intramuscular, or subcutaneous administration of MSM to the medium susceptible to microbial contamination. In other embodiments, contacting the medium includes spraying or scrubbing the medium susceptible to microbial contamination with a disclosed MSM composition / formulation. For example, a surface can include any surface susceptible to contamination, including, but not limited to, a domestic surface, an industrial surface (such as surfaces in public toilets, door handles, floors, walls, roof rails, shopping carts and the like), bedding, coatings, industrial equipment or surfaces, blood, skin, or a combination thereof. For example, a domestic surface can include a door handle, a door knob, a waste bin, a counter, a floor, a toilet seat, or any surface that is normally touched or exposed to potential contaminants.
Unintended microbial growth can occur in many cosmetics, health and beauty supplements, topical products, and oral products. Acute or continued use of products with microbial contamination can lead to adverse health effects for the user. Contamination can occur, for example, during manufacturing, packaging, or repeated consumer use, including repeated opening and closing of containers, contact with hands, skin, or mucous membranes, or removal / administration. repeated single doses. In the absence of antimicrobial properties, these products can allow the unintended growth of many different, and potentially harmful, microorganisms.
Antimicrobial preservatives can be added to products to protect against microbial growth. Commonly used antimicrobial preservatives include calcium propionate, sodium nitrate, sodium nitrite, sulfites (sulfur dioxide, sodium bisulfite, potassium hydrogen sulfite, etc.), and disodium EDTA. Cosmetic preservatives include formaldehyde, potassium sorbate, methyl paraben, and methylchloroisothiazolinone.
In many cases, preservatives have to be added at a minimum effective concentration, since adverse reactions can occur at certain concentrations or doses. Therefore, although preservatives can inhibit microbial growth, they also have the potential to cause chemical burns and / or irritate the skin and mucous membranes. Some modern synthetic preservatives are controversial because they have been shown to cause respiratory and other health problems. The addition of certain preservatives to commercial products can present unique complications in terms of solubility, pH limits, deactivation by some polyethylene glycol (PEG) compounds, and a change in the color, consistency or fragrance of a product. Some preservatives have only limited activity against particular classes of microorganisms.
Methods for inhibiting microbial activity in a consumer product are also disclosed. In one example, the method includes selecting a medium that is susceptible to microbial contamination, such as a consumer product, and adding MSM to the medium to affect microbial contamination by inhibiting microbial activity. According to one embodiment, the MSM is provided at a concentration of at least 10% (eg, 10-16%, 16-20%, 20-30%, 30-40%, 40-50%, 50- 75% or higher, and the intervals in between). In some embodiments, the medium is devoid of preservatives.
In some examples, methods are provided for inhibiting microbial activity in a cosmetic cream at room temperature. In one example, the method includes selecting a medium that is susceptible to microbial contamination; and adding MSM to the medium to affect microbial contamination by inhibiting microbial activity. The MSM is added to a concentration of at least the MSM according to one example (e.g. 5-10%, 10-16%, 16-20%, 20-30%, 30-40%, 40-50% , 50-75% or greater, and the intermediate ranges thereof). In some embodiments, the medium is devoid of preservatives. In some embodiments, the medium includes a cosmetic cream. In one example, MSM inhibits microbial activity by at least 50% in cosmetic cream at room temperature.
In some examples, the medium includes one or more of the following: cosmetics, broths, agar, cultures, foods, beverages, cell suspensions, biological tissue, biological fluids, inorganic surfaces, organic surfaces, substrates, living cells, host cells, assays diagnostics, and other solid, liquid, matrix, gelatinous or gaseous environments. For example, in one embodiment, the medium includes an optical product or an oral health or hygiene product. The medium can also include a body fluid or tissue, such as blood. In one example, the medium is sterilized before adding MSM and / or after adding MSM. In other examples, sterilization is not needed. In some examples, the antimicrobial properties of MSM reduce or eliminate the need for sterilization.
In some examples, microbial contamination is caused by bacteria, such as gram-positive and / or gram-negative bacteria, fungi, parasites, yeast, mold, virus, or combinations thereof (eg, bacteria and mold, or other combinations). In various embodiments, the microbial contamination is caused by one or more of the following genera: Candida, Aspergillus, Escherichia, Pseudomonas, Staphylococcus, and Streptococcus, or combinations thereof. In another embodiment, the microbial contamination is caused by
ES 2 616 630 T3 an infectious disease, including any of the infectious diseases described herein.
In various examples, methods for treating an infectious disease including, but not limited to, H1N1, herpes simplex virus, or HIV are disclosed. In one example, the method includes administering a therapeutically effective amount of a therapeutic agent and only DMSO, only MSM, or a combination of DMSO and MSM. The DMSO and / or MSM concentration ranges from about 6% to about 17% in a composition.
In some examples, MSM inhibits microbial activity by reducing the growth rate of one or more microbes by more than 50%, which in turn increases the shelf life of the medium. It is contemplated that MSM may confer a therapeutic and / or aesthetic benefit. In some examples, the therapeutic and / or aesthetic benefit is not related to microbial inhibition.
In some examples, the disclosed methods for inhibiting microbial activity inhibit microbial activity at temperatures that result in microbial activity, including 20-25 ° C, 25-30 ° C, 30-40 ° C, 40-50. ° C and above (and the ranges in between). In some examples, MSM inhibits microbial activity at humidity levels favorable to microbial activity, including 50-60%, 60-70%, 70-80%, 80-95%, and higher (and the ranges in between thereof).
MSM is particularly advantageous in several examples because it can be used at higher concentrations than other preservatives, which when used even at low concentrations can cause adverse effects. For example, preservatives have been linked to atopic dermatitis, eczema, rashes, abdominal pain, nausea, asthma, rhinitis, muscle aches, joint pain, fatigue, numbness, migraines, attention deficit hyperactivity disorder, palpitations, and arrhythmias. In contrast, MSM is known not to cause such effects at the concentrations provided according to the preferred examples herein. Furthermore, the MSM has a dual function according to some embodiments. Not only does MSM inhibit the growth of unwanted microorganisms, MSM also beneficially affects the product where it is added in various embodiments.
In some examples, the disclosed methods for inhibiting microbial activity not only inhibit microbial activity, but provide one or more beneficial effects, including, but not limited to, reduction of muscle cramps, skin irritation, reduction of pain, lubrication of the joints, reduction of inflammation, treatment of rheumatoid arthritis and osteoarthritis, cardiovascular improvements, lubrication of the skin, improved wound healing, and improved scalp, hair, cuticles, and nails.
In some examples, the disclosed methods for inhibiting microbial activity are used to prevent or minimize the formation of new microbes. In other examples, the methods are used to kill or reduce existing microbes. In one example, MSM can convert a contaminated unusable product to a usable product.
According to several examples, the methods instantly inhibit microbial activity. In other examples, the methods inhibit microbial activity a and up to 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, 1 month, 3 months, 6 months, 1 year, 2 years. , 3 years, 4 years, 5 years or more.
In several examples, MSM is added to cleaning agents to enhance antimicrobial activity (eg, to inhibit the activity of microorganisms). In some examples, MSM is added to a soap formulation. In some examples, the product is a dry soap, while in other embodiments, the product is a liquid soap. In some examples, MSM is added to a gel formulation to provide a sanitizer. For example, methods of inhibiting microbial activity include methods of sanitizing a surface, such as the body, equipment, floors, materials, walls, etc. In certain examples, the resulting sanitizer is an instant sanitizer. In other examples, the sanitizer acts non-instantaneously (eg, it is effective over time). In some examples, the sanitizer is applied to the body. In still other embodiments, the product is applied to a surface. Surfaces include, but are not limited to, commercial surfaces, medical devices, medical surfaces, production equipment, production floors, and food preparation surfaces. Surfaces can include, but are not limited to, household surfaces, vehicles, computers, clothing, and toys.
In some additional examples, methods to inhibit microbial activity include spraying or incorporating MSM (eg, at from about 5% to about 50% in face masks or filters. Filters may include, but are not limited to, filters for air conditioning, air filters, water filters Environments with recycled air, such as airplanes, can especially benefit from MSM filtration systems. Water treatment and filtration plants can also incorporate MSM to inhibit microbial activity. In some examples, MSM is provided to reduce microbial contamination in bouquets and garden products (such as fertilizers and substrates).
In some examples, methods of inhibiting microbial activity include inhibiting the microbial activity of a
ES 2 616 630 T3 microorganism present in animal feed and to prevent microbial growth during storage or processing of feed. Types of animal feed include, but are not limited to, compound feed, pasture, or forage. Animal feeds can consist of raw materials and / or additives. The raw feed can be provided in the form of hay or grains. Alternatively, the raw material can be processed and provided in the form of a food, granules or flakes. In some examples, MSM is applied to animal feed to reduce mold growth. In other embodiments, the MSM is applied to animal feed to reduce fungal growth. In some examples, MSM is applied to raw food materials and therefore incorporated into a finished food product. In additional examples, the product is applied to the food during or after its manufacture. In some examples, the product is applied to food for long-term storage.
V. Methods for producing products that include MSM
Methods for producing products that include MSM are disclosed herein. In some embodiments, MSM is incorporated in a step that reduces the crystallization of the MSM. In one embodiment, MSM is incorporated into a product prior to emulsifying said product. In another embodiment, the MSM is encapsulated (eg, in a lipid, polymer, or other material) prior to its addition to a product. Microencapsulated MSM, according to some embodiments, can be designed for timed or dose release of MSM. In still other embodiments, MSM is combined with the aqueous portion of a product prior to mixing the wet and dry ingredients. In one embodiment, MSM in dry powder form is mixed in a matrix with an aqueous or polar liquid to activate the MSM.
In yet another embodiment, MSM is added to a product at an elevated temperature (eg, greater than 25 ° C, 30 ° C, 40 ° C, 50 ° C, 75 ° C, or greater). In some embodiments, MSM is materially unaffected by heat, and can be added prior to heating. Solutions that are at temperatures greater than about 35 ° C support MSM concentrations greater than 50% in some embodiments. In various embodiments, MSM does not have a substantial impact on the pH of the product to which it is added. In one embodiment, the hygroscopic solid products and other low moisture content products comprise MSM in the range of about 15% or more.
Also disclosed herein are methods of making a product having a reduced concentration of preservatives. In one embodiment, the method includes providing a medium that is susceptible to microbial contamination, wherein the medium comprises a preservative, and adding MSM to the medium, wherein MSM affects microbial contamination by inhibiting microbial growth. According to one embodiment, the MSM is added at a concentration of at least about 5% to about 20% (eg, 5-8%, 8-12%, 12-15%, 15-20%, or greater, and the intermediate intervals thereof). In one embodiment, the MSM and the preservative inhibit microbial growth by at least 50% in the medium at room temperature, and the MSM supplements or enhances the ability of the preservative to inhibit microbial growth, thereby reducing the concentration of preservative. necessary to inhibit microbial growth. In one embodiment, the medium is emulsified or otherwise mixed. In one embodiment, MSM is added to the medium prior to emulsifying (or otherwise mixing).
The following ingredients are provided to illustrate certain particular features and / or embodiments. These examples are not to be construed as limiting the disclosure to the particular features or embodiments described.
Examples
Example 1
Modulation of microbial activity based on MSM
This example describes MSM-based modulation of microbial activity, such as enhancement or inhibition of microbial growth depending on the concentration of MSM.
Adjacent microbial growth studies were carried out in media supplemented with MSM at a concentration of 0.1% to 10% and a control sample containing 0% MSM. The microorganisms evaluated were Aspergillus niger, Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella cholerasuis. All microorganisms were grown in tryptic soy broth (TSB) and with the exception of Candida and Aspergillus, all were successfully transferred to fresh medium every day for 4 consecutive days before inoculation to keep the organisms in an exponential growth phase. . Candida and Aspergillus had 48-58 hours of growth in TSB before inoculation to the test medium. Aspergillus was also grown on multiple potato dextrose agar (PDA) plates for 48-58 hours. The Aspergillus inoculum was prepared by extracting a TSB surface rinse from PDA plates with an Aspergillus lane, and then added to the culture for 48-58 hours until cloudy. For each test microorganism, 90 ml aliquots were prepared with 10% or 0% MSM. Once each set had been placed
ES 2 616 630 T3 of sterility test media were inoculated at a level of 5 µl of inoculum per 10 ml of broth (inoculum dilution at 1: 2000) with each respective microorganism. Bacterial organisms were incubated at 30 ° C ± 2 ° C and fungal organisms were incubated at 25 ° C ± 2 ° C.
The fungal organisms were plated daily on PDA on day 0 through day 7 every 24 hours. Preparation and plating were carried out at room temperature. The fungal plates were incubated at 25 ° C ± 2 ° C for at least 3 days. Test samples were placed in triplicate on each test date and averages are indicated. Data are expressed as recovered colony forming units per milliliter (cfu / ml).
The effects of MSM on the growth of Aspergillus nigery on the growth of Candida albicans are shown in Tables 1-1 (a) and 1-1 (b), respectively. Ten percent MSM inhibited Aspergillus niger growth on day 4 of treatment, as represented by a dramatic reduction in colony formation on that date. A reduction in growth was also observed in the Candida albicans samples treated with MSM; however, the reduction was not as drastic compared to Aspergillus niger. For example, medium supplemented with 10% MSM resulted in reduced yeast viability compared to lower MSM concentrations as early as 2 days (plating day 2 for Candida). Candida growth slowed early in the study, with substantial reductions in the fungal population on day 4. After these time points, divergence in growth curves continued during the Candida study. These data indicate that a 10% MSM concentration provides a significantly negative effect on the growth of various fungal organisms over time.
Table 1-1 (a). Effect of MSM on the growth of Aspergillus niger
<td>Aspergillus niger</td><td>0% MSM</td><td>0.1% MSM</td><td>0.5% MSM</td><td>1.0% MSM</td><td>10% MSM</td>
<td>Day 0</td><td>1.0 x 10<sup>3</sup></td><td>9.1 x 10<sup>2</sup></td><td>7.6 x 10<sup>2</sup></td><td>8.9 x 10<sup>2</sup></td><td>1.2 x 10<sup>3</sup></td>
<td>Day 1</td><td>1.3 x 10<sup>3</sup></td><td>2.3 x 10<sup>3</sup></td><td>2.7 x 10<sup>3</sup></td><td>2.0 x 10<sup>3</sup></td><td>7.0 x 10<sup>2</sup></td>
<td>Day 2</td><td>4.3 x 10<sup>3</sup></td><td>3.0 x 10<sup>2</sup></td><td>4.0 x 10<sup>3</sup></td><td>2.0 x 10<sup>3</sup></td><td>1.6 x 10<sup>2</sup></td>
<td>Day 3</td><td>2.0 x 10<sup>3</sup></td><td>7.5 x 10<sup>2</sup></td><td>1.3 x 10<sup>3</sup></td><td>1.0 x 10<sup>3</sup></td><td>4.6 x 10<sup>2</sup></td>
<td>Day 4</td><td>1.4 x 10<sup>4</sup></td><td>5.0 x 10<sup>3</sup></td><td>3.7 x 10<sup>3</sup></td><td>3.3 x 10<sup>3</sup></td><td> 20</td>
<td>Day 5</td><td>1.3 x 10<sup>4</sup></td><td>4.3 x 10<sup>3</sup></td><td>9.7 x 10<sup>3</sup></td><td>6.0 x 10<sup>3</sup></td><td> 10</td>
<td>Day 6</td><td>4.4 x 10<sup>4</sup></td><td>1.1 x 10<sup>4</sup></td><td>7.0 x 10<sup>3</sup></td><td>4.7 x 10<sup>3</sup></td><td> 3</td>
<td>Day 7</td><td>4.1 x 10<sup>4</sup></td><td>1.3 x 10<sup>4</sup></td><td>7.0 x 10<sup>3</sup></td><td>5.7 x 10<sup>3</sup></td><td> 3</td>
Table 1-1 (b). Effect of MSM on the growth of Candida albicans.
<td>Candida albicans</td><td>0% MSM</td><td>0.1% MSM</td><td>0.5% MSM</td><td>1.0% MSM</td><td>10% MSM</td>
<td>Day 0</td><td>2.2 x 10<sup>4</sup></td><td>2.1 x 10<sup>4</sup></td><td>1.8 x 10<sup>4</sup></td><td>2.5 x 10<sup>4</sup></td><td>2.6 x 10<sup>4</sup></td>
<td>Day 1</td><td>1.0 x 10<sup>5</sup></td><td>4.7 x 10<sup>6</sup></td><td>4.9 x 10<sup>6</sup></td><td>5.0 x 10<sup>6</sup></td><td><1.0 x 10<sup>5</sup></td>
<td>Day 2</td><td>1.0 x 10<sup>7</sup></td><td>9.8 x 10<sup>6</sup></td><td>1.1 x 10<sup>7</sup></td><td>9.8 x 10<sup>6</sup></td><td>4.0 x 10<sup>3</sup></td>
<td>Day 3</td><td>1.3 x 10<sup>7</sup></td><td>1.2 x 10<sup>7</sup></td><td>1.3 x 10<sup>7</sup></td><td>1.2 x 10<sup>7</sup></td><td><1.0 x 10<sup>5</sup></td>
<td>Day 4</td><td>1.9 x 10<sup>7</sup></td><td>1.3 x 10<sup>7</sup></td><td>1.4 x 10<sup>7</sup></td><td>1.2 x 10<sup>7</sup></td><td>7.0 x 10<sup>4</sup></td>
<td>Day 5</td><td>1.6 x 10<sup>7</sup></td><td>1.3 x 10<sup>7</sup></td><td>1.4 x 10<sup>7</sup></td><td>1.3 x 10<sup>7</sup></td><td>1.7 x 10<sup>4</sup></td>
<td>Day 6</td><td>8.8 x 10<sup>6</sup></td><td>1.3 x 10<sup>7</sup></td><td>1.4 x 10<sup>7</sup></td><td>1.6 x 10<sup>7</sup></td><td>2.8 x 10<sup>3</sup></td>
<td>Day 7</td><td>1.7 x 10<sup>7</sup></td><td>1.4 x 10<sup>7</sup></td><td>1.6 x 10<sup>7</sup></td><td>1.7 x 10<sup>7</sup></td><td>3.3 x 10<sup>3</sup></td>
The effect of MSM on the growth of Staphylococcus aureus is illustrated in Table 1-2. A difference in viability was observed in the presence of higher concentrations of MSM. In particular, 10% MSM appeared to reduce the growth rate and maximum population size of Staphylococcus aureus.
Table 1-2. Effect of MSM on the growth of Staphylococcus aureus.
<td>Staphylococcus aureus</td><td>0% MSM</td><td>0.1% MSM</td><td>0.5% MSM</td><td>1.0% MSM</td><td>10% MSM</td>
<td>Day 0</td><td>2.4 x 10<sup>5</sup></td><td>2.4 x 10<sup>5</sup></td><td>2.5 x 10<sup>5</sup></td><td>2.3 x 10<sup>5</sup></td><td>2.5 x 10<sup>5</sup></td>
<td>Day 1</td><td>3.6 x 10<sup>8</sup></td><td>4.6 x 10<sup>8</sup></td><td>4.1 x 10<sup>8</sup></td><td>6.0 x 10<sup>8</sup></td><td>5.3 x 10<sup>7</sup></td>
<td>Day 2</td><td>7.5 x 10<sup>8</sup></td><td>8.5 x 10<sup>8</sup></td><td>7.8 x 10<sup>8</sup></td><td>8.2 x 10<sup>8</sup></td><td>3.0 x 10<sup>8</sup></td>
<td>Day 3</td><td>9.6 x 10<sup>8</sup></td><td>9.1 x 10<sup>8</sup></td><td>8.4 x 10<sup>8</sup></td><td>9.6 x 10<sup>8</sup></td><td>5.1 x 10<sup>8</sup></td>
<td>Day 4</td><td>6.4 x 10<sup>8</sup></td><td>7.9 x 10<sup>8</sup></td><td>4.7 x 10<sup>8</sup></td><td>4.5 x 10<sup>8</sup></td><td>3.7 x 10<sup>8</sup></td>
<td>Day 7</td><td>2.6 x 10<sup>8</sup></td><td>1.8 x 10<sup>8</sup></td><td>1.7 x 10<sup>8</sup></td><td>3.3 x 10<sup>8</sup></td><td>9.0 x 10<sup>8</sup></td>
ES 2 616 630 T3
The effect of MSM on the growth of Pseudomonas aeruginosa is illustrated in Table 1-3. Supplementation of the medium with ten percent MSM resulted in a substantial divergence in viability of Pseudomonas aeruginosa over time. For example, the medium supplemented with 10% MSM produced a population reduction that lasted for the first 4 days of the study, but did not persist beyond that time.
_Table 1-3. Effect of 10% MSM on the growth of Pseudomonas aeruginosa._
<td>Pseudomonas aeruginosa</td><td>0% MSM</td><td>0.1% MSM</td><td>0.5% MSM</td><td>1.0% MSM</td><td>10% MSM</td>
<td>Day 0</td><td>4.0 x 10<sup>5</sup></td><td>4.8 x 10<sup>5</sup></td><td>4.8 x 10<sup>5</sup></td><td>4.2 x 10<sup>5</sup></td><td>4.0 x 10<sup>5</sup></td>
<td>Day 1</td><td>4.4 x 10<sup>8</sup></td><td>5.1 x 10<sup>8</sup></td><td>5.4 x 10<sup>8</sup></td><td>5.8 x 10<sup>8</sup></td><td>1.0 x 10<sup>8</sup></td>
<td>Day 2</td><td>1.1 x 10<sup>9</sup></td><td>6.0 x 10<sup>8</sup></td><td>9.2 x 10<sup>8</sup></td><td>5.7 x 10<sup>8</sup></td><td>5.8 x 10<sup>8</sup></td>
<td>Day 3</td><td>1.6 x 10<sup>8</sup></td><td>1.4 x 10<sup>9</sup></td><td>1.2 x 10<sup>9</sup></td><td>1.0 x 10<sup>9</sup></td><td>5.3 x 10<sup>8</sup></td>
<td>Day 4</td><td>1.6 x 10<sup>8</sup></td><td>2.0 x 10<sup>9</sup></td><td>1.4 x 10<sup>9</sup></td><td>1.9 x 10<sup>9</sup></td><td>3.7 x 10<sup>6</sup></td>
<td>Day 7</td><td>1.4 x 10<sup>9</sup></td><td>2.1 x 10<sup>9</sup></td><td>1.6 x 10<sup>9</sup></td><td>1.2 x 10<sup>9</sup></td><td>6.0 x 10<sup>6</sup></td>
The effect of MSM on the growth of Pseudomonas aeruginosa is illustrated in Table 1-4. Supplementation of the medium with 10 percent MSM resulted in substantially less growth of E. coli over time.
Table 1-4. Effect of 10% MSM on the growth of Escherichia coli.
<td>Escherichia coli</td><td>0% MSM</td><td>0.1% MSM</td><td>0.5% MSM</td><td>1.0% MSM</td><td>10% MSM</td>
<td>Day 0</td><td>6.9 x 10<sup>5</sup></td><td>6.4 x 10<sup>5</sup></td><td>7.0 x 10<sup>5</sup></td><td>6.7 x 10<sup>5</sup></td><td>6.8 x 10<sup>5</sup></td>
<td>Day 1</td><td>9.2 x 10<sup>8</sup></td><td>1.0 x 10<sup>9</sup></td><td>1.4 x 10<sup>9</sup></td><td>1.2 x 10<sup>9</sup></td><td>3.0 x 10<sup>4</sup></td>
<td>Day 2</td><td>1.3 x 10<sup>9</sup></td><td>1.4 x 10<sup>9</sup></td><td>1.6 x 10<sup>9</sup></td><td>3.2 x 10<sup>9</sup></td><td>1.2 x 10<sup>5</sup></td>
<td>Day 3</td><td>1.6 x 10<sup>9</sup></td><td>2.0 x 10<sup>9</sup></td><td>1.9 x 10<sup>9</sup></td><td>1.8 x 10<sup>9</sup></td><td>3.9 x 10<sup>7</sup></td>
<td>Day 4</td><td>1.4 x 10<sup>9</sup></td><td>1.4 x 10<sup>9</sup></td><td>1.4 x 10<sup>9</sup></td><td>1.4 x 10<sup>9</sup></td><td>1.2 x 10<sup>8</sup></td>
<td>Day 7</td><td>1.3 x 10<sup>8</sup></td><td>1.2 x 10<sup>9</sup></td><td>2.8 x 10<sup>9</sup></td><td>1.5 x 10<sup>9</sup></td><td>7.0 x 10<sup>7</sup></td>
The effect of MSM on the growth of Salmonella cholerasuis is illustrated in Table 1-5. The medium supplemented with 10% MSM reduced the growth of Salmonella cholerasuis in most of the time points of the study.
Table 1-5. Effect of 10% MSM on the growth of Salmonella cholerasuis.
<td>Salmonella cholerasuis</td><td>0% MSM</td><td>0.1% MSM</td><td>0.5% MSM</td><td>1.0% MSM</td><td>10% MSM</td>
<td>Day 0</td><td>6.8 x 10<sup>5</sup></td><td>8.7 x 10<sup>5</sup></td><td>5.9 x 10<sup>5</sup></td><td>5.5 x 10<sup>5</sup></td><td>7.6 x 10<sup>5</sup></td>
<td>Day 1</td><td>9.6 x 10<sup>8</sup></td><td>1.2 x 10<sup>9</sup></td><td>9.7 x 10<sup>8</sup></td><td>1.1 x 10<sup>9</sup></td><td>1.7 x 10<sup>6</sup></td>
<td>Day 2</td><td>1.3 x 10<sup>9</sup></td><td>1.0 x 10<sup>9</sup></td><td>1.2 x 10<sup>9</sup></td><td>1.3 x 10<sup>9</sup></td><td>7.7 x 10<sup>7</sup></td>
<td>Day 3</td><td>1.2 x 10<sup>9</sup></td><td>1.2 x 10<sup>9</sup></td><td>1.5 x 10<sup>9</sup></td><td>1.5 x 10<sup>9</sup></td><td>3.2 x 10<sup>8</sup></td>
<td>Day 4</td><td>7.8 x 10<sup>8</sup></td><td>6.0 x 10<sup>8</sup></td><td>7.1 x 10<sup>8</sup></td><td>8.0 x 10<sup>8</sup></td><td>1.9 x 10<sup>8</sup></td>
<td>Day 7</td><td>3.0 x 10<sup>8</sup></td><td>3.4 x 10<sup>8</sup></td><td>3.6 x 10<sup>8</sup></td><td>9.2 x 10<sup>8</sup></td><td>2.3 x 10<sup>8</sup></td>
These studies indicate that certain concentrations of MSM inhibit growth, including the growth of Aspergillus niger, Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa, and E. coli.
Example 2
Antimicrobial efficacy testing of media supplemented with MSM
This example describes the results of antimicrobial efficacy testing of media supplemented with MSM.
Compounds or formulated products that have antimicrobial activity can be evaluated with the United States Pharmacopeia (USP) Antimicrobial Efficacy Test (AET). TEA involves the addition of specific microorganisms (Candida albicans, Aspergillus niger, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) directly to a test product at relatively high concentrations to stimulate contamination. The product is kept for a month, analyzing the levels of microorganisms weekly. Depending on the route of administration of a product, meeting the TEA requires a 1 to 3 log reduction in bacteria from baseline levels, which should occur within one to two weeks, with no subsequent increases in bacteria after two weeks. For yeasts and molds, the lack of increase with respect to the initial inoculum level is allowed. Successfully meeting the AET criteria shows that a product, optionally supplemented with an antimicriobial compound under evaluation, can withstand an inoculation of up to one million microorganisms per gram of product without contamination. AET demonstrates the efficacy of a preservative system in a
ES 2 616 630 T3 product and / or can be used as part of a stability study to determine if a preservative will affect the shelf life of a product.
TEA was carried out by adding the specified microorganisms directly to the test medium supplemented with MSM at concentrations that simulate microbial contamination. Standardized fresh active cultures were added to the MSM supplemented media at a concentration between 100,000 and 1,000,000 cells per ml of the test product. Inoculations were carried out using Candida albicans, Aspergillus niger, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Tryptic soy broth (TSB) was used as the culture medium. MSM was diluted 1/1, 1/5, 1/10, 1/100, and 1/1000 and each was used to supplement the medium. The inoculated medium was kept for one month, during which time the added microorganisms were enumerated weekly to determine if they were growing, dying, or remained close to the initial level of inoculation. Data points were measured in triplicate at 48 hours, 3, 5, 14, 20, 28, and 30 days. The results of these studies are shown in Tables 2-1 through 2-8. Acceptance criteria for antimicrobial efficacy are described in detail in the USP.
Table 2-1. Results of the AET test for the 1: 1 dilution of MSM.
<td>Test body</td><td>Initial inoculum</td><td>48 h</td><td>3 days</td><td>5 days</td><td>14 days</td><td>20 days</td><td>28 days</td><td>30 days</td>
<td>Aspergillus Niger</td><td>3 x 10<sup>5</sup></td><td>3.5 X 105</td><td>3.2 x 10<sup>5</sup></td><td>3.1 x 10<sup>5</sup></td><td>2.3 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Candida Albicans</td><td>2.5 x 10<sup>5</sup></td><td>2.8 X 10<sup>5</sup></td><td>2.6 x 10<sup>5</sup></td><td>2.4 x 10<sup>5</sup></td><td>1.2 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Escherichia coli</td><td>1.3 x 10<sup>5</sup></td><td>1.3 X 10<sup>5</sup></td><td>1.2 x 10<sup>5</sup></td><td>1.1 x 10<sup>5</sup></td><td>1.8 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Pseudomonas aeruginosa</td><td>1.5 x 10<sup>5</sup></td><td>1.8 X 105</td><td>1.9 x 10<sup>5</sup></td><td>1.6 x 10<sup>5</sup></td><td>4.0 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Staphylococcus aureus</td><td>5.5 x 10<sup>5</sup></td><td>5.4 X 105</td><td>5.6 x 10<sup>5</sup></td><td>5.2 x 10<sup>5</sup></td><td>3.7 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Salmonella typhimurium</td><td>4.6 x 10<sup>5</sup></td><td>4.9 X 105</td><td>5.1 x 10<sup>5</sup></td><td>4.7 x 10<sup>5</sup></td><td>2.5 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
Table 2-2. Logarithmic reduction from the initial microorganism inoculum with a 1: 1 dilution of MSM.
<td>Test body</td><td>14 days</td><td>28 days</td>
<td>Aspergillus Niger</td><td> 3,4</td><td> 4,5</td>
<td>Candida Albicans</td><td> 3,0</td><td> 4,4</td>
<td>Escherichia coli</td><td> 3,3</td><td> 4,1</td>
<td>Pseudomonas aeruginosa</td><td> 3,6</td><td> 4,2</td>
<td>Staphylococcus aureus</td><td> 3,6</td><td> 4,7</td>
<td>Salmonella typhimurium</td><td> 3,4</td><td> 4,7</td>
Table 2-3. AET test results for 1: 5 dilution of MSM.
<td>Test body</td><td>Inoculum initial</td><td>48 h</td><td>3 days</td><td>5 days</td><td>14 days</td><td>20 days</td><td>28 days</td><td>30 days</td>
<td>Aspergillus Niger</td><td>3 x 10<sup>5</sup></td><td>4.0 X 105</td><td>4.2 x 10<sup>5</sup></td><td>3.8 x 10<sup>5</sup></td><td>3 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
ES 2 616 630 T3
<td>Candida Albicans</td><td>2.5 x 10<sup>5</sup></td><td>2.9 X 105</td><td>3.1 x 10<sup>5</sup></td><td>3.2 x 10<sup>5</sup></td><td>2 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Escherichia coli</td><td>1.3 x 10<sup>5</sup></td><td>1.4 X 10<sup>5</sup></td><td>1.7 x 10<sup>5</sup></td><td>2 x 10<sup>5</sup></td><td>1 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Pseudomonas aeruginosa</td><td>1.5 x 10<sup>5</sup></td><td>1.6 X 105</td><td>2.0 x 10<sup>5</sup></td><td>2.2 x 10<sup>5</sup></td><td>1.2 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Staphylococcus aureus</td><td>5.5 x 10<sup>5</sup></td><td>5.7 X 105</td><td>6.0 x 10<sup>5</sup></td><td>5.9 x 10<sup>5</sup></td><td>2.3 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Salmonella typhimurium</td><td>4.6 x 10<sup>5</sup></td><td>5.0 X 105</td><td>5.2 x 10<sup>5</sup></td><td>5.4 x 10<sup>5</sup></td><td>3.2 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
Table 2-4. Logarithmic reduction from the initial microorganism inoculum with a 1: 5 dilution of MSM.
<td>Test body</td><td>14 days</td><td>28 days</td>
<td>Aspergillus Niger</td><td> 3,5</td><td> 4,5</td>
<td>Candida Albicans</td><td> 3,3</td><td> 4,4</td>
<td>Escherichia coli</td><td> 3,0</td><td> 4,1</td>
<td>Pseudomonas aeruginosa</td><td> 3,2</td><td> 4,2</td>
<td>Staphylococcus aureus</td><td> 3,4</td><td> 4,7</td>
<td>Salmonella typhimurium</td><td> 3,5</td><td> 4,7</td>
Table 2-5. AET test results for 1:10 dilution of MSM.
<td>Test body</td><td>Inoculum initial</td><td>48 h</td><td>3 days</td><td>5 days</td><td>14 days</td><td>20 days</td><td>28 days</td><td>30 days</td>
<td>Aspergillus Niger</td><td>3 x 10<sup>5</sup></td><td>5x10<sup>5</sup></td><td>5.2 x 10<sup>5</sup></td><td>5.4 x 10<sup>5</sup></td><td>2 x 10<sup>4</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Candida Albicans</td><td>2.5 x 10<sup>5</sup></td><td>3 x 10<sup>5</sup></td><td>3.4 x 10<sup>5</sup></td><td>4 x 10<sup>5</sup></td><td>1.5 x 10<sup>4</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Escherichia coli</td><td>1.3 x 10<sup>5</sup></td><td>2 x 10<sup>5</sup></td><td>2.3 x 10<sup>5</sup></td><td>3.2 x 10<sup>5</sup></td><td>2.5 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Pseudomonas aeruginosa</td><td>1.5 x 10<sup>5</sup></td><td>1.9 X 105</td><td>2.2 x 10<sup>5</sup></td><td>2.9 x 10<sup>5</sup></td><td>4 x 10<sup>5</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Staphylococcus aureus</td><td>5.5 x 10<sup>5</sup></td><td>5.9 X 10<sup>5</sup></td><td>6.1 x 10<sup>5</sup></td><td>6.3 x 10<sup>5</sup></td><td>2.9 x 10<sup>4</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Salmonella typhimurium</td><td>4.6 x 10<sup>5</sup></td><td>5.0 X 105</td><td>5.2 x 10<sup>5</sup></td><td>5.5 x 10<sup>5</sup></td><td>3.2 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
Table 2-6. Logarithmic reduction with respect to the inoculum of microorganisms, initial with a 1:10 dilution of MSM.
<td>Test body</td><td>14 days</td><td>28 days</td>
<td>Aspergillus Niger</td><td> 4,3</td><td> 4,5</td>
<td>Candida Albicans</td><td> 4,2</td><td> 4,4</td>
ES 2 616 630 T3
<td>Escherichia coli</td><td> 3,4</td><td> 4,1</td>
<td>Pseudomonas aeruginosa</td><td> 3,6</td><td> 4,2</td>
<td>Staphylococcus aureus</td><td> 4,5</td><td> 4,7</td>
<td>Salmonella typhimurium</td><td> 3,5</td><td> 4,7</td>
Table 2-7. AET test results for 1: 100 dilution of MSM.
<td>Test body</td><td>Initial inoculum</td><td>48 h</td>
<td>Aspergillus Niger</td><td>3 x 10<sup>5</sup></td><td>TNTC *</td>
<td>Candida Albicans</td><td>2.5 x 10<sup>5</sup></td><td>TNTC</td>
<td>Escherichia coli</td><td>1.3 x 10<sup>5</sup></td><td>TNTC</td>
<td>Pseudomonas aeruginosa</td><td>1.5 x 10<sup>5</sup></td><td>TNTC</td>
<td>Staphylococcus aureus</td><td>5.5 x 10<sup>5</sup></td><td>TNTC</td>
<td>Salmonella typhimurium</td><td>4.6 x 10<sup>5</sup></td><td>TNTC</td>
<td colspan="3">* - Colonies too numerous to count (TNTC)</td>
Table 2-8. Results of the AET test for the 1: 1000 dilution of MSM.
<td>Test body</td><td>Initial inoculum</td><td>48 h</td>
<td>Aspergillus Niger</td><td>3 x 10<sup>5</sup></td><td>TNTC *</td>
<td>Candida Albicans</td><td>2.5 x 10<sup>5</sup></td><td>TNTC</td>
<td>Escherichia coli</td><td>1.3 x 10<sup>5</sup></td><td>TNTC</td>
<td>Pseudomonas aeruginosa</td><td>1.5 x 10<sup>5</sup></td><td>TNTC</td>
<td>Staphylococcus aureus</td><td>5.5 x 10<sup>5</sup></td><td>TNTC</td>
<td>Salmonella typhimurium</td><td>4.6 x 10<sup>5</sup></td><td>TNTC</td>
The 1: 1, 1: 5, and 1:10 dilutions of MSM (Tables 2-1 to 2-6, above) indicate that these concentrations of MSM in the media killed the organisms and do not simply have a static effect on the media. increase. Based on the culture populations on day 5, the bactericidal effects were unexpected, as the culture populations were stable or showed signs of increased growth. However, at 14 days, reductions in the initial inoculation levels were observed, and at 20 days, a total elimination of the microorganisms was observed using MSM at least 10%. These results were confirmed by adding a 90 ml TSB blank with 10 ml of the dilution matrix tested (eg medium that is considered to no longer contain live microorganisms). None of the microorganisms could be cultured and no contamination was observed. These results demonstrate that MSM, at certain concentrations, is bactericidal for these organisms.
Example 3
Bactericidal effects of sterile and non-sterile MSM on Escherichia coli
This example describes the bactericidal effects of sterile and non-sterile MSM on the growth of E. coli.
The USP AET <51> assay methodology described in Example 2 was used as the basis for assessing the lethality to Escherichia coli (strain ATCC 8739) of various concentrations of MSM in the range of 5 to 16% in TSB or saline. . USP (United States Pharmacopeia) AET <51> is a compendium of a test method for antimicrobial efficacy to determine whether a preservative is effective based on a verified and validated methodology. The parameters of the AET have been described previously. In this study, after the designated incubation period, the cultures were visually evaluated and then streaked and grown on selective MacConkey agar for qualitative analysis of the effects of various concentrations of MSM. This study also evaluated the effect of the previous sterilization of the MSM (by means of steam autoclaving at 121 ° C for 15 minutes) before the preparation of the medium. Assay media was prepared by weighing a suitable amount of MSM and adding it to 25 ml of TSB medium or saline. Media compositions were coded as provided in Table 3-1.
ES 2 616 630 T3
Table 3-1. Compositions of media tested against E. coli.
<td>Middle code</td><td>Preparation</td>
<td>NS</td><td>Sterile saline added to non-sterile MSM</td>
<td>NSA</td><td>Saline + MSM, then sterilized</td>
<td>TSB</td><td>Sterile TSB added to non-sterile MSM</td>
<td>TSBA</td><td>TSB + MSM, then sterilized</td>
<td>TSBC</td><td>TSB without MSM, seeded with E. coli</td>
<td>(-) TSBC</td><td>negative control (no E. coli)</td>
<td>NSC</td><td>Saline without MSM, seeded with E. coli</td>
<td>(-) NSC</td><td>negative control (no E. coli)</td>
All tubes except negative controls were seeded with 250 μl of a 1.2 x 10 culture.<sup>8</sup>, which provides an initial E. coli population density of 1.2 x 10<sup>6</sup>/ ml. The tubes were incubated at 25 ° C.
At 24 hours, visible signs of growth were observed in the TSB / TSBA medium with 5-9% MSM (see Table 3-2 below). In contrast, no signs of growth were observed in tubes with 10-16% MSM TSB / TSBA medium (see Table 3-2 below). The saline tubes showed no signs of growth. When seeded in MacConkey medium, a strong growth result was obtained on all media containing 5-9% MSM, while fewer colonies were detected on plates seeded from tSb / TSBA medium with 10% MSM. Little or no growth was obtained from seeding tSb / TSBA media with 11-16% MSM. Growth was detected in the positive control of TSB and saline (TSB or saline without MSM, seeded with E. coli), while no growth was detected in the plates seeded from the medium without addition.
Table 3-2. Growth profile of E. coli in MSM containing medium after 24 hours.
<td>MSM concentration</td><td>TSB</td><td>TSBA</td><td>NS</td><td>NSA</td>
<td> 5 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 6 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 7 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 8 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 9 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 10 %</td><td>Moderate</td><td>Moderate</td><td>High</td><td>High</td>
<td> 11 %</td><td>Scarce</td><td>Scarce</td><td>High</td><td>High</td>
<td> 12 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 13 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 14 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 15 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 16 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
As shown in Table 3-3, signs of strong growth were observed at 48 hours in the culture tubes of TSB / TSBA with 5-10% MSM. Obvious growth was observed in the culture tubes with TSB / TSBA with 11% MSM. Similarly at the 24 hour time point, little or no signs of growth were observed in the 12-15% MSM TSB / TSBA culture tubes. After seeding, strong bacterial growth occurred on all media containing 5-10% MSM. TSB / TSBA with 11% MSM allowed moderate growth, while the same concentration of MSM added to saline allowed high growth. At concentrations of 12-16% MSM in TSB / TSBA, little to no growth was detected on the plates. Moderate growth was observed from similar concentrations of MSM in saline medium.
ES 2 616 630 T3
Table 3-3. Growth profile of E. coli in MSM containing medium after 48 hours.
<td>MSM concentration</td><td>TSB</td><td>TSBA</td><td>NS</td><td>NSA</td>
<td> 5 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 6 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 7 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 8 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 9 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 10 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 11 %</td><td>Moderate</td><td>Moderate</td><td>High</td><td>High</td>
<td> 12 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 13 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 14 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 15 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 16 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
After 72 hours of culture, signs of strong growth were observed in the culture tubes of TSB / TSBA with 5-10% MsM (Table 3-4). Obvious growth was observed in the culture tubes with TSB / TSBA with MSM at 11
%. Similarly at the 24 hour time point, little or no signs of growth were observed in the 12-15% MSM TSB / TSBA culture tubes. After seeding, strong bacterial growth occurred on all media containing 5-10% MSM. TSB / TSBA with 11% MSM allowed moderate growth, while the same concentration of MSM added to saline allowed high growth. At concentrations of 12-16% MSM in TSB / TSBA, little to no growth was detected on the plates. Moderate growth was observed from similar concentrations of MSM in saline medium.
Table 3-4. Growth profile of E. coli in MSM containing medium after 72 hours.
<td>MSM concentration</td><td>TSB</td><td>TSBA</td><td>NS</td><td>NSA</td>
<td> 5 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 6 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 7 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 8 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 9 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 10 %</td><td>High</td><td>High</td><td>High</td><td>High</td>
<td> 11 %</td><td>Moderate</td><td>Moderate</td><td>High</td><td>High</td>
<td> 12 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 13 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 14 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 15 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
<td> 16 %</td><td>Scarce</td><td>Scarce</td><td>Moderate</td><td>Moderate</td>
These results demonstrate that MSM concentrations of approximately 10-16% are effective in killing bacteria at certain times. At 24 hours, 10% MSM reduced the viable bacterial population, while at 48-72 hours the higher concentrations were effective in eliminating most of the bacterial population. The 10-16% concentrations of MSM in TSB / TSBA were more effective than the same concentration in saline-based medium. The data further suggests that steam sterilization has no inherent impact on the effectiveness of MSM.
ES 2 616 630 T3
Example 4
Comparison of the bactericidal efficacy of MSM in a saline-based medium or tryptic soy broth (TSB)
This example compares the bactericidal efficacy of MSM in saline-based and TSB-based medium.
As presented in Examples 2 and 3, USP AET <51> assay methodology was used as the basis for evaluating the E. coli lethality of various concentrations of MSM (flake or Microprill) in the range of 5 to 16% in TSB or saline. Each composition of medium was inoculated with 1.25 x 10<sup>6</sup>/ ml, of E. coli and then cultured for seven days at 35 ° C. At the end of the incubation period, the cultures were visually evaluated and then grown on trypto-soy agar at serially diluted concentrations to ensure bacterial growth (if any) at a density that could be quantified. Plated cultures were grown for 24 hours at 35 ° C prior to analysis. The medium compositions were coded as shown in Table 4-1 and the results of these studies are provided in Table 4-2.
Table 4-1. Media compositions
<td>Middle code</td><td>Preparation</td>
<td>$</td><td>Microprill MSM added to saline</td>
<td>FS</td><td>Flaked MSM added to saline</td>
<td>PTSB</td><td>Microprill MSM added to TSB</td>
<td>FTSB</td><td>Flaked MSM added to TSB</td>
<td>TSBC</td><td>TSB without MSM, seeded with E. coli</td>
<td>(-) TSBC</td><td>negative control (no E. coli)</td>
<td>NSC</td><td>Saline without MSM, seeded with E. coli</td>
<td>(-) NSC</td><td>negative control (no E. coli)</td>
Table 4-2. Logarithmic growth of E. coli in different media with different concentrations of MSM.
<td>MSM concentration</td><td>FTSB</td><td>FS</td><td>PTSB</td><td>$</td>
<td> 16 %</td><td> 1</td><td> 4,5</td><td> 1</td><td> 4,8</td>
<td> 15 %</td><td> 2,6</td><td> 4,8</td><td> 2,3</td><td> 5,1</td>
<td> 14 %</td><td> 2,9</td><td> 4,9</td><td> 2,1</td><td> 5,6</td>
<td> 13 %</td><td> 2,6</td><td> 5,5</td><td> 3</td><td> 5,6</td>
<td> 12 %</td><td> 2,7</td><td> 5,1</td><td> 3</td><td> 5,8</td>
<td> 11 %</td><td> 3,6</td><td> 5,4</td><td> 3,3</td><td> 5,9</td>
<td> 10 %</td><td> 6</td><td> 5,5</td><td> 6</td><td> 5,8</td>
<td> 9 %</td><td> 6</td><td> 5,6</td><td> 6</td><td> 5,9</td>
<td> 8 %</td><td> 6</td><td> 5,7</td><td> 6</td><td> 6</td>
<td> 7 %</td><td> 6</td><td> 5,9</td><td> 6</td><td> 6</td>
<td> 6 %</td><td> 6</td><td> 5,9</td><td> 6</td><td> 6,2</td>
<td> 5 %</td><td> 6</td><td> 5,7</td><td> 6</td><td> 6,4</td>
MSM concentrations in the range of 11-16% have a negative impact on the growth of E. coli in culture for 7 days. As the concentration of MSM increased above 10% in the FTSB or PTSB media, the growth of E. coli was reduced. Both forms of MSM showed efficacy in inhibiting bacterial growth.
ES 2 616 630 T3
Example 5
Effect of the medium without sodium chloride on the bactericidal effect of MSM
This example shows the effect of the medium without sodium chloride on the bactericidal effects of MSM.
A study was carried out using Müller-Hinton broth medium, which does not contain NaCl. Standard Müller-Hinton medium was compared with Müller-Hinton medium supplemented with NaCl at the same level as the saline-based medium of Example 4. MSM was added to each medium at concentrations in the range of 5-16%. After inoculation of each type of medium containing MSM with 1, x 10<sup>7</sup> cfu / ml of E. coli, the cultures were incubated at 35 ° C for seven days. Aliquots of each culture were taken at 24 and 48 hours, as well as at 7 days. The aliquots were then grown on trypto-soy agar at serially diluted concentrations to ensure bacterial growth (if any) at a density that could be quantified. Plated cultures were grown for 24 hours at 35 ° C prior to analysis. Media compositions were coded as provided in Table 5-1. The results of these studies are provided in Tables 5-2 through 5-4.
Table 5-1. Media compositions
<td>Middle code</td><td>Preparation</td>
<td>PMHS</td><td>Microprill MSM added to Müller-Hinton medium plus NaCl</td>
<td>FMHS</td><td>Flaked MSM added to Müller-Hinton medium plus NaCl</td>
<td>PMH</td><td>Microprill MSM added to Müller-Hinton medium</td>
<td>FMH</td><td>Flaked MSM added to Müller-Hinton medium</td>
<td>MHC</td><td>Müller-Hinton medium without MSM, seeded with E. coli</td>
<td>(-) MHC</td><td>negative control (no E. coli)</td>
<td>MHNSC</td><td>Müller-Hinton medium plus NaCl without MSM, seeded with E. coli</td>
<td>(-) MHNSC</td><td>negative control (no E. coli)</td>
After 24 hours in culture, a reduction of approximately 1 log from the initial inoculum was detected in all media that had MSM concentrations greater than 13% (Table 5-2). In addition, at 12% MSM, all medium compositions reduced the growth of E. coli, except for the PMHS composition. At 11% MSM, only FMH medium reduced the growth of E. coli.
Table 5-2. Logarithmic growth of E. coli in Müller-Hinton medium supplemented with MSM or Müller-Hinton (plus NaCl) after 24 hours
<td>MSM concentration</td><td>PMHS</td><td>FMHS</td><td>PMH</td><td>FMH</td>
<td> 16 %</td><td> 6,2</td><td> 6,1</td><td> 6,1</td><td> 6,2</td>
<td> 15 %</td><td> 6,0</td><td> 6,1</td><td> 6,0</td><td> 6,2</td>
<td> 14 %</td><td> 6,0</td><td> 6,0</td><td> 5,9</td><td> 6,3</td>
<td> 13 %</td><td> 6,3</td><td> 6,2</td><td> 5,8</td><td> 5,9</td>
<td> 12 %</td><td> 7,2</td><td> 5,7</td><td> 5,8</td><td> 5,9</td>
<td> 11 %</td><td> 7,9</td><td> 7,8</td><td> 7,4</td><td> 5,9</td>
<td> 10 %</td><td> 8,7</td><td> 8,2</td><td> 7,9</td><td> 7,8</td>
<td> 9 %</td><td> 8,3</td><td> 8,3</td><td> 8,3</td><td> 8,2</td>
<td> 8 %</td><td> 8,2</td><td> 8,4</td><td> 8,4</td><td> 8,4</td>
<td> 7 %</td><td> 8,3</td><td> 8,4</td><td> 8,4</td><td> 8,5</td>
<td> 6 %</td><td> 8,1</td><td> 8,4</td><td> 8,5</td><td> 8,5</td>
<td> 5 %</td><td> 8,2</td><td> 8,4</td><td> 8,6</td><td> 8,6</td>
After 48 hours in culture, medium compositions with MSM concentrations greater than 13% reduced the growth of E. coli by 1-2 log. Certain concentrations of MSM are effective in reducing the
ES 2 616 630 T3 bacterial growth, which is surprising since other concentrations of MSM are effective in supporting increased bacterial activity.
Table 5-3. Logarithmic growth of E. coli in Müller-Hinton medium supplemented with MSM or Müller-Hinton (plus 5 __NaCl) after 48 hours __
<td>MSM concentration</td><td>PMHS</td><td>FMHS</td><td>PMH</td><td>FMH</td>
<td> 16 %</td><td> 6,0</td><td> 5,9</td><td> 6,0</td><td> 5,9</td>
<td> 15 %</td><td> 6,0</td><td> 5,9</td><td> 5,8</td><td> 4,6</td>
<td> 14 %</td><td> 5,7</td><td> 5,9</td><td> 5,4</td><td> 5,3</td>
<td> 13 %</td><td> 5,9</td><td> 5,9</td><td> 5,4</td><td> 5,2</td>
<td> 12 %</td><td> 6,7</td><td> 6,9</td><td> 5,6</td><td> 5,3</td>
<td> 11 %</td><td> 7,8</td><td> 7,8</td><td> 7,2</td><td> 7,1</td>
<td> 10 %</td><td> 7,9</td><td> 8,0</td><td> 7,9</td><td> 8,2</td>
<td> 9 %</td><td> 8,2</td><td> 8,1</td><td> 8,1</td><td> 8,2</td>
<td> 8 %</td><td> 8,1</td><td> 8,1</td><td> 8,2</td><td> 8,3</td>
<td> 7 %</td><td> 8,2</td><td> 8,2</td><td> 8,0</td><td> 8,4</td>
<td> 6 %</td><td> 8,1</td><td> 8,2</td><td> 8,4</td><td> 8,4</td>
<td> 5 %</td><td> 8,4</td><td> 8,3</td><td> 8,3</td><td> 8,4</td>
After 7 days in culture, medium compositions containing as little as 12% MSM substantially inhibited the growth of E. coli (Table 5-4). FMHS medium was the most efficient with 12% MSM, providing a 3 log reduction in E. coli.
Table 5-4. Logarithmic growth of E. coli in Müller-Hinton medium supplemented with MSM or Müller-Hinton (plus NaCl) after 7 days.
<td>MSM concentration</td><td>PMHS</td><td>FMHS</td><td>PMH</td><td>FMH</td>
<td> 16 %</td><td> 4,4</td><td> 4,7</td><td> 3,9</td><td> 7,8</td>
<td> 15 %</td><td> 4,7</td><td> 4,7</td><td> 3,8</td><td> 7,9</td>
<td> 14 %</td><td> 4,9</td><td> 4,7</td><td> 3,3</td><td> 7,6</td>
<td> 13 %</td><td> 3,9</td><td> 4,4</td><td> 3,2</td><td> 7,2</td>
<td> 12 %</td><td> 6,0</td><td> 4,1</td><td> 6,0</td><td> 6,5</td>
<td> 11 %</td><td> 6,8</td><td> 6,2</td><td> 6,6</td><td> 7,0</td>
<td> 10 %</td><td> 6,8</td><td> 6,9</td><td> 7,0</td><td> 7,0</td>
<td> 9 %</td><td> 6,9</td><td> 7,0</td><td> 7,0</td><td> 6,5</td>
<td> 8 %</td><td> 7,2</td><td> 7,0</td><td> 7,8</td><td> 7,2</td>
<td> 7 %</td><td> 7,5</td><td> 6,8</td><td> 6,7</td><td> 7,6</td>
<td> 6 %</td><td> 8,0</td><td> 8,0</td><td> 7,5</td><td> 7,6</td>
<td> 5 %</td><td> 8,0</td><td> 8,1</td><td> 8,0</td><td> 7,8</td>
Example 6
Bactericidal effect of MSM in the medium low in protein and without sodium chloride
This example shows the bactericidal effect of MSM in the low protein medium without sodium chloride.
Lactose broth without NaCl and proteins was used as the medium in this experiment. MSM was added to the lactose broth at concentrations in the 5-16% range. A duplicate set of media containing MSM was supplemented with DMSO to a final concentration of 1%. Each composition of medium was initially inoculated with 6.75 x 10<sup>6</sup> cfu / ml
ES 2 616 630 T3
E. coli. The cultures were inoculated at 25 ° C for seven days. Aliquots of each culture were taken after 24 hours of culture and at the end of the seven days in culture. Aliquots were serially diluted (with modified Letheen's diluent) and plated on trypto-soy agar plates. The cultured plates were grown for 24 to 35 ° C and then analyzed. Media compositions were coded as shown in Table 6-1. The results of these studies are shown in Tables 6-2 and 6-3.
Table 6-1. Media compositions
<td>Middle code</td><td>Preparation</td>
<td>LBM</td><td>MSM added to lactose broth</td>
<td>LBMD</td><td>MSM added to lactose broth supplemented with 1% DMSO</td>
<td>LB</td><td>Lactose broth without MSM seeded with E. coli.</td>
<td>(-) LB</td><td>negative control (no E. coli)</td>
Lactose broth containing 11-16% MSM reduced bacterial growth from approximately 1 log (16-10% MSM) to a maximum of 2.2 log (11% MSM) as shown in Table 6- two. Bacterial growth inhibition was reduced by 1 log or more from 9-16% MSM.
Table 6-2. Log of E. coli growth at 24 hours in MSM-lactose broth with or without. DMSO
<td>MSM concentration</td><td>LBM</td><td>LBMD</td>
<td> 16 %</td><td> 5,8</td><td> 5,3</td>
<td> 15 %</td><td> 5,6</td><td> 5,5</td>
<td> 14 %</td><td> 5,5</td><td> 5,3</td>
<td> 13 %</td><td> 5,1</td><td> 5</td>
<td> 12 %</td><td> 5,5</td><td> 4,9</td>
<td> 11 %</td><td> 4,6</td><td> 5,6</td>
<td> 10 %</td><td> 7</td><td> 4,8</td>
<td> 9 %</td><td> 7,1</td><td> 5,7</td>
<td> 8 %</td><td> 8</td><td> 7,8</td>
<td> 7 %</td><td> 8</td><td> 9,2</td>
<td> 6 %</td><td> 8,1</td><td> 8,4</td>
<td> 5 %</td><td> 8,4</td><td> 8,8</td>
After 7 days of culture, a more defined pattern of inhibition of bacterial growth was evident (Table 6-3). 10% MSM in lactose broth kept the E. coli population roughly equivalent to the initial inoculum. Table 6-3. E. coli growth log at 7 days in MSM-lactose broth with or without .DMSO
<td>MSM concentration</td><td>LBM</td><td>LBMD</td>
<td> 16 %</td><td> 4</td><td> 4</td>
<td> 15 %</td><td> 3,9</td><td> 3,6</td>
<td> 14 %</td><td> 4</td><td> 3,6</td>
<td> 13 %</td><td> 3,3</td><td> 3,2</td>
<td> 12 %</td><td> 3,4</td><td> 2,9</td>
<td> 11 %</td><td> 2,5</td><td> 3,1</td>
<td> 10 %</td><td> 6,9</td><td> 4,2</td>
<td> 9 %</td><td> 7,9</td><td> 7,9</td>
<td> 8 %</td><td> 8,2</td><td> 8,4</td>
<td> 7 %</td><td> 8,4</td><td> 8,6</td>
<td> 6 %</td><td> 8,4</td><td> 8,6</td>
ES 2 616 630 T3
<td> 5 %</td><td> 8,5</td><td> 8,6</td>
Example 7
Evaluation of the bactericidal effect of MSM in cosmetics
This example shows the bactericidal effect of MSM in cosmetics.
An initial evaluation of the bactericidal effect of MSM was made on a cosmetic matrix. The cosmetic matrix was a cream base (jojoba) that is used in many cosmetic products. MSM was incorporated into the cream at concentrations in the range of 5-16% MSM. Then, each of these concentrations was seeded with E. coli at a level of 4.6 x 10<sup>5</sup> cfu / ml and incubated at 25 ° C for 48 hours. After 48 hours, aliquots of each culture were diluted and plated on tryptic soy agar, which was then incubated at 35 ° C for 24 hours before counting. The results of these studies are shown in Table 7-1.
Table 7-1. 48 hour culture log of E. coli in cosmetic matrix containing MSM
<td>MSM concentration</td><td>Cream</td>
<td> 16 %</td><td> 1</td>
<td> 15 %</td><td> 1</td>
<td> 14 %</td><td> 1</td>
<td> 13 %</td><td> 1</td>
<td> 12 %</td><td> 1</td>
<td> 11 %</td><td> 0,78</td>
<td> 10 %</td><td> 1,5</td>
<td> 9 %</td><td> 1,9</td>
<td> 8 %</td><td> 1,8</td>
<td> 7 %</td><td> 2,5</td>
<td> 6 %</td><td> 2</td>
<td> 5 %</td><td> 3,16</td>
These data indicate that bacteria growing on cosmetic cream are particularly sensitive to MSM. Surprisingly, lower concentrations of MSM (eg, the 5-9% concentration range) substantially inhibited bacterial growth in this study. Therefore, in various embodiments, MSM is used in concentrations greater than 5% to inhibit microbial activity.
Example 8
Evaluation of the bactericidal activity of 10% MSM in a cosmetic base with or without preservative for 28 days
This example shows the bactericidal activity of 10% MSM in a cosmetic base with or without a preservative for a period of 28 days.
To evaluate the ability of MSM to function as a long-acting antimicrobial agent in a cosmetic base, 10% MSM was incorporated into a cosmetic cream matrix seeded with E. coli, which was evaluated over a time period of 28 days using the AET <51> protocol of the USP. The cosmetic cream matrix MSN was incorporated into had no preservatives. It was also seeded with E. coli and an additional cream, with a preservative, was evaluated. The results of these studies are shown in Tables 8-1 through 8-4.
Table 8-1. Effect of 10% MSM on microbial growth in a preservative-free cosmetic cream
<td>Test body</td><td>Initial inoculum</td><td>48 h</td><td>7 days</td><td>14 days</td><td>28 days</td>
<td>Aspergillus Niger</td><td>1.1 x 10<sup>5</sup></td><td>8x10<sup>3</sup></td><td>8x10<sup>3</sup></td><td>6 x 10<sup>3</sup></td><td>5x10<sup>2</sup></td>
<td>Candida Albicans</td><td>2.1 x 10<sup>5</sup></td><td> <10</td><td> <10</td><td> <10</td><td> <10</td>
ES 2 616 630 T3
<td>Escherichia coli</td><td>4.8 x 10<sup>6</sup></td><td> <10</td><td> <10</td><td> <10</td><td> <10</td>
<td>Pseudomonas aeruginosa</td><td>1.89 x 10<sup>6</sup></td><td> <10</td><td> <10</td><td> <10</td><td> <10</td>
<td>Staphylococcus aureus</td><td>4.0 x 10<sup>6</sup></td><td> <10</td><td> <10</td><td> <10</td><td> <10</td>
Table 8-2. Logarithmic reduction with respect to the initial microorganism inoculum with 10% MSM in dilution of MSM in jojoba cosmetic without preservatives.
<td>Test body</td><td>14 days</td><td>28 days</td>
<td>Aspergillus Niger</td><td> 1,2</td><td> 2,3</td>
<td>Candida Albicans</td><td> 4,3</td><td> 4,3</td>
<td>Escherichia coli</td><td> 5,7</td><td> 5,7</td>
<td>Pseudomonas aeruginosa</td><td> 5,3</td><td> 5,3</td>
<td>Staphylococcus aureus</td><td> 5,6</td><td> 5,6</td>
Table 8-3. Microbial growth in a cosmetic cream that contains a preservative
<td>Test body</td><td>Initial inoculum</td><td>48 h</td><td>7 days</td><td>14 days</td><td>28 days</td>
<td>Aspergillus Niger</td><td>1.1 x 10<sup>5</sup></td><td>2 x 10<sup>6</sup></td><td>1.6 x 10<sup>2</sup></td><td>18 x 10<sup>1</sup></td><td>3 x 10<sup>1</sup></td>
<td>Candida Albicans</td><td>2.1 x 10<sup>5</sup></td><td> <10</td><td> <10</td><td> <10</td><td> <10</td>
<td>Escherichia coli</td><td>4.8 x 10<sup>6</sup></td><td> <10</td><td> <10</td><td> <10</td><td> <10</td>
<td>Pseudomonas aeruginosa</td><td>1.89 x 10<sup>6</sup></td><td> <10</td><td> <10</td><td> <10</td><td> <10</td>
<td>Staphylococcus aureus</td><td>4.0 x 10<sup>6</sup></td><td>3 x 10<sup>4</sup></td><td>1.6 x 10<sup>4</sup></td><td> <10</td><td> <10</td>
Table 8-4. Logarithmic reduction relative to the initial microorganism inoculum in cosmetic jojoba cream containing a preservative
<td>Test body</td><td>14 days</td><td>28 days</td>
<td>Aspergillus Niger</td><td> 2,7</td><td> 3,5</td>
<td>Candida Albicans</td><td> 4,3</td><td> 4,3</td>
<td>Escherichia coli</td><td> 5,7</td><td> 5,7</td>
<td>Pseudomonas aeruginosa</td><td> 5,3</td><td> 5,3</td>
<td>Staphylococcus aureus</td><td> 5,6</td><td> 5,6</td>
These studies demonstrate that a cosmetic cream base containing MSM is effective in substantially inhibiting microbial growth for a period of 28 days. Furthermore, these studies demonstrated that under certain conditions, MSM is a more effective antimicrobial agent than a conventional cosmetic preservative. For example, 10% MSM reduced microbial load to a greater degree after 48 hours compared to a cream containing preservative. Furthermore, S. aureus was reduced to virtually undetectable levels at 48 hours in the cream containing MSM. In contrast, the cream containing preservative showed a modest bacterial population of 3 x 10<sup>4</sup> bacteria after 48 hours. Despite a less robust initial phase, the bacterial load of the preservative-containing cream was reduced to the same extent at the end of the study, compared to the preservative-containing cream.
Example 9
Evaluation of the antimicrobial activity of MSM in two cosmetic compositions without preservatives
This example describes the antimicrobial activity of MSM in two representative preservative-free cosmetic compositions.
As described in Example 8, above, 10% MSM was incorporated into the cosmetic matrices, which were seeded with various initial microbial inoculations. According to the USP AET <51> test, these cultures seeded with microbes were incubated for 28 days, with samples withdrawn at 48 hours, 7 days, 14 days, and 28 days for plating and subsequent colony counting. The results of these studies are shown in the
ES 2 616 630 T3 Tables 9-1 to 9-4 below.
Table 9-1. Effect of 10% MSM on bacterial growth in the composition, cosmetic without preservatives # 1
<td>Test body</td><td>Initial inoculum</td><td>48 h</td><td>7 days</td><td>14 days</td><td>28 days</td>
<td>Aspergillus Niger</td><td>8.0 x 10<sup>5</sup></td><td>9.0 x 10<sup>3</sup></td><td>4.0 x 10<sup>3</sup></td><td>5.0 x 10<sup>1</sup></td><td> <10</td>
<td>Candida Albicans</td><td>2.0 x 10<sup>6</sup></td><td>2.1 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Escherichia coli</td><td>5.8 x 10<sup>6</sup></td><td>5.4 x 10<sup>4</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Pseudomonas aeruginosa</td><td>5.7 x 10<sup>6</sup></td><td>7.3 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Staphylococcus aureus</td><td>5.3 x 10<sup>6</sup></td><td>1.9 x 10<sup>4</sup></td><td> <10</td><td> <10</td><td> <10</td>
Table 9-2. Log reduction from initial microorganism inoculum with 10% MSM in Preservative Free Cosmetic Composition # 1
<td>Test body</td><td>14 days</td><td>28 days</td>
<td>Aspergillus Niger</td><td> 4,2</td><td> 4,9</td>
<td>Candida Albicans</td><td> 5,3</td><td> 5,3</td>
<td>Escherichia coli</td><td> 5,8</td><td> 5,8</td>
<td>Pseudomonas aeruginosa</td><td> 5,8</td><td> 5,8</td>
<td>Staphylococcus aureus</td><td> 5,7</td><td> 5,7</td>
Table 9-3. Effect of 10% MSM on Bacterial Growth in Preservative-Free Cosmetic Composition # 2
<td>Test body</td><td>Initial inoculum</td><td>48 h</td><td>7 days</td><td>14 days</td><td>28 days</td>
<td>Aspergillus Niger</td><td>8.0 x 10<sup>5</sup></td><td>9.0 x 10<sup>3</sup></td><td>3.0 x 10<sup>3</sup></td><td>1.3 x 10<sup>3</sup></td><td>6.0 x 10<sup>1</sup></td>
<td>Candida Albicans</td><td>2.0 x 10<sup>6</sup></td><td>9.0 x 10<sup>2</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Escherichia coli</td><td>5.8 x 10<sup>6</sup></td><td>1.7 x 10<sup>5</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Pseudomonas aeruginosa</td><td>5.7 x 10<sup>6</sup></td><td>2.1 x 10<sup>3</sup></td><td> <10</td><td> <10</td><td> <10</td>
<td>Staphylococcus aureus</td><td>5.3 x 10<sup>6</sup></td><td>1.5 x 10<sup>5</sup></td><td> <10</td><td> <10</td><td> <10</td>
Table 9-4. Log reduction from initial microorganism inoculum with 10% MSM in Preservative Free Cosmetic Composition # 2
<td>Test body</td><td>14 days</td><td>28 days</td>
<td>Aspergillus Niger</td><td> 2,8</td><td> 5,1</td>
<td>Candida Albicans</td><td> 5,3</td><td> 5,3</td>
<td>Escherichia coli</td><td> 5,8</td><td> 5,8</td>
<td>Pseudomonas aeruginosa</td><td> 5,8</td><td> 5,8</td>
<td>Staphylococcus aureus</td><td> 5,7</td><td> 5,7</td>
These studies demonstrate that MSM exhibited effective antimicrobial properties in the absence of a preservative.
Example 10
Selected concentrations of MSM support microbial activity
This example demonstrates that selected concentrations of MSM support microbial activity.
Adjacent growth studies in medium fortified with MSM at an MSM concentration of 0, 0.04, 0.1, 0.2, 0.4 and 1% were compared to the growth curve of various microorganisms with a control sample. with a MSM concentration of 0%. Each organism (Lactobacillus rhamnosus, Lactobacillus acidophilus, and Bifidobacterium bifidum) was grown in MRS bacterial growth medium (broth) and plated on MRS agar at different time intervals. Results are expressed in colony forming units per milliliter (cfu / ml).
ES 2 616 630 T3
For each test organism, 100 ml aliquots of MRS broth were prepared with the respective concentration of MRS as indicated. Initially, a test solution of 1%, 0.4%, and 0.2% MSM (+/- 0.01%) was prepared by adding 1 g or 0.45 g of MSM in 110 g of MRS broth and 0 , 20 g of MSM in 100 g, respectively. The 0.1% and 0.04% test concentrations were prepared by preparing a 1:10 dilution of the 1% and 0.4% test solutions. Once each set of test media had been plated for sterility, they were inoculated at a level of 100 µl of inoculum per 100 g or ml of test broth (inoculum dilutions at 1: 1000) with each respective microorganism. All bacterial organisms were incubated at 35 ° C +/- 2 ° C for the entire duration of the study.
All samples were plated on MRS agar at time 0, 12, 36, 48, 60 and 72 hours (+/- 45 minutes). All preparations and plating were carried out at room temperature. All plating events were incubated at 35 ° C +/- 2 ° C for at least 2 days or 3 days for Bifidobacterium. Test samples were placed in triplicate on each test date and averages are indicated. The results of these studies are provided in Tables 10-1 through 10-3.
For Lactobacillus rhamnosus samples (Table 10-1), in the first 12 hours, all MSM samples had recovered at least 12% or more than the 0% control. The 0.2% and 1% concentrations were 41% and 47% higher, respectively, in the first 12 hours. All test values were online 24 hours before leveling off, the cultures were highly cloudy, suggesting that the organism had entered the stationary phase. However, after leveling off slightly at 36 and 48 hours, the counts in the MSM samples continued to increase while the 0% control began to decline.
Table 10-1. Lactobacillus rhamnosus growth
<td>Time (hours)</td><td>0% MSM</td><td>0.04% MSM</td><td>0.1% MSM</td><td>0.2% MSM</td><td>0.4% MSM</td><td>1% MSM</td>
<td> 0</td><td>1.4 x 10<sup>6</sup></td><td>1.7 x 10<sup>6</sup></td><td>1.7 x 10<sup>6</sup></td><td>1.9 x 10<sup>6</sup></td><td>2.2 x 10<sup>6</sup></td><td>1.5 x 10<sup>6</sup></td>
<td> 12</td><td>1.7 x 10<sup>7</sup></td><td>2.1 x 10<sup>7</sup></td><td>1.9 x 10<sup>7</sup></td><td>2.4 x 10<sup>7</sup></td><td>2.2 x 10<sup>7</sup></td><td>2.5 x 10<sup>7</sup></td>
<td> 24</td><td>1.4 x 10<sup>9</sup></td><td>1.5 x 10<sup>9</sup></td><td>1.3 x 10<sup>9</sup></td><td>1.4 x 10<sup>9</sup></td><td>1.4 x 10<sup>9</sup></td><td>1.4 x 10<sup>9</sup></td>
<td> 36</td><td>2.0 x 10<sup>9</sup></td><td>1.8 x 10<sup>9</sup></td><td>1.8 x 10<sup>9</sup></td><td>2.0 x 10<sup>9</sup></td><td>2.1 x 10<sup>9</sup></td><td>2.7 x 10<sup>9</sup></td>
<td> 48</td><td>2.0 x 10<sup>9</sup></td><td>2.1 x 10<sup>9</sup></td><td>2.3 x 10<sup>9</sup></td><td>2.3 x 10<sup>9</sup></td><td>2.1 x 10<sup>9</sup></td><td>2.4 x 10<sup>9</sup></td>
<td> 60</td><td>3.0 x 10<sup>9</sup></td><td>2.6 x 10<sup>9</sup></td><td>3.0 x 10<sup>9</sup></td><td>2.3 x 10<sup>9</sup></td><td>2.8 x 10<sup>9</sup></td><td>2.6 x 10<sup>9</sup></td>
<td> 72</td><td>2.7 x 10<sup>9</sup></td><td>2.6 x 10<sup>9</sup></td><td>3.0 x 10<sup>9</sup></td><td>3.2 x 10<sup>9</sup></td><td>3.0 x 10<sup>9</sup></td><td>3.8 x 10<sup>9</sup></td>
These studies suggest that MSM concentrations of approximately 0.1% to approximately 1% enhance Lactobacillus rhamnosus growth / function, with microbial levels in the range 11% to 41% higher after 72 hours.
For Lactobacillus acidophilus samples (Table 10-2), concentrations of MSM 0.04% and 0.1% were the first to provide growth, followed by MSM 0.2% and 0.4% at at 36 hours and the 1% MSM sample at 48 hours. Growth did not recover from the 0% control, suggesting that MSM had a positive impact on the recovery. Lower MSM concentrations revealed a shorter recovery time than higher MSM concentrations. The 0.04% and 0.4% MSM samples resulted in high levels of growth for Lactobacillus acidophilus. These studies indicate that MSM affects microbial metabolism in a way that promotes microbial resilience and recovery.
Table 10-2. Lactobacillus acidophilus growth
<td>Time (hours)</td><td>0% MSM</td><td>0.04% MSM</td><td>0.1% MSM</td><td>0.2% MSM</td><td>0.4% MSM</td><td>1% MSM</td>
<td> 0</td><td>1.0 x 10<sup>3</sup></td><td>1.0 x 10<sup>3</sup></td><td>1.0 x 10<sup>3</sup></td><td>1.0 x 10<sup>3</sup></td><td>1.0 x 10<sup>3</sup></td><td>1.0 x 10<sup>3</sup></td>
<td> 12</td><td>1.0 x 10<sup>3</sup></td><td>1.0 x 10<sup>3</sup></td><td>1.0 x 10<sup>3</sup></td><td>1.0 x 10<sup>3</sup></td><td>1.0 x 10<sup>3</sup></td><td>1.0 x 10<sup>3</sup></td>
<td> 24</td><td>1.0 x 10<sup>3</sup></td><td>1.5 x 10<sup>4</sup></td><td>1.2 x 10<sup>4</sup></td><td>1.0 x 10<sup>5</sup></td><td>1.0 x 10<sup>5</sup></td><td>1.0 x 10<sup>5</sup></td>
<td> 36</td><td>1.0 x 10<sup>3</sup></td><td>8.7 x 10<sup>7</sup></td><td>3.4 x 10<sup>7</sup></td><td>7.3 x 10<sup>5</sup></td><td>1.6 x 10<sup>8</sup></td><td>1.0 x 10<sup>3</sup></td>
<td> 48</td><td>1.0 x 10<sup>3</sup></td><td>2.2 x 10<sup>8</sup></td><td>2.1 x 10<sup>8</sup></td><td>7.4 x 10<sup>6</sup></td><td>2.2 x 10<sup>8</sup></td><td>4.7 x 10<sup>4</sup></td>
<td> 60</td><td>1.0 x 10<sup>5</sup></td><td>3.4 x 10<sup>8</sup></td><td>1.7 x 10<sup>8</sup></td><td>1.7 x 10<sup>7</sup></td><td>3.1 x 10<sup>8</sup></td><td>2.8 x 10<sup>7</sup></td>
<td> 72</td><td>1.0 x 10<sup>5</sup></td><td>4.7 x 10<sup>8</sup></td><td>2.7 x 10<sup>8</sup></td><td>8.9 x 10<sup>6</sup></td><td>3.6 x 10<sup>8</sup></td><td>1.6 x 10<sup>8</sup></td>
ES 2 616 630 T3
Bifidobacterium bifidum, a common microbe used in probiotics, was also tested. All Bifidobacterium samples were incubated under anaerobic conditions. Oxygen indicators were used to verify anaerobic conditions between plating intervals for Bifidobacterium test samples and plating events.
At 48 hours, the 0.04% and 0.2% MSM samples were 1 log greater than the 0% MSM control. The 0.2% MSM sample had the highest level of growth for Bifidobacterium bifidum, followed by the 0.04% MSM sample.
As observed with Lactobacillus rhamnosus (Table 10-2), the MSM samples from 0.1% to 1% continued to grow while the control was doomed to a phase of decreasing stationary growth (Table 10-3). Increases of one and 2 log of Bifidobacterium were observed with the concentrations of MSM at 0.04% and 0.2%, respectively (Table 10-3).
Table 10-3. Bifidobacterium bifidum growth
<td>Time (hours)</td><td>0% MSM</td><td>0.04% MSM</td><td>0.1% MSM</td><td>0.2% MSM</td><td>0.4% MSM</td><td>1% MSM</td>
<td> 0</td><td>8.7 x 10<sup>4</sup></td><td>6.6 x 10<sup>4</sup></td><td>7.0 x 10<sup>4</sup></td><td>7.0 x 10<sup>4</sup></td><td>1.1 x 10<sup>5</sup></td><td>6.8 x 10<sup>4</sup></td>
<td> 12</td><td>2.0 x 10<sup>3</sup></td><td>3.5 x 10<sup>3</sup></td><td>4.4 x 10<sup>4</sup></td><td>3.1 x 10<sup>4</sup></td><td>2.0 x 10<sup>3</sup></td><td>2.3 x 10<sup>4</sup></td>
<td> 24</td><td>1.4 x 10<sup>5</sup></td><td>1.2 x 10<sup>5</sup></td><td>1.3 x 10<sup>5</sup></td><td>1.3 x 10<sup>5</sup></td><td>1.0 x 10<sup>5</sup></td><td>2.0 x 10<sup>5</sup></td>
<td> 36</td><td>2.4 x 10<sup>5</sup></td><td>2.3 x 10<sup>5</sup></td><td>2.0 x 10<sup>5</sup></td><td>2.3 x 10<sup>5</sup></td><td>2.3 x 10<sup>5</sup></td><td>4.0 x 10<sup>5</sup></td>
<td> 48</td><td>2.6 x 10<sup>5</sup></td><td>5.2 x 10<sup>6</sup></td><td>2.3 x 10<sup>5</sup></td><td>5.8 x 10<sup>6</sup></td><td>4.0 x 10<sup>5</sup></td><td>3.1 x 10<sup>5</sup></td>
<td> 60</td><td>3.3 x 10<sup>5</sup></td><td>3.4 x 10<sup>7</sup></td><td>4.7 x 10<sup>5</sup></td><td>4.8 x 10<sup>7</sup></td><td>8.7 x 10<sup>5</sup></td><td>3.7 x 10<sup>5</sup></td>
<td> 72</td><td>1.6 x 10<sup>6</sup></td><td>8.0 x 10<sup>7</sup></td><td>5.3 x 10<sup>5</sup></td><td>1.2 x 10<sup>8</sup></td><td>1.2 x 10<sup>6</sup></td><td>5.7 x 10<sup>5</sup></td>
The general growth characteristics of probiotic organisms in supplemented medium such as MSM and without MSM were also tested by observation. Colony sizes of Bacillus coagulans grown in medium containing 0% and 5% MSM were compared (see Example 13 for a detailed description).
Example 11
Evaluation of the influence of MSM on the shelf life
This example describes the effect of MSM on the shelf life of milk.
MSM as an additive has been shown to increase the growth and recovery of beneficial microorganisms in a product. This example examined whether MSM modified microorganisms that affect shelf life stability of a product based on microbial count. Milk, which has a relatively short shelf life, was used as the product to be evaluated in this study. Milk with fat concentrations was analyzed to study the effects of how the concentration of solids in the product can affect MSM. The conventional shelf life for milk is 18 to 21 days, the study was carried out up to 28 days. A shelf life study was carried out on dairy products fortified with 0.0%, 0.5%, 1.0%, 2.5%, 5.0% and 10% MSM. The time intervals for sowing the solutions in days were on day 0, 7, 14, 21, 24 and 28. The influences of the percentage of solids on the MSM concentrations were evaluated in the following percentages: 0.0%, 1.0%, 2%, 10.5% and 40%. The growth curves of the colony forming units per milliliter (cfu / ml) of the microorganisms were compared between the MSM concentrations, with the 0% MSM concentration as a control sample. The stock of MSM powder was supplied by Bergstrom Nutrition with Certificate of Analysis. The powder was the Microprill formula, lot # 0806809, with an expiration date 10/31/13. All MSM media, water, and powder stock were tested for sterility prior to study. Working concentrations of MSM were prepared from a single 10.0% MSM solution and diluted accordingly with bottled milk to obtain the desired final concentration of MSM. The product was supplied by a local milk processing plant. Samples were collected and the study began on the processing day. The product included two bottles of each product for concentration of MSM for each day of analysis. The total number of bottles for one type of product was 72 for the entire experiment. All bottles of the same product type came from a production batch.
The microorganisms analyzed were the normal flora in the product after its processing. Product samples were kept at 4 ° C for the duration of the study. All preparation and seeding was carried out at room temperature. Each concentration of MSM was carried out in duplicate. Each dilution was plated in duplicate for the time interval sampled. To capture suitable colonies per milliliter, each organism was plated at each time interval in three dilutions. All plates were incubated at 37 ° C ± 0.5 ° C for 48 hours prior to examination. The appropriate dilution plate was used for enumeration and averaged for reporting. The plate suitable for enumeration contained between 25 and 250 cfu / ml.
ES 2 616 630 T3
The MSM stock sample and all MSM-prepared media were tested for background levels of microorganisms on tryptic soy agar. The MSM pool was <10 cfu / g and all test media were negative in all cases before inoculation. All time intervals for seeding included negative control plates during pouring for quality control purposes. All negative control plates were devoid of microorganism growth. The results of these studies are shown in Tables 11.1-15.1 below.
Table 11.1 Logarithmic growth in skimmed milk
<td></td><td colspan="6">Time (days)</td>
<td>MSM conc.</td><td> 0</td><td> 7</td><td> 14</td><td> 21</td><td> 24</td><td> 28</td>
<td> 0 %</td><td> 1,50</td><td> 2,05</td><td> 0,94</td><td> 4,40</td><td> 1,44</td><td> 2,20</td>
<td> 0,5 %</td><td> 0,00</td><td> 1,00</td><td> 0,70</td><td> 4,41</td><td> 4,68</td><td> 3,79</td>
<td> 1 %</td><td> 0,74</td><td> 0,70</td><td> 0,00</td><td> 4,56</td><td> 1,28</td><td> 2,02</td>
<td> 2,5 %</td><td> 0,74</td><td> 0,70</td><td> 1,65</td><td> 4,37</td><td> 3,69</td><td> 0,00</td>
<td> 5 %</td><td> 0,00</td><td> 0,70</td><td> 0,00</td><td> 1,15</td><td> 3,20</td><td> 0,00</td>
<td> 10 %</td><td> 0,92</td><td> 0,70</td><td> 0,59</td><td> 0,00</td><td> 0,00</td><td> 0,00</td>
Table 12.1. Logarithmic growth in 1% fat milk
<td rowspan="2">MSM conc.</td><td colspan="6">Time (days)</td>
<td> 0</td><td> 7</td><td> 14</td><td> 21</td><td> 24</td><td> 28</td>
<td> 0,0 %</td><td> 0,35</td><td> 1,36</td><td> 0,00</td><td> 4,44</td><td> 4,44</td><td> 5,74</td>
<td> 0,5 %</td><td> 1,05</td><td> 0,50</td><td> 0,70</td><td> 4,37</td><td> 3,27</td><td> 6,09</td>
<td> 1,0 %</td><td> 0,35</td><td> 1,23</td><td> 0,70</td><td> 4,37</td><td> 5,27</td><td> 5,36</td>
<td> 2,5 %</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 4,28</td><td> 4,28</td><td> 2,53</td>
<td> 5,0 %</td><td> 0,00</td><td> 2,55</td><td> 0,59</td><td> 0,59</td><td> 1,28</td><td> 0,00</td>
<td> 10,0 %</td><td> 0,70</td><td> 0,00</td><td> 0,50</td><td> 0,85</td><td> 0,50</td><td> 0,00</td>
Table 13.1. Logarithmic growth in 2% fat milk
<td rowspan="2">MSM conc.</td><td colspan="6">Time (days)</td>
<td> 0</td><td> 7</td><td> 14</td><td> 21</td><td> 24</td><td> 28</td>
<td> 0,0 %</td><td> 1,90</td><td> 1,60</td><td> 2,44</td><td> 4,45</td><td> 4,44</td><td> 4,93</td>
<td> 0,5 %</td><td> 1,26</td><td> 1,59</td><td> 2,09</td><td> 4,43</td><td> 3,27</td><td> 5,48</td>
<td> 1,0 %</td><td> 1,04</td><td> 3,21</td><td> 0,00</td><td> 4,07</td><td> 5,27</td><td> 6,56</td>
<td> 2,5 %</td><td> 1,06</td><td> 0,00</td><td> 0,70</td><td> 2,96</td><td> 4,28</td><td> 5,81</td>
<td> 5,0 %</td><td> 1,39</td><td> 0,35</td><td> 0,00</td><td> 3,97</td><td> 1,28</td><td> 0,59</td>
<td> 10,0 %</td><td> 0,35</td><td> 0,50</td><td> 0,70</td><td> 1,97</td><td> 0,50</td><td> 1,66</td>
Table 14.1. Logarithmic growth in milk with fat at 10.5%
<td rowspan="2">MSM conc.</td><td colspan="6">Time (days)</td>
<td> 0</td><td> 7</td><td> 14</td><td> 21</td><td> 24</td><td> 28</td>
<td> 0,0 %</td><td> 0,35</td><td> 0,42</td><td> 1,98</td><td> 4,43</td><td> 2,94</td><td> 4,7</td>
<td> 0,5 %</td><td> 0,00</td><td> 1,09</td><td> 0,00</td><td> 4,36</td><td> 2,73</td><td> 3,84</td>
<td> 1,0 %</td><td> 0,50</td><td> 0,00</td><td> 0,94</td><td> 3,30</td><td> 2,24</td><td> 3,91</td>
<td> 2,5 %</td><td> 0,35</td><td> 0,35</td><td> 0,00</td><td> 2,34</td><td> 3,14</td><td> 1,84</td>
<td> 5,0 %</td><td> 0,00</td><td> 0,85</td><td> 0,00</td><td> 0,35</td><td> 1,99</td><td> 0,00</td>
ES 2 616 630 T3
<td> 10,0 %</td><td> 1,00</td><td> 0,85</td><td> 0,35</td><td> 2,14</td><td> 0,35</td><td> 0,00</td>
Table 15.1. Logarithmic growth in milk with 40% fat
<td rowspan="2">MSM conc.</td><td colspan="6">Time (days)</td>
<td> 0</td><td> 7</td><td> 14</td><td> 21</td><td> 24</td><td> 28</td>
<td> 0,0 %</td><td> 0,00</td><td> 1,89</td><td> 2,08</td><td> 4,47</td><td> 4,23</td><td> 4,75</td>
<td> 0,5 %</td><td> 0,00</td><td> 0,35</td><td> 2,32</td><td> 4,46</td><td> 4,02</td><td> 1,95</td>
<td> 1,0 %</td><td> 0,74</td><td> 0,00</td><td> 2,37</td><td> 4,35</td><td> 3,33</td><td> 3,86</td>
<td> 2,5 %</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 2,16</td><td> 3,79</td><td> 0,95</td>
<td> 5,0 %</td><td> 0,00</td><td> 0,80</td><td> 0,50</td><td> 1,25</td><td> 1,03</td><td> 0,35</td>
<td> 10,0 %</td><td> 0,00</td><td> 0,00</td><td> 1,95</td><td> 4,45</td><td> 0,85</td><td> 0,35</td>
When milk is evaluated without the MSM, there was an increase in the counts on day 21. This is a conventional peak in dairy products. The increase in normal flora reaches a 2 log point and there is an onset of product degradation. At 4 log, the shelf life of the product is questionable and sensory factors make the product undesirable.
The evaluation study takes into consideration the nature of the product being used. The product was taken from the one-day production batch. Microbial counts for a single batch of dairy products can range from 0.5 to
1.5 log. Observing the growth on day 0, the intervals for each product are 1.5 log from each other.
Day 7 shows that for control and MSM concentrations there was a slight increase in microbial counts. There was one microbial count that was higher than the other for each sample, except for the 10.5% fat milk product. The microbial counts of the 10.5% product were all within 0.75 log of each other, (control and MSM concentrations). The 40% fat-free products had an increase in the control sample. Although the 1% dairy product had an increase in the 5% MSM sample and the 2% milk product had an increase in the 1% MSM sample.
On day 14, the products show normal microbial counts and growth rates. No abnormal growth is observed in the products. Products with lower milk fat are among the expected microbial load variabilities, when the blank is compared with MSM concentrations. The 40% milk product demonstrates a lower microbial count for the 5.00% and 2.50% MSM concentrations, although the blank and all other MSM concentrations are between 0.40 log in the microbial counts . The 10.5% dairy product demonstrates that the blank is 1 log greater than the MSM concentrations, with the 1.00% and 10.0% MSM being the only two with a microbial count.
On day 21, the microbial product counts were separated with reference to blank and MSM concentrations. 1% nonfat milk products indicated that MSM in the highest concentrations (5.0% and 10.0%) slowed the growth of normal flora. While the microbial counts of the control concentrations and the minor ones of MSM increased to 4 log. In the 2% milk product, the concentration of MSM at 10.0% and the concentration of MSM at 2.50% slowed down the growth rate of the normal flora. Microbial counts for MSM 0.50%, 1.00%, 5.00%, and blank control were 4 log. The 10.5% dairy product showed the 5.00% MSM at 0.35 log, showing the growth rate compared to the control, which was 4.43 log. The concentration of 0.50% MSM was 4.36 log, 1.00% MSM was 3.30 log, 2.50% MSM was 2.34 and 10.0% MSM was 2 , 14 log. With the increase in MSM concentrations, the microbial counts decreased, with the exception of MSM at 5.00%. The microbial load of the 40% dairy product was relatively equal in its count for the control MSM, at 0.5%, 1.0% and 10%. The 2.5% MSM concentration was two log lower than the control at 2.16 log, while the 5.0% MSM was 3.22 log lower.
On day 24, the control and the 1.0% MSM for the non-fat dairy product dropped to approximately 1.3 log, while the 0.50% MSM maintained the microbial counts. Microbial counts of 2.50% MSM decreased, while 5.0% MSM increased. No growth was observed in MSM at 10.0%. The 1% dairy product had a reduced microbial count in the 0.50% MSM, increased in the 1.00% MSM, and there was no alteration in the control and the 2.5% MSM. The 5.0% MSM increased and the 10.0% MSM decreased. These two higher MSM concentrations were kept at a low microbial count. For the 2% dairy product, all MSM concentrations and the control continued to increase in microbial load. The 10% MSM continued to lag behind the microbial count. The 10.5% dairy product demonstrated a reduction in control and MSM concentrations; 0.5%, 1.0%, and 10.0%. The 2.5% MSM and 5.0% MSM continued to increase. The dairy product at 40% showed a slight reduction in growth for the control, MSM at 5.0% and the two
ES 2 616 630 T3 lower concentrations of MSM. Microbial counts of 2.5% MSM increased at 24 hours, while 10.0% MSM had a significant reduction in microbial growth. MSM 5.0% and MSM 10.0% had a count close to the microbial loads on day 0.
On day 28, nonfat dairy products were greater than 2 log microbial load. The concentration of 0.5% MSM was 3.79 log. Higher concentrations of MSM; 2.50%, 5.0% and 10.0% had no growth by day 28. 1% dairy product shows an increase for the control, 0.5% and 1.0% MSM. The MSM sample at
2.5% had a reduction in microbial load, while MSM at 5.0% and 10.0% had no growth. The 2% dairy product had an increase for the 1.0% MSM, a slight reduction for the control, 0.50% MSM and the
2.5%. The 5.0% MSM and 10.0% MSM were reduced to 0.59 log and 1.66 log, respectively. The fattiest dairy products, 10.5% and 40%, show that control continues to increase in microbial counts. Both products have an increase with MSM at 1.0%, while the product at 10.5% also had the increase of MSM at 0.5% in microbial counts, the product at 40% had a reduction of MSM at 0, 5 %. The 2.5% MSM reduced the microbial load for both products. At 5.0% and 10% there was no growth for the 10.5% dairy product. The 40% product had a microbial count of 0.35 log for the 5.0% and 10.0% MSM concentrations.
These studies indicate that the use of MSM as an additive to milk does not adversely affect the shelf life of the milk. In particular, on day 21 there was no MSM concentration that had a higher microbial count than the control. Furthermore, these studies indicate that in certain dairy products, a concentration of MSM at 5.0% or MSM at 10.0% actually kept the microbial load significantly lower than that of the control. These studies suggest that MSM at such concentrations can be used to increase the shelf life of a product, such as milk.
Example 12
Growth of Milk Acidophilus and Bacillus coagulans in simulated gastric acid supplemented with MSM
This example describes the growth of Milk Acidophilus and Bacillus coagulans in simulated gastric acid supplemented with MSM.
To analyze the effects of methylsulfonylmethane (MSM) on the growth of probiotic microorganisms in medium fortified with MSM in a simulated stomach fluid. Previous studies have shown that the addition of MSM to the culture medium helps in the growth rate of microorganisms. The study will measure the effect of MSM-fortified probiotic growth in simulated gastric acid fluid.
Microbial growth studies were carried out in the presence of 0%, 0.25%, 2.0%, and 5%. The plating time intervals were used every 3 hours for 15 hours, then 24 and 48 hours. The growth curves of colony forming units per milliliter (cfu / ml) of the microorganisms between the MSM concentrations were compared with the 0% MSM concentration as a control sample for each microorganism. The stock of MSM powder was supplied by Bergstrom Nutrition with Certificate of Analysis. The powder was the Microprill formula, lot # 0806809, with an expiration date 10/31/13. All media and the MSM powder stock were tested for sterility prior to study. The study was carried out in two organisms over a period of two weeks. The microorganisms tested included milk Lactobacillus acidophilus and Bacillus coagulans (15BB lot # 90BC004A1MZ supplied by Ganeden).
For Lactobacillus acidophilus milk, 11 ml of milk with a count of 81,000 cfu / ml, 99 ml of simulated gastric acid was added. For Bacillus coagulans, 1 gram of powder was added to 99 ml of a tryptic soy broth (TSB) to obtain a count of 108,000 cfu per ml of Bacillus coagulans. Eleven millilisters of Bacillus coagulans TSB were added to 99 ml of simulated gastric acid. Working concentrations of MSM were prepared from a single 5.0% MSM solution and diluted accordingly with milk or TSB to obtain the desired final concentration of MSM. The sterility of all solutions was verified before proceeding with the study. The working simulated gastric acid was incubated at 35.0 ± 0.2 ° C during the study. The simulated gastric acid pH was 1.2.
Lactobacillus acidophilus milk was inoculated on MRS agar and the times listed above. Bacillus coagulans was inoculated on tryptic soy agar (TSA) at the times listed above. All preparation and seeding was carried out at room temperature. Each concentration of MSM in simulated gastric acid was run in duplicate. Each dilution for each organism was plated in triplicate for each time interval sampled. To capture the proper colonies per milliliter, each organism was plated at each time interval at six different dilutions. All plates were incubated at 35 ° C ± 0.5 ° C for 72 hours for all organisms, except for Bacillus which was incubated for 48 hours, prior to examination. The appropriate dilution plate was used for enumeration and averaged for reporting. The plate suitable for enumeration contains between 25 and 250 cfu / ml.
The MSM stock sample and all MSM-prepared media were tested for background levels of microorganisms on MRS agar and TSA. MSM stock was <10 cfu / g and all assay media were <1 cfu / ml
ES 2 616 630 T3 in all cases before inoculation (see table below). All time intervals for seeding included negative control plates during pouring for quality control purposes. All negative control plates were devoid of microorganism growth. The results of these studies are provided in Tables 16.1 to 18.2.
Table 16.1. Stock culture control numbers prior to inoculation of the test sample
<td></td><td>Lactobacillus acidophilus milk</td><td>Bacillus coagulans</td>
<td>cfu / ml, inoculum</td><td>8.1 x 10<sup>4</sup></td><td>1.08 x 10<sup>5</sup></td>
<td>cfu added to 99 ml</td><td>8.1 x 10<sup>5</sup></td><td>1.08 x 10<sup>6</sup></td>
<td>cfu / ml in medium at time 0</td><td>8.1 x 10<sup>3</sup></td><td>1.08 x 10<sup>4</sup></td>
Control numbers are derived from the growth of specific organisms in the appropriate medium. The inoculation fluids were placed for enumeration in the appropriate medium. To capture the proper colonies per milliliter, each liquid was plated in triplicate at fourteen different dilutions. The appropriate dilution plate was used for enumeration and averaged for reporting. The plate suitable for enumeration contains between 25 and 250 cfu / ml.
Table 17.1. Logarithmic growth of L. acidophilus in milk in duplicate in o
or
-I— <
C
Φ 'E o
<p or
Concentration in percent of MSM
<td>Time in (hours)</td><td> 0</td><td> 0</td><td> 0,25</td><td> 0,25</td><td> 2,5</td><td> 2,5</td><td> 5</td><td> 5</td>
<td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0,82</td>
<td> 3</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 1,3</td><td> 1</td><td> 0</td><td> 0,82</td>
<td> 6</td><td> 0</td><td> 0</td><td> 0,52</td><td> 0,52</td><td> 0</td><td> 0</td><td> 0,82</td><td> 1</td>
<td> 9</td><td> 0</td><td> 0</td><td> 0,82</td><td> 0</td><td> 0,82</td><td> 0</td><td> 0,52</td><td> 0,52</td>
<td> 12</td><td> 0</td><td> 1,00</td><td> 1,00</td><td> 0</td><td> 1,22</td><td> 0,82</td><td> 0,82</td><td> 1,37</td>
<td> 15</td><td> 0,52</td><td> 1,42</td><td> 1,56</td><td> 1,3</td><td> 1,3</td><td> 1,37</td><td> 1,43</td><td> 1,43</td>
<td> 24</td><td> 0,52</td><td> 0,82</td><td> 1,37</td><td> 1,27</td><td> 1,67</td><td> 1,6</td><td> 1,67</td><td> 1,64</td>
<td> 48</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 1,3</td><td> 1,22</td><td> 1,22</td><td> 1,43</td>
Table 17.2. Mean log growth of L. acidophilus in milk
Concentration in percent of MSM
<td>Time in (hours)</td><td> 0</td><td> 0,25</td><td> 2,5</td><td> 5</td>
<td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0,41</td>
<td> 3</td><td> 0</td><td> 0</td><td> 1,15</td><td> 0,41</td>
<td> 6</td><td> 0</td><td> 0,52</td><td> 0</td><td> 0,91</td>
<td> 9</td><td> 0</td><td> 0,41</td><td> 0,41</td><td> 0,52</td>
<td> 12</td><td> 0,5</td><td> 0,5</td><td> 1,02</td><td> 1,10</td>
<td> 15</td><td> 0,98</td><td> 1,43</td><td> 1,34</td><td> 1,43</td>
<td> 24</td><td> 0,67</td><td> 1,32</td><td> 1,64</td><td> 1,66</td>
<td> 48</td><td> 0</td><td> 0</td><td> 1,26</td><td> 1,33</td>
Acidophilus milk placed in simulated gastric acid was assisted by MSM in the recovery of Lactobacillus acidophilus. The initial recovery was less than the detection limit of the method. 5.0% MSM had an initial recovery of 0.41. Hour 3 showed an increase in recovery for MSM at 2.5%, while maintaining logarithmic growth with MSM at 5.0%. At hour 6, the 0.25% MSM had a 0.52 log recovery, 5.0% MSM showed a 0.91 log recovery. 2.5% MSM had a reduction in growth until not detectable. At hour 9, there was significant detection for all MSM concentrations. The 0% control remained below the detectable limit. At hour 12, the control grew to 0.5 log coinciding with MSM at 0.25%. The 2.5% and 5.0% MSM samples had growth rates of 1.02 and 1.10 log, respectively. Hour 15 demonstrated continuous growth with MSM concentrations at 1.43, 1.34, and 1.43 log. The 0% MSM control increased from 0.48 log to 0.98 log. 0% MSM and 0.25% MSM at 24 hours reduced growth by 0.31 log and 0.11 log, respectively. The 0.25% MSM increased by 0.30 log and the 5.0% MSM increased by 0.23 log. In the hour
ES 2 616 630 T3
48, the 0.25% MSM and the 0% MSM control were reduced below detectable limits. The 2.5% MSM decreased by 0.38 log and the 5.0% MSM decreased by 0.33 log.
Table 18.1. Logarithmic growth of Bacillus coagulans in duplicate
Concentration in percent of MSM
<td>Time in (hours)</td><td> 0</td><td> 0</td><td> 0,25</td><td> 0,25</td><td> 2,5</td><td> 2,5</td><td> 5</td><td> 5</td>
<td> 0</td><td> 0</td><td> 0,52</td><td> 0,52</td><td> 0</td><td> 0</td><td> 0</td><td> 0,52</td><td> 0</td>
<td> 3</td><td> 0</td><td> 0</td><td> 0,52</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td> 6</td><td> 0</td><td> 0</td><td> 052</td><td> 0,52</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td> 9</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td> 12</td><td> 0</td><td> 0</td><td> 1,12</td><td> 0,52</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td> 15</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0,52</td><td> 0</td>
<td> 24</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td> 48</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
Table 18.2. Logarithmic growth mean of Bacillus coagulans
Concentration in percent of MSM
<td>Time in (hours)</td><td> 0</td><td> 0,25</td><td> 2,5</td><td> 5</td>
<td> 0</td><td> 0</td><td> 0,26</td><td> 0</td><td> 0,26</td>
<td> 3</td><td> 0</td><td> 0,26</td><td> 0</td><td> 0</td>
<td> 6</td><td> 0</td><td> 0,52</td><td> 0</td><td> 0</td>
<td> 9</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td> 12</td><td> 0</td><td> 0,82</td><td> 0</td><td> 0</td>
<td> 15</td><td> 0</td><td> 0</td><td> 0</td><td> 0,26</td>
<td> 24</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
<td> 48</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>
The initial recovery of Bacillus coagulans did not indicate recovery. However, the 0.25% and 5.0% MSM samples had a mean of 0.26 log. Low recovery was observed throughout the study for MSM at 0.25% and at hour 15 for MSM at 5.0%. The recovery was too low to draw a conclusion about the gastric acid study for Bacillus coagulans. It is possible that the initial 3 hour exposure killed the organisms.
These studies reveal that when it comes to Acidophilus milk, there was a positive impact on bacterial growth with milk containing MSM. In the first 3 to 6 hours, there was a slight increase in the log phase growth of each L. acidophilus at the 0.25%, 2.5% and 5.0% MSM concentrations. At hour 9, there is a significant recovery of L. acidophilus from milk in MSM concentrations versus control. Hour 12 is when the first indication of recovery of L. acidophilus occurred in the control milk at 0.5 log, coinciding with MSM at 0.25%. MSM concentrations of 2.5% and 5.0% are at a recovery growth rate of 1 log. At hour 15, the control maximizes its growth at 0.98 log. The 0.25% MSM concentration is maximized at 1.43 log. 2.5% and 5.0% MSM maximized at hour 24 at 1.64 and 1.66 log, respectively. Hour 24 shows a kill for the control and 0.25% MSM. At 48 hours, the control and 0.25% MSM are below the detectable limit for the method and 2.5% and 5.0% MSM are still above 1 log of organism. MSM appears to aid this process by speeding up adaptation and allowing microorganisms to adapt faster to environmental stressors.
The samples treated with MSM showed an increase in the log phase of growth. This increase in log phase is most easily observed at the 5.0% MSM concentration. The 5.0% MSM is 0.45 log greater than the control at hour 15, which is the maximum growth recovery for the control. The 5.0% MSM peaked at 1.66 lg or 0.68 log higher than the control. This indicates a daughter cell survival rate at a higher percentage than the 0% control sample. Therefore, suggesting that the MSM environment leads to cell multiplication and survival.
MSM also affected the stationary phase and the death phase. The stationary control phase was shorter than the
ES 2 616 630 T3 2.5% and 5.0% MSM stationary phase. From hours 15 to 24, the samples not only maintained the growth rate, but continued to increase in log by a minimum of 0.24 log. These results indicate that MSM as an additive allowed L. acidophilus to thrive longer, allowing the body to establish itself for better health benefit. The control was not detectable at hour 48. Growth with 2.5% and 5.0% MSM was still above 1 log. This indicates that the survival capacity of L. acidophilus in milk was higher with the MSM additive.
The Bacillus coagulans study did not indicate recovery. This was possibly due to the exposure time in gastric fluid. A shorter exposure time could be beneficial for the survivability of Bacillus coagulans. The difference between the Lactobacillus acidophilus study and the Bacillus coagulans study was the matrix. The milk provided a sufficient buffer to allow the survival of L. acidophilus in gastric fluid.
Example 13
Bacillus coagulans viability measurement supplemented with MSM
This example describes the effect of MSM on the viability and colony formation of Bacillus coagulans.
Robust microbial colony formation studies were conducted in the presence of 0%, 1.0%, 2.0%, and 5% MSM. The microorganisms were cultured for 72 hours in 30 milliliters of tryptic soy broth. At the end of 72 hours, the broth was measured for colony formation, with photographic documentation of colony formation on tryptic soy agar. The percent transmittance in a 25mm x 25mm area of tryptic soy agar placed between two microscope slides was also measured on a spectrophotometer.
The growth curves of colony forming units per milliliter (cfu / ml) of the microorganisms between the MSM concentrations were compared with the 0% MSM concentration as a control sample for each microorganism. The stock of MSM powder was supplied by Bergstrom Nutrition with Certificate of Analysis. The powder was the Microprill formula, lot # 0806809, with an expiration date 10/31/13. All media and the MSM powder stock were tested for sterility prior to study. Microorganisms tested included Bacillus coagulans 9BB, Lot # 0109E002 supplied by Ganeden.
For Bacillus coagulans, the organism was isolated and cultured for 24 hours before harvest. The collected microorganism was placed in a sterile 100 ml bottle called dilution A. Dilution A was further diluted in a working solution, with a count of 210 Bacillus coagulans per 1 ml, called dilution B. One milliliter of dilution was used B to inoculate the 30 ml TSB concentrations indicated above. Working concentrations of MSM were prepared from a single 5.0% MSM solution and diluted accordingly with TSB to obtain the desired final concentration of MSM. The sterility of all solutions was verified before proceeding with the study.
Bacillus coagulans was inoculated on tryptic soy agar (TSA) at 35 ° C ± 0.5 ° C for 72 hours for verification of colonies and population density. All preparation and seeding was carried out at room temperature. Each concentration of MSM in the study was carried out in duplicate. Each dilution for the microorganism was plated in triplicate for each sample. To capture the proper colonies per milliliter, the microorganism was plated at six different dilutions. All plates were incubated at 35 ° C ± 0.5 ° C for 48 hours for the microorganism. The appropriate dilution plate was used for enumeration and averaged for reporting. The plate suitable for enumeration contained between 25 and 250 cfu / ml.
The MSM stock sample and all MSM-prepared media were tested for background levels of microorganisms on MRS agar and TSA. The MSM pool was <10 cfu / g and all test media were <1 cfu / ml in all cases before inoculation. All time intervals for seeding included negative control plates during pouring for quality control purposes. All negative control plates were devoid of microorganism growth. The results of these studies are provided below.
Table 19.1. Stock culture control numbers prior to inoculation of the test sample
<td></td><td>Bacillus coagulans</td>
<td>cfu / ml, inoculum</td><td>2.1 x 10<sup>4</sup></td>
<td>cfu added to 99 ml</td><td>2.1 x 10<sup>4</sup></td>
<td>cfu / ml in medium at time 0</td><td>2.1 x 10<sup>2</sup></td>
Control numbers are derived from the growth of specific organisms in the appropriate medium. The inoculation fluids were placed for enumeration in the appropriate medium. To capture the proper colonies per milliliter, each liquid was plated in triplicate at fourteen different dilutions. The appropriate dilution plate was used for enumeration and averaged for reporting. The plate suitable for enumeration contains between 25 and 250
ES 2 616 630 T3 cfu / ml.
Table 20.1. Bacillus coagulans population table Concentration in percent of MSM Average population count
4.3 x 10<sup>10</sup>
1.0 1.01 x 10<sup>11</sup>
2.5 3.3 x 10<sup>11</sup>
5.0 2.8 x 10<sup>11</sup>
Population counting was based on dilution of tryptic soy broth after 72 hours and inoculation on tryptic soy agar plates. The plates were incubated for 48 hours and numbered.
Table 21.1. Bacillus coagulans weight
Concentration in percentage of MSM Average weight of the population
0,0586
1,0 0,1363
2,5 0,9828
5,0 0,1260
The tryptic soy broth was centrifuged 72 hours later in a conical vial. The supernatant was removed and the pellet was washed. Centrifugation and washing was repeated three times. At the end of the third wash, the vials with sediment were weighed. Each vial was weighed empty and recorded. Then, the weight of the corresponding vial was subtracted from the final weight of the sediment and the vial, to obtain the weight of the Bacillus coagulans population. A 25 x 25 mm section was cut from each plate at the end of the 72 hour period and placed between two microscope slides. The vials were sealed to prevent the agar plug from dislodging. The wavelength was set at 546 nm, and two empty slides were used as blanks. When the plates were examined, a Bacillus coagulans colony was observed, the largest and most robust being observed with the 5.0% MSM compared to the 0% MSM. The weights indicated greater growth of a colony that was larger by adding to the weight of the biomass.
Table 22.1. Bacillus coagulans transmittance percentage
Concentration in percent of MSM Percentage of transmittance
<td>Agar</td><td> 61,1 %</td>
<td> 0,0</td><td> 52,3 %</td>
<td> 1,0</td><td> 51,5 %</td>
<td> 2,5</td><td> 49,8 %</td>
<td> 5,0</td><td> 45,6 %</td>
The percent transmittance was used to indicate the size of the Bacillus coagulans colonies, in which a reduction in transmittance indicates an increase in the size of the colonies as the colony inhibited the passage of light through the agar. The addition of 1 to 5% MSM appeared to cause a reduction in the percent transmittance. It was observed that the result of the percentage of transmittance could be influenced by the size of the sample, the location of the sample and the agar variables.
From a visual observation, it was observed that the colonies were larger in size after MSM treatment compared to the blank control. The colonies also resulted in a higher total weight when the biomass of the MSM-treated samples was measured compared to the blank control. These two results combined with the percentage of transmittance measured indicate that MSM as an additive (at certain concentrations) positively influences the viability, health and size of Bacillus coagulans.
Example 14
Effect of MSM on the growth and recovery of Lactobacillus acidophilus in a simulated intestinal tract environment
This example describes the effect of MSM on the growth of Lactobacillus acidophilus in a simulated intestinal tract environment.
MSM-fortified microbial growth studies were carried out at the following concentrations: 0%, 0.25%, 2.0%, and 5%. The time intervals for sowing the solutions in hours were 0, 3, 8, 24, 30, 36, 48, 54, 60 and 72. The growth curves of colony-forming units per milliliter (cfu / ml) of the microorganisms between the MSM concentrations with the 0% MSM concentration as a control sample for each microorganism. The stock of MSM powder was supplied by Bergstrom Nutrition with Certificate of Analysis. The powder was the Microprill formula, lot # 0806809, with an expiration date 10/31/13. All the
ES 2 616 630 T3 media, water, and MSM powder stock were tested for sterility prior to study. The simulated gastric acid pH was 1.2. The pH of the simulated intestinal fluid was 6.8. The microorganisms tested included Lactobacillus acidophilus, ATCC # 4356.
For Lactobacillus acidophilus, bottled milk was used as the product. One milliliter of a 9 log solution was placed in 99 ml of bottled milk and mixed by hand shaking. This was repeated for each concentration of MSM. The suspension was numbered for each MSM concentration and this is cited as the starting inoculum.
Ten milliliters of each concentration of MSM and Lactobacillus acidophilus milk were placed in 90 ml of simulated gastric acid for 20 minutes. The gastric acid was preheated to 35 ° C and held at 35 ° C for the course of 20 minutes. At the end of the 20 minute period, 10 ml of the mock gastric acid, Lactobacillus acidophilus and milk mixture was placed in 90 ml of mock intestinal fluid. This simulated intestinal fluid was preheated to 35 ° C and held at 35 ° C for the duration of the study. Working concentrations of MSM were prepared from a single 5.0% MSM solution and diluted accordingly with bottled milk to obtain the desired final concentration of MSM. The sterility of all solutions was verified before proceeding with the study.
Lactobacillus acidophilus intestinal solution was inoculated on MRS agar at the times listed above. All preparation and seeding was carried out at room temperature. Each concentration of MSM was carried out in duplicate. Each dilution for each organism was plated in triplicate for each time interval sampled. To capture the proper colonies per milliliter, each organism was plated at each time interval at four dilutions. All plates were incubated at 37 ° C ± 0.5 ° C for 72 hours in a CO2 environment, prior to examination. The appropriate dilution plate was used for enumeration and averaged for reporting. The plate suitable for enumeration contained between 25 and 250 cfu / ml.
The MSM stock sample and all MSM-prepared media were tested for background levels of microorganisms on MRS agar and tryptic soy agar. The MSM pool was <10 cfu / g and all test media were negative in all cases before inoculation. All time intervals for seeding included negative control plates during pouring for quality control purposes. All negative control plates were devoid of microorganism growth. The results of these studies are provided in the tables below.
Table 23.1. Logarithmic growth of Lactobacillus acidophilus
<td colspan="5">MSM concentration</td>
<td>Weather</td><td>0% MSM</td><td>0.25% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>
<td>Starting inoculum</td><td> 6,83</td><td> 6,86</td><td> 6,86</td><td> 7,02</td>
<td>20 min gastric</td><td> <1,00</td><td> <1,00</td><td> <1,00</td><td> <1,00</td>
<td>Hour 3</td><td> 1,33</td><td> 1,30</td><td> 1,46</td><td> 1,62</td>
<td>Hour 8</td><td> 2,22</td><td> 2,18</td><td> 2,30</td><td> 2,33</td>
<td>Hour 24</td><td> 4,47</td><td> 4,40</td><td> 5,94</td><td> 6,39</td>
<td>Hour 30</td><td> 4,10</td><td> 4,66</td><td> 5,39</td><td> 6,63</td>
<td>Hour 36</td><td> 4,00</td><td> 4,38</td><td> 5,32</td><td> 8,65</td>
<td>Hour 48</td><td> 4,59</td><td> 8,03</td><td> 11,17</td><td> 10,17</td>
<td>Hour 54</td><td> 8,97</td><td> 8,02</td><td> 13,23</td><td> 13,33</td>
<td>Hour 60</td><td> 9,23</td><td> 9,14</td><td> 11,27</td><td> 12,76</td>
<td>Hour 72</td><td> 9,21</td><td> 9,34</td><td> 12,87</td><td> 12,78</td>
Reviewing the data, there is a benefit with the addition of MSM to the product on the growth and recovery of Lactobacillus acidophilus. Comparing the 0% MSM control vs. 5.0% MSM, there is a significant increase in the log phase of growth without Lactobacillus acidophilus. In the first 24, the 5.0% MSM was 1.81 log higher than the control. In the following 12 hours the control, 0.25% and 2.5% were reduced. 5.0% MSM continued to increase over the same time period. At the end of the time frame, the 5.0% MSM was 8.65 log, 4.65 log higher than the control. From hour 36 to hour 48, MSM concentrations grew at a faster rate than the blank control. 0.25% MSM increased by 3.65 log and 2.5% MSM increased by 5.85 log compared to 0.59 log for 0.0% MSM. This trend changed in the next 8 hours, with the control increasing by 4.38 logs. The 0.25% MSM was reduced, while the 2.5% and 5.0% MSM did not increase as significantly as the control, there was an increase of 2.07 and 3.16 log. From hour 60 to hour 72 the control of
ES 2 616 630 T3 blank decreased as MSM concentrations increased.
Analyzing the data, another 24-hour trial period could have helped better predict a death stage. At 72 hours, the graph indicates that Lactobacillus acidophilus reaches the stationary phase. Without an indication of a death stage, it is difficult to predict whether the concentration of MSM could prolong the life of the population more than the control. What is observed is an increase in the speed of growth, achieving a larger population with the MSM at 2.5% and 5.0%. MSM as an additive to Lactobacillus acidophilus products may increase the likelihood of the body to establish itself in the intestinal tract. More organisms and faster could increase the benefit of taking a probiotic.
With the addition of MSM there is a benefit in the recovery of Lactobacillus acidophilus after a reduction in population growth. From hour 24 to hour 36, there is a reduction in growth for MSM at 0.25%, MSM at 2.5% and MSM at 0.0%. While 0.25% MsM and 2.5% MSM recovered within twelve hours with a significant increase in growth rate, 0.0% MSM took an additional twelve hours to show significant growth rate. For the 5.0% MSM, there was no reduction in recovery for this frame, only a slight reduction in growth rate. The 5.0% MSM took 6 hours to produce a substantial growth rate increase after the slight reduction in growth rate at 24 hour. This demonstrates that MSM influences recovery time for Lactobacillus acidophilus. Increased recovery time for Lactobacillus acidophilus could be a benefit in helping you establish an intestinal colony earlier, increasing the health benefits.
The study needs to be extended to 96 hours and beyond to see if MSM as an additive can extend the Lactobacillus acidophilus population longer. A population that can establish itself for a longer time in the intestinal tract could be an added benefit for probiotic products and for the people who consume them.
MSM as a supplement with Lactobacillus acidophilus helps the microorganism to establish faster, grow faster and reach a larger population. These attributes could benefit people who consume Lactobacillus acidophilus as a probiotic.
Example 15
Effect of MSM on grass growth and nutritional value
This example describes the effect of MSM on grass growth and the nutritional value of grass.
The effect of MSM on grass growth and nutritional value was evaluated by controlling grass growth under the following conditions: (1) fertilizer only; (2) mSm only (OptiMSM® GNC - Lot # 0922904, 1: 500 rate or 2 pounds per 1,000 square feet); and (3) fertilizer and MsM (MSM at the rate of 1: 500 or 2 pounds per 1,000 square feet in the presence of fertilizer) applied to the same field, but through a separate application. The fertilizer tested was urea (45-0-0) and the pass type included the following herb mix (tall fescue, perennial rye grass, orchard grass, Timothy, white clover, medium red clover, and intermediate rye grass; the seeds for this formulation are commercially available on the World Wide Web at the web address oregroseeds.com/allnatdairy.html). The field to be tested was measured and then marked to indicate different levels of MSM application and control. Using a diffuser for controlled diffusion, MSM and / or fertilizer were applied. The field was irrigated in a conventional manner (sprinkler irrigation every four days). The grass was grown for seven weeks and three days of being cut for testing. The sampled grass was cut 2.5 cm from the ground and placed in plastic bags for drying prior to shipment. The results of these tests on nutritional value are shown in table 23.2 below.
Table 23.2
<td rowspan="2"></td><td colspan="4">Results at 1: 500 indicated on a dry basis</td>
<td>Percentage of MSM / Fert</td><td>Percentage of MSM</td><td>Fertilizer percentage</td><td>AOAC method</td>
<td>Dry material</td><td> 43,26</td><td> 39,57</td><td> 44,62</td><td> 934,02</td>
<td>Humidity</td><td> 56,74</td><td> 60,43</td><td> 55,33</td><td> 934,02</td>
<td>Raw protein</td><td> 20,03</td><td> 24,36</td><td> 22,26</td><td> 2001,11</td>
<td>Acid detergent fiber</td><td> 30,95</td><td> 25,77</td><td> 29,44</td><td> 973,18</td>
<td>Neutral detergent fiber</td><td> 52,84</td><td> 35,23</td><td> 48,44</td><td> 2002,04</td>
<td>Soluble cells</td><td> 47,16</td><td> 64,71</td><td> 51,56</td><td>Calcium</td>
<td>Lignin</td><td> 4,21</td><td> 4,62</td><td> 3,98</td><td> 973,18</td>
ES 2 616 630 T3
<td>Ash</td><td> 10,89</td><td> 11,25</td><td> 11,26</td><td> 942,05</td>
<td>Estimated TDN / DDM</td><td> 64,47</td><td> 68,39</td><td> 65,61</td><td>Calculation</td>
<td>Net lact energy (Mcal / lb)</td><td> 0,66</td><td> 0,71</td><td> 0,67</td><td>NFTA calc</td>
<td>Net energy est. (Mcal / lb)</td><td> 0,55</td><td> 0,6</td><td> 0,57</td><td>NFTA calc</td>
<td>Calcium</td><td> 0,47</td><td> 0,96</td><td> 0,55</td><td> 968,08</td>
<td>Match</td><td> 0,46</td><td> 0,43</td><td> 0,42</td><td> 964,06</td>
<td>Magnesium</td><td> 0,16</td><td> 0,28</td><td> 0,17</td><td> 968,08</td>
<td>Potassium</td><td> 3,97</td><td> 3,48</td><td> 4,09</td><td> 968,08</td>
<td>Sodium</td><td> 0,03</td><td> 0,3</td><td> 0,05</td><td> 983,04</td>
<td>Copper</td><td> 14,57</td><td> 8,82</td><td> 8,18</td><td> 968,08</td>
<td>Iron</td><td> 94,51</td><td> 157,77</td><td> 128,15</td><td> 968,08</td>
<td>Zinc</td><td> 25,75</td><td> 24,79</td><td> 25,76</td><td> 968,08</td>
<td>Manganese</td><td> 40,23</td><td> 33,08</td><td> 33,73</td><td> 968,08</td>
<td>Selenium</td><td> 2,61</td><td> 3,23</td><td> 2,87</td><td> 996,17</td>
<td>Quantitative nitrate</td><td> 0,3</td><td> 0,05</td><td> 0,64</td><td> 968,07</td>
<td>Relative feed value</td><td> 114,06</td><td> 181,73</td><td> 126,68</td><td>NFTA calc</td>
<td>RFV-ash corrected est</td><td></td><td> 191,08</td><td> 132</td><td>Calculation</td>
<td>Chloride</td><td> 0,94</td><td> 0,62</td><td> 0,34</td><td> 915,01</td>
<td>Sulfur</td><td> 0,32</td><td> 0,25</td><td> 0,36</td><td> 923,01</td>
<td>Protein solubility</td><td> 46,48</td><td> 59,52</td><td> 52,56</td><td> 923,04</td>
<td>Non-structural carbon</td><td> 12,74</td><td> 25,66</td><td> 14,54</td><td>Calculation</td>
Likewise, it was observed that although all the evaluated grasses grew at equal speeds, the rye grass grew 5 to 8 cm taller in the areas treated with MSM. Furthermore, it was observed that there was no visible color variation between the grass treated with MSM and not treated with MSM. Furthermore, it was observed that the horses preferred the pasture grass treated with MSM over the grass not treated with MSM.
These studies indicate that MSM can alter the nutritional value of the grass (for example, it can increase the relative nutritional value compared to fertilizer alone), possibly the flavor of the grass as well as the height of the grass, depending on the type of grass. .
Example 16
Effect of 0.5% MSM on fermentation efficiency relative to beer production (Scottish beer)
This example describes the effect of 0.5% MSM on fermentation efficiency in relation to beer production, in particular Scottish beer.
Herein, MSM at certain concentrations has been shown to have a positive effect on microorganisms, including the growth of microorganisms. This positive impact includes organisms such as fungi, yeasts, and bacteria. Yeast cultures are involved in the production of beer during the fermentation process to produce ethanol and carbon dioxide. This study determined whether 0.5% by weight MSM had a positive impact, such as increasing the efficiency of the fermentation process. MSM was added to the yeast primer (1000 ml H2O, 100 g dry malt extract, 1 vial of Edimburg Ale yeast from White Labs) and to the wort. The must is the liquid extracted from the maceration process during the fermentation of beer or whiskey. Wort contains sugars that are fermented by fermentation yeast to produce alcohol.
First, the yeast starter was prepared according to methods known to those skilled in the art except for MSM added at 0.5% to a treatment group and without the addition of MSM to a control group. This process of
ES 2 616 630 T3 detailed below. Materials included the following: 2, 1900 ml glass jars; funnel; 2 standard type beer closures; 5.0 grams of MSM; 2000 ml of water; 200 grams of dry malt extract (DME); 2-vials of WLPO28 Edinburgh Ale yeast extract from White Labs; and fermentation sanitizer (San Star).
The preparation of the treatment initiation batch included the following steps: (1) the glass jugs, closures and funnel were thoroughly washed and then rinsed with a beer sanitizer; (2) 1000 ml of water was boiled and then 100 grams of DME was added; (3) the sample was boiled for 10 minutes; (4) the sample was removed from the heat and 5.0 grams of MSM was added; and (5) the solution was allowed to cool to 22 ° C. The treatment initiation batch was then placed in a 1900 ml glass jar using White Labs Edinburgh Ale yeast vial. The closure was applied and the entire glass was placed in a dark room at room temperature for 48 hours.
Preparation of the control starter batch included the following steps: (1) the glass jars, closures, and funnel were thoroughly washed and then rinsed with a beer sanitizer; (2) 1000 ml of water was boiled and then 100 grams of DME was added; (3) the sample was boiled for 10 minutes; (4) the sample was removed from the heat; and (5) the solution was allowed to cool to 22 ° C. The treatment initiation batch was then placed in a 1900 ml glass jar using White Labs Edinburgh Ale yeast vial. The closure was applied and the entire glass was placed in a dark room at room temperature for 48 hours.
The start of MSM treatment showed signs of activity (bubbling through the trap) at approximately 2 hours after the yeast bloomed. The control starter showed no signs of activity until about 10 hours after the yeast bloom.
On the day of making (2 days after preparing the starter yeast) the dough was prepared. Materials to prepare the dough included the following: 8.16 kg of American 2-Row base grain; 1.36 lg of special grain Crystal Malt 40L; 0.45 kg of Cara-Pils Malt special grain; and water. A mash keg (a beaker used in the mashing process to convert the starches in the ground grains into sugars for fermentation) and the fermentation heater were cleaned with powdered brewery wash and rinsed thoroughly. Afterwards, the mash tank was sanitized (San Star mash sanitizer). The following grains were crushed and ground for maceration: 8.16 kg of American 2-Row base grain; 1.36 lg of special grain Crystal Malt 40L; and 0.45 kg of Cara-Pils Malt specialty grain. 26 liters of water were heated to 72 ° C and then combined with the preheated dough vat. Subsequently, the ground grains were added and the solution was mixed thoroughly. The lid was attached and the solution was allowed to steep for 60 minutes. After 60 minutes, 16 liters of work were drained from the dough vat into the fermentation heater. The water was preheated to 75 ° C, added to the dough vat and mixed thoroughly with grain. The mixture was allowed to incubate for 10 minutes. This process was repeated two more times until a pre-boiling volume of 48 liters was obtained in the fermentation heater.
After preparing the dough, the fermentation process started. The following materials were used for the fermentation process: 85 ml of Cascade hops; 2 tablespoons of Irish moss; 105 grams of MSM; fermentation heater containing 48 liters of broth; wort cooler; 2 fermentation glasses; refractometer; Fermentation Sanitizer, Powdered Brewer Wash (PBW); and a stone of filtered air. The equipment was extensively cleaned with PBW. The wort cooler and filtered air stone were sanitized with a brewer's sanitizer (San Star Brewer's Sanitizer). The wort (48 liters) was brought to a boil in a fermentation heater and a 1<sup>to</sup> aliquot of Cascade hops (44 milliliters) to the solution. After 30 minutes of boiling, a second aliquot (14.7 ml) of Cascade hops was added. After 40 minutes of boiling, a third aliquot (14.7 ml) of Cascade hops was added as well as 2 tablespoons of Irish moss. After 50 minutes of boiling, a fourth aliquot (14.7 ml) of Cascade hops was added. The wort was decanted from the fermentation heater to the wort cooler and cooled to 23 ° C. The wort was then divided into two fermenters (each 21 liters in volume). 0.5% MSM (105 grams) was added to the treatment fermenter. The Brix reading of both fermenters was recorded and the baseline was recorded (Treatment fermenter = 15 Brix; control fermenter = 14.75 Brix). Each fermenter was tested for Brix every 24 hours for 21 days. Both fermenters were aerated for 25 minutes with sanitized filtered air stone. MSM-infused yeast was added to the treatment fermenter beaker while undisturbed yeast was added to the control fermentor beaker. Blow tubes were attached to both fermentation and fermentation was allowed to occur for 21 days. The results of these studies are provided in Table 23.3 below.
Table 23.3
<td>Day</td><td>MSM FG (Adjusted for Alc and Temp)</td><td>Control FG (Adjusted for Alc and Temp)</td>
<td></td><td></td><td></td>
<td> 1</td><td> 1,026</td><td> 1,028</td>
<td> 2</td><td> 1,018</td><td> 1,020</td>
<td> 3</td><td> 1,016</td><td> 1,018</td>
<td> 4</td><td> 1,016</td><td> 1,017</td>
ES 2 616 630 T3
<td> 5</td><td> 1,015</td><td> 1,017</td>
<td> 6</td><td> 1,015</td><td> 1,015</td>
<td> 7</td><td> 1,015</td><td> 1,015</td>
<td> 8</td><td> 1,015</td><td> 1,015</td>
<td> 9</td><td> 1,015</td><td> 1,015</td>
<td> 10</td><td> 1,015</td><td> 1,015</td>
<td> 11</td><td> 1,015</td><td> 1,015</td>
<td> 12</td><td> 1,015</td><td> 1,015</td>
<td> 13</td><td> 1,015</td><td> 1,015</td>
<td> 14</td><td> 1,015</td><td> 1,015</td>
<td> 15</td><td> 1,015</td><td> 1,015</td>
<td> 16</td><td> 1,015</td><td> 1,015</td>
<td> 17</td><td> 1,015</td><td> 1,015</td>
<td> 18</td><td> 1,015</td><td> 1,015</td>
<td> 19</td><td> 1,015</td><td> 1,015</td>
<td> 20</td><td> 1,015</td><td> 1,015</td>
<td> 21</td><td> 1,015</td><td> 1,015</td>
When preparing a yeast starter, the faster the activation of the yeast culture occurs the better the efficiency and the potential environmental contamination against unwanted airborne microorganisms will be minimized to the maximum. The MSM starter batch showed activity 80% faster than the control (2 hours compared to 10 hours). The study also indicated that MSM helped the fermentation process. As in the yeast starter, the faster the activation of the yeast fermentation process, the better the efficiency and the more environmental contamination against unwanted airborne microorganisms will be minimized. The MSM-treated fermenter showed activity 58% faster than the control (3.5 hours compared to 9 hours). The MSM treatment batch also reached a maximum fermentation in 5 days, while the control batch took 6 days (a completion time 17-25% earlier).
These results indicate that MSM is useful in the beer fermentation process.
Example 17
Lactobacillus acidophilus growth in Acidophilus milk supplemented with MSM
This example describes the growth of Lactobacillus acidophilus in Acidophilus milk supplemented with MSM.
Microbial growth studies were carried out in Acidophilus milk fortified with 0%, 0.5%, 2.5% and 5% MSM. The time intervals for evaluation were at 8 and 16 hours for a total of 104 hours. Afterwards, samples were evaluated every 7 days for a total of 28 days. The growth curves of the colony forming units per milliliter (cfu / ml) of the microorganisms between Acidophilus milk with MSM concentrations were compared with Acidophilus milk with 0% MSM concentration as a control sample. The stock of MSM powder was supplied by Bergstrom Nutrition with Certificate of Analysis. The powder was the Microprill formula, lot # 0806809, with an expiration date 10/31/13. The milks were purchased from a local store. Acidophilus milk was low fat (Darigold). Acidophilus plus Bifidus milk contained 2% milk fat (Lucerne). Acidophilus plus Bifidus milk was run concurrently at 2.5% and 0% MSM concentration as a product containing two microorganisms. The working solutions were kept at 4 ° C during the study. MSM milk working solutions were run in duplicate.
All preparation and seeding was carried out at room temperature. All dilutions for all solutions were plated in triplicate for all time intervals sampled. To capture the proper colonies per milliliter, all organisms at all time intervals were plated at three different dilutions. All plates were incubated at 35 ° C ± 0.5 ° C in CO<sub>2</sub> for 72 hours for all solutions. The appropriate dilution plate was used for enumeration and averaged for reporting. The plate suitable for enumeration contains between 25 and 250 cfu / ml.
ES 2 616 630 T3
The MSM stock sample and all MSM-prepared media were tested for background levels of microorganisms on MRS agar and TSA. The MSM pool was <10 cfu / g and all test media were <1 cfu / ml in all cases before inoculation. All time intervals for seeding included negative control plates during pouring for quality control purposes. All control plates were clean of microorganism growth. At 72 hours, the concentrations of MSM and negative control solutions were verified to be negative for contamination. The results of these studies are provided in Table 24 below.
MSM concentration in percentage in Acidophilus milk
<td>Weather</td><td>Milk acid</td><td>A / B 0</td><td>A / B 2.5%</td><td> 0,50 %</td><td> 0,50 %</td><td> 2,50 %</td><td> 2,50 %</td><td> 5 %</td><td> 5 %</td>
<td> 0</td><td> 8,82</td><td> 5,33</td><td> 6,41</td><td> 7,33</td><td> 6,62</td><td> 6,67</td><td> 6,7</td><td> 6,56</td><td> 6,47</td>
<td> 8</td><td> 9,08</td><td> 8,36</td><td> 8,67</td><td> 9,06</td><td> 9,00</td><td> 9,06</td><td> 8,97</td><td> 8,73</td><td> 9,08</td>
<td> 24</td><td> 9,17</td><td> 8,19</td><td> 9,16</td><td> 9,41</td><td> 9,35</td><td> 9,24</td><td> 9,01</td><td> 9,32</td><td> 9,26</td>
<td> 32</td><td> 8,85</td><td> 8,73</td><td> 8,87</td><td> 9,2</td><td> 9,12</td><td> 9,06</td><td> 9,11</td><td> 9,33</td><td> 9,19</td>
<td> 48</td><td> 8,54</td><td> 8,15</td><td> 8,3</td><td> 8,55</td><td> 8,69</td><td> 8,93</td><td> 8,77</td><td> 8,78</td><td> 8,72</td>
<td> 56</td><td> 8,46</td><td> 8,51</td><td> 8,56</td><td> 8,77</td><td> 8,69</td><td> 8,7</td><td> 8,81</td><td> 8,88</td><td> 8,7</td>
<td> 72</td><td> 8,65</td><td> 8,45</td><td> 8,45</td><td> 8,56</td><td> 8,67</td><td> 8,71</td><td> 8,6</td><td> 8,75</td><td> 8,63</td>
<td> 80</td><td> 10,09</td><td> 9,66</td><td> 9,82</td><td> 9,92</td><td> 9,51</td><td> 9,52</td><td> 9,96</td><td> 9,87</td><td> 9,71</td>
<td> 96</td><td> 10,02</td><td> 9,68</td><td> 8,61</td><td> 9,93</td><td> 10,25</td><td> 10,18</td><td> 10,31</td><td> 10,1 4</td><td> 10,2 6</td>
<td> 104</td><td> 9,45</td><td> 8,83</td><td> 8,92</td><td> 9,47</td><td> 9,52</td><td> 9,8</td><td> 9,45</td><td> 9,74</td><td> 9,38</td>
<td>Day 7</td><td> 10,29</td><td> 10,1</td><td> 10,42</td><td> 10,64</td><td> 10,72</td><td> 10,4</td><td> 10,59</td><td> 10,8 9</td><td> 10,5 2</td>
<td>Day 14</td><td> 6,96</td><td> 6,83</td><td> 5,70</td><td> 6,81</td><td> 5,63</td><td> 8,00</td><td> 6,66</td><td> 7,82</td><td> 8,26</td>
<td>Day 21</td><td> 5,30</td><td> 5,60</td><td> 5,37</td><td> 5,37</td><td> 5,37</td><td> 5,56</td><td> 5,37</td><td> 5,64</td><td> 5,48</td>
<td>Day 28</td><td> 4,00</td><td> 3,82</td><td> 3,94</td><td> 2,52</td><td> 3,48</td><td> 3,43</td><td> 3,22</td><td> 2,52</td><td> 2,52</td>
At hour 0, there was a 1 log greater growth of Lactobacillus acidophilus in milk without MSM compared to milk fortified with MSM. The significance is, at hour 8, that the MSM fortified milk showed a minimum of a 1.73 log increase in growth, while the non-MSM milk showed a 0.26 increase in growth rate. MSM in the first 8 hours of growth provided a significant increase compared to the control. The maximum control increase is the 5% MSM with a mean log increase of 2.39, while the 2.5% MSM had a mean 2.33 log increase. Hour 24 shows a growth rate that balanced between control and MSM concentrations. The control had a 0.32 log reduction in growth at hour 32. MSM concentrations of 2.5% and 5% had a reduction of 0.04 and 0.03 log in growth, while at 0.5% it was reduced by 0.22 log at hour 32. In At hour 48, the control has a reduction of 0.31 log, while the reduction of MSM is 0.54 log for 0.5%, 0.24 log for 2.5% and 0.51 log for 5%. The MSM concentrations maintained a higher recovery rate compared to the control. The MSM concentration of the
2.5% was on average 0.31 log greater and 5% was 0.21 log greater. Hour 56 did not show a significant change in growth increase or decrease. At hour 72, the control increased by 0.19 log, while the MSM concentrations were stable. The 80th hour had a significant increase in growth. The control showed a 1.44 log increase. The samples treated with MSM showed an increase in growth of 0.5% (1.1 log), 2.5% (1.09 log), and 5% (1.1 log). At 96 hours the control stabilized. All MSM concentrations increased on average (0.5%, 0.38 log; 2.5%, 0.51 log; and 5.0%, 0.41 log) by hour 96. At hour 96, the concentration of MSM at
2.5% was 0.23 log greater than the control. At hour 104, the reduction in log growth was comparable between control and MSM concentrations (control decreased 0.57 log; MSM 0.5% decreased 0.60 log; MsM 2.5% 0.62 log decreased; and 5.0% MSM decreased 0.64 log). When comparing the final growth recovery between the control and the MSM concentrations, the study showed the MSM at 0.5% at 0.05 log greater than the control, the MSM at 2.5% was 0.18 log greater than the control, and 5% MSM was 0.11 log greater than the control.
Day 7 showed increased growth from hour 104 for all working solutions. The control increased 0.84 log, MSM 0.5% increased 1.19 log, MSM 2.5% increased 0.87 log, and MSM 5.0% increased 1.15 log. 0.5% MSM was 0.39 log greater than the control, 2.5% MSM was 0.21 log greater than the control, and 5.0% MSM was 0.42 log greater than the control. Day 14 showed a significant reduction in growth. The largest reduction in growth was MSM at 0.5% at 4.46 log. The control followed with a 3.33 log reduction, 2.5% MSM to 3.17 and 5% MSM to 2.67 log. 0.5% MSM was 0.74 log lower than the control, while 2.5% MSM was 0.37 log higher. The 5.0% MSM sample was a complete log greater than the control at 1.08 log. On the 21st the
ES 2 616 630 T3 reduction in growth. The control was 1.66 log lower, MSM at 0.5% was 0.85 log lower, 2.5% was 1.87 lower, and MSM at 5.0% was 2.48 log lower. 0.5% MSM and the control were equal in log growth, with 2.5% MSM being 0.17 log greater than the control and 5.0% MSM 0.26 log greater than the control. On day 28, reduced growth continued with a 1.3 log reduction for the control, 2.37 log for 0.5% MSM, 2.14 log for 2.5% MSM, and 3.04 log for 5.0% MSM. Growth for the negative control on day 28 was 1.00 log greater than 0.5% MSM, 0.68 log greater than 2.5% MSM, and 1.48 log greater than 5.0% MSM.
Acidophilus milk plus Bifidus over the course of the study demonstrated similar growth rates. From hour 0 to hour 8 they showed a significant increase in growth. At hour 24, the control decreased 0.17 log, while the 2.5% MSM increased 0.49 log, giving the 2.5% MSM a 0.97 log higher count than the control. At hour 32, the 2.5% MSM decreased 0.29 log and the control increased 0.54 log, with the 2.5% MSM having a 0.14 log higher than the control. From hour 48 to hour 72 there was a pattern of continuous increase and a reduction in growth, with MSM at 2.5% having a growth increase of 0.15 and 0.5 log compared to the control. At hour 72, the growth was equal between the control and MSM at 2.5%. Hour 80 showed a 1.21 log increase in growth for the control and 1.37 log for the 2.5% MSM, with the 2.5% MSM having a 0.16 log increase in growth. At hour 96, there was a significant reduction in growth for the 2.5% MSM of 1.21 log. The control did not show a significant difference from hour 80, resulting in 1.07 log higher growth for the control compared to 2.5% MSM. At hour 104, the control growth decreased by 0.85 log and the 2.5% MSM samples increased by 0.31 log. Hour 104 showed the 2.5% MSM samples 0.09 log larger than the control. On day 7 there was a 1.27 log increase for milk and 1.5 log for 2.5% MSM, with 2.5% MSM 0.32 log higher than milk. Day 14 showed a reduction in growth, 3.27 log for milk, 4.72 log for 2.5% MSM. Milk had an increase of 1.13 compared to MSM at 2.5%. On day 21, the reduction was reduced, the milk was reduced by 1.23 log and MSM at 2.5% by 0.33 log, the milk being 0.23 log greater in growth than the MSM. Milk on day 28 was reduced by 1.78 log and MSM at 2.5% was reduced by 1.43 log, with MSM 0.12 log higher than milk without MSM. Table 25 shows the data averaging the duplicates.
Table 25. Average growth of Lactobacillus acidophilus in milk fortified with MSM.
<td colspan="7">MSM concentration in percentage in acidophilus milk</td>
<td>Weather</td><td>Acid Milk</td><td>A / B</td><td>A / B 2.5%</td><td> 0,50 %</td><td> 2,50 %</td><td> 5,00 %</td>
<td> 0</td><td> 8,82</td><td> 5,33</td><td> 6,41</td><td> 6,98</td><td> 6,69</td><td> 6,52</td>
<td> 8</td><td> 9,08</td><td> 8,36</td><td> 8,67</td><td> 9,03</td><td> 9,02</td><td> 8,91</td>
<td> 24</td><td> 9,17</td><td> 8,19</td><td> 9,16</td><td> 9,38</td><td> 9,13</td><td> 9,29</td>
<td> 32</td><td> 8,85</td><td> 8,73</td><td> 8,87</td><td> 9,16</td><td> 9,09</td><td> 9,26</td>
<td> 48</td><td> 8,54</td><td> 8,15</td><td> 8,3</td><td> 8,62</td><td> 8,85</td><td> 8,75</td>
<td> 56</td><td> 8,46</td><td> 8,51</td><td> 8,56</td><td> 8,73</td><td> 8,76</td><td> 8,79</td>
<td> 72</td><td> 8,65</td><td> 8,45</td><td> 8,45</td><td> 8,62</td><td> 8,66</td><td> 8,69</td>
<td> 80</td><td> 10,09</td><td> 9,66</td><td> 9,82</td><td> 9,72</td><td> 9,74</td><td> 9,79</td>
<td> 96</td><td> 10,02</td><td> 9,68</td><td> 8,61</td><td> 10,09</td><td> 10,25</td><td> 10,20</td>
<td> 104</td><td> 9,45</td><td> 8,83</td><td> 8,92</td><td> 9,50</td><td> 9,63</td><td> 9,56</td>
<td>Day 7</td><td> 10,29</td><td> 10,1</td><td> 10,42</td><td> 10,68</td><td> 10,50</td><td> 10,71</td>
<td>Day 14</td><td> 6,96</td><td> 6,83</td><td> 5,7</td><td> 6,22</td><td> 7,33</td><td> 8,04</td>
<td>Day 21</td><td> 5,3</td><td> 5,6</td><td> 5,37</td><td> 5,37</td><td> 5,465</td><td> 5,56</td>
<td>Day 28</td><td> 4,00</td><td> 3,82</td><td> 3,94</td><td> 3,00</td><td> 3,325</td><td> 2,52</td>
Table 26. Growth of cfu per ml of non-probiotic organisms in Acidophilus milk. Concentration in percent of MSM_
<td colspan="10">Concentration in percent of MSM</td>
<td>Day</td><td>Acid Milk</td><td>A / B 0</td><td>A / B 2.5%</td><td> 0,50 %</td><td> 0,50 %</td><td> 2,50 %</td><td> 2,50 %</td><td> 5 %</td><td> 5 %</td>
<td> 0</td><td> 10</td><td> 300</td><td> 370</td><td> 20</td><td> 30</td><td> 10</td><td> 10</td><td> 10</td><td> 20</td>
<td> 3</td><td> 20</td><td> 830</td><td> 480</td><td> 10</td><td> 10</td><td> 30</td><td> 10</td><td> 10</td><td> 10</td>
ES 2 616 630 T3
<td> 7</td><td> 10</td><td> 1250</td><td> 570</td><td> 10</td><td> 10</td><td> 20</td><td> 10</td><td> 10</td><td> 20</td>
<td> 14</td><td> 20</td><td> 2500</td><td> 1460</td><td> 20</td><td> 20</td><td> <10</td><td> <10</td><td> 20</td><td> 10</td>
<td> 21</td><td> 20</td><td> 8000</td><td> 3500</td><td> 20</td><td> 40</td><td> 60</td><td> 170</td><td> 20</td><td> 10</td>
<td> 28</td><td> 650</td><td> 120000</td><td> 110000</td><td> 30</td><td> 2250</td><td> 2460</td><td> 2700</td><td> 10</td><td> 10</td>
Table 26 shows the data for standard plate counts tested in the working solutions. This was done to see how MSM might affect the normal flora found in milk. Day 0 was the day the samples were prepared for the start of the study. Acidophilus plus Bifidus milk started with a higher count on day 0 than normally expected. This caused the final values to be high. The Acidophilus dairy product on day 0 was at expected values. Acidophilus milk maintained adequate growth rates throughout the study and were equivalent to typical growth rates in dairy products. The 5.0% MSM did not allow significant growth during the study.
MSM as an additive to this product played a significant role in increasing the population of the probiotic, Lactobacillus acidophilus in a product. In the first eight hours after fortifying a product with MSM, there was a significant influence on the probiotic in the product. There was a significant increase in the growth rate of the probiotic. Acidophilus milk without MSM had a 0.26 log increase in growth in the first 8 hours. Although the Acidophilus milk with MSM had a minimum of 1.73 log growth. The milk sample with MSM at
2.5% had an increase of 2.27 and 2.39 log. The 5.0% MSM sample had an increase of 2.17 and 2.61 log.
During the study, there was a continuous increase in growth when the Acidophilus milk control was compared to the MSM fortified Acidophilus milk. The increase in growth speed varies from 0.04 to 1.08 compared to the control. Only at two time points were there data that showed growth control greater than the MSM solutions, hour 80 and day 28. When the data is analyzed, the reason for the greater growth at hour 80 was due to the peak of the curve growth. Acidophilus milk without MSM peaked before MSM milk. Therefore, the MSM was still in the growth phase, while the Acidophilus milk had reached the maximum of its growth. As the increase in growth was due to MSM, there was a higher mortality rate at the end of the study. Therefore, day 28 showed less growth for the MSM solutions than the control.
The growth peak was reached with Acidophilus milk fortified with 5.0% MSM, with a log of 10.89. Acidophilus milk reached a growth peak of 10.29 log. Each concentration of MSM outpaced the growth of Acidophilus milk. 2.5% MSM had a 10.59 log growth peak and 0.5% MSM had a 10.72 log growth rate peak. This further establishes the influence of MSM on a probiotic. The product, once fortified with MSM, outpaced the growth of the product without MSM. As the growth peak was greater, an increase in growth was observed on day 14, a full week after the growth rate peak. Acidophilus milk growth was 6.96 log; MSM fortified milk was 7.82 and 8.26 log, an increase of 0.86 and 1.3 log, respectively.
The probiotic efficacy is based on three points; ability to survive, colonize and produce lactic acid. MSM demonstrates the ability to affect probiotic bacteria's ability to survive and colonize. In the first eight hours, the ability to colonize was observed with increasing growth rate. On day 14, the ability to survive with increased log growth was observed. The ability to increase lactic acid production was the third component of a probiotic's efficacy to be studied. In this study, there was an observed reaction of increased foam production in the MSM solutions.
The claim that MSM is a beneficial dietary supplement is supported by this study. The microbial flora of the gastrointestinal tract can be positively affected by the addition of MSM to the human diet. There was an increase in the growth of probiotic bacteria, with an increase in the ability to survive. Increases the likelihood that MSM, when used in a probiotic product, could increase consumer benefit.
Example 18
Recovery of Lactobacillus acidophilus in Acidophilus milk supplemented with MSM
This example shows the recovery of Lactobacillus acidophilus in acidophilus milk supplemented with MSM.
To analyze the effects of MSM on the recovery of Lactobacillus acidophilus in Acidophilus milk, after a specified incubation time a diluted portion of the original growth solutions was transferred to the appropriate broth and sampled at time intervals analyzing the recovery rates. . Microbial growth was determined in Acidophilus milk fortified with MSM at 0%, 0.5%, 2.5% and 5%. On day 7, 14, 21 and 28, the MSM fortified Acidophilus milk samples were diluted and transferred to the appropriate broths are
ES 2 616 630 T3
MSM for the recovery study. The plating time intervals were used every 24 hours for a 72 hour period. The growth curves of the colony forming units per milliliter (cfu / ml) of the microorganisms between Acidophilus milk with MSM concentrations were compared with Acidophilus milk with 0% MSM concentration as a control sample. All media and the MSM powder stock were tested for sterility prior to study. The study was carried out in four organisms over a period of two weeks. The microorganisms were separated in two runs, each one week long, analyzing two microorganisms each week.
Acidophilus milk was low fat (Darigold). Acidophilus plus Bifidus milk contained 2% milk fat (Lucerne). Acidophilus plus Bifidus milk was run concurrently with 2.5% and 0% MSM concentration as a product containing two microorganisms. The working solutions were kept at 4 ° C during the study. MSM milk working solutions were run in duplicate. All preparations and seeding were carried out at room temperature. All dilutions for all solutions were plated in triplicate for all time intervals sampled. To capture the proper colonies per milliliter, all organisms at all time intervals were plated at three different dilutions. All plates were incubated at 35 ° C ± 0.5 ° C in CO2 for 72 hours for all solutions. The appropriate dilution plate was used for enumeration and averaged for reporting. The plate suitable for enumeration contains between 25 and 250 cfu / ml.
The MSM stock sample and all MSM-prepared media were tested for background levels of microorganisms on MRS agar and TSA. The MSM pool was <10 cfu / g and all test media were <1 cfu / ml in all cases before inoculation. All time intervals for seeding included negative control plates during pouring for quality control purposes. All control plates were clean of microorganism growth. At 72 hours, the concentrations of MSM and negative control solutions were verified to be negative for contamination.
The study looks at the effect of MSM on the recovery of Lactobacillus Acidophilus from Acidophilus milk fortified with MSM. The recovery study was run in parallel with the study conducted on the effects of MSM on the growth of Lactobacillus acidophilus in MSM-fortified Acidophilus milk. The growth recovery rate was analyzed on day 7, day 14, day 21 and day 28. For Table 27, the growth for day x no time is calculated from the initial growth study with the dilution factor in the logarithmic growth. For table 28, the growth was calculated from the log growth on day x without time subtracted from the log growth for the dates analyzed later, for example, on day 28 it had a result of 4.00 log, calculating the dilution, the value day 28 day 0 is 2.00 log for table 27. Table 28 takes the value of 2.00 as the starting value. Subsequent data for the hours analyzed takes the counts in log and subtracts the initial value of 2.00, for example, day 28 24 is 11.29 log, subtracting 2.00 log, the increase in growth speed is 9, 29 log.
Table 27. Delayed recovery of Lactobacillus acidophilus in milk fortified with MSM on day 7
Concentration in percent of MSM
<td>Day hour</td><td> 0 %</td><td>A / B 0%</td><td>A / B 2.5%</td><td> 0,50 %</td><td> 0,5 %</td><td> 2,50 %</td><td> 2,5 %</td><td> 5 %</td><td> 5 %</td>
<td>Day 7 - 24</td><td> 11,29</td><td> 12,88</td><td> 13,44</td><td> 13,1</td><td> 12,78</td><td> 13,16</td><td> 13,15</td><td> 12,12</td><td> 13,29</td>
<td>Day 7 - 48</td><td> 12,71</td><td> 13,42</td><td> 12,5</td><td> 12,7</td><td> 12,67</td><td> 12,04</td><td> 12,67</td><td> 13,09</td><td> 13,25</td>
<td>Day 7 - 72</td><td> 12,17</td><td> 13,47</td><td> 12,77</td><td> 12,4</td><td> 13,74</td><td> 11,92</td><td> 13,28</td><td> 13,1</td><td> 12,87</td>
Table 28. Recovery of Lactobacillus acidophilus in milk fortified with MSM on day 7 with mean recovery per percentage of MSM with initial starting data
<td colspan="7">Concentration in percent of MSM</td>
<td>Day hour</td><td>Acidophilus milk</td><td>Ac / Bf milk</td><td>Ac / Bf with 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>
<td>Day 7</td><td> 8,29</td><td> 8,1</td><td> 8,42</td><td> 8,68</td><td> 8,50</td><td> 8,71</td>
<td>Day 7 - 24</td><td> 11,29</td><td> 12,88</td><td> 13,44</td><td> 12,94</td><td> 13,16</td><td> 12,71</td>
<td>Day 7 - 48</td><td> 12,71</td><td> 13,42</td><td> 12,50</td><td> 12,69</td><td> 12,36</td><td> 13,17</td>
<td>Day 7 - 72</td><td> 12,17</td><td> 13,47</td><td> 12,77</td><td> 13,07</td><td> 12,60</td><td> 12,99</td>
Recovery from day 7 shows that all MSM concentrations within the first 24 hours of growth have an increase of more than 1 log versus control, MSM at 0.5% of 1.65, MSM at 2.5% of 1.87, and 5.0% MSM of 1.42. 48 hour, control was slightly higher in growth compared to 0.5% MSM
ES 2,616,630 T3 at 0.03 log, and 0.36 log greater than 2.5% MSM, but 0.46 log less than 5.0% MSM. Hour 72, MSM concentrations exceed control growth; 0.5% MsM at 0.9 log, 2.5% MsM at 0.43 log, and 5.0% at 0.82 log.
The acidophilus plus bifidus control in the first 24 hours was at 12.88 log for growth, while acidophilus plus bifidus with 2.5% MSN was at 13.44 log for growth. The 2.5% MSM bed was 0.56 log greater than the control. In the next two 24 hour periods, the Acidophilus plus bifidus control had a growth of 13.42 log and 13.47 log. Acidophilus plus bifidus with 2.5% MSM had growth at 12.50 log and 12.77 log during the same time period. The control was 0.92 log greater than the 2.5% MSM in the second 24-hour period and was 0.7 log greater in the third 24-hour period.
Table 29. Recovery of Lactobacillus acidophilus in MSM-fortified milk on day 7 in log increase in recovery growth rate from zero start time.
<td colspan="7">Concentration in percent of MSM</td>
<td>Hour</td><td>Acidophilus milk</td><td>Ac / Bf milk</td><td>Ac / Bf with 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>
<td> 0</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td>
<td> 24</td><td> 3,00</td><td> 4,78</td><td> 5,02</td><td> 4,26</td><td> 4,66</td><td> 4,00</td>
<td> 48</td><td> 1,42</td><td> 0,54</td><td> -0,94</td><td> -0,26</td><td> -0,8</td><td> 0,47</td>
<td> 72</td><td> -0,54</td><td> 0,05</td><td> 0,27</td><td> 0,39</td><td> 0,25</td><td> -0,19</td>
Analyzing the recovery on day 7 based on the increase in log growth rate, there was a significant increase in the first 24 hours of growth. The control increased by 3 log from the initial inoculation, while MSM concentrations increased by 4.26 log for 0.5% MSM, 4.66 log for 2.5% MSM, and 4.00 log for MSM at 5.0. In the second twenty-four hours, there was a reduction in growth relative to MSM at 0.5% and MSM at 2.5%. The growth control increased by 1.42 log and the 5.0% MSM growth increased by 0.47 log. In the third twenty-four hour period, the growth control was reduced by 0.54 log and the growth of 5.0% MSM was reduced by 0.19 log. The 0.5% MSM and 2.5% MSM increased by log, 0.39 and 0.25 log growth, respectively.
The Acidophilus plus bifidus control milk showed a 4.78 log increase in the first 24 hours, compared to a 5.02 increase for the 2.5% MSM fortified Acidophilus plus bifidus milk. In the second 24-hour period, acidophilus plus bifidus control increased by 0.54 log, while milk with 2.5% MSM decreased by 0.94 log. In the third 24-hour period, the Acidophilus plus bifidus control increased by 0.05 log, while the 2.5% MSM milk increased by 0.27 log.
Table 30. Delayed recovery of Lactobacillus acidophilus in milk fortified with MSM on day 14.
Concentration in percent of MSM
<td>Day hour</td><td> 0 %</td><td>A / B 0%</td><td>A / B 2.5</td><td> 0,5 %</td><td> 0,5 %</td><td> 2,50 %</td><td> 2,50 %</td><td> 5,0 %</td><td> 5,0 %</td>
<td>Day 14 - 24</td><td> 10,62</td><td> 13,38</td><td> 13,35</td><td> 10,19</td><td> 10,10</td><td> 10,00</td><td> 9,73</td><td> 9,6</td><td> 9,6</td>
<td>Day 14 - 48</td><td> 12,37</td><td> 13,52</td><td> 13,47</td><td> 12,95</td><td> 12,72</td><td> 12,43</td><td> 12,2</td><td> 12,99</td><td> 13,04</td>
<td>Day 14 - 72</td><td> 12,43</td><td> 13,31</td><td> 13,43</td><td> 13,02</td><td> 12,70</td><td> 12,51</td><td> 12,63</td><td> 12,65</td><td> 12,21</td>
Table 31. Recovery of Lactobacillus acidophilus in milk fortified with MSM on day 14 with mean recovery per percentage of MSM with initial starting data
<td colspan="7">Concentration in percent of MSM</td>
<td>Day hour</td><td>Acidophilus milk</td><td>Ac / Bf milk</td><td>Ac / Bf with 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>
<td>Day 14 - 0</td><td> 4,96</td><td> 4,83</td><td> 3,70</td><td> 4,22</td><td> 5,33</td><td> 6,04</td>
<td>Day 14 - 24</td><td> 10,62</td><td> 13,38</td><td> 13,35</td><td> 10,15</td><td> 9,87</td><td> 9,60</td>
<td>Day 14 - 48</td><td> 12,37</td><td> 13,52</td><td> 13,47</td><td> 12,84</td><td> 12,32</td><td> 13,02</td>
<td>Day 14 - 72</td><td> 12,43</td><td> 13,31</td><td> 13,43</td><td> 12,86</td><td> 12,57</td><td> 12,43</td>
On day 14, the control exceeded the growth of MSM concentrations in the first 24 hours. The control was 0.48 log greater than 0.5% MSM, 0.75 log greater than 2.5% MSM, and 1.02 log greater than 5.0% MSM. At 48
ES 2,616,630 T3 hours, the control solution was 0.05 log greater than 2.5% MSM. 0.5% MSM was 0.47 log greater than the control and 5.0% MSM was 0.65 log greater than the control. At 72 hours, the 0.5% MSM was 0.43 log higher, 2.5% MSM was 0.14 log higher than the control, and 5.0% MSM was equal to the control.
Acidophilus plus bifidus fortified with 2.5% MSM was 0.03 less than the Acidophilus plus bifidus control at 24 hours. At 48 hours, the Acidophilus plus bifidus control was 0.05 log greater than Acidophilus plus bifidus with 2.5% MSM. At 72 hours, the Acidophilus plus bifidus with 2.5% MSM was 0.12 log greater than the Acidophilus plus bifidus control.
Table 32. Recovery of Lactobacillus acidophilus in MSM fortified milk on day 14 in log increase in recovery growth rate from zero start time.
<td colspan="7">Concentration in percent of MSM</td>
<td>Hour</td><td>Acidophilus milk</td><td>Ac / Bf milk</td><td>Ac / Bf with 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>
<td> 0</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td>
<td> 24</td><td> 5,66</td><td> 8,55</td><td> 9,65</td><td> 5,93</td><td> 4,54</td><td> 3,56</td>
<td> 48</td><td> 1,75</td><td> 0,14</td><td> 0,12</td><td> 2,69</td><td> 2,45</td><td> 3,42</td>
<td> 72</td><td> 0,06</td><td> -0,21</td><td> -0,04</td><td> 0,03</td><td> 0,26</td><td> -0,59</td>
Analyzing the recovery on day 14 based on the log increase in growth rate, the following was observed; control increased by 5.66 log from initial inoculation, while MSM concentrations increased by 5.93 log for 0.5% MSM, 4.54 log for 2.5% MSM, and 3.56 log for MSM at 5.0. In the second twenty-four hours, the growth control increased by 1.75 log while the MSM at 0.5% increased by 2.69 log, the MSM at 2.5% increased by 2.45 log and the MSM at 5 , 0% increased by 3.42 log. In the third twenty-four hour period, the growth control increased by 0.06 log and the MSM growth at 5.0% decreased by 0.59 log. The 0.5% MSM and the 2.5% MSM increased by log growth, 0.03 and 0.26 log, respectively.
Acidophilus plus bifidus control milk showed an 8.55 log increase in the first 24 hours, compared to a 9.65 increase for Acidophilus plus bifidus milk fortified with 2.5% MSM. In the second 24-hour period, acidophilus plus bifidus control increased by 0.14 log, while milk with 2.5% MSM decreased by 0.12 log. In the third 24-hour period, control of Acidophilus plus bifidus was reduced by
0.21 log, while milk with 2.5% MSM decreased by 0.04 log.
Table 33. Delayed recovery of Lactobacillus acidophilus in milk fortified with MSM on day 21
Concentration in percent of MSM
<td>Day hour</td><td> 0 %</td><td>A / B 0%</td><td>A / B 2.5%</td><td> 0,50 %</td><td> 0,50 %</td><td> 2,50 %</td><td> 2,50 %</td><td> 5 %</td><td> 5,00 %</td>
<td>Day 21 - 24</td><td> 12,94</td><td> 12,43</td><td> 13,08</td><td> 12,89</td><td> 12,93</td><td> 10,62</td><td> 10,95</td><td> 9,52</td><td> 9,85</td>
<td>Day 21 - 48</td><td> 13,02</td><td> 12,16</td><td> 12,08</td><td> 12,94</td><td> 13,1</td><td> 12,44</td><td> 13,12</td><td> 13,08</td><td> 13,07</td>
<td>Day 21 - 72</td><td> 10,32</td><td> 10,86</td><td> 12,33</td><td> 12,29</td><td> 10,31</td><td> 13,14</td><td> 13,05</td><td> 13,26</td><td> 13,04</td>
Table 34. Recovery of Lactobacillus acidophilus in milk fortified with MSM on day 21 with average recovery 30 _by percentage of MSM with initial starting data_
<td colspan="7">Concentration in percent of MSM</td>
<td>Day hour</td><td>Acidophilus milk</td><td>Ac / Bf milk</td><td>Ac / Bf with 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>
<td>Day 21</td><td> 3,30</td><td> 3,60</td><td> 3,37</td><td> 3,37</td><td> 3,47</td><td> 3,56</td>
<td>Day 21 - 24</td><td> 12,94</td><td> 12,43</td><td> 13,08</td><td> 12,91</td><td> 10,79</td><td> 9,69</td>
<td>Day 21 - 48</td><td> 13,02</td><td> 12,16</td><td> 12,08</td><td> 13,02</td><td> 12,78</td><td> 13,08</td>
<td>Day 21 - 72</td><td> 10,32</td><td> 10,86</td><td> 12,33</td><td> 11,30</td><td> 13,10</td><td> 13,15</td>
On day 21, the Acidophilus control in the first 24 hours had a log growth of 12.94. For 0.5% MSM the growth was 12.91 log, for 2.5% MSM it was 10.79 log and for 5.0% MSM f and e 9.69. In the following 24 hour period, the control was equal in growth to milk with 0.5% MSM, 0.24 log greater than 2.5% MSM and
0.05 log less than 5.0% MSM. The final 24-hour period shows a significant increase in
ES 2 616 630 T3 MSM concentrations compared to control. 0.5% MSM was 0.98 log greater than the control, 2.5% MSM was 2.78 log greater than the control, and 5.0% MSM was 2.83 log greater than the control.
The Acidophilus plus bifidus with 2.5% MSM was 0.65 log higher than the Acidophilus plus bifidus control in the first 24 hours. At hour 48, the acidophilus plus bifidus control was 0.08 log higher. In the final 24 hours, acidophilis plus bifidus with 2.5% MSM outpaced the growth of the Acidophilos plus bifidus control by 1.47 log.
Table 35. Recovery of Lactobacillus acidophilus in MSM fortified milk on day 21 with log increase in recovery growth rate from zero start time.
<td colspan="7">Concentration in percent of MSM</td>
<td>Hour</td><td>Acidophilus milk</td><td>Ac / Bf milk</td><td>Ac / Bf with 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>
<td> 0</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td>
<td> 24</td><td> 9,64</td><td> 8,83</td><td> 9,71</td><td> 9,54</td><td> 7,32</td><td> 6,13</td>
<td> 48</td><td> 0,08</td><td> -0,27</td><td> -1,00</td><td> 0,11</td><td> 2,00</td><td> 3,39</td>
<td> 72</td><td> -2,70</td><td> -1,30</td><td> 0,25</td><td> -1,72</td><td> 0,32</td><td> 0,08</td>
When reviewing the log increase for day 21, in the first 24 hours the acidophilic control milk had a 9.64 log increase. 0.5% MSM had a 9.54 log increase, 2.5% MSM had a 7.32 log increase, and 5.0% MSM had a 6.13 log increase. In the second 24 hours the control increased by 0.08 log. 0.5% MSM increased by 0.11 log, 2.5% MSM increased by 2.00, and 5.0% MSM increased by 3.39 log. The final 24 hours show that the control is reduced by 2.70 logs. 0.5% MSM was reduced by 1.72 log. 2.5% MSM increased by 0.32 log and 5.0% MSM increased by 0.08 log.
Acidophilus plus bifidus with 2.5% MSM increased by 9.71 log and Acidophilus plus bifidus control increased by 8.83 log. In the final two 24-hour periods, the Acidophilus plus bifidus control had a reduction of 0.27 log and 1.30 log. Acidophilus plus bifidus with 2.5% MSM decreased 1.00 log in the second 24-hour period and increased
0.25 log in the final 24 hour period.
Table 36. Delayed recovery of Lactobacillus acidophilus in milk fortified with MSM on day 28.
Concentration in percent of MSM
<td>Day hour</td><td> 0 %</td><td>A / B 0</td><td>A / B 2.5%</td><td> 0,50 %</td><td> 0,50 %</td><td> 2,50 %</td><td> 2,50 %</td><td> 5,0 %</td><td> 5,0 %</td>
<td>Day 28 - 24</td><td> 13,1</td><td> 13,41</td><td> 12,67</td><td> 6,11</td><td> 6,47</td><td> 6,37</td><td> 6,37</td><td> 5,64</td><td> 5,48</td>
<td>Day 28 - 48</td><td> 11,04</td><td> 13,42</td><td> 13,19</td><td> 13,66</td><td> 12,99</td><td> 12,52</td><td> 12,62</td><td> 12,58</td><td> 13,16</td>
<td>Day 28 - 72</td><td> 12,47</td><td> 10,31</td><td> 12,00</td><td> 12,99</td><td> 13,01</td><td> 12,98</td><td> 12,34</td><td> 12,49</td><td> 12,47</td>
Table 37. Recovery of Lactobacillus acidophilus in milk fortified with MSM on day 28 with mean recovery 25 _by percentage of MSM with initial baseline data_
<td colspan="7">Concentration in percent of MSM</td>
<td>Day hour</td><td>Acidophilus milk</td><td>Ac / Bf milk</td><td>Ac / Bf with 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>
<td>Day 28</td><td> 2,00</td><td> 1,82</td><td> 1,94</td><td> 1,00</td><td> 1,33</td><td> 0,52</td>
<td>Day 28 - 24</td><td> 13,10</td><td> 13,41</td><td> 12,67</td><td> 6,29</td><td> 6,37</td><td> 5,56</td>
<td>Day 28 - 48</td><td> 11,04</td><td> 13,42</td><td> 13,19</td><td> 13,33</td><td> 12,57</td><td> 12,87</td>
<td>Day 28 - 72</td><td> 12,47</td><td> 10,31</td><td> 12,00</td><td> 13,00</td><td> 12,66</td><td> 12,48</td>
The recovery data from day 28 shows that in the first 24 hour period the Acidophilus milk control had a log growth of 13.10. The MSM concentrations were: 6.29 log for 0.5% MSM, 6.37 log for 2.5% MSM, and 5.56 log for 5.0% MSM. At 48 hours, the log growth of 0.5% MSM was 13.33, of 2.5% MsM was 12.57 and of 5.0% MSM was 12.87 log. The control at 48 hours was 12.71 log. The control was reduced to 12.17 at 72 hours. 0.5% MSM decreased to 13.00 log and 5.0% MSM decreased to 12.48 log. MSM to
2.5% improved to 12.66 log. This was an increase of 0.53 over the control.
The control of Acidophilus plus bifidus was 13.41 log at 24 hours, 0.74 log higher than Acidophilus plus bifidus with
ES 2 616 630 T3
2.5% MSM. At hour 48, the difference was smaller, with the control 0.23 log greater than Acidophilus plus bifidus with 2.5% MSM. At hour 72, Acidophilus plus bifidus with 2.5% MSM was 1.69 logs higher than the control, which was 10.31 log.
Table 38. Recovery of Lactobacillus acidophilus in MSM fortified milk on day 28 in log increase in recovery growth rate from zero start time.
<td colspan="7">Concentration in percent of MSM</td>
<td>Weather</td><td>Acidophilus milk</td><td>Ac / Bf milk</td><td>Ac / Bf with 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>
<td> 0</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td><td> 0,00</td>
<td> 24</td><td> 11,10</td><td> 11,59</td><td> 10,73</td><td> 5,29</td><td> 5,04</td><td> 5,04</td>
<td> 48</td><td> -2,06</td><td> 0,01</td><td> 0,52</td><td> 7,04</td><td> 6,20</td><td> 7,31</td>
<td> 72</td><td> 1,43</td><td> -3,11</td><td> -1,19</td><td> -0,32</td><td> 0,09</td><td> -0,39</td>
On day 28, the growth rate increased in which the Acidophilus milk control in the first 24 hour period increased 11.10 log, MSM at 0.5% increased 5.29 log, MSM at 2.5% increased 5.04 log, and 5.0% MsM increased 5.04 log. In the second 24-hour period, there was a change, reducing the control by 2.06 log, while the concentrations of milk fortified with MSM increased for MSN at 0.5% by 7.04 log, 2.5% by 6 , 20 log, and 5.0% at 7.31 log. The final 24 hour period showed that the control increased by 1.43, the 2.5% MSM increased by 0.09 log. 0.5% MSM decreased by 0.32 log and 5.0% MSM decreased by 0.39 log.
The Acidophilus plus bifidus milk control increased 11.59 log in the first 24 hours and Acidophilos plus bifidus with 2.5% MSM increased 10.73 log. In the second 24 hour period, the Acidophilus plus bifidus control increased 0.01 log and Acidophilus plus bifidus with 2.5% MSM increased 0.52 log. In the final 24 hour period, the Acidophilus plus bifidus control decreased by 3.11 log, while the Acidophilus plus bifidus with 2.5% MSM decreased by 1.19 log.
These studies demonstrate that MSM as an additive to this product played a significant role in the recovery of the probiotic, Lactobacillus acidophilus. In each recovery case, there was an increase in the growth rate of Lactobacillus Acidophilus with the product fortified with MSM versus the product without MSM.
Recovery data from day 7 showed in the first 24 hours that MSM had an increase from 0.99 log to 1.66 log compared to the control. In the second period of 24 hours for 7 days, the growth rate was lower than the control, the general growth numbers were higher for the MSM at 5.0%, 0.46 log higher. In the third 24-hour period to day 7, the growth rate with MSM was higher than the control, at 0.36 log, 0.79 log and 0.93 log.
On day 14, only the 0.5% MSM outpaced the control by 0.27 log of growth rate in the first 24 hours. The 2.5% MSM and 5.0% MSM samples were 1.13 and 2.10 log, respectively, lower than the control. This is where the control began to outgrow MSM concentrations in the first 24 hours. On days 21 and 28, the control exceeded the growth of all concentrations in the first 24 hours.
The second 24 hours for each data point that were collected after day 7 showed that the MSM concentrations exceeded the control. The data from the second period of day 14 showed growth rates with MSM at 0.70, 0.94, and 1.67 log higher than the control. Data from the second period of day 21 showed growth rates with MSM 0.03, 1.92, and 3.31 log higher than the control. Data from the second period of day 28 showed growth rates with MSM at 9.10, 8.26, and 9.37 log higher than the control. This increase in growth rate did not always translate into a higher concentration of Lactobacillus acidophilus in the recovery broth. On day 14, the 0.5% and 5.0% MSM concentrations were higher than the control, while the 2.5% MSM was lower. On day 21, only the 5.0% MSM was higher. On day 28, the three MSM concentrations were significantly higher than the control, at 2.29, 1.53, and 1.83 log.
The third data period for day 14 showed that only the growth rate at the concentration of MSM at
2.5% was higher, by 0.20 log. Even with the lower growth rates, a higher log growth was observed for the MSM concentrations, except for the 5.0% MSM, which was equal to the control. On day 21, the third data period showed that the growth rate of MSM concentrations exceeded the control by 0.98, 3.02, and 2.78 log. This increased growth rate translated into a higher concentration of Lactobacillus acidophilus for the MSM-fortified samples. The growth recovery counts were 0.98, 2.78, and 2.83 log higher than the control. The control exceeded the MSM concentrations for the growth rate of day 28 in the third period. Although the growth recovery counts for the
ES 2,616,630 T3 MSM concentrations of 0.5, 2.5, and 0.5% were 0.53, 0.19, and 0.01 log, respectively, higher than the control.
With bacterial growth curves there is an initial delay phase where the bacteria adjust to the environment, before entering the exponential or logarithmic phase, where cells are duplicated. After the log phase, a stationary phase occurs where the growth rate slows down. In this phase, peaks and troughs are observed as growth curves. Finally, the death phase occurs, where bacteria deplete nutrients and die.
This study provides indicators of how MSM helps in the lag phase, logarithmic phase, stationary phase, and death phase. MSM in different stages shortens the lag phase, such that probiotic bacteria start the logarithmic phase earlier. The log phase extended beyond the control in this study, such that the product with the MSM additive had a higher peak value. The stationary phase was affected by MSM as there was an extension of the higher values over a longer period of time. Death speed decreased with MSM. At different points, there was a slower rate of reduction in growth. These different observations show that MSM as an additive positively affects probiotic bacteria. The benefit of ingesting an MSM-fortified probiotic product could be a faster response time with a longer-lasting effect. The consumer could obtain a product that increases their body responses to the added benefits of probiotic bacteria.
MSM consistently aided the recovery and growth of probiotic bacteria in the product studied. In the first 24 hours of culture, there was an increase in the recovery rate, indicating that the stressed microorganisms responded better to a new environment with MSM as an additive.
Example 19
Growth of Bifidobacterium bifidum in MSM-fortified medium
This example shows the effects of MSM on the growth of Bifidobacterium bifidum in MSM-fortified microbial growth medium.
Microbial growth studies were carried out on media fortified with 0%, 0.125%, 0.25%, 0.5%, 1.0%, 2.5%, and 5% MSM. The plating time intervals were used every 8 hours for a total of 96 hours. The growth curves of colony forming units per milliliter (cfu / ml) of the microorganisms between the MSM concentrations were compared with the 0% MSM concentration as a control sample for each microorganism. The stock of MSM powder was supplied by Bergstrom Nutrition with Certificate of Analysis. The powder was the Microprill formula, lot # 0806809, with an expiration date 10/31/13. All media and the MSM powder stock were tested for sterility prior to study. The microorganism analyzed was Bifidobacterium bifidum ATCC No. 29521.
Bifidobacterium bifidum (99 ml of MRS broth with the addition of 0.05% L-cysteine) was prepared with the respective concentrations of MSM. Working concentrations of MSM were prepared from a single 5% MSM solution in MRS broth and diluted accordingly with MRS broth to obtain the desired final concentration of MSM. The sterility of the solutions was verified before proceeding with the study.
The working solutions were inoculated at a level of 1.5 to 2 log of microorganism per 100 ml of broth. Bifidobacterium bifidum was incubated under anaerobic conditions at 35 ° C ± 0.5 ° C for 72 hours. Oxygen indicators were used to verify anaerobic conditions between plating intervals for the Bifidobacterium test samples.
Bifidobacteruim was inoculated on MRS agar supplemented with L-cysteine and the times listed above to reduce the oxidation-reduction potential of the medium. All preparation and seeding was carried out at room temperature. All dilutions for all organisms were plated in triplicate for all time intervals sampled. To capture the proper colonies per milliliter, all organisms at all time intervals were plated at six different dilutions. All plates were incubated at 35 ° C ± 0.5 ° C for 72 hours. The appropriate dilution plate was used for enumeration and averaged for reporting. The plate suitable for enumeration contains between 25 and 250 cfu / ml. The MSM stock sample and all MSM-prepared media were tested for background levels of microorganisms on MRS agar and TSA. The MSM pool was <10 cfu / g and all test media were <1 cfu / ml in all cases before inoculation. All time intervals for seeding included negative control plates during pouring for quality control purposes. All negative control plates were devoid of microorganism growth. At 72 hours, the concentrations of MSM and control solutions were verified to be negative for contamination by strains and the strains were verified to be original species.
ES 2 616 630 T3
Table 39. Stock Culture Control Numbers Before Test Sample Inoculation
<td></td><td>Bifidobacterium bifidum</td>
<td>cfu / ml inoculum</td><td>1.48 x 10<sup>4</sup></td>
<td>CFU added to 100 ml</td><td>1.48 x 10<sup>4</sup></td>
<td>cfu / ml in medium at time 0</td><td>1.48 x 10<sup>2</sup></td>
Control numbers were derived from the growth of specific organisms in the appropriate medium. After incubation, cells were washed out of the medium and captured in a sterile vial. The vial was used as the starting solution for the number control (stock). The stock solution was then diluted to obtain a suitable reading on a spectrophotometer, using a 420 nm wavelength with percent light transmission. Bacterial concentrations were determined according to AOAC method 960.09, table 960.09A. The preparation of the culture suspension from the stock culture was determined by spectrophotometric readings or comparison with the Mc Farland standard.
Table 40. Log growth of Bifidobacteruim bifidum in medium fortified with MSM. Concentration in percent of MSM
Concentration in percent of MSM
<td>H</td><td> 0</td><td> 0,125</td><td> 0,25</td><td> 0,5</td><td> 1</td><td> 2,5</td><td> 5</td>
<td> 0</td><td> 1,22</td><td> 1,12</td><td> 1,00</td><td> 1,00</td><td> 1,00</td><td> 1,30</td><td> 1,12</td>
<td> 8</td><td> 2,55</td><td> 2,89</td><td> 2,93</td><td> 2,84</td><td> 2,73</td><td> 2,71</td><td> 2,04</td>
<td> 16</td><td> 7,16</td><td> 7,85</td><td> 8,07</td><td> 7,45</td><td> 7,73</td><td> 7,56</td><td> 6,10</td>
<td> 24</td><td> 8,94</td><td> 9,06</td><td> 8,49</td><td> 8,81</td><td> 8,81</td><td> 8,64</td><td> 8,43</td>
<td> 32</td><td> 10,81</td><td> 11,45</td><td> 11,41</td><td> 11,22</td><td> 11,49</td><td> 11,56</td><td> 11,13</td>
<td> 40</td><td> 11,03</td><td> 10,80</td><td> 10,31</td><td> 12,09</td><td> 11,19</td><td> 11,73</td><td> 11,78</td>
<td> 48</td><td> 11,54</td><td> 8,22</td><td> 8,10</td><td> 9,39</td><td> 10,43</td><td> 10,72</td><td> 10,70</td>
<td> 56</td><td> 10,64</td><td> 7,25</td><td> 7,22</td><td> 9,51</td><td> 10,57</td><td> 11,34</td><td> 10,59</td>
<td> 64</td><td> 9,97</td><td> 8,86</td><td> 6,77</td><td> 10,34</td><td> 10,66</td><td> 12,26</td><td> 14,32</td>
<td> 72</td><td> 8,56</td><td> 6,59</td><td> 6,39</td><td> 8,35</td><td> 8,34</td><td> 10,26</td><td> 8,65</td>
<td> 80</td><td> 10,56</td><td> 9,12</td><td> 8,52</td><td> 10,38</td><td> 9,43</td><td> 9,52</td><td> 12,20</td>
<td> 88</td><td> 10,64</td><td> 9,12</td><td> 8,52</td><td> 9,70</td><td> 10,41</td><td> 10,64</td><td> 12,31</td>
<td> 96</td><td> <6,00</td><td> <6,00</td><td> <6,00</td><td> 8,82</td><td> <6,00</td><td> <6,00</td><td> 9,60</td>
The growth observed with Bifidobacterium bifidum shows a 0.2 to 0.4 log increase in growth rate for MSM concentrations of 0.125% to 2.5% at 8 hours. MSM concentrations from 0.125% to 2.5% at 16 hours increased to 0.3 to 0.7 log. At 24 hours, MSM concentrations of 0.125% to 2.5% were shown that were reduced to be equal to or less than the control. At a MSM concentration of 5%, a slower growth rate was observed compared to the control during the first 24 hours. At hour 32, there was an increase in growth rate in the range of 0.3 to 0.75 log for all MSM concentrations compared to the control. At hour 40, MSM concentrations of 0.125% and 0.25% showed a constant reduction in growth rate, to the point that they were below control from hour 40 to hour 96. The hour 40 showed the sample with MSM at 0.5% a complete log greater in growth than the control. 0.5% MSM from hour 48 to hour 96 slowed its growth rate until it was 2 to 3 full logs below the growth rate of the control. MSM at 1% concentration equaled the growth rate of growth from hour 40 to hour 96, except for hour 48 and hour 80 where it was a lower full log. 2.5% MSM at hour 40 was 0.7 log higher in growth rate compared to control. Hour 48 showed a 0.7 log reduction in growth rate compared to the control. From hour 56 to hour 72, 2.5% MSM had a growth rate that was 0.7 to 2.29 log greater than that of the control. Hour 80 showed a 1 log lower growth rate for the 2.5% MSM sample compared to the control and at hours 88 and 96, the growth rate was equivalent. 5% MSM at hour 40 had a growth rate of 0.7 log greater than that of the control. At hour 48, it was reduced to 0.7 log lower than that of the control and at hour 56, the growth rate was equivalent to that of the control. Hour 64 shows an increase in growth rate of 4.35 log for the 5% MSM versus the control. The 72 hour showed
ES 2 616 630 T3 a reduction in growth rate, with a return to a 1.6 log increase in growth rate at hour 80 and hour 88. Hour 96 showed MSM growth rate at 5 % which was approximately
3.6 log greater than that of the control.
Bifidobacterium bifidum showed significant benefit by having MSM as an additive to influence growth. All concentrations increased growth rate to the point that Bifidobacterium bifidum peaked 16 hours earlier than the control. The control peaked at 11.54 log growth at hour 48. This maximum growth was reached by all MSM concentrations at hour 32. The MSM concentrations of 0.125% and 0.25% show a reduction in growth from hour 40 to hour 96, not reaching maximum growth again. 0.5% MSM increased growth 0.5 log greater than the control maximum. 0.5% MSM showed growth reduction from hour 48 to hour 96. The 0.5% MSM delayed the death stage to the point that at hour 96 there was a growth loss of 8.82 log, which was approximately 2 log greater than that of the control. 1% MSM did not increase the growth of bacteria compared to the control, but did reduce the death stage. From hour 40 to hour 64, the 1% MSM did not show a great reduction in growth, there was a slow reduction of 0.5 log for hour 48, but there was no reduction for hours 56 and 64. At hour 72, there was a 2 log reduction in growth, but at hour 80, there was a 1 log increase in growth and at hour 88 there was another 1 log increase in growth. At hour 96, growth was outside the accounting range and was estimated to be less than 6 log growth. Continuing for another 8 hours, there may have been another increase in growth to more than 6 log. 2.5% MSM at hour 40 reached a growth of 11.73 log, with a reduction of 1 log at hour 48. There was a gradual increase in growth at hour 56 and hour 64, reaching a maximum of 12 , 26 log, 0.72 log greater than control. At hour 72, there was a 2 log reduction, with a 0.7 log fall at hour 80 for the 2.5% MSM. At hour 88, the 2.5% MSM increased growth by 1 log, before falling below the counting interval at hour 96. MSM at 5% concentration took longer to increase growth rate compared to the other concentrations of MSM. At hour 32, the growth was 11.13 log and at hour 40, the growth was 11.78 log. At hour 48, the growth was reduced by 1 log and at hour 56, there was a reduction of 0.1 log. At hour 64, growth reached the highest of all MSM concentrations of 14.32 log for the 5% MSM. There was a 6 log reduction at hour 72, but at hour 80, the growth increased 4 log to 12.20. At hour 88, an increase of 0.1 log was observed, before falling 9.60 log of growth at hour 96. MSM at 5% slowed the death rate considerably, extending the stationary phase up to 40 hours . Once the stationary phase was reached, there was a continuous increase and decrease in growth, moving toward a lower growth pattern. These studies indicate that MSM leads to stationary phase faster for all concentrations, extending the stationary phase for concentrations above 0.5% MSM and increasing peak growth for concentrations of 2.5% and 5% MSM. .
Example 20
Effect of bromocresol purple on E. coli when added to the MSM matrix
This example shows the effects of bromocresol purple on E-coli when MSM is added to the matrix.
To investigate whether MSM functions as a carrier / transporter, the ability of MSM to transport Bromocresol into E. coli was evaluated. Bromocresol purple is an indicator dye that turns yellow in the presence of E. coli bacteria. It is not toxic to the body. To minimize potential ionic interference, lactose broth was selected as the preferred medium for this study because it lacks both NaCl and protein. The USP <51> of antimicrobial efficacy for assays was used as a template to display CL100 (lethal concentration). MSM concentrations of 5% -16% were used in 1% increments. All concentrations were plated at dilutions up to 10<sup>7</sup> to determine the log reduction.
Materials included the following: OptiMSM Flake lot number 0604751; Escherichia coli strain ATCC 8739 lot: 57762704; 30 ml borosilicate glass culture tubes were used for all OptiMSM material; MacConkey Broth from Accumedia (MB) batch: 100,974A; The diluent used was modified Letheen broth from Alpha Biosciences (MLB) batch: I08-09; Tryptic Soy Agar with Lecithin from Alpha Biosciences; and Tween 80 (TSA) lot: F08-42.
Flake OptiMSM was weighed using a certified Mettler Toledo AG245 balance, NS: 1115210833 and aliquoted for each concentration. The material was placed in 30 ml glass borosilicate culture tubes. The material was calculated in a volume of 10 ml. Material was added to each tube as follows: 5% (0.5g), 6% (0.6g), 7% (0.7g), 8% (0.8g), 9% (0.9g), 10% (1.0g), 11% (1.1g), 12% (1.2g), 13% (1.3g), 14% (1.4g), 15% (1.5g), and 16 % (1.6g). MacConkey broth was aliquoted into 10ml for each tube and then sterilized for 20 minutes at 121 ° C. The tubes were cooled to room temperature, which was apparently 20 ° C. All tubes were then seeded with the same dilution of Eschericia coli which provided a level of 6 colony forming units at 6.0 x 10 / ml (6.8). The tubes were then incubated at 25 ° C. A daily observation of the color change was made during the first seven days. The tubes were periodically mixed to ensure that the OptiMSM was well balanced at all times. A positive and negative control were determined.
The results of these studies are as follows:
ES 2,616,630 T3 (1) Day one: showed the color change of the broth to yellow for the concentration of 5-7%; 8% showed a slight color lightening; and 9-16% showed no signs of change.
(2) Day two: showed the same signs as day one.
(3) Day three: showed a change in concentration at 8% that turned to the typical yellow color.
(4) Day 4 to day 6: showed no significant signs of change.
(5) Day 7: It showed a color change from 9% to a yellow color. No color change at 10% -16%.
(6) Day 14: Did not show signs for the 10% -16% concentration range.
The concentration tubes were seeded on MacConkey agar to see if the organism could recover. No microorganisms were observed after the 72 hour incubation. Day 30 showed no signs of change for the 10-16% concentration range. The positive control was seeded for each time point and showed signs of organisms demonstrated by a classic isolation streak.
This qualitative test indicates that OptiMSM has some type of carrier effect and reduces or eliminates the body. This is demonstrated by the absence of yellow color in MSM concentrations lower than those demonstrated in previous studies using culture medium. Color showed a reduction at concentrations as low as 8% versus 11% in the growth medium studies.
Example 21
Antimicrobial study of MSM and DMSO in Streptococcus organisms
This example shows the effects of MSM and DMSO on the growth of Streptococcus organisms.
Specific concentrations of MSM (such as 10% to 16% MSM) have been shown here to kill microorganisms. Dimethylsulfoxide has also been shown to kill microorganisms at concentrations of 30-50%. This study evaluated the bactericidal properties of both compounds, alone or in combination, as well as their efficacy when used with a low level of penicillin.
Streptococcus pyogenes (Lancefield group A) has a hyaluronic acid capsule and Streptococcus pneumoniae (no Lancefield group identified to date) has a distinct polysaccharide capsule. These two organisms are responsible for many types of human streptococcal infections and exhibit two different types of encapsulation. These two organisms were used in this in vitro study. In particular, this study determined the antimicrobial effects of MSM and DMSO, both individually and in combination, with Streptococcus pyogenes and Streptococcus pneumoniae. This study also determined the most effective concentrations for antimicrobial properties for both compounds in combination and whether combining MSM and DMSO reduces the concentrations of any compound necessary to achieve microbial reduction. Furthermore, the efficacy of using MSM, DMSO and the combination of the two together with an antibiotic agent was evaluated.
Streptococcus pneumoniae (# 10341 ™) and Streptococcus pyogenes (Lancefield Group A, # 10096 ™) were purchased from the ATCC. MSM (# 41631) and DMSO (# D8418) were purchased from Sigma-Aldrich. Penicillin was purchased from Henry Schein. Bacterial culture medium was purchased from Becton-Dickinson and Company (# 297963). The Bioluminescent ATP Assay Kit from Promega (# G8230). Streptococcus Pyogenes was cultured in brain heart infusion broth (BD 237500, # 44 booth) overnight. Equal amounts of broth containing bacteria were used for the studies. Streptococcus pneumoniae was also cultured in brain heart infusion broth.
Evaluation of bacterial viability:
The bioluministence test kit for ATP was used to evaluate bacterial activity, based on the following reaction:
ATP + D-Luciferin + O<sub>2</sub> Oxyluciferin + AMP + pyrophosphate + CO<sub>2</sub> + light (560 nm).
Bacterial ATP can be measured by direct lysis of the bacteria with a suitable detergent; then this released ATP is free to react with luciferin / luciferase and give rise to the emission of light. The intensity of the emitted light is proportional to the ATP concentration. Measurement of light intensity using a luminometer allows direct quantification of ATP, which is the universal indicator of viability for living microorganisms.
Both S. pyogenes and S. pneumoniae were grown under various conditions to determine optimal conditions for evaluating MSM, DMSO, and / or penicillin. MSM, DMSO, and penicillin were diluted in culture medium, according to Table 45-1. Bacteria were cultured for 7 hours for Streptococcus pneumoniae and 18 hours for Streptococcus pyogenes, respectively. Then, the bacterial viability was evaluated by means of the bioluminescent ATP test kit. The test was carried out in triplicate.
ES 2 616 630 T3
T able 41. MSM, DMSO and penicillin concentrations evaluated.
<td>MSM (%)</td><td>DMSO (%)</td><td>Penicillin (pg / l)</td>
<td> 20</td><td> 20</td><td> 100</td>
<td> 10</td><td> 10</td><td> 50</td>
<td> 5</td><td> 5</td><td> 25</td>
<td> 2,5</td><td> 2,5</td><td> 12,5</td>
<td> 1,25</td><td> 1,25</td><td> 6,25</td>
<td> 0,625</td><td> 0,625</td><td> 3,125</td>
<td> 0,3125</td><td> 0,3125</td><td> 1,5625</td>
<td> 0</td><td> 0</td><td> 0</td>
MSM and DMSO were diluted in culture medium, according to Table 42 (for Streptococcus pneumoniae, lower left) and Table 43 (Streptococcus pyogenes, lower right).
Table 42
<td>DMSO (%)</td><td>MSM (%)</td>
<td rowspan="4"> 0</td><td> 0</td>
<td> 5</td>
<td> 10</td>
<td> 20</td>
<td rowspan="4"> 5</td><td> 0</td>
<td> 5</td>
<td> 10</td>
<td> 20</td>
<td rowspan="4"> 10</td><td> 0</td>
<td> 5</td>
<td> 10</td>
<td> 20</td>
<td rowspan="5"> 20</td><td> 0</td>
<td> 5</td>
<td> 10</td>
<td> 20</td>
<td></td>
Table 43
<td>DMSO (%)</td><td>MSM (%)</td>
<td rowspan="4"> 0</td><td> 0</td>
<td> 2,5</td>
<td> 5</td>
<td> 10</td>
ES 2 616 630 T3
<td rowspan="4"> 2,5</td><td> 0</td>
<td> 2,5</td>
<td> 5</td>
<td> 10</td>
<td rowspan="4"> 5</td><td> 0</td>
<td> 2,5</td>
<td> 5</td>
<td> 10</td>
<td rowspan="5"> 8</td><td> 0</td>
<td> 2,5</td>
<td> 5</td>
<td> 10</td>
<td></td>
To determine the efficacy of using MSM, and DMSO in conjunction with penicillin, MSM, DMSO, and penicillin were dissolved in culture medium according to Table 44-1 (S. pneumoniae) and Table 44-2 (S. pyogenes).
Table 44-1
<td>DMSO</td><td>MSM</td><td>Penicillin (pg / l)</td>
<td rowspan="3"> 5</td><td> 5</td><td rowspan="9"> 25</td>
<td> 10</td>
<td> 20</td>
<td rowspan="3"> 10</td><td> 5</td>
<td> 10</td>
<td> 20</td>
<td rowspan="3"> 20</td><td> 5</td>
<td> 10</td>
<td> 20</td>
Table 44-2
<td>DMSO</td><td>MSM</td><td>Penicillin (pg / l)</td>
<td rowspan="6"> 2,5</td><td> 2,5</td><td rowspan="3"> 3,125</td>
<td> 5</td>
<td> 10</td>
<td> 2,5</td><td rowspan="3"> 6,25</td>
<td> 5</td>
<td> 10</td>
<td rowspan="2"> 5</td><td rowspan="4"> 2,5</td><td> 3,125</td>
<td> 6,25</td>
<td rowspan="2"> 8</td><td> 3,125</td>
<td> 6,25</td>
ES 2 616 630 T3
The IC50 of DMSO, MSM and penicillin in Streptococcus pneumonia was 12.86%, 15.97% and 68.54g / l, respectively. DMSO and MSM had synergistic effects at doses of 5% to 20% (for both drugs) to inhibit the growth of Streptococcus pneumoniae. DMSO and penicillin also had a synergistic effect at doses of 10% to 20% (for DMSO) and 25 mg / L (for penicillin) to inhibit the growth of
Streptococcus pneumoniae. Furthermore, MSM and penicillin also had a synergistic effect at doses of 5% (for MSM) and 25 mg / l (for penicillin) to inhibit the growth of Streptococcus pneumoniae. When penicillin, DMSO and MSM were used together, the greatest synergistic effect was the result of DMSO + only MSM instead of penicillin + DMSO + MSM.
The CI<sub>50</sub> of DMSO, MSM and penicillin in Streptococcus pyogenes was 9.07%, 10.26% and 15.25 mg / l, respectively. DMSO and MSM had synergistic effects at doses of 2.5% to 5% (for both drugs) to inhibit the growth of Streptococcus pyogenes. DMSO and penicillin also had a synergistic effect at doses of 5% (for DMSO) and 6.25 mg / l (for penicillin) to inhibit the growth of Streptococcus pyogenes. MSM and penicillin had a synergistic effect at doses between 2.5% to k 5% (for MSM) and 3.125 to
6.25mg / l (for Penicillin penicillin) to inhibit the growth of Streptococcus Pyogenes. When penicillin, DMSO and MSM were used together, the synergistic effect was the result of DMSO + only MSM instead of penicillin + DMSO + MSM.
Table 45-1. Viability of S. pneumoniae after exposure to DMSO
<td>DMSO concentration</td><td>S. pneumoniae viability (%)</td>
<td> 5</td><td> 75,45</td>
<td> 10</td><td> 40,18</td>
<td> 20</td><td> 27,19</td>
Table 45-2. S. pneumoniae viability after exposure to MSM
<td>MSM concentration</td><td>S. pneumoniae viability (%)</td>
<td> 5</td><td> 95,34</td>
<td> 10</td><td> 48,57</td>
<td> 20</td><td> 39,08</td>
Table 45-3. Viability of S. pneumoniae after exposure to various concentrations of MSM in 5% DMSO
<td>DMSO (%)</td><td>MSM (%)</td><td>S. pneumoniae viability (%)</td>
<td> 5</td><td> 0</td><td> 75,45</td>
<td> 5</td><td> 5</td><td> 50,94*</td>
<td> 5</td><td> 10</td><td> 45,40</td>
<td> 5</td><td> 20</td><td> 27,22</td>
Table 45-4. Viability of S. pneumoniae after exposure to various concentrations of MSM in 10% DMSO
<td>DMSO (%)</td><td>MSM (%)</td><td>S. pneumoniae viability (%)</td>
<td> 10</td><td> 0</td><td> 40,18</td>
<td> 10</td><td> 5</td><td> 47,81</td>
<td> 10</td><td> 10</td><td> 37,42</td>
<td> 10</td><td> 20</td><td> 11,95</td>
Table 45-5. Viability of S. pneumoniae after exposure to various concentrations of MSM in 20% DMSO
<td>DMSO (%)</td><td>MSM (%)</td><td>S. pneumoniae viability (%)</td>
<td> 20</td><td> 0</td><td> 27,19</td>
<td> 20</td><td> 5</td><td> 17,60*</td>
ES 2 616 630 T3
<td> 20</td><td> 10</td><td> 7,76</td>
<td> 20</td><td> 20</td><td> 5,15</td>
Table 45-6. Viability of S. pneumoniae after. exposure to various concentrations of penicillin
<td>Penicillin (pg / l)</td><td>S. pneumoniae viability (%)</td>
<td> 25</td><td> 79,82</td>
<td> 50</td><td> 42,70</td>
<td> 100</td><td> 40,93</td>
Table 45-7. S. pneumoniae viability after exposure to 25 mg / L penicline with various concentrations of DMSO
<td>Penicillin (pg / l)</td><td>DMSO (%)</td><td>S. pneumoniae viability (%)</td>
<td> 25</td><td> 0</td><td> 79,82</td>
<td> 25</td><td> 5</td><td> 46,13</td>
<td> 25</td><td> 10</td><td> 39,78</td>
<td> 25</td><td> 20</td><td> 22,08</td>
Table 45-8. Viability of S. pneumoniae after exposure to 50 mg / L penicline with various concentrations of DMSO
<td>Penicillin (pg / l)</td><td>DMSO (%)</td><td>S. pneumoniae viability (%)</td>
<td> 50</td><td> 0</td><td> 42,70</td>
<td> 50</td><td> 5</td><td> 46,27</td>
<td> 50</td><td> 10</td><td> 37,09</td>
<td> 50</td><td> 20</td><td> 19,14</td>
Table 45-9. Viability of S. pneumoniae after exposure to 100 mg / L penicline with various concentrations of DMSO
<td>Penicillin (pg / l)</td><td>DMSO (%)</td><td>S. pneumoniae viability (%)</td>
<td> 100</td><td> 0</td><td> 40,93</td>
<td> 100</td><td> 5</td><td> 45,09</td>
<td> 100</td><td> 10</td><td> 35,80</td>
<td> 100</td><td> 20</td><td> 21,76</td>
The combination of 5% MSM with 25 mg / L penicillin showed a synergistic reduction in S. pneumoniae viability, resulting in only 41% viability (see Table 10). Synergy compared to expected results based on only MSM and only penicillin is indicated in the tables by a Conversely, only 5% MSM reduced viability by only about 5%, while penicillin alone 25 mg / l reduced viability by approximately 21%. Therefore, the combination of 5% MSM / 25 mg / L penicillin was unexpectedly more effective than expected based on the results obtained with only MSM or penicillin.
In addition, as in the case of DMSO, certain concentrations of MSM allowed lower concentrations of penicillin to reduce bacterial viability almost as effectively as higher concentrations. For example, 20% MSM with 100 mg / L penicillin reduced the viability of S. pneumoniae to 21.37%, MSM 20% with 50 mg / L penicillin reduced the viability of S. pneumoniae to 20.75% . Therefore, with the use of 20% MSM, the required concentration of penicillin was reduced by half. Continuing this trend is the combination of 20% MSM with 25 mg / L penicillin, which reduced the viability of S. pneumoniae to approximately 25%. Similarly, although with a less robust reduction in bacterial viability, 5% MSM allowed 25 mg / L penicillin to have a nearly identical yield to 100 mg / L penicillin (compare Tables 45-10 to 45-12 for 25 mg / l penicillin).
ES 2 616 630 T3
Table 45-10. S. pneumoniae viability after exposure to 25 mg / L penicline with various concentrations of MSM
<td>Penicillin (pg / l)</td><td>MSM (%)</td><td>S. pneumoniae viability (%)</td>
<td> 25</td><td> 0</td><td> 79,82</td>
<td> 25</td><td> 5</td><td> 41,23*</td>
<td> 25</td><td> 10</td><td> 41,83</td>
<td> 25</td><td> 20</td><td> 25,36</td>
Table 45-11. Viability of S. pneumoniae after exposure to 50 mg / L penicline with various concentrations of MSM
<td>Penicillin (pg / l)</td><td>MSM (%)</td><td>S. pneumoniae viability (%)</td>
<td> 50</td><td> 0</td><td> 42,70</td>
<td> 50</td><td> 5</td><td> 41,23</td>
<td> 50</td><td> 10</td><td> 47,47</td>
<td> 50</td><td> 20</td><td> 20,75</td>
Table 45-12. S. pneumoniae viability after exposure to 100 mg / L penicline with various concentrations of MSM
<td>Penicillin (pg / l)</td><td>MSM (%)</td><td>S. pneumoniae viability</td>
<td> 100</td><td> 0</td><td> 40,93</td>
<td> 100</td><td> 5</td><td> 41,75</td>
<td> 100</td><td> 10</td><td> 36,67</td>
<td> 100</td><td> 20</td><td> 21,37</td>
Based on the synergy results observed in certain combinations of MSM or DMSO with penicillin, the present study was carried out to identify the various combinations of MSM, DMSO, and penicillin that provided synergistic reductions in bacterial viability compared to the combination of the effects of combining DMSO, MSM, and penicillin on bacterial activity. This study was also designed to identify combinations of the three compounds that advantageously reduce one or more of the compounds, and still effectively reduce bacterial viability.
DMSO at 5, 10 and 20% was combined individually with MSM at one of 5, 10 or 20% and penicillin at one of 25, 50 or 100 mg / L. Viability was evaluated as described above. Viability data are presented in Table 45-13. The symbol * represents synergistic results compared to the corresponding combination of DMSO and penicillin. The symbol ψ represents synergistic results compared to the corresponding combination of MSM and penicillin. The values for the reduction of bacterial viability were summed to determine the reduction threshold for synergy. For example, 5% DMSO reduces viability by approximately 25% and 25 mg / L penicillin reduces viability by approximately 21%, with a combined total reduction of approximately 46% expected. This represents a 64% viability. Therefore, in case the combination of 5% MSM, 5% DMSO, and 25 mg / ml penicillin results in less than 64% viability, synergy between the compounds is identified.
Various combinations of MSM, DMSO, and penicillin provide synergistic improvements in bacterial reduction. For example, the combination of 5% DMSO, 5% MSM, and 25 mg / L penicillin reduces bacterial viability to approximately 52% (see Table 45-13). Five percent DMSO in combination with 25 mg / L penicillin reduced cell viability to approximately 64% (i.e., a reduction of approximately 46%, based on the individual reduction observed with 5% DMSO, see table 45-1, and the individual reduction observed with 25 mg / l penicillin). Therefore, the combination of all three compounds reduced bacterial viability by an additional 12%. Similarly, the combination of 5% MSM with 25 mg / L penicillin resulted in a bacterial viability of approximately 74%, while the combination of all three compounds reduced the viability by approximately a further 22%.
In some combinations, synergistic results were detected with respect to both DMSO and penicillin as well as MSM plus penicillin. For example, 10% DMSO in combination with 20% MSM and 25 mg / L penicillin provides enhanced synergistic antimicrobial activity compared to both combinations of
ES 2 616 630 T3 reference. In other combinations, synergy was detected only with respect to DMSO plus penicillin or MSM plus penicillin. For example, the combinations of 5% MSM with 10% DMSO and 25 mg / L penicillin were synergistic with respect to MSM plus penicillin, but not with respect to DMSO plus penicillin.
In addition to the synergistic effects described above, there are several cases where specific combinations of DMSO, MSM and penicillin allow a reduction in the effective concentration of penicillin. For example, as shown in Table 45-13, the combination of 5% DMSO with 20% MSM provides very similar overall bacterial viability over the range of penicillin concentrations tested (with -25% viability with 25 mg / l penicillin -18% with 100 mg / l penicillin). Furthermore, 10% DMSO with 20% MSM resulted in virtually identical bacterial viability throughout the penicillin concentration range.
Similar results are seen with 20% DMSO in combination with 5, 10, or 20% MSM and any concentration of penicillin. These results reveal a slightly greater range of bacterial viability across the different concentrations of penicillin, however, since the reduction in all cases is close to approximately 90 to 95%, these combinations remain effective.
Table 45-13. Viability of S. pneumoniae after exposure to various combinations of DMSO, MSM, and penicillin
<td>DMSO (%)</td><td>MSM (%)</td><td>Penicillin (pg / l)</td><td>S. pneumoniae viability (%)</td>
<td> 5</td><td> 5</td><td> 25</td><td>52.11 *, ψ</td>
<td> 5</td><td> 5</td><td> 50</td><td> 43,36</td>
<td> 5</td><td> 5</td><td> 100</td><td> 53,03</td>
<td> 5</td><td> 10</td><td> 25</td><td> 51,82*</td>
<td> 5</td><td> 10</td><td> 50</td><td> 44,52</td>
<td> 5</td><td> 10</td><td> 100</td><td> 31,33</td>
<td> 5</td><td> 20</td><td> 25</td><td> 24,91*</td>
<td> 5</td><td> 20</td><td> 50</td><td> 19,20</td>
<td> 5</td><td> 20</td><td> 100</td><td> 18,12</td>
<td> 10</td><td> 5</td><td> 25</td><td>44.41 ψ</td>
<td> 10</td><td> 5</td><td> 50</td><td> 38,24</td>
<td> 10</td><td> 5</td><td> 100</td><td> 36,19</td>
<td> 10</td><td> 10</td><td> 25</td><td> 39,38</td>
<td> 10</td><td> 10</td><td> 50</td><td> 33,73</td>
<td> 10</td><td> 10</td><td> 100</td><td> 25,98</td>
<td> 10</td><td> 20</td><td> 25</td><td> 11,87 *, <sup>ψ</sup></td>
<td> 10</td><td> 20</td><td> 50</td><td> 11,03</td>
<td> 10</td><td> 20</td><td> 100</td><td> 10,96</td>
<td> 20</td><td> 5</td><td> 25</td><td>12.74 *, ψ</td>
<td> 20</td><td> 5</td><td> 50</td><td> 13,39 <sup>ψ</sup></td>
<td> 20</td><td> 5</td><td> 100</td><td> 9,28 <sup>ψ</sup></td>
<td> 20</td><td> 10</td><td> 25</td><td> 7,69 *, <sup>ψ</sup></td>
<td> 20</td><td> 10</td><td> 50</td><td> 7,74</td>
<td> 20</td><td> 10</td><td> 100</td><td> 5,58</td>
<td> 20</td><td> 20</td><td> 25</td><td> 4,93 *, <sup>ψ</sup></td>
<td> 20</td><td> 20</td><td> 50</td><td> 5,60</td>
<td> 20</td><td> 20</td><td> 100</td><td> 1,80</td>
ES 2 616 630 T3
As described above, the structure of S. pyogenes differs from that of S. pneumoniae, and therefore additional experiments were carried out to evaluate the synergistic effects of various concentrations of DMSO and MSM, as well as combinations of DMSO, MSM, and penicillin.
DMSO was added to S. pyogenesa cultures, final concentrations of 0.31, 0.63, 1.25, 2.50, 5.00, 10.0, or 20.0. At these concentrations, DMSO resulted in reductions in bacterial viability in a dose-dependent manner. See Table 45-14. MSM alone was added to S. pyogenes cultures at final concentrations of 0.31, 0.63, 1.25, 2.50, 5.00, 10.0, or 20.0. At these concentrations, MSM also resulted in reductions in bacterial viability in a dose-dependent manner. See table 45-15.
Table 45-14. Viability of S. pyogenes after exposure to DMSO
<td>DMSO concentration</td><td>Viability of S. pyogenes (%)</td>
<td> 0,31</td><td> 100</td>
<td> 0,63</td><td> 100</td>
<td> 1,25</td><td> 100</td>
<td> 2,50</td><td> 100</td>
<td> 5,00</td><td> 96,66</td>
<td> 10,0</td><td> 14,50</td>
<td> 20,0</td><td> 5,14</td>
Table 45-15. Viability of S. pyogenes after exposure to MSM
<td>MSM concentration</td><td>Viability of S. pyogenes (%)</td>
<td> 0,31</td><td> 100</td>
<td> 0,63</td><td> 100</td>
<td> 1,25</td><td> 100</td>
<td> 2,50</td><td> 100</td>
<td> 5,00</td><td> 95,88</td>
<td> 10,0</td><td> 23,94</td>
<td> 20,0</td><td> 15,18</td>
MSM and DMSO in combination were evaluated for their antibacterial effects on S. pyogenes. 2.5%, 5%, and 8% DMSO was combined with 0% MSM (DMSO only control), 2.5%, 5%, and 10%. As shown in Tables 16, 17, and 18 certain combinations of MSM with DMSO are synergistic compared to the effects of DMSO or mSm alone. Synergistic results compared to DMSO or MSM alone are indicated by a For example, the addition of 2.5% MSM to 2.5% DMSO reduced bacterial viability to approximately 65% (see Table 16), while that no effect could be expected from these concentrations of MSM and DMSO, since individually, none of the compounds reduced bacterial viability. The synergistic effect is also observed with DMSO 2.5% and MSM at .5%, where bacterial viability is reduced by approximately 83% (compared to an expected reduction of 4% based on the effects of the compounds individually). . Synergy is also observed with 5% DMSO in combination with any concentration of MSM. Therefore, in some embodiments, 5% DMSO induces synergistic reductions in bacterial viability in combination with any concentration of MSM between 2.5% and 10%. In some embodiments, 2.5% DMSO and MSM at concentrations between 2.5% and 5% are advantageously and unexpectedly synergistic in reducing bacterial viability.
Table 45-16. Viability of S. pyogenes after exposure to various concentrations of MSM in DMSO. 2.5%
<td>DMSO (%)</td><td>MSM (%)</td><td>Viability of S. pyogenes (%)</td>
<td> 2,5</td><td> 0</td><td> 100</td>
<td> 2,5</td><td> 2,5</td><td> 65,06*</td>
<td> 2,5</td><td> 5,0</td><td> 17,71*</td>
ES 2 616 630 T3
<td> 2,5</td><td> 10,0</td><td> 16,37</td>
Table 45-17. Viability of S. pyogenes after exposure to various concentrations of MSM in 5% DMSO
<td>DMSO (%)</td><td>MSM (%)</td><td>Viability of S. pyogenes (%)</td>
<td> 5,0</td><td> 0</td><td> 96,66</td>
<td> 5,0</td><td> 2,5</td><td> 36,21*</td>
<td> 5,0</td><td> 5,0</td><td> 7,87*</td>
<td> 5,0</td><td> 10,0</td><td> 7,64*</td>
Table 45-18. Viability of S. pyogenes after exposure to various concentrations of MSM in 8% DMSO
<td>DMSO (%)</td><td>MSM (%)</td><td>Viability of S. pyogenes (%)</td>
<td> 8,0</td><td> 0</td><td> 9,96</td>
<td> 8,0</td><td> 2,5</td><td> 14,37</td>
<td> 8,0</td><td> 5,0</td><td> 5,97</td>
<td> 8,0</td><td> 10,0</td><td> 5,60</td>
Various concentrations of penicillin alone were evaluated for their ability to reduce the viability of S. pyogenes. As shown in Table 45-19, penicillin reduced bacterial viability in a dose-dependent manner.
Table 45-19. Viability of S. pyogenes after exposure to various concentrations of .penicillin
<td>Penicillin (pg / l)</td><td>Viability of S. pyogenes (%)</td>
<td> 1,56</td><td> 100</td>
<td> 3,13</td><td> 100</td>
<td> 6,25</td><td> 100</td>
<td> 12,5</td><td> 13,16</td>
<td> 25,0</td><td> 9,07</td>
<td> 50</td><td> 9,57</td>
<td> 100</td><td> 9,40</td>
Due to the highly effective nature of penicillin concentrations at or above 25 pg / L, DMSO was combined with penicillin concentrations that were less effective (in the range of 3.125 to 12.5 pg / ml). As such, the identification of the synergy between DMSO and penicillin would be less likely to be mathematically obscured.
As shown in Tables 45-20, 45-21, and 45-22 (identified by *) various combinations of DMSO and penicillin provided synergistic results. For example, 5% DMSO in combination with 3.125 mg / L penicillin, based on the efficacy of just the two compounds, could be expected to reduce bacterial viability by approximately 4%. However, when combined, the actual reduction was approximately 10 times greater (viability reduced to -61%, see Table 45-20). Synergistic effects were observed when 5% DMSO was combined with 6.25 mg / L or 12.5 mg / L penicillin (see Tables 45-21 and 45-22, respectively).
Table 45-20. Viability of S. pyogenes after exposure to 3.13 mg / L penicline with various concentrations of DMSO
<td>Penicillin (pg / l)</td><td>DMSO (%)</td><td>Viability of S. pyogenes (%)</td>
<td> 3,13</td><td> 0</td><td> 100</td>
<td> 3,13</td><td> 2,5</td><td> 100</td>
<td> 3,13</td><td> 5,0</td><td> 60,85*</td>
<td> 3,13</td><td> 8,0</td><td> 12,90</td>
ES 2 616 630 T3
Table 45-21. S. pyogenes viability after exposure to 6.25 mg / L penicline with various concentrations of DMSO
<td>Penicillin (pg / l)</td><td>DMSO (%)</td><td>Viability of S. pyogenes (%)</td>
<td> 6,25</td><td> 0</td><td> 100</td>
<td> 6,25</td><td> 2,5</td><td> 100</td>
<td> 6,25</td><td> 5,0</td><td> 60,23*</td>
<td> 6,25</td><td> 8,0</td><td> 6,91*</td>
Table 45-22. Viability of S. pyogenes after exposure to 12.5 mg / L penicline with various concentrations of DMSO
<td>Penicillin (pg / l)</td><td>DMSO (%)</td><td>Viability of S. pyogenes (%)</td>
<td> 12,5</td><td> 0</td><td> 13,16</td>
<td> 12,5</td><td> 2,5</td><td> 19,63</td>
<td> 12,5</td><td> 5,0</td><td> 14,77*</td>
<td> 12,5</td><td> 8,0</td><td> 6,43</td>
Studies similar to those using DMSO were carried out by combining MSM as penicillin in the range of 3.125 to 12.5 mg / L. The results are shown in Tables 45-23, 45-24, and 45-25. The synergy is shown by a As in the case of DMSO, the previous ineffective concentrations of MSM and penicillin were effective in combination to reduce bacterial viability. When taken individually, an effect of 3.13 mg / L penicillin with 2.5% MSM could not be expected, however an 8% reduction in viability was observed (see Table 45-23). These effects are more pronounced with the combination of 6.25 mg / L penicillin with MSM. For example, 5% MSM with 6.25 mg / L penicillin could be expected to produce a 96% viable bacterial population (see Table 45-24). However, the data indicates that viability was reduced to approximately 17%, a reduction of approximately 80% from expected results. No synergy was detected when 12.5 mg / L penicillin was used, due to the efficacy of that concentration of penicillin individually.
Table 45-23. Viability of S. pyogenes after exposure to 3.13 mg / L penicline with various concentrations of MSM
<td>Penicillin (pg / l)</td><td>MSM (%)</td><td>Viability of S. pyogenes (%)</td>
<td> 3,13</td><td> 0</td><td> 100</td>
<td> 3,13</td><td> 2,5</td><td> 92,89*</td>
<td> 3,13</td><td> 5,0</td><td> 78,31*</td>
<td> 3,13</td><td> 8,0</td><td> 9,91*</td>
Table 45-24. Viability of S. pyogenes after exposure to 6.25 mg / L penicline with various concentrations of MSM
<td>Penicillin (pg / l)</td><td>MSM (%)</td><td>Viability of S. pyogenes (%)</td>
<td> 6,25</td><td> 0</td><td> 100</td>
<td> 6,25</td><td> 2,5</td><td> 90,11*</td>
<td> 6,25</td><td> 5,0</td><td> 17,42*</td>
<td> 6,25</td><td> 8,0</td><td> 10,77*</td>
Table 45-25. Viability of S. pyogenes after exposure to 12.5 mg / L penicline with various concentrations of MSM
<td>Penicillin (pg / l)</td><td>MSM (%)</td><td>Viability of S. pyogenes (%)</td>
<td> 12,5</td><td> 0</td><td> 13,16</td>
<td> 12,5</td><td> 2,5</td><td> 16,33</td>
<td> 12,5</td><td> 5,0</td><td> 12,85</td>
ES 2 616 630 T3
<td> 12,5</td><td> 8,0</td><td> 16,02</td>
As with S. pneumoniae, various concentrations of DMSO, MSM, and penicillin were evaluated for their effects on bacterial viability and possible synergistic activity compared to MSM with penicillin or DMSO with penicillin. The results are shown in Table 45-26. Synergy compared to DMSO and penicillin is indicated by a *, while synergy compared to MSM and penicillin is indicated by a ψ. As can be seen from the data in Table 45-26, substantial synergy was detected between the various compound concentrations. Most of the DMSO and MSM combinations showed a dose response curve based on the concentration of penicillin used. Based on the efficacy of only 12.5 mg / L, it is not expected that combinations of that concentration of penicillin with DMSO and MSM should be more effective. Interestingly, previously ineffective concentrations of penicillin are made effective in a dose-dependent manner by combining it with DMSO and MSM. For example, 2.5% DMSO with 5% MSM and 3.125 mg / L penicillin could be expected to reduce bacterial viability to between 100% and 96% (compared to DMSO + penicillin and MSM + penicillin, respectively ). However, the combination of the three reduced bacterial viability to approximately 19%. The expected results are similar for combinations with 6.25 mg / L penicillin, but the combination actually reduced bacterial viability even more, to about 13%. Increasing the concentration of the various compounds does not result in further reductions in bacterial viability. For example, the combination of 8% DMSO with 2.5% MSM and 3.125 mg / L penicillin appears to be more effective than 8% DMSO with 2.5% MSM and 12.5 mg / L penicillin.
Table 45-26. Viability of S. pneumoniae after exposure to various combinations of DMSO, MSM, and penicillin.
<td>DMSO (%)</td><td>MSM (%)</td><td>Penicillin (pg / l)</td><td>Viability of S. pyogenes (%)</td>
<td> 2,5</td><td> 2,5</td><td> 3,125</td><td>91.74 *, ψ</td>
<td> 2,5</td><td> 2,5</td><td> 6,25</td><td>60.55 *, ψ</td>
<td> 2,5</td><td> 2,5</td><td> 12,5</td><td>8.08 *, ψ</td>
<td> 2,5</td><td> 5</td><td> 3,125</td><td>18.72 *, ψ</td>
<td> 2,5</td><td> 5</td><td> 6,25</td><td>13.38 *, ψ</td>
<td> 2,5</td><td> 5</td><td> 12,5</td><td> 9,41*</td>
<td> 2,5</td><td> 10</td><td> 3,125</td><td>16.05 *, ψ</td>
<td> 2,5</td><td> 10</td><td> 6,25</td><td>11.78 *, ψ</td>
<td> 2,5</td><td> 10</td><td> 12,5</td><td> 11,77*</td>
<td> 5</td><td> 2,5</td><td> 3,125</td><td>14.60 *, ψ</td>
<td> 5</td><td> 2,5</td><td> 6,25</td><td> 10,44 *, <sup>ψ</sup></td>
<td> 5</td><td> 2,5</td><td> 12,5</td><td> 9,55 *, <sup>ψ</sup></td>
<td> 8</td><td> 2,5</td><td> 3,125</td><td> 9,55 *, <sup>ψ</sup></td>
<td> 8</td><td> 2,5</td><td> 6,25</td><td> 10,28 <sup>ψ</sup></td>
<td> 8</td><td> 2,5</td><td> 12,5</td><td> 15,55 <sup>ψ</sup></td>
These studies indicate that at certain concentrations of MSM, DMSO or a combination thereof, Streptococcus pyogenes and Streptococcus pneumoniae can be inhibited, supporting a possible use of said substances to prevent or inhibit the growth of Streptococcus pyogenes and Streptococcus pneumoniae.
Example 22
Probiotic growth in medium supplemented with MSM
This example describes probiotic growth in medium supplemented with MSM.
Lactobacillus Acidophilus, Bifidobacterium bifidum, Lactobacillus delbrueckii, and Bacillus coagulans growth media were supplemented with 0, 0.125, 0.25, 0.5, 1.0, 2.5, and 5% MSM. A single stock of MRS broth was prepared with 5% MSM and used to prepare each medium composition. Medium for Lactobacillus organisms was prepared by adding the appropriate amount of MSM to 99 ml of MRS broth. For Bifidobacterium bifidum,
ES 2 616 630 T3 prepared 99 ml of MRS broth with the respective concentrations of MSM and 0.05% L-cysteine. For Bacillus coagulans, 99 ml of tryptic soy broth was supplemented with the appropriate amount of MSM.
These medium solutions were inoculated with each probiotic organism and incubated at 35 ° C ± 0.5 ° C in CO2 for a total of 72 hours for all solutions, except for Bifidobacterium bifidum, which was cultured under anaerobic conditions. Samples of each medium were collected at 0, 8, 16, 24, 32, 40, 48, 56, 64, and 72 hours. Lactobacillus samples were plated on MRS agar, Bifidobacterium bifidum samples were plated on MRS + L-cysteine agar, and Bacillus coagulans samples were plated on tryptic soy agar. The plates were incubated at 35 ° C ± 0.5 ° C in CO2 for a total of 72 hours for all solutions, except for Bacillus coagulans, which was cultured for 48 hours. The plates were then counted. Negative controls (stock medium and plating controls) were free of microbial growth. Data are presented in cfu / ml. The results of these studies are presented in the tables below.
Table 46. Lactobacillus growth. acidophilus in medium fortified with MSM
<td>Weather</td><td>0% MSM</td><td>0.125% MSM</td><td>0.2% MSM</td><td>0.50% MSM</td><td>1% MSM</td><td>2.5% MSM</td><td>5% MSM</td>
<td> 0</td><td> 1,48</td><td> 1,37</td><td> 1,37</td><td> 1,30</td><td> 1,48</td><td> 1,48</td><td> 1,52</td>
<td> 8</td><td> 1,48</td><td> 2,19</td><td> 1,43</td><td> 2,01</td><td> 2,25</td><td> 2,20</td><td> 1,37</td>
<td> 16</td><td> 4,87</td><td> 4,83</td><td> 5,74</td><td> 3,82</td><td> 3,79</td><td> 4,24</td><td> 2,69</td>
<td> 24</td><td> 6,97</td><td> 7,14</td><td> 8,19</td><td> 6,47</td><td> 6,77</td><td> 5,78</td><td> 5,36</td>
<td> 32</td><td> 9,47</td><td> 9,15</td><td> 9,85</td><td> 9,15</td><td> 9,05</td><td> 9,16</td><td> 8,91</td>
<td> 40</td><td> 7,08</td><td> 9,53</td><td> 9,50</td><td> 9,58</td><td> 9,49</td><td> 9,35</td><td> 9,25</td>
<td rowspan="3"> 48</td><td></td><td></td><td></td><td></td><td rowspan="3"> 10,9 8</td><td></td><td></td>
<td> 7,27</td><td> 9,59</td><td> 9,32</td><td> 10,45</td><td> 10,16</td><td> 10,88</td>
<td></td><td></td><td></td><td></td><td></td><td></td>
<td rowspan="3"> 56</td><td></td><td></td><td></td><td></td><td rowspan="3"> 10,9 0</td><td></td><td></td>
<td> 7,20</td><td> 10,16</td><td> 9,26</td><td> 9,33</td><td> 11,10</td><td> 10,01</td>
<td></td><td></td><td></td><td></td><td></td><td></td>
<td rowspan="3"> 64</td><td></td><td></td><td></td><td></td><td rowspan="3"> 10,3 4</td><td></td><td></td>
<td> 7,29</td><td></td><td></td><td></td><td> 11,58</td><td> 8,95</td>
<td></td><td></td><td></td><td></td><td></td><td></td>
<td> 72</td><td> 7,19</td><td> 8,56</td><td> 8,36</td><td> 8,57</td><td> 8,68</td><td> 8,44</td><td> 6,66</td>
Table 47. Growth of .Lactobacillus bulgaricus in medium fortified with MSM
<td>Weather</td><td>0% MSM</td><td>0.125% MSM</td><td>0.2% MSM</td><td>0.50% MSM</td><td>1% MSM</td><td>2.5% MSM</td><td>5% MSM</td>
<td> 0</td><td> 2,22</td><td> 2,29</td><td> 2,25</td><td> 2,23</td><td> 2,29</td><td> 2,26</td><td> 2,19</td>
<td> 8</td><td> 3,56</td><td> 4,15</td><td> 4,08</td><td> 4,42</td><td> 4,83</td><td> 4,57</td><td> 3,28</td>
<td> 16</td><td> 8,09</td><td> 8,22</td><td> 8,34</td><td> 8,28</td><td> 8,36</td><td> 8,09</td><td> 7,39</td>
<td> 24</td><td> 8,71</td><td> 9,04</td><td> 9,10</td><td> 9,05</td><td> 9,03</td><td> 9,01</td><td> 8,50</td>
<td> 32</td><td> 9,29</td><td> 8,55</td><td> 9,60</td><td> 9,54</td><td> 9,31</td><td> 9,15</td><td> 9,29</td>
<td> 40</td><td> 9,32</td><td> 9,29</td><td> 9,11</td><td> 9,40</td><td> 9,34</td><td> 9,27</td><td> 9,37</td>
<td> 48</td><td> 10,81</td><td> 10,94</td><td> 11,07</td><td> 10,82</td><td> 11,23</td><td> 11,37</td><td> 10,92</td>
<td> 56</td><td> 7,69</td><td> 8,00</td><td> 8,79</td><td> 9,14</td><td> 8,11</td><td> 8,23</td><td> 10,07</td>
<td> 64</td><td> 8,78</td><td> 8,59</td><td> 8,79</td><td> 8,80</td><td> 6,50</td><td> 8,75</td><td> 10,96</td>
<td> 72</td><td> 6,56</td><td> 6,74</td><td> 6,72</td><td> 6,72</td><td> 6,45</td><td> 6,51</td><td> 8,62</td>
ES 2 616 630 T3
Table .48. Growth, of Bacillus coagulans in medium fortified with MSM
<td>Weather</td><td>0% MSM</td><td>0.125% MSM</td><td>0.2% MSM</td><td>0.50% MSM</td><td>1% MSM</td><td>2.5% MSM</td><td>5% MSM</td>
<td> 0</td><td> 1,43</td><td> 1,52</td><td> 1,67</td><td> 1,48</td><td> 1,30</td><td> 1,43</td><td> 1,56</td>
<td> 8</td><td> 5,05</td><td> 4,81</td><td> 4,94</td><td> 4,42</td><td> 4,98</td><td> 5,13</td><td> 4,61</td>
<td> 16</td><td> 6,75</td><td> 6,95</td><td> 7,19</td><td> 6,94</td><td> 7,29</td><td> 7,05</td><td> 7,56</td>
<td> 24</td><td> 10,34</td><td> 9,87</td><td> 10,34</td><td> 10,29</td><td> 10,30</td><td> 10,22</td><td> 10,48</td>
<td> 32</td><td> 10,70</td><td> 11,06</td><td> 11,25</td><td> 11,05</td><td> 11,42</td><td> 11,70</td><td> 11,55</td>
<td> 40</td><td> 10,70</td><td> 11,72</td><td> 11,34</td><td> 10,25</td><td> 11,02</td><td> 10,55</td><td> 10,85</td>
<td> 48</td><td> 11,07</td><td> 11,56</td><td> 9,94</td><td> 10,40</td><td> 10,38</td><td> 10,88</td><td> 10,22</td>
<td> 56</td><td> 11,35</td><td> 9,60</td><td> 11,45</td><td> 10,76</td><td> 10,86</td><td> 10,86</td><td> 11,1</td>
<td> 64</td><td> 11,01</td><td> 12,13</td><td> 11,37</td><td> 10,45</td><td> 10,40</td><td> 10,97</td><td> 10,75</td>
<td> 72</td><td> 10,92</td><td> 10,14</td><td> 10,94</td><td> 10,86</td><td> 10,70</td><td> 11,05</td><td> 11,81</td>
Table 49. Growth of Bifidobacterium bifidum in medium fortified with MSM
<td>Weather</td><td>0% MSM</td><td> 0,125 %</td><td>0.2% MSM</td><td>0.50% MSM</td><td>1% MSM</td><td>2.5% MSM</td><td>5% MSM</td>
<td> 0</td><td> 1,67</td><td> 1,64</td><td> 1,82</td><td> 1,85</td><td> 1,48</td><td> 1,64</td><td> 1,00</td>
<td> 8</td><td> 2,30</td><td> 1,73</td><td> 2,08</td><td> 1,99</td><td> 1,70</td><td> 1,60</td><td> 2,29</td>
<td> 16</td><td> 5,33</td><td> 6,55</td><td> 5,22</td><td> 6,53</td><td> 6,81</td><td> 6,21</td><td> 6,71</td>
<td> 24</td><td> 5,86</td><td> 2,70</td><td> 6,15</td><td> 3,14</td><td> 2,75</td><td> 2,52</td><td> 5,72</td>
<td> 32</td><td> 8,80</td><td> 3,37</td><td> 5,03</td><td> 3,52</td><td> 3,37</td><td> 3,37</td><td> 10,32</td>
<td> 40</td><td> 9,71</td><td> 4,19</td><td> 8,14</td><td> 4,62</td><td> 3,48</td><td> 3,52</td><td> 12,02</td>
<td> 48</td><td> 10,60</td><td> 6,41</td><td> 8,55</td><td> 4,51</td><td> 3,90</td><td> 3,95</td><td> 10,54</td>
<td> 56</td><td> 10,42</td><td> 9,97</td><td> 9,00</td><td> 6,05</td><td> 8,32</td><td> 8,35</td><td> 10,92</td>
<td> 64</td><td> 10,65</td><td> 11,34</td><td> 10,19</td><td> 9,55</td><td> 8,02</td><td> 8,30</td><td> 12,04</td>
<td> 72</td><td> 11,21</td><td> 10,00</td><td> 9,10</td><td> 7,52</td><td> 9,52</td><td> 10,12</td><td> 12,43</td>
These studies indicate that MSM can enhance the growth of probiotic organisms, depending on the concentration of MSM used.
Example 23
Effect of MS on H1N1 and herpes simplex virus
This example demonstrates the ability of MSM to enhance or reduce the infectivity of the swine influenza A virus strain H1N1 A / California / 04/2009 (CDC ID # 2009712047), rhinovirus type 14 (ATCC # VR-284), and herpes simplex virus type 1 (ATCC No. VR-260). The study was conducted as a pre-treatment test of eight concentrations of MSM. The virus production reduction / increase test and subsequent virus titration were carried out in three replicates. Inhibitory concentrations of MSM (IC50 or IC90 - the concentration at which growth or activity is inhibited by 50% or 90%) were also determined in this study.
The cytotoxicity of MSM was determined prior to testing. Eight concentrations of MSM (16%, 14%, 12%, 10%, 8.0%, 6.0%, 1.0%, and 0.5%) were tested in MDCK cells (ATCC # CCL- 3. 4). MSM concentrations of 16% to 8% were toxic to MDCK cells and completely destroyed the cell monolayers. Concentrations of 6% to 0.5% did not produce a visible cytotoxic effect. The CT50 (concentration at which the compound alone kills 50% of the uninfected cells) was determined to be approximately 7%. Therefore, this concentration was the first lowest non-cytotoxic dilution used in the assay.
A total of eight concentrations of MSM were included in the test: 7% (~ 74.365 mM); 6% (~ 63.742 mM); 5% (~ 53.118mM); 4% (~ 42.494mM); 3% (~ 31.871mM); 2% (~ 21.247mM); 1% (~ 10.624mM); and 0.5% (~ 5.312mM). A detailed description of the materials and methods is provided below.
ES 2 616 630 T3
Host cells. Madin Darby cells from canine kidney (MDCK [ATCC # CCL-34]), MRC-5 cells (human lung fibroblasts; [ATCC # CCL-171]), and Vero cells (African green monkey kidney [ATCC # CCL-81]) were maintained as monolayers in disposable cell culture labware and used for pretreatment in the antiviral test of the swine influenza A H1N1 virus strain A / California / 04/2009, rhinovirus type 14 (ATCC No.Vr-284), and HSV-1 (ATCC No. VR-260), respectively. Before testing, the host cell cultures were seeded into the appropriate cell culture plates. The cell monolayers were 80 to 90% confluent and less than 48 hours old prior to virus inoculation. The culture medium (GM) and the maintenance medium (MM) were 1X EMEM and / or advanced MEM with the appropriate supplements.
Determination of the cytotoxicity of the test product. The highest non-cytotoxic concentration of the test product was determined prior to testing. The MDCK cell culture was washed with phosphate buffered saline (PBS) and incubated with the following dilutions of a product: 16%, 14%, 12%, 10%, 8.0%, 6.0%, 1 , 0%, and 0.5%. Incubation was for 1 hour at 37 ° C ± 2 ° C in a CO2 incubator. After incubation, the treated cells were coated with MM. The plates were incubated in a CO2 incubator for 3 days at 37 ± 2 ° C. Toxicity was monitored using a complex inverted microscope. A cytotoxicity test performed as outlined in the study protocol showed that product concentrations of 16% to 8% were toxic to MDCK cells and destroyed cell monolayers. Product concentrations of 6% to 0.5% did not produce a visible cytotoxic effect. The CT50 (concentration at which the compound alone kills 50% of the uninfected cells) was determined to be approximately 7%.
A. Pre-treatment test. The test product stock solution was prepared as follows. 35.0 grams of product were diluted in 100 ml of PBS and heated at 40 ° C until dissolved. The 35% solution was kept at 40 ° C until higher dilutions were prepared (see project notes [Form # 95-G-001] in Appendix VI of this final report). The MDCK, MRC-5, and Vero cell cultures were washed with PBS and incubated with the following product dilutions: 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0.5 %. Incubation was for 1 hour at 37 ° C ± 2 ° C in a CO2 incubator. After the incubation was complete, approximately 300-1000 IU (infectious units) of each of the test viruses were added to the appropriate treated cells. The test was carried out in three replicates. The plates were incubated in a CO2 incubator for 6 days at the appropriate temperature for each virus. CPE was monitored using an inverted complex microscope. All the data resulting from the test are included in Appendix IV of this final report (Forms No.: 95-G-001,91-L-002, and 07-L-002).
B. Toxicity control for pre-treatment tests. The MDCK, MRC-5 and Vero cell cultures were washed with PBS and incubated with the 7% to 0.5% product dilutions. Incubation was for 1 hour at 37 ° C ± 2 ° C in a CO2 incubator. After incubation, the treated cells were coated with MM. The plates were incubated in a CO incubator.<sub>2</sub> for 6 days at the appropriate temperature for each virus. Toxicity was monitored using a complex inverted microscope. The results of the cytotoxicity tests are presented in Table 50.
C. Virus control. The MDCK, MRC-5 and Vero cell cultures were washed with PBS and incubated with MM. Incubation was for 1 hour at 37 ° C ± 2 ° C in a CO2 incubator. After the incubation was completed, approximately 300-1000 IU (infectious units) of each of the test viruses were added to the cells. Three replicates of virus control were performed. The plates were incubated in a CO2 incubator for 6 days at the appropriate temperature for each virus. CPE was monitored using an inverted complex microscope.
D. Negative control. Intact cell culture monolayers served as negative control. GM was changed to MM in all negative control wells.
E. Determination of the reduction and / or enhancement of virus production. After the virus control had reached the maximum cytopathic effect (complete destruction of the monolayer), samples were taken from the test wells and virus control wells for titration. Factor ten dilutions were made in MM and plated in susceptible cells in four replicates. The results of the virus production reduction / enhancement tests are presented in Tables 51 to 91.
Analysis of data. The titer of the virus population in the cell cultures was expressed as the -logw of the 50% titer endpoint for infectivity. To calculate the virus titer, a 50% tissue culture infectious dose calculation (TCID<sub>50</sub>) - the Quantal test (Spearman-Karber method).
log TCID50 = I - d (s - 0.5)
Where:
l = -Iog10 of the lower dilution;
d = difference between dilution steps;
s = sum of the proportions of positive wells.
1.1 The highest product concentration producing a cytotoxic effect was determined as 50% of the toxic product concentration (CT50).
1.2 The percentage reduction was calculated as follows:
ES 2 616 630 T3 ϊ Cf tvnfrní tíe Finta
1.3 The TCID50 of the virus population recovered from the test and the virus control was used to calculate the reduction or enhancement of viral infectivity. IC50 was determined using GraphPad Prlsm 5, Inc. software. IC90 was determined experimentally, when present.
Test acceptance criteria. A valid test requires that: 1) the cells in the negative control wells are viable and attached to the bottom of the well; 2) the medium is free of contamination in all wells of the plate; and 3) the virus control shows the presence of virus-specific CPE.
Reductions in the virus population were observed for all three viruses tested. MSM at a concentration of 7% produced the following mean reductions: 1.16 log-io reduction (93.08% reduction) of swine influenza A H1N1 virus; 2.50 logio reduction (99.68% reduction) of herpes simplex virus 1 (HSV-1); 1.25 log reduction (94.38% reduction) in rlnovlrus type 14. MSM at a concentration of 6% produced the following mean reductions: 1.00 log reduction<sub>10</sub> (90.00% reduction) of the swine influenza A H1N1 virus; 1.00 log reduction<sub>10 </sub>(90.00% reduction) HSV-1; 0.67 log reduction<sub>10</sub> (78.62% reduction) of type 14 rlnovlrus.
MSM at a concentration of 5% produced the following mean reductions: 0.41 log-io reduction (61.10% reduction) of swine influenza A H1N1 virus; 1.34 log-io reduction (95.43% reduction) of HSV-1; reduction of 0.09 log-io (18.72% reduction) of rhinovirus type 14. MSM at a concentration of 4% produced the following mean reductions: 0.16 log-io reduction (30.82% reduction) of swine influenza A H1N1 virus; reduction of 1.59 log-io (97.43% reduction) of HSV-1; 0.28 log-io reduction (47.52% reduction) of rhinovirus type 14. MSM at a concentration of 3% produced the following mean reductions: 0.00 log reduction<sub>10</sub> (00.00% reduction) of the swine influenza A H1N1 virus; 1.00 logio reduction (90.00% reduction) of HSV-1; 0.11 log-io reduction (22.38% reduction) of rhinovirus type 14. MSM at a concentration of 2% produced the following mean reductions: 0.41 log-io reduction (61.10% reduction ) of the swine influenza A H1N1 virus; 0.84 log-io reduction (85.55% reduction) of HSV-1; 0.42 log-io reduction (61.98% reduction) of rhinovirus type 14. MSM at 1% concentration produced the following mean reductions: 0.25 log-io reduction (43.77% reduction) of swine influenza A H1N1 virus; reduction of 0.67 log-io (78.62% reduction) of HSV-1; 0.14 log-io reduction (27.56% reduction) of rhinovirus type 14. MSM at a concentration of 0.5% produced the following mean reductions: 0.66 log reduction<sub>10</sub> (78.12% reduction) of the swine influenza A H1N1 virus; reduction of 0.25 log-io (43.77% reduction) of HSV-1; 0.40 log reduction<sub>10</sub> (60.19% reduction) of rhinovirus type 14.
Potentiation / stimulation of viral infectivity was observed for swine influenza A H1N1 virus treated with 3% MSM. The mean potentiation of the virus population was 0.17 log-io (32.39%). A total of three concentrations of MSM enhanced the infectivity of rlnovlrus type 14. MSM at a concentration of 5% produced a mean enhancement of 0.053 log-io (11.49%). Three percent MSM produced a mean enhancement of 0.11 log-io (22.38%); and 1% MSM produced a mean potentiation of 0.11 log<sub>10</sub> (22.38%). All enhancements / stimulations of viral infectivity determined in this study were within the Range of Normal Variation for the virus population and were not significant. An Inhibitory concentration of MSM at which growth or infectivity is inhibited by 50% (IC50) was calculated using non-linear dose-response regression (GraphPad Prism 5, software). MSM IC50 and IC50 best fit values with 95% confidence intervals were calculated for each test virus. For swine H1N1 influenza A virus, the MSM IC50 best fit value was 5.114 mM. The IC50 with a 95% confidence interval ranged from 0.008038 mM to 3253 mM. For HSV-1, the best fit value of the MSM IC50 was determined at 10.13mM with a 95% confidence interval for the CI<sub>50</sub> which ranged from 7.144mM to 14.37mM. For rhinovirus type 14, the best fit value for IC<sub>50</sub> MSM was 38.16mM. The IC50 with a 95% confidence interval was in the range of 13.07mM to 111.4mM. The ci<sub>90 </sub>(1.0 logio reduction) were determined experimentally for HSV-1 and swine influenza A H1N1 virus. However, due to the interception of multiple MSM concentrations with the 90% reduction axis, the experimental IC90 values cannot be considered accurate.
MSM tested at four different concentrations against HSV-1, swine influenza A H1N1 and rhinovirus produced U-shaped dose response curves. For example: 4% MSM (1.00 logio reduction) was more effective against HSV-1 than MSM at 6% (1.59 log-ιο reduction); 0.5% MSM (0.66 log reduction) was more or equally effective against swine influenza A H1N1 than 5% MSM (0.41 log reduction<sub>10</sub>); concentrations of 4% to 0.5% were more or equally effective against rhinovirus than MSM at 5%. It is possible, if confirmed in further research, that the U-shaped MSM effects represent a stable event.
This study indicates that MSM can be used as an antiviral product. Non-toxic concentrations of 7% and 6% reduced the population of enveloped viruses, such as HSV-1 and swine influenza A H1N1 virus by more than 1.0 logi0. Tables 50 to 91 include the results for the aforementioned studies.
Table 50 presents the cytotoxicity test for eight product concentrations carried out in parallel with
ES 2 616 630 T3 test pretreatment using MDCK, MRC-5, and Vero cell cultures.
TABLE 50
<td colspan="9">Test Product: Methylsulfonylmethane, Lot # 0902951</td>
<td rowspan="2">Cell culture name</td><td colspan="8">Cytotoxicity of the test product</td>
<td> 7 %</td><td> 6 %</td><td> 5 %</td><td> 4 %</td><td> 3 %</td><td> 2 %</td><td> 1 %</td><td> 0,5 %</td>
<td>Vero</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td>
<td>MDCK</td><td> ++</td><td> ++</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td>
<td>MRC-5</td><td> ++</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td>
<td colspan="9">+ = CPE present 0 = CPE not present</td>
Tables 51 to 58 present the virus control infectivity (TCID50), the mean infectivity I (TCID50), and the 5 log1Q and percentage reductions observed in the pretreatment test of the test product, Methylsulfonylmethane (lot no. 0902951), and swine influenza A H1N1 virus strain A / California / 04/2009 (CDC ID # 2009712047).
TABLE 51 Reduction of infectivity
<td colspan="8">Test Product: Methylsulfonylmethane, 7% (Lot # 0902951) Virus: Swine influenza A H1N1 strain A / California / 04/2009 virus, CDC ID # 2009712047 Host cell line: Host cell line from MDcK, ATCC # CCL-34</td>
<td rowspan="2">Dilutions (-log<sub>1Q</sub>)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"> 0000</td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 00+0</td><td> 0000</td><td> 0+00</td>
<td> -5</td><td> 00+0</td><td> +000</td><td> +0+0</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCID50</td><td>4.75log1Q</td><td>4.75 log1Q</td><td>5.00 log1Q</td><td>3.75 log1Q</td><td>3.50 log1Q</td><td>3.75 log1Q</td>
<td>TCID50 mean</td><td colspan="3">4.83 log1Q</td><td colspan="3">3.67 log<sub>1Q</sub></td>
<td>Log reduction *</td><td colspan="3" rowspan="4"></td><td>1.08 log<sub>1Q</sub></td><td>1.33 log<sub>1Q</sub></td><td>1.08 log<sub>1Q</sub></td>
<td>Average log reduction</td><td colspan="3">1.16 log1Q</td>
<td>Reduction Percentage</td><td> 91,68 %</td><td> 95,32 %</td><td> 91,68 %</td>
<td>Average reduction percentage **</td><td colspan="3"> 93,08 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
ES 2 616 630 T3
TABLE 52 Reduction of infectivity
<td colspan="8">Test Product: Methylsulfonylmethane, 6% (Lot # 0902951) Virus: Swine influenza A H1N1 strain A / California / 04/2009 virus, CDC ID # 2009712047 Host cell line: host cell line from MDCK, ATCC # CCL-34</td>
<td rowspan="2">Dilutions (-log10)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 0+00</td><td> 000+</td><td> +00+</td>
<td> -5</td><td> 00+0</td><td> +000</td><td> +0+0</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCID50</td><td>4.75 log<sub>1</sub>Q</td><td>4.75 log1Q</td><td>5.00 log1Q</td><td>3.75 log1Q</td><td>3.75 log1Q</td><td>4.00 log1Q</td>
<td>TCID50 mean</td><td colspan="3">4.83 log1Q</td><td colspan="3">3.67 log<sub>1Q</sub></td>
<td>Log reduction *</td><td colspan="3" rowspan="4"></td><td>1.08 log<sub>1Q</sub></td><td>1.08 log<sub>1Q</sub></td><td>0.83 log<sub>1Q</sub></td>
<td>Average log reduction</td><td colspan="3">1.00 log1Q</td>
<td>Reduction percentage</td><td> 91,68 %</td><td> 91,68 %</td><td> 85,21 %</td>
<td>Average reduction percentage **</td><td colspan="3"> 90,00 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
TABLE 53
Reduced Infectivity Test Product: Methylsulfonylmethane, 5% (Lot # 0902951) Virus: Swine A / California / 04/2009 influenza A H1N1 virus, CDC ID # 2009712047 Host cell line : MDCK host cell line, ATCC # CCL-34
<td rowspan="2">Dilutions (-log1o)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 0+++</td><td> +++0</td><td></td>
<td> -5</td><td> 00+0</td><td> +000</td><td> +0+0</td><td> 0000</td><td> +000</td><td> 0000</td><td></td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
<td>TCIDsq</td><td>4.75 log1Q</td><td>4.75 log1Q</td><td>5.00 log1Q</td><td>4.50 log1Q</td><td>4.50 log1Q</td><td>4.25 log1Q</td><td></td>
<td>TCID<sub>5Q</sub> half</td><td colspan="3">4.83 log<sub>1Q</sub></td><td colspan="3">4.42 log<sub>1</sub>Q</td><td></td>
<td>Log reduction *</td><td colspan="3"></td><td>0.33 log<sub>1Q</sub></td><td>0.33 log<sub>1Q</sub></td><td>0.58 log<sub>1Q</sub></td><td></td>
ES 2 616 630 T3
<td>Average log reduction</td><td rowspan="3"></td><td>0.41 log1Q</td><td rowspan="3"></td>
<td>Reduction Percentage</td><td> 53,23 % 53,23 % 73,70 %</td>
<td>Average reduction percentage **</td><td> 61,10 %</td>
<td colspan="4">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean ACID50 of virus control - ACID50 of assay replica ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / ACIDsq reduction) * 100</td>
TABLE 54
<td colspan="8">Reduction of infectivity Test Product: Methylsulfonylmethane, 4% (Lot # 0902951) Virus: Swine influenza A H1N1 strain A / California / 04/2009 virus, CDC ID # 2009712047 Host cell line: host cell line from MDCK, ATCC # CCL-34</td>
<td rowspan="2">Dilutions (-log10)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="10"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 0+++</td><td> ++++</td>
<td> -5</td><td> 00+0</td><td> +000</td><td> +0+0</td><td> 00+0</td><td> 000+</td><td> 000+</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCID<sub>5</sub>what</td><td>4.75 log<sub>1</sub>Q</td><td>4.75 log<sub>1</sub>Q</td><td>5.00 log<sub>1Q</sub></td><td>4.75 log<sub>1</sub>Q</td><td>4.50 log<sub>1Q</sub></td><td>4.75 log<sub>1</sub>Q</td>
<td>TCID1Q mean</td><td colspan="3">4.83 log1Q</td><td colspan="3">4.67 log1Q</td>
<td>Log reduction *</td><td colspan="3" rowspan="2"></td><td>0.08 log1Q</td><td>0.33 log1Q</td><td>0.08 log1Q</td>
<td>Average log reduction</td><td colspan="3">0.16 log1Q</td>
<td>Reduction Percentage</td><td rowspan="2"></td><td> 16,82 %</td><td> 53,23 %</td><td> 16,82 %</td><td colspan="3" rowspan="2"></td>
<td>Average Reduction Percentage **</td><td colspan="3"> 30,82 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCIDsq of virus control - TCIDsq of assay replicate ** -% mean reduction (calculated from the log reduction<sub>1Q</sub> mean) = 100- (1 / Reduction TCID<sub>5</sub>q) * 100</td>
ES 2 616 630 T3
TABLE 55
<td colspan="8">Reduction of infectivity Test Product: Methylsulfonylmethane, 3% (Lot # 0902951) Virus: Swine influenza A H1N1 strain virus A / California / 04/2009, CDC ID # 2009712047 Host cell line: MDcK host cell line, ATCC # CCL-34</td>
<td rowspan="2">Dilutions (-log<sub>1Q</sub>)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> 00+0</td><td> +000</td><td> +0+0</td><td> 00++</td><td> +00+</td><td> 0++0</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCID50</td><td>4.75log1Q</td><td>4.75 log1Q</td><td>5.00 log1Q</td><td>5.00 log<sub>1Q</sub></td><td>5.00 log<sub>1Q</sub></td><td>5.00 log<sub>1Q</sub></td>
<td>TCID50 mean</td><td colspan="3">4.83 log1Q</td><td colspan="3">5.00 log1Q</td>
<td>Log reduction</td><td colspan="3" rowspan="4"></td><td>0.00 log<sub>1Q</sub></td><td>0.00 log<sub>1Q</sub></td><td>0.00 log<sub>1Q</sub></td>
<td>Average log reduction</td><td colspan="3">0.00 log1Q</td>
<td>Reduction Percentage</td><td> 00,00 %</td><td> 00,00 %</td><td> 00,00 %</td>
<td>Average Reduction Percentage</td><td colspan="3"> 00,00 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = TCID<sub>50</sub> virus control media - TCID<sub>50</sub> of the test replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
_TABLE 56_
Reduction of infectivity
Test Product: Methylsulfonylmethane, 2% (Lot # 0902951) Virus: Swine influenza A H1N1 strain virus
A / California / 04/2009, CDC ID # 2009712047 Host cell line: MDcK host cell line, ATCC # CCL-34
<td rowspan="2">Dilutions (-logw)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="6"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> +000</td>
<td> -5</td><td> 00+0</td><td> +000</td><td> +0+0</td><td> 0000</td><td> 000+</td><td> 0+00</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
ES 2 616 630 T3
<td>TCIDsq</td><td>4.75log1Q</td><td>4.75 log1Q</td><td>5.00 log1Q</td><td>4.50 log1Q</td><td>4.75 log1Q</td><td>4.00 log1Q</td><td></td>
<td>TCID50 mean</td><td colspan="3">4.83 log1Q</td><td colspan="3">4.42 log1Q</td><td></td>
<td>Log reduction *</td><td></td><td></td><td></td><td>0.33 log<sub>1Q</sub></td><td>0.08 log<sub>1Q</sub></td><td>0.83 log<sub>1Q</sub></td><td></td>
<td>Average log reduction</td><td></td><td></td><td></td><td colspan="3">0.41 log1Q</td><td></td>
<td>Reduction Percentage</td><td></td><td></td><td></td><td> 53,23 %</td><td> 16,82 %</td><td> 85,21 %</td><td></td>
<td>Average reduction percentage **</td><td></td><td></td><td></td><td colspan="3"> 61,10 %</td><td></td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
TABLE 57
<td colspan="8">Reduction of infectivity Test Product: Methylsulfonylmethane, 1% (Lot # 0902951) Virus: Swine influenza A H1N1 strain A / California / 04/2009 virus, CDC ID # 2009712047 Host cell line: host cell line from MDcK, ATCC # CCL-34</td>
<td rowspan="2">Dilutions (-log<sub>1Q</sub>)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td rowspan="4"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++0+</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> 00+0</td><td> +000</td><td> +0+0</td><td> 00+0</td><td> 000+</td><td> 0000</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td rowspan="8"></td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCIDsq</td><td>4.75 log1Q</td><td>4.75 log1Q</td><td>5.00 log1Q</td><td>4.50 log1Q</td><td>4.75 log1Q</td><td>4.50 log1Q</td>
<td>TCIDsq mean</td><td colspan="3">4.83 log1Q</td><td colspan="3">4.58 log1Q</td>
<td>Log reduction *</td><td colspan="3" rowspan="4"></td><td>0.33 log1Q</td><td>0.08 log1Q</td><td>0.33 log1Q</td>
<td>Average log reduction</td><td colspan="3">0.25 log1Q</td>
<td>Reduction Percentage</td><td> 53,23 %</td><td> 16,82 %</td><td> 53,23 %</td>
<td>Average reduction percentage **</td><td colspan="3"> 43,77 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCIDsq of virus control - TCIDsq of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
ES 2 616 630 T3
TABLE 58
<td colspan="8">Reduction of infectivity Test Product: Methylsulfonylmethane, 0.5% (Lot # 0902951) Virus: Swine influenza A H1N1 strain A / California / 04/2009 virus, CDC ID # 2009712047 Host cell line: line MDCK host cell, ATCC # CCL-34</td>
<td>Dilutions</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>(-log10)</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> +000</td><td> ++++</td><td> +++0</td>
<td> -5</td><td> 00+0</td><td> +000</td><td> +0+0</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCID50</td><td>4.75 log10</td><td>4.75 log10</td><td>5.00 log10</td><td>3.75 log<sub>10</sub></td><td>4.50 log<sub>10</sub></td><td>4.25 log10</td>
<td>TCID50 mean</td><td colspan="3">4.83 log10</td><td colspan="3">4.17 log10</td>
<td>Log reduction *</td><td colspan="3" rowspan="4"></td><td>1.08 log10</td><td>0.33 log10</td><td>0.58 log10</td>
<td>Average log reduction</td><td colspan="3">0.66 log<sub>10</sub></td>
<td>Reduction Percentage</td><td> 91,68 %</td><td> 53,23 %</td><td> 73,70 %</td>
<td>Half of</td><td colspan="3"> 78,12 %</td>
<td>Reduction Percentage **</td><td></td><td></td><td colspan="5"></td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of assay replicate ** -% mean reduction (calculated from mean logw reduction) = 100- (1 / TCIDs0 reduction) * 100</td>
Tables 59 to 67 present the virus control infectivity (TCID50), the mean infectivity (TCID50), and the 5 log-ιο and percentage reductions observed in the pre-treatment test of the test product, Methylsulfonylmethane (# of lot 0902951), and herpes simplex virus type 1 (ATCC No. VR-260).
TABLE 59
<td colspan="8">Reduction of infectivity Test Product: Methylsulfonylmethane, 7% (Lot # 0902951) Virus: Herpes Simplex Virus, HF ATCC strain # VR-260 Host cell line: Vero ATCC host cell line # CCL-81</td>
<td rowspan="2">Dilutions (-log10)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -1</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
ES 2 616 630 T3
<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> +000</td><td> 0+0+</td><td rowspan="12"></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 0000</td><td> 00+0</td><td> 0000</td>
<td> -5</td><td> ++++</td><td> +++0</td><td> ++++</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -6</td><td> 0000</td><td> 00+0</td><td> +0+0</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td>NT</td><td>NT</td><td>NT</td>
<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td>NT</td><td>NT</td><td>NT</td>
<td>TCID50</td><td>5.50 log<sub>1Q</sub></td><td>5.50 log<sub>1Q</sub></td><td>6.00 log<sub>1Q</sub></td><td>3.50 log<sub>1Q</sub></td><td>3.00 log<sub>1Q</sub></td><td>3.00 log<sub>1Q</sub></td>
<td>TCID50 mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">3.17 log1Q</td>
<td>Log reduction *</td><td colspan="3" rowspan="4"></td><td>2.17 log1Q</td><td>2.67 log1Q</td><td>2.67 log1Q</td>
<td>Average log reduction</td><td colspan="3">2.50 log1Q</td>
<td>Reduction Percentage</td><td> 99,32 %</td><td> 99,79 %</td><td> 99,79 %</td>
<td>Average Reduction Percentage **</td><td colspan="3"> 99,68 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
TABLE 60
<td colspan="8">Reduction of infectivity Test Product: Methylsulfonylmethane, 6% (Lot # 0902951) Virus: Herpes Simplex Virus, HF ATCC strain # VR-260</td>
<td></td><td colspan="7">Host cell line: Vero ATCC host cell line # CCL-81</td>
<td rowspan="2">Dilutions (-logw)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td>Control of</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>cells</td>
<td colspan="7"></td><td> 0000</td>
<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> +++0</td><td></td>
<td> -5</td><td> ++++</td><td> +++0</td><td> ++++</td><td> 000+</td><td> 0+00</td><td> +000</td><td></td>
<td> -6</td><td> 0000</td><td> 00+0</td><td> +0+0</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
<td>TCIDsq</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>4.75log1Q</td><td>4.75 log1Q</td><td>4.50 log1 0</td><td></td>
<td>TCIDsq mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">4.67 log1Q</td><td></td>
<td>Log reduction *</td><td></td><td></td><td></td><td>0.92 log1Q</td><td>0.92 log1Q</td><td>1.17 log1Q</td><td></td>
<td>Average log reduction</td><td></td><td></td><td></td><td></td><td>1.00 log1Q</td><td></td><td></td>
<td>Reduction Percentage</td><td></td><td></td><td></td><td> 87,98 %</td><td> 87,98 %</td><td> 93,24 %</td><td></td>
ES 2 616 630 T3
<td>Average reduction percentage **</td><td></td><td> 90,00 %</td><td></td>
<td colspan="4">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = TCID<sub>50</sub> virus control media - TCID<sub>50</sub> of the test replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
TABLE 61
<td colspan="8">Reduction of infectivity Test Product: Methylsulfonylmethane, 5% (Lot # 0902951) Virus: Herpes Simplex Virus, HF ATCC strain # VR-260 Host cell line: Vero ATCC host cell line # CCL-81</td>
<td rowspan="2">Dilutions (-log1o)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++0+</td><td> ++++</td><td> 000+</td>
<td> -5</td><td> ++++</td><td> +++0</td><td> ++++</td><td> 0+00</td><td> 0000</td><td> 0000</td>
<td> -6</td><td> 0000</td><td> 00+0</td><td> +0+0</td><td> 000+</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCIDsq</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>4.75log1Q</td><td>4.50 log1Q</td><td>3.75 log1Q</td>
<td>TCID mean<sub>5</sub>what</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">4.33 log1Q</td>
<td>Log reduction *</td><td colspan="3" rowspan="4"></td><td>0.92 log1Q</td><td>1.17 log1Q</td><td>1.92 log1Q</td>
<td>Average log reduction</td><td colspan="3">1.34 log1Q</td>
<td>Reduction Percentage</td><td> 87,98 %</td><td> 93,24 %</td><td> 98,80</td>
<td>Average Reduction Percentage **</td><td colspan="3"> 95,43 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCIDsq of virus control - TCIDsq of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
TABLE 62 Reduction of infectivity
<td colspan="8">Test Product: Methylsulfonylmethane, 4% (Lot # 0902951) Virus: Herpes Simplex Virus, HF ATCC strain # VR-260 Host cell line: Vero ATCC host cell line # CCL-81</td>
<td>Dilutions</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td>Control of</td>
<td>(-log1Q)</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>cells</td>
<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0000</td>
100
ES 2 616 630 T3
<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 00+0</td><td> +0+0</td><td> +0+0</td>
<td> -5</td><td> ++++</td><td> +++0</td><td> ++++</td><td> 0000</td><td> +00+</td><td> 0000</td>
<td> -6</td><td> 0000</td><td> 00+0</td><td> +0+0</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCID50</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>3.75 log1Q</td><td>4.50 log1Q</td><td>4.00 log1Q</td>
<td>TCID50 mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">4.08 log<sub>1Q</sub></td>
<td>Log reduction *</td><td colspan="3" rowspan="4"></td><td>1.92 log1Q</td><td>1.17 log1Q</td><td>1.67 log1Q</td>
<td>Average log reduction</td><td colspan="3">1.59 log1Q</td>
<td>Reduction Percentage</td><td> 98,80 %</td><td> 93,24 %</td><td> 97,86 %</td>
<td>Average Reduction Percentage **</td><td colspan="3"> 97,43 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / ACIDsq reduction) * 100</td>
_TABLE 63_
Reduction of infectivity
Test Product: Methylsulfonylmethane, 3% (Lot # 0902951) Virus: Herpes Simplex Virus, HF ATCC strain # VR-260
Host cell line: Vero ATCC host cell line # CCL-81
<td rowspan="2">Dilutions (-log1Q)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 00++</td><td> ++++</td><td> ++++</td><td></td>
<td> -5</td><td> ++++</td><td> +++0</td><td> ++++</td><td> 00+0</td><td> 0000</td><td> 00++</td><td></td>
<td> -6</td><td> 0000</td><td> 00+0</td><td> +0+0</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 000+</td><td></td>
<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
<td>TCIDsq</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>4.25 log1Q</td><td>4.50 log1Q</td><td>5.25 log1Q</td><td></td>
<td>TCIDsq mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">4.67 log<sub>1Q</sub></td><td></td>
101
ES 2 616 630 T3
<td>Log reduction *</td><td rowspan="4"></td><td>142 iog<sub>1</sub>Q</td><td>1.17 log10</td><td>0.42 log<sub>10</sub></td><td rowspan="4"></td>
<td>Average log reduction</td><td colspan="3">1.00 log<sub>10</sub></td>
<td>Reduction Percentage</td><td> 96,20 %</td><td> 93,24 %</td><td> 61,98 %</td>
<td>Average Reduction Percentage **</td><td colspan="3"> 90,00 %</td>
<td colspan="6">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = TCID<sub>50</sub> virus control media - TCID<sub>50</sub> of the test replicate ** -% mean reduction (calculated from the log reduction<sub>10</sub> mean) = 100- (1 / TCID reduction<sub>50</sub>)*100</td>
TABLE 64
<td colspan="8">Reduction of infectivity Test Product: Methylsulfonylmethane, 2% (Lot # 0902951) Virus: Herpes Simplex Virus, HF ATCC strain # VR-260 Host cell line: Vero ATCC host cell line # CCL-81</td>
<td rowspan="2">Dilutions (-log10)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> ++++</td><td> +++0</td><td> ++++</td><td> 0+++</td><td> 0000</td><td> 00+0</td>
<td> -6</td><td> 0000</td><td> 00+0</td><td> +0+0</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCID<sub>5</sub>0</td><td>5.50 log10</td><td>5.50 log10</td><td>6.00 log10</td><td>5.25 log10</td><td>4.50 log10</td><td>4.75 log10</td>
<td>TCID mean<sub>5</sub>0</td><td colspan="3">5.67 log<sub>10</sub></td><td colspan="3">4.83 log10</td>
<td>Log reduction</td><td colspan="3" rowspan="4"></td><td>0.42 log10</td><td>1.17 log10</td><td>0.92 log10</td>
<td>Average log reduction</td><td colspan="3">0.84 log<sub>10</sub></td>
<td>Reduction Percentage</td><td> 61,98 %</td><td> 93,24 %</td><td> 87,98 %</td>
<td>Average Reduction Percentage</td><td colspan="3"> 85,55 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCIDs0 of virus control - TCIDs0 of the assay replicate ** -% mean reduction (calculated from the log reduction<sub>10</sub> mean) = 100- (1 / TCID5 reduction<sub>Q</sub>)*100</td>
102
ES 2 616 630 T3
TABLE 65
<td colspan="8">Reduction of infectivity Test Product: Methylsulfonylmethane, 1% (Lot # 0902951) Virus: Herpes Simplex Virus, HF ATCC strain # VR-260 Host cell line: Vero ATCC host cell line # CCL-81</td>
<td rowspan="2">Dilutions (-log10)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="3"></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> ++++</td><td> +++0</td><td> ++++</td><td> 00++</td><td> 00++</td><td> 00++</td>
<td> -6</td><td> 0000</td><td> 00+0</td><td> +0+0</td><td> 0000</td><td> 0000</td><td> 0000</td><td rowspan="9"></td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCID50</td><td>5.50 log<sub>1Q</sub></td><td>5.50 log<sub>1Q</sub></td><td>6.00 log<sub>1Q</sub></td><td>5.00 log<sub>1Q</sub></td><td>5.00 log<sub>1Q</sub></td><td>5.00 log<sub>1Q</sub></td>
<td>TCID50 mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">5.00 log<sub>1Q</sub></td>
<td>Log reduction</td><td colspan="3" rowspan="4"></td><td>0.67 log<sub>1Q</sub></td><td>0.67 log<sub>1Q</sub></td><td>0.67 log<sub>1Q</sub></td>
<td>Average log reduction</td><td colspan="3">0.67 log1Q</td>
<td>Reduction Percentage</td><td> 78,62 %</td><td> 78,62 %</td><td> 78,62 %</td>
<td>Average Reduction Percentage</td><td colspan="3"> 78,62 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
_TABLE 66_
Reduction of infectivity
Test Product: Methylsulfonylmethane, 0.5% (Lot # 0902951) Virus: Herpes Simplex Virus, HF ATCC strain # VR-260
Host cell line: Vero ATCC host cell line # CCL-81
<td rowspan="2">Dilutions (-log1Q)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="6"></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> ++++</td><td> +++0</td><td> ++++</td><td> ++++</td><td> ++0+</td><td> ++0+</td>
<td> -6</td><td> 0000</td><td> 00+0</td><td> +0+0</td><td> 0000</td><td> 0000</td><td> 000+</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
103
ES 2 616 630 T3
<td>TCID50</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>5.50 log1Q</td><td>5.25 log1Q</td><td>5.50 log1Q</td><td rowspan="4"></td>
<td>TCID50 mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">5.42 log1Q</td>
<td>Log reduction</td><td colspan="3"></td><td>0.17 log<sub>1Q</sub></td><td>0.42 log<sub>1Q</sub></td><td>0.17 log<sub>1Q</sub></td>
<td>Average log reduction</td><td colspan="3"></td><td colspan="3">0.25 log1Q</td>
<td>Reduction Percentage</td><td rowspan="2"></td><td> 32,39 %</td><td> 61,98 %</td><td> 32,39 %</td><td colspan="3" rowspan="2"></td>
<td>Average Reduction Percentage</td><td colspan="3"> 43,77 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
TABLE 67
<td colspan="8">Reduction of infectivity Test product: Methylsulfonylmethane, 7% (lot # 0902951) Virus: Rhinovirus type 14, strain 1059 ATCC # VR-284 Host cell line: MRC-5 ATCC host cell line # CCL-171</td>
<td rowspan="2">Dilutions (-log1Q)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td></td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++0+</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0+0+</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCIDsq</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>4.25 log1Q</td><td>4.50 log1Q</td><td>4.50 log1Q</td>
<td>TCID mean<sub>5</sub>what</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">4.42 log1Q</td>
<td>Log reduction</td><td colspan="3" rowspan="4"></td><td>1.42 log1Q</td><td>1.17 log1Q</td><td>1.17 log1Q</td>
<td>Average log reduction</td><td colspan="3">1.25 log1Q</td>
<td>Reduction Percentage</td><td> 96,20 %</td><td> 93,24 %</td><td> 93,24 %</td>
<td>Average Reduction Percentage</td><td colspan="3"> 94,38 %</td>
104
ES 2 616 630 T3
<td colspan="4">Reduction of infectivity Test product: Methylsulfonylmethane, 7% (lot # 0902951) Virus: Rhinovirus type 14, strain 1059 ATCC # VR-284 Host cell line: MRC-5 ATCC host cell line # CCL-171</td>
<td rowspan="2">Dilutions (-log10)</td><td>Virus control</td><td>Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1 Rep. 2 Rep. 3</td><td>Rep. 1 Rep. 2 Rep. 3</td>
<td colspan="4">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
Tables 68 to 74 present the virus control infectivity (TCID50), the mean infectivity (TCID50), and the logw and percent reductions observed in the pre-treatment run of the test product, Methylsulfonylmethane (lot # 0902951). , and rhinovirus type 14 (ATCC # VR-284).
TABLE 68
<td colspan="8">Reduction of infectivity Test product: Methylsulfonylmethane, 6% (lot # 0902951) Virus: Rhinovirus type 14, strain 1059 ATCC # VR-284 Host cell line: MRC-5 ATCC host cell line # CCL-171</td>
<td rowspan="2">Dilutions (-log1o)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="8"> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> ++++</td><td> ++++</td><td> ++++</td><td> +000</td><td> +0+0</td><td> +0++</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0+0+</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td></td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>4.75log1Q</td><td>5.00 log1Q</td><td>5.25 log1Q</td>
<td>Half of</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">5.00 log1Q</td>
<td>Log reduction</td><td colspan="3" rowspan="4"></td><td>0.92 log<sub>1Q</sub></td><td>0.67 log<sub>1Q</sub></td><td>0.42 log<sub>1Q</sub></td>
<td>Average log reduction</td><td colspan="3">0.67 log1Q</td>
<td>Reduction Percentage</td><td> 87,98 %</td><td> 78,62 %</td><td> 61,98 %</td>
<td>Average Reduction Percentage</td><td colspan="3"> 78,62 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = TCID<sub>5Q</sub> virus control media - TCID<sub>5Q</sub> of the test replicate ** -% mean reduction (calculated from the log reduction<sub>1Q</sub> mean) = 100- (1 / TCID reduction<sub>5Q</sub>)*100</td>
105
ES 2 616 630 T3
TABLE 69
<td colspan="8">Reduction of infectivity Test Product: Methylsulfonylmethane, 5% (Lot # 0902951) Virus: Rhinovirus Type 14, Strain 1059 ATCC # VR-284 Host Cell Line: MRC-5 ATcC Host Cell Line # CCL-171</td>
<td rowspan="2">Dilutions (-log10)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 0+++</td><td> ++++</td><td> ++++</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0+0+</td><td> 0000</td><td> 0000</td><td> +00+</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCID50</td><td>5.50 log<sub>1Q</sub></td><td>5.50 log<sub>1Q</sub></td><td>6.00 log<sub>1Q</sub></td><td>5.25 log<sub>1Q</sub></td><td>5.50 log<sub>1Q</sub></td><td>6.00 log<sub>1Q</sub></td>
<td>TCID50 mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">5.58 log1Q</td>
<td>Log reduction</td><td colspan="3" rowspan="4"></td><td>0.09 log1Q</td><td>0.17 log1Q</td><td>0.00 log1Q</td>
<td>Average log reduction</td><td colspan="3">0.09 log1Q</td>
<td>Reduction Percentage</td><td> 18,72 %</td><td> 32,39 %</td><td> 00,00 %</td>
<td>Average Reduction Percentage</td><td colspan="3"> 18,72 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
TABLE 70
Reduction of infectivity
Test Product: Methylsulfonylmethane, 4% (Lot # 0902951) Virus: Rhinovirus Type 14, Strain 1059 ATCC # VR-284 Host Cell Line: MRC-5 ATcC Host Cell Line # CCL-171
<td rowspan="2">Dilutions (-log1Q)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="3"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 00++</td><td> ++++</td><td> ++++</td><td rowspan="3"></td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0+0+</td><td> 0000</td><td> 0000</td><td> 0+00</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
106
ES 2 616 630 T3
<td>TCID50</td><td>5.50 log<sub>1Q</sub></td><td>5.50 log<sub>1Q</sub></td><td>6.00 log<sub>1Q</sub></td><td>5.00 log<sub>1Q</sub></td><td>5.50 log<sub>1Q</sub></td><td>5.75 log<sub>1Q</sub></td><td></td>
<td>TCID50 mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">5.42 log<sub>1</sub>Q</td><td></td>
<td>Log reduction</td><td></td><td></td><td></td><td>0.67 log<sub>1Q</sub></td><td>0.17 log<sub>1Q</sub></td><td>0.00 log<sub>1Q</sub></td><td></td>
<td>Average log reduction</td><td></td><td></td><td></td><td colspan="3">0.28 log1Q</td><td></td>
<td>Reduction Percentage</td><td></td><td></td><td></td><td> 78,62 %</td><td> 32,39 %</td><td> 00,00 %</td><td></td>
<td>Average Reduction Percentage</td><td></td><td></td><td></td><td colspan="3"> 47,52 %</td><td></td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of assay replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
TABLE 71
<td colspan="8">Reduction of infectivity Test product: Methylsulfonylmethane, 3% (lot # 0902951) Virus: Rhinovirus type 14, strain 1059 ATCC # VR-284 Host cell line: MRC-5 ATCC host cell line # CCL-171</td>
<td>Dilutions</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td>Cell</td>
<td>(-log1Q)</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Control</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -5</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++0+</td><td> ++++</td><td> ++++</td><td></td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0+0+</td><td> 000+</td><td> 0000</td><td> 000+</td><td></td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> +000</td><td></td>
<td>TCIDsq</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td></td>
<td>TCID mean<sub>5</sub>what</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">5.67 log<sub>1Q</sub></td><td></td>
<td>Log reduction</td><td></td><td></td><td></td><td>0.17 log<sub>1Q</sub></td><td>0.17 log<sub>1Q</sub></td><td>0.00 log<sub>1Q</sub></td><td></td>
<td>Average log reduction</td><td></td><td></td><td></td><td colspan="3">0.11 log1Q</td><td></td>
<td>Reduction Percentage</td><td></td><td> 32,39 %</td><td> 32,39 %</td><td> 00,00 %</td><td></td><td></td><td></td>
<td>Average Reduction Percentage</td><td></td><td colspan="3"> 22,38 %</td><td></td><td></td><td></td>
107
ES 2 616 630 T3 + = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = TCID<sub>50</sub> virus control media - TCID<sub>50</sub> of test replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100
TABLE 72
<td colspan="8">Reduction of infectivity Test product: Methylsulfonylmethane, 2% (lot # 0902951) Virus: Rhinovirus type 14, strain 1059 ATCC # VR-284 Host cell line: MRC-5 ATCC host cell line # CCL-171</td>
<td rowspan="2">Dilutions (-log10)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 0+++</td><td> 0000</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0+0+</td><td> 0000</td><td> 00+0</td><td> 00+0</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCIDsq</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>4.75 log1Q 0</td>
<td>TCIDsq mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">5.25 log<sub>1Q</sub></td>
<td>Log reduction</td><td colspan="3" rowspan="4"></td><td>0.17 log1Q</td><td>0.17 log1Q</td><td>0.92 log1Q</td>
<td>Average log reduction</td><td colspan="3">0.42 log1Q</td>
<td>Reduction Percentage</td><td> 32,39 %</td><td> 32,39 %</td><td> 87,98 %</td>
<td>Average Reduction Percentage</td><td colspan="3"> 61,98 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = TCID<sub>5Q</sub> virus control media - TCID<sub>5Q</sub> of the test replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
TABLE 73
<td colspan="8">Reduction of infectivity Test product: Methylsulfonylmethane, 1% (lot # 0902951) Virus: Rhinovirus type 14, strain 1059 ATCC # VR-284 Host cell line: MRC-5 ATCC host cell line # CCL-171</td>
<td>Dilutions</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td>Control of</td>
<td>(-log1Q)</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>cells</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
108
ES 2 616 630 T3
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -5</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++0+</td><td> ++++</td><td></td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0+0+</td><td> +0+0</td><td> 0000</td><td> 000+</td><td></td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
<td>TCIDsq</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>6.00 log1Q</td><td>5.25 log1Q</td><td>5.75 log1Q 0</td><td></td>
<td>TCIDsq mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">5.67 log<sub>1Q</sub></td><td></td>
<td>Reduction</td><td></td><td></td><td></td><td>0.00 log1Q</td><td>0.42 log1Q</td><td>0.00 log1Q</td><td></td>
<td>Average log reduction</td><td></td><td></td><td></td><td colspan="3">0.14 log1Q</td><td></td>
<td>Reduction Percentage</td><td></td><td></td><td></td><td> 00,00 %</td><td> 61,98 %</td><td> 00,00 %</td><td></td>
<td>Average Reduction Percentage</td><td></td><td></td><td></td><td colspan="3"> 27,56 %</td><td></td>
+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of test replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100 _TABLE 74_
Reduction of infectivity
Test product: Methylsulfonylmethane, 0.5% (lot # 0902951) Virus: Rhinovirus type 14, strain 1059 ATCC # VR-284
Host cell line: MRC-5 ATCC host cell line # CCL-171
<td rowspan="2">Dilutions (-log1Q)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -5</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 0000</td><td></td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0+0+</td><td> 0000</td><td> +000</td><td> 0000</td><td></td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
<td>TCIDsq</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>5.50 log1Q</td><td>5.75 log1Q</td><td>4.50 log1 0</td><td></td>
<td>TCIDsq mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">5.25 log1Q</td><td></td>
<td>Log reduction</td><td colspan="3"></td><td>0.17 log1Q</td><td>0.00 log1Q</td><td>1.17 log1Q</td><td></td>
109
ES 2 616 630 T3
<td>Average log reduction</td><td rowspan="2"></td><td colspan="3">0.40 log1Q</td><td rowspan="2"></td>
<td>Reduction Percentage</td><td> 32,39 %</td><td> 00,00 %</td><td> 93,24 %</td>
<td>Average Reduction Percentage</td><td></td><td colspan="3"> 60,19 %</td><td></td>
<td colspan="6">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Log reduction = mean TCID50 of virus control - TCID50 of test replicate ** -% mean reduction (calculated from mean log1Q reduction) = 100- (1 / TCIDsq reduction) * 100</td>
Table 75 presents the control virus infectivity (TCID50), the mean infectivity (TCID50), and the log10 and percent improvements observed in the pretreatment test of the test product, Methylsulfonylmethane (lot # 0902951), and swine influenza A H1N1 virus strain A / California / 04/2009 (CDC ID # 2009712047).
TABLE 75
<td colspan="8">Improved infectivity Test Product: Methylsulfonylmethane, 3% (Lot # 0902951) Virus: Swine influenza A H1N1 strain A / California / 04/2009 virus, CDC ID # 2009712047 Host cell line: host cell line from MDCK, ATCC # CCL-34</td>
<td rowspan="2">Dilutions (-log1Q)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -5</td><td> 00+0</td><td> +000</td><td> +0+0</td><td> 00++</td><td> +00+</td><td> 0++0</td><td></td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
<td>TCIDsq</td><td>4.75 log1Q</td><td>4.75 log1Q</td><td>5.00 log1Q</td><td>5.00 log1Q</td><td>5.00 log1Q</td><td>5.00 log1Q</td><td></td>
<td>TCID mean<sub>5</sub>what</td><td colspan="3">4.83 log1Q</td><td colspan="3">5.00 log1Q</td><td></td>
<td>Log stimulation</td><td></td><td>0.17 log<sub>1Q</sub></td><td>0.17 log1Q</td><td>0.17 log<sub>1Q</sub></td><td></td><td></td><td></td>
<td>Mean log of stimulation</td><td></td><td colspan="3">0.17 log1Q</td><td></td><td></td><td></td>
<td>Percentage of stimulation</td><td></td><td> 32,39 %</td><td> 32,39 %</td><td> 32,39 %</td><td></td><td></td><td></td>
<td>Average Percentage of stimulation</td><td></td><td colspan="3"> 32,39 %</td><td></td><td></td><td></td>
110
ES 2 616 630 T3 + = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Stimulation log = mean TCIDsq of the test - TCIDsq of the virus control replicate ** -% of mean stimulation (calculated at from mean log1Q stimulation) = 100- (1 / TCIDsq stimulation) * 100
Tables 76 to 78 present the virus control infectivity (TCID<sub>sq</sub>), mean infectivity (TCID<sub>sq</sub>), and improvements log<sub>1Q</sub> and percentages observed in the pre-treatment test of the test product, Methylsulfonylmethane (lot # 0902951), and rhinovirus type 14 (ATCC # VR-284).
TABLE 76
<td colspan="8">Improved infectivity Test product: Methylsulfonylmethane, 5% (lot # 0902951) Virus: Rhinovirus type 14, strain 1059 ATCC # VR-284 Host cell line: MRC-5 ATCC host cell line # CCL-171</td>
<td rowspan="2">Dilutions (-log1Q)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell Control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="10"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> ++++</td><td> ++++</td><td> ++++</td><td> 0+++</td><td> ++++</td><td> ++++</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0+0+</td><td> 0000</td><td> 0000</td><td> +00+</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>
<td>TCIDsq</td><td>5.50 log-io,</td><td>5.50 log ™</td><td>6.00 log ™</td><td>5.25 log ™,</td><td>5.50 log ™,</td><td>6.00 log ™</td>
<td>TCIDsq mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">5.58 log ™</td>
<td>Stimulation log</td><td colspan="3" rowspan="2"></td><td>0.08 log ™</td><td>0.08 log ™</td><td>0.00 log ™</td>
<td>Mean log of stimulation</td><td colspan="3">0.053 log ™</td>
<td>Percentage of stimulation</td><td rowspan="2"></td><td> 16,82 %</td><td> 16,82 %</td><td> 00,00 %</td><td colspan="3" rowspan="2"></td>
<td>Average Percentage of stimulation</td><td></td><td> 11,49 %</td><td></td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Stimulation log = TCID<sub>sq</sub> test mean - TCID<sub>sq</sub> of virus control replication ** -% mean stimulation (calculated from mean logw stimulation) = 100- (1 / TCIDsq stimulation) * 100</td>
111
ES 2 616 630 T3
TABLE 77
<td colspan="8">Improved infectivity Test product: Methylsulfonylmethane, 3% (lot # 0902951) Virus: Rhinovirus type 14, strain 1059 ATCC # VR-284 Host cell line: MRC-5 ATCC host cell line # CCL-171</td>
<td rowspan="2">Dilutions (-log1Q)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td rowspan="12"></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td>
<td> -5</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++0+</td><td> ++++</td><td> ++++</td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0+0+</td><td> 000+</td><td> 0000</td><td> 000+</td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> +000</td>
<td>TCIDsq</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td><td>5.50 log1Q</td><td>5.50 log1Q</td><td>6.00 log1Q</td>
<td>TCIDsq mean</td><td colspan="3">5.67 log<sub>1Q</sub></td><td colspan="3">5.67 log<sub>1Q</sub></td>
<td>Stimulation log</td><td colspan="3" rowspan="4"></td><td>0.17 log1Q</td><td>0.17 log1Q</td><td>0.00 log1Q</td>
<td>Mean log of stimulation</td><td colspan="3">0.11 log<sub>1Q</sub></td>
<td>Percentage of stimulation</td><td> 32,39 %</td><td> 32,39 %</td><td> 00,00 %</td>
<td>Average Percentage of stimulation</td><td colspan="3"> 22,38 %</td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Stimulation log = mean TCIDsq of the test - TCIDsq of the virus control replica ** -% mean stimulation (calculated from mean log1Q stimulation) = 100- (1 / TCIDsq stimulation) * 100</td>
_TABLE 78_
Improved infectivity
Test product: Methylsulfonylmethane, 1% (lot # 0902951) Virus: Rhinovirus type 14, strain 1059 ATCC # VR-284
Host cell line: MRC-5 ATCC host cell line # CCL-171
<td rowspan="2">Dilutions (-log1Q)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>
<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>
<td colspan="7"></td><td> 0000</td>
<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>
<td> -5</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++++</td><td> ++0+</td><td> ++++</td><td></td>
<td> -6</td><td> 0000</td><td> 0000</td><td> 0+0+</td><td> +0+0</td><td> 0000</td><td> 000+</td><td></td>
<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>
112
ES 2 616 630 T3
<td>TCID50</td><td>5.50 log10</td><td>5.50 log10</td><td>6.00 log10</td><td>6.00 log10</td><td>5.25 log10</td><td>5.75 log10</td><td></td>
<td>TCID50 mean</td><td colspan="3">5.67 log<sub>10</sub></td><td colspan="3">5.67 log<sub>10</sub></td><td></td>
<td>Log stimulation</td><td></td><td></td><td></td><td>0.17 log10</td><td>0.17 log10</td><td>0.00 log10</td><td></td>
<td>Mean log of stimulation</td><td></td><td></td><td></td><td colspan="3">0.11 log10</td><td></td>
<td>Percentage of stimulation</td><td></td><td></td><td></td><td> 32,39 %</td><td> 32,39 %</td><td> 00,00 %</td><td></td>
<td>Average Percentage of stimulation</td><td></td><td></td><td></td><td colspan="3"> 22,38 %</td><td></td>
<td colspan="8">+ = CPE present 0 = CPE not detected NT = not tested Rep = Replicated * - Stimulation log = TCID<sub>50</sub> test mean - TCID<sub>50</sub> of virus control replication ** -% mean stimulation (calculated from mean logw stimulation) = 100- (1 / TCIDs0 stimulation) * 100</td>
Nonlinear regression, dose versus response
Dose-response (inhibition) analyzes were carried out for the concentrations of test product converted to mM (molecular weight of test product = 94.13). Nonlinear regression analyzes were as follows: log (inhibitor) vs. normalized response - variable slope. The concentrations are presented in the table
79.
TABLE 79
<td>Concentration, %</td><td>Concentration, mM</td>
<td> 7 %</td><td> 74,365</td>
<td> 6 %</td><td> 63,742</td>
<td> 5 %</td><td> 53,118</td>
<td> 4 %</td><td> 42,494</td>
<td> 3 %</td><td> 31,871</td>
<td> 2 %</td><td> 21,247</td>
<td> 1 %</td><td> 10,624</td>
<td> 0,5 %</td><td> 5,312</td>
Table 80 presents the data entry for the herpes simplex virus.
TABLE 80
<td>Dose, mM</td><td colspan="3">Response,% reduction</td>
<td> 74,365</td><td> 99,320</td><td> 99,790</td><td> 99,790</td>
<td> 63,742</td><td> 87,980</td><td> 87,980</td><td> 93,240</td>
<td> 53,118</td><td> 87,980</td><td> 93,240</td><td> 98,800</td>
<td> 42,494</td><td> 98,800</td><td> 93,240</td><td> 97,860</td>
<td> 31,871</td><td> 96,200</td><td> 93,240</td><td> 61,980</td>
<td> 21,247</td><td> 61,980</td><td> 93,240</td><td> 87,980</td>
113
ES 2 616 630 T3
<td> 10,624</td><td> 78,620</td><td> 78,620</td><td> 78,620</td>
<td> 5,312</td><td> 32,390</td><td> 61,980</td><td> 32,390</td>
Table 81 presents the transformation (log dose = X = Log (X)) of data for herpes simplex virus.
TABLE 81
<td>Dose, mM</td><td colspan="3">Response,% reduction</td>
<td> 1,871369</td><td> 99,320</td><td> 99,790</td><td> 99,790</td>
<td> 1,804426</td><td> 87,980</td><td> 87,980</td><td> 93,240</td>
<td> 1,725242</td><td> 87,980</td><td> 93,240</td><td> 98,800</td>
<td> 1,628328</td><td> 98,800</td><td> 93,240</td><td> 97,860</td>
<td> 1,503396</td><td> 96,200</td><td> 93,240</td><td> 61,980</td>
<td> 1,327298</td><td> 61,980</td><td> 93,240</td><td> 87,980</td>
<td> 1,02628</td><td> 78,620</td><td> 78,620</td><td> 78,620</td>
<td> 0,7252581</td><td> 32,390</td><td> 61,980</td><td> 32,390</td>
Table 82 presents the transformation of the normalized data for herpes simplex virus. The percent reduction was normalized as follows: 32.39% becomes 0% for the entire data set; 99.79% becomes 100% for the entire data set.
TABLE 82
<td>Dose, mM</td><td colspan="3">Response,% reduction</td>
<td> 1,871369</td><td> 99,30267</td><td> 100,000</td><td> 100,000</td>
<td> 1,804426</td><td> 82,47775</td><td> 82,47775</td><td> 90,2819</td>
<td> 1,725242</td><td> 82,47775</td><td> 90,2819</td><td> 98,53116</td>
<td> 1,628328</td><td> 98,53116</td><td> 90,2819</td><td> 97,1365</td>
<td> 1,503396</td><td> 94,67358</td><td> 90,2819</td><td> 43,90208</td>
<td> 1,327298</td><td> 43,90208</td><td> 90,2819</td><td> 82,47775</td>
<td> 1,02628</td><td> 68,59051</td><td> 68,59051</td><td> 68,59051</td>
<td> 0,7252581</td><td> 0,000</td><td> 43,90208</td><td> 0,000</td>
The calculation of the CIsq for herpes simplex virus is presented in Table 83. The best fit value for the herpes simplex virus CIsq was determined at 10.13 mM. However, due to the significant variation in virus reduction, CIsq values in the range 7.144mM to 14.37mM can be considered a more plausible approximation.
TABLE 83
<td>log (inhibitor) vs. normalized response - Variable slope</td><td></td>
<td>Best fit values</td><td></td>
<td>LogCI50</td><td> 1,006</td>
<td>Hill slope</td><td> 1,523</td>
<td>CIsq</td><td> 10,13</td>
<td>Typical error</td><td></td>
<td>LogCI50</td><td> 0,07314</td>
<td>Hill slope</td><td> 0,3281</td>
<td>95% confidence intervals</td><td></td>
114
ES 2 616 630 T3
<td>LogCI50</td><td>0.8539 to 1.157</td>
<td>Hill slope</td><td>0.8428 to 2.204</td>
<td>CI5Q</td><td>7.144 to 14.37</td>
<td>Goodness of fit</td><td></td>
<td>Degrees of freedom</td><td> 22</td>
<td>R square</td><td> 0,6761</td>
<td>Absolute sum of squares</td><td> 6312</td>
<td>Sy.x</td><td> 16,94</td>
<td>Number of points</td><td></td>
<td>Analyzed</td><td> 24</td>
Table 84 presents the data entry for the swine influenza A H1N1 virus.
TABLE 84
<td>Dose, mM</td><td colspan="3">Response,% reduction</td>
<td> 74,365</td><td> 91,680</td><td> 91,680</td><td> 85,210</td>
<td> 63,742</td><td> 91,680</td><td> 91,680</td><td> 85,210</td>
<td> 53,118</td><td> 53,230</td><td> 53,230</td><td> 73,700</td>
<td> 42,494</td><td> 16,820</td><td> 53,230</td><td> 16,820</td>
<td> 31,871</td><td> 0,000</td><td> 0,000</td><td> 0,000</td>
<td> 21,247</td><td> 53,230</td><td> 16,820</td><td> 85,210</td>
<td> 10,624</td><td> 53,230</td><td> 16,820</td><td> 53,230</td>
<td> 5,312</td><td> 91,680</td><td> 53,230</td><td> 73,700</td>
Table 85 presents the transformation [log dose = X = Log (X)] of the data for swine influenza A H1N1 virus.
TABLE 85
<td>Dose, mM</td><td colspan="3">Response,% reduction</td>
<td> 1,871369</td><td> 91,680</td><td> 91,680</td><td> 85,210</td>
<td> 1,804426</td><td> 91,680</td><td> 91,680</td><td> 85,210</td>
<td> 1,725242</td><td> 53,230</td><td> 53,230</td><td> 73,700</td>
<td> 1,628328</td><td> 16,820</td><td> 53,230</td><td> 16,820</td>
<td> 1,503396</td><td> 0,000</td><td> 0,000</td><td> 0,000</td>
<td> 1,327298</td><td> 53,230</td><td> 16,820</td><td> 85,210</td>
<td> 1,02628</td><td> 53,230</td><td> 16,820</td><td> 53,230</td>
<td> 0,7252581</td><td> 91,680</td><td> 53,230</td><td> 73,700</td>
Table 86 presents the transformation of the normalized data for swine influenza A H1N1 virus. The percent reduction was normalized as follows: 0% becomes 0% for the entire data set; 91.68% becomes 100% for the entire data set.
TABLE 86
<td>Dose, mM</td><td colspan="3">Response,% reduction</td>
<td> 1,871369</td><td> 100,000</td><td> 100,000</td><td> 92,94284</td>
115
ES 2 616 630 T3
<td>Dose, mM</td><td colspan="3">Response,% reduction</td>
<td> 1,804426</td><td> 100,000</td><td> 100,000</td><td> 92,94284</td>
<td> 1,725242</td><td> 58,06065</td><td> 58,06065</td><td> 80,38831</td>
<td> 1,628328</td><td> 18,34642</td><td> 58,06065</td><td> 18,34642</td>
<td> 1,503396</td><td> 0,000</td><td> 0,000</td><td> 0,000</td>
<td> 1,327298</td><td> 58,06065</td><td> 18,34642</td><td> 92,94284</td>
<td> 1,02628</td><td> 58,06065</td><td> 18,34642</td><td> 58,06065</td>
<td> 0,7252581</td><td> 100,000</td><td> 58,06065</td><td> 80,38831</td>
The calculation of the ICs0 for the swine influenza A H1N1 virus is presented in Table 87. The best fit value for the IC<sub>50</sub> for the swine H1N1 influenza virus A it was determined at 5,114 mM. CI values<sub>50</sub> with 95% confidence intervals they ranged from 0.008038 mM to 3253 mM. In view of the inconsistency of virus reduction (U-shaped curve), MSM IC50's were determined to a significant approximation. IC90 values cannot be calculated from this data set.
TABLE 87
<td>log (inhibitor) vs. normalized response - Variable slope</td><td></td>
<td>Best fit values</td><td></td>
<td>LogCI50</td><td> 0,7087</td>
<td>Hill slope</td><td> 0,2135</td>
<td>CI<sub>5</sub>0</td><td> 5,114</td>
<td>Typical error</td><td></td>
<td>LogCI50</td><td> 1,352</td>
<td>Hill slope</td><td> 0,3534</td>
<td>95% confidence intervals</td><td></td>
<td>LogCI50</td><td>-2,095 to 3,512</td>
<td>Hill slope</td><td>-0.5194 to 0.9464</td>
<td>CI<sub>5</sub>0</td><td>0.008038 to 3253</td>
<td>Goodness of fit</td><td></td>
<td>CI<sub>5</sub>0</td><td>0.008038 to 3253</td>
<td>Degrees of freedom</td><td> 22</td>
<td>R square</td><td> 0,01810</td>
<td>Absolute sum of squares</td><td> 29296</td>
<td>Sy.x</td><td> 36,49</td>
<td>Number of points</td><td></td>
<td>Analyzed</td><td> 24</td>
Table 88 presents the data entry for Rhinovirus type 14.
TABLE 88
<td>Dose, mM</td><td colspan="3">Response,% reduction</td>
<td> 74,365</td><td> 96,200</td><td> 93,240</td><td> 93,240</td>
<td> 63,742</td><td> 87,980</td><td> 78,620</td><td> 61,980</td>
116
ES 2 616 630 T3
<td> 53,118</td><td> 18,720</td><td> 32,390</td><td> 0,000</td>
<td> 42,494</td><td> 78,620</td><td> 32,390</td><td> 0,000</td>
<td> 31,871</td><td> 32,390</td><td> 32,390</td><td> 0,000</td>
<td> 21,247</td><td> 32,390</td><td> 32,390</td><td> 87,980</td>
<td> 10,624</td><td> 0,000</td><td> 61,980</td><td> 0,000</td>
<td> 5,312</td><td> 32,390</td><td> 0,000</td><td> 93,240</td>
Table 89 presents the transformation [log dose = X = Log (X)] of data for rhinovirus type 14.
TABLE 89
<td>Dose, mM</td><td colspan="3">Response,% reduction</td>
<td> 1,871369</td><td> 96,200</td><td> 93,240</td><td> 93,240</td>
<td> 1,804426</td><td> 87,980</td><td> 78,620</td><td> 61,980</td>
<td> 1,725242</td><td> 18,720</td><td> 32,390</td><td> 0,000</td>
<td> 1,628328</td><td> 78,620</td><td> 32,390</td><td> 0,000</td>
<td> 1,503396</td><td> 32,390</td><td> 32,390</td><td> 0,000</td>
<td> 1,327298</td><td> 32,390</td><td> 32,390</td><td> 87,980</td>
<td> 1,02628</td><td> 0,000</td><td> 61,980</td><td> 0,000</td>
<td> 0,7252581</td><td> 32,390</td><td> 0,000</td><td> 93,240</td>
Table 90 presents the transformation of the normalized data for rhinovirus type 14. The percent reduction was normalized as follows: 0% becomes 0% for the entire data set; 96.20% becomes 100% for the entire data set.
TABLE 90
<td>Dose, mM</td><td colspan="3">Response,% reduction</td>
<td> 1,871369</td><td> 100,000</td><td> 96,92308</td><td> 96,92308</td>
<td> 1,804426</td><td> 91,45531</td><td> 81,72558</td><td> 64,42828</td>
<td> 1,725242</td><td> 19,45946</td><td> 33,66944</td><td> 0,000</td>
<td> 1,628328</td><td> 81,72558</td><td> 33,66944</td><td> 0,000</td>
<td> 1,503396</td><td> 33,66944</td><td> 33,66944</td><td> 0,000</td>
<td> 1,327298</td><td> 33,66944</td><td> 33,66944</td><td> 91,45531</td>
<td> 1,026288</td><td> 0,000</td><td> 64,42828</td><td> 0,000</td>
<td> 0,7252581</td><td> 33,66944</td><td> 0,000</td><td> 96,92308</td>
Calculating the IC<sub>5Q</sub> for rhinovirus type 14 is presented in Table 91. The best fit value of IC 50 for rhinovirus type 14 was determined to be 38.16 mM. IC50 values with 95% confidence intervals ranged from 13.07 mM to 111.4 mM. In view of the inconsistency of virus reduction (U-shaped curve), the IC5Q of MSM were determined to a significant approximation. CI9Q values cannot be calculated from this data set.
TABLE 91
<td>log (inhibitor) vs. normalized response - Variable slope</td><td></td>
<td>Best fit values</td><td></td>
<td>LogCI50</td><td> 1,582</td>
<td>Hill slope</td><td> 0,6280</td>
117
ES 2 616 630 T3
<td>log (inhibitor) vs. normalized response - Variable slope</td><td></td>
<td>Best fit values</td><td></td>
<td>CIsq</td><td> 38,16</td>
<td>Typical error</td><td></td>
<td>LogCI50</td><td> 0,2244</td>
<td>Hill slope</td><td> 0,4179</td>
<td>95% confidence intervals</td><td></td>
<td>LogCI50</td><td>1,116 to 2,047</td>
<td>Hill slope</td><td>-0.2387 to 1.495</td>
<td>CIsq</td><td>13.07 to 111.4</td>
<td>Goodness of fit</td><td></td>
<td>Degrees of freedom</td><td> 22</td>
<td>R square</td><td> 0,1044</td>
<td>Absolute sum of squares</td><td> 29118</td>
<td>Sy.x</td><td> 36,38</td>
<td>Number of points</td><td></td>
<td>Analyzed</td><td> 24</td>
Example 24
Effect of MSM on algae
This example shows the effects of MSM on algae activity.
The growth of two species of Chlorella, Chlorella sorokiniana, a freshwater species, and Chlorella minutíssima, a marine species, were examined. The study measured the effect on algae growth in a freshwater and saltwater environment with the addition of MSM, in which MSM was added at the following concentrations: 0%, 0.25%, 2%, 5% , 10% and 20%. Growth was measured on day 0, 1,2, 3, 4, 5, 6, 7, 8, 9 and 10. The growth curves of the percentage of transmittance of the algae were compared between the concentrations of MSM, with the concentration of MSM at 0% as a control sample for each microorganism. The stock of MSM powder was supplied by Bergstrom Nutrition with Certificate of Analysis. The powder was the microprill formula, lot # 0806809. All media, water, and MSM powder stock were tested for sterility prior to study. The following media were purchased from the UTEX Culture Collection of Algae: Saltwater Enriched Medium and Volvox Dextrose Medium.
The algae were cultured for 48 hours in the appropriate medium. The initial suspension for each alga was numbered and cited as the initial inoculums. Chlorella sorokiniana was present at 381 million cells per milliliter and Chlorella minutíssima was present at 19 million cells per milliliter. One milliliter of the algae solution was placed in 9 ml of medium and mixed by vortexing. This was repeated for each concentration of mixture of medium and MSM. The algae and MSM tubes were incubated at room temperature with exposure to light. Working concentrations of MSM were prepared from a single 20.0% MSM solution and diluted accordingly with medium to obtain the desired final concentration of MSM. The sterility of all solutions was verified before proceeding with the study. Each MSM dilution was adjusted for each organism and analyzed in triplicate for each measured time interval. Samples were measured by percent transmittance on a UV / VIS spectrometer at a wavelength of 750 nm. The reserve of medium was tested against the percentage of transmittance background levels at each measured time interval. The results of these studies are provided in Tables 92 and 93 below. The lower percentage of transmittance indicated a high growth factor. These studies show that MSM treatment can increase algae growth.
118
ES 2 616 630 T3
Table 92 - Growth of Chlorella sorokiniana
MSM concentrations
<td></td><td> 0</td><td> 0,5</td><td>I</td><td> 2,5</td><td> 5</td><td> 10</td><td> 20</td><td>Half</td>
<td> 0</td><td> 80,3</td><td> 47,3</td><td> 48,7</td><td> 40,6</td><td> 35,6</td><td> 31,2</td><td> 19,3</td><td> 30,2</td>
<td> 1</td><td> 50,6</td><td> 41,8</td><td> 42,5</td><td> 43,1</td><td> 31,2</td><td> 31,3</td><td> 21,9</td><td> 40,3</td>
<td> 2</td><td> 24,5</td><td> 29,3</td><td> 37,5</td><td> 44,9</td><td> 27,4</td><td> 29,7</td><td> 24,1</td><td> 82,4</td>
<td> 3</td><td> 29,9</td><td> 29,6</td><td> 37,5</td><td> 43,8</td><td> 21,4</td><td> 28,4</td><td> 28,1</td><td> 91,2</td>
<td> 4</td><td> 25,4</td><td> 19,0</td><td> 17,3</td><td> 26,1</td><td> 18,2</td><td> 29,0</td><td> 31,4</td><td> 94,5</td>
<td> 5</td><td> 10,6</td><td> 12,4</td><td> 13,5</td><td> 10,3</td><td> 15,7</td><td> 28,7</td><td> 33,6</td><td> 93,3</td>
<td> 6</td><td> 10,5</td><td> 12,5</td><td> 13,0</td><td> 10,9</td><td> 15,9</td><td> 27,1</td><td> 36,8</td><td> 97,8</td>
<td> 7</td><td> 10,0</td><td> 11,9</td><td> 12,5</td><td> 11,0</td><td> 16,3</td><td> 27,6</td><td> 40,6</td><td> 34,3</td>
<td> 8</td><td> 10,0</td><td> 12,2</td><td> 12,4</td><td> 11,2</td><td> 17,0</td><td> 27,1</td><td> 40,8</td><td> 32,2</td>
<td> 9</td><td> 8,5</td><td> 7,5</td><td> 7,8</td><td> 8,4</td><td> 13,7</td><td> 76,4</td><td> 84,8</td><td> 30,4</td>
<td> 10</td><td> 7,4</td><td> 6,4</td><td> 6,6</td><td> 6,0</td><td> 12,8</td><td> 93,8</td><td> 96,7</td><td> 18,8</td>
<td></td><td></td><td>Table 93</td><td colspan="4">- Chlorella growth minutíssima</td><td></td><td></td>
<td></td><td></td><td></td><td colspan="3">Percentage of MSM</td><td></td><td></td><td></td>
<td></td><td> 0,0</td><td> 0,5</td><td> 1,0</td><td> 2,5</td><td> 5,0</td><td> 10,0</td><td> 20,0</td><td>Half</td>
<td> 0</td><td> 72,4</td><td> 93,5</td><td> 91,1</td><td> 82,8</td><td> 68,9</td><td> 49,6</td><td> 30,0</td><td> 105,1</td>
<td> 1</td><td> 74,6</td><td> 70,1</td><td> 80,5</td><td> 75,1</td><td> 87,4</td><td> 49,2</td><td> 33,8</td><td> 105,1</td>
<td> 2</td><td> 51,5</td><td> 45,4</td><td> 40,0</td><td> 59,5</td><td> 76,5</td><td> 50,8</td><td> 51,1</td><td> 105,1</td>
<td> 3</td><td> 33,4</td><td> 32,1</td><td> 31,0</td><td> 45,4</td><td> 62,0</td><td> 51,9</td><td> 54,8</td><td> 105,1</td>
<td> 4</td><td> 28,2</td><td> 27,6</td><td> 27,9</td><td> 33,6</td><td> 52,9</td><td> 52,0</td><td> 57,3</td><td> 105,1</td>
<td> 5</td><td> 26,4</td><td> 26,6</td><td> 26,5</td><td> 32,4</td><td> 52,2</td><td> 51,9</td><td> 57,5</td><td> 105,1</td>
<td> 6</td><td> 25,6</td><td> 25,1</td><td> 25,4</td><td> 30,0</td><td> 50,7</td><td> 54,9</td><td> 57,4</td><td> 105,1</td>
<td> 7</td><td> 24,3</td><td> 23,6</td><td> 24,4</td><td> 28,6</td><td> 51,0</td><td> 56,1</td><td> 57,1</td><td> 106,4</td>
<td> 8</td><td> 24,1</td><td> 22,8</td><td> 23,7</td><td> 27,7</td><td> 51,9</td><td> 58,6</td><td> 55,8</td><td> 107,0</td>
<td> 9</td><td> 18,0</td><td> 20,4</td><td> 21,0</td><td> 23,3</td><td> 47,1</td><td> 41,4</td><td> 45,1</td><td> 109,3</td>
<td> 10</td><td> 14,9</td><td> 18,9</td><td> 19,4</td><td> 21,1</td><td> 44,1</td><td> 36,7</td><td> 30,2</td><td> 112,0</td>
Example 25
The absorption of MSM in topical formulations is within recognized safety levels
This example demonstrates that the absorption of MSM in topical formulations is within recognized safety levels.
New Zealand White rabbits, which are an accepted animal model for dermal absorption studies, were used to assess the absorption and resulting blood levels of MSM. Rabbits were obtained from Charles River Canada (Saint-Constant, Quebec). Five male rabbits, aged 12-13 weeks and weighing in the range 2.6 kg to 2.7 kg were used for dermal absorption studies. Rabbits were used due to the higher permeability of their skin compared to rats, pigs or humans. Therefore, testing in rabbits is a more conservative strategy for the safety of topical products for human use. The size of the rabbits was based on the ethical restriction of collecting more than 6 ml / kg of blood body weight in a period of two weeks. The total volume of blood to be drawn during this study was 10 ml in a single day. One animal per group was used to minimize the number of animals required. Animals were housed individually in stainless steel cages with 12 hour light / dark cycles. The environment of the animal house was monitored daily (target ranges: 18-26 ° C and relative humidity 25-50%). Fresh air was supplied to the room at a rate sufficient to provide approximately 15-17 ambient air changes per hour. Clinical observations were carried out for all animals to ensure that they were in good health prior to dosing. Observations of mortality and morbidity were also carried out during the study period.
The treatment groups were as shown in table 94.
119
ES 2 616 630 T3
Table 94: Study 1 design
<td>Group</td><td>Essay article</td><td>Exposed surface area</td><td>Volume Applied</td><td>Number of animals</td><td>Blood collection times (min)</td>
<td>TO</td><td>10% MSM + 90% water</td><td>6 cm<sup>2</sup></td><td>0.5ml</td><td> 1</td><td>0 (before dose), 10, 30, 120,480 minutes</td>
<td>B</td><td>50% DMSO + 50% water</td><td>6 cm<sup>2</sup></td><td>0.5ml</td><td> 1</td><td>0 (before dose), 10, 30, 120,480 minutes</td>
<td>C</td><td>70% DMSO + 30% water</td><td>6 cm<sup>2</sup></td><td>0.5ml</td><td> 1</td><td>0 (before dose), 10, 30, 120, 480 minutes</td>
<td>D</td><td>10% MSM + 50% DMSO + 40% water</td><td>6 cm<sup>2</sup></td><td>0.5ml</td><td> 1</td><td>0 (before dose), 10, 30, 120,480 minutes</td>
<td>AND</td><td>10% MSM + 70% DMSO + 20% water</td><td>6 cm<sup>2</sup></td><td>0.5ml</td><td> 1</td><td>0 (before dose), 10, 30, 120,480 minutes</td>
One day before the study, each rabbit's back was shaved using a hair clipper. An area of 6cm was measured<sup>2</sup> and was marked to ensure equivalence in the application of the various compositions. Each product was applied by pipetting 0.5 ml of each composition into the center of the test zone and spread to cover the entire test zone. After an exposure time of 5 minutes, the compositions were removed with a wipe, the test area was rinsed and dried.
Before blood collection, animals were tranquilized with Accepromazine (1 mg / kg) by intramuscular injection into the muscle of the right hind paw, after which EMLA (lidocaine / prilocaine) cream was applied to both ears along the length of the ear artery. Blood was drawn by inserting a 21 gauge needle (with the hub removed) into the artery of the ear. Approximately 2 ml of whole blood was drawn into 4 ml Vacuutainer tubes (Becton Dickinson, Mississauga, ON) containing K<sub>2</sub>EDTA. The tubes were inverted to mix with the anticoagulant and stored under refrigeration until the plasma was removed by centrifugation. Plasma was separated from whole blood by centrifugation at 3000 xg for 10 minutes. Plasma was collected, transferred and stored in a cryogenization vial at -70 ° C until further processing for MSM analysis.
After the 5 minute exposure time to the various test products (see Table 1), blood was drawn after 10 minutes, 30 minutes, 2 hours and 8 hours. Before the 2 and 8 hour blood draws, EMLA cream was applied to the ears (approximately 30 minutes before each of these blood draws) because the anesthetic effects of the EMLA cream last approximately 1 to 2 hours . Both EMLA and Accepromazine were used due to ethical considerations and to ensure the welfare of the animals used in the study.
Plasma MSM concentrations were quantified by gas chromatography-mass spectrometry (GC / MS) based on established methods. Briefly, 450 µl of plasma sample was mixed with 50 µl of physiological saline and vortexed for 30 seconds. After this, 1 ml of acetonitrile (Fisher, HPLC grade) was added to the mixture. The solution was vortexed vigorously for 30 seconds and centrifuged at 2000 rpm for 5 minutes. One microliter of the clear supernatant was introduced into the GC / MS system (GC / MS QP20108 EI, Shimadzu, Kyoto, Japan). Analysis was performed on a Shimadzu SHR5XLB column (0.25mm ID X 30m long, 0.25um film, Kyoto, Japan). The retention time of MSM was 6.1-6.3 minutes. MSM was detected with MS and m / z 79 (M + -15) was used to monitor the MSM ion SIM profiles. Helium gas was used as carrier gas, the inlet pressure was 0.25kg / cm<sup>2</sup>, the make-up gas was 30 ml / min, the column temperature was 80 ° C, the injector temperature was 120 ° C, the separator temperature was 200 ° C, and the ionization source temperature was 250. ° C. The ionization energy was 70eV. An external standard plot was prepared with MSM dissolved in acetonitrile at the following concentrations: 62.5 pg / ml, 31.3 pg / ml, 15.6 pg / ml, 7.8 pg / ml, 3.9 pg / ml , 1.9 pg / ml, 0.98 pg / ml and 0.49 pg / ml. The concentration of MSM in plasma samples was calculated from the slope of the standard curve. The best fitted graph was linear, with an R2 value of 0.998.
All animals were observed before the start of the study and all were found to be in good health. During the course of the study and after the study, all the animals showed good health. Morbidity, mortality, and injuries were assessed twice daily. None of the animals showed morbidity, mortality, or injury.
The results of the absorption study are summarized in Table 95. The initial plasma concentrations of MSM (before exposure to test articles) ranged from 4.2 pg / ml and 104.2 pg / ml. The variation from baseline is within the normal range of natural MSM concentrations that have been determined in previous studies. After exposure to the various test articles, the peak plasma MSM concentrations measured were less than or equal to about 140 pg / ml. This maximum concentration is the result of exposure to 10% MSM + 70% DMSO + 20% water. When corrected for the
120
ES 2,616,630 T3 natural variation in basal MSM concentrations, the largest change in plasma MSM was detected in the 70% DMSO + 30% water group. These data suggest that the variations in MSM, either due to the absorption or metabolism of DMSO; they are within the natural range of MSM concentrations.
_Table 95: MSM concentration in plasma after exposure to MSM and DMSO_
<td>Treatment</td><td>Time point (minute)</td><td>MSM concentration (mg / ml)</td>
<td rowspan="5">10% MSM + 90% water</td><td> 0</td><td> 25,6</td>
<td> 10</td><td> 17,6</td>
<td> 30</td><td> 16,3</td>
<td> 120</td><td> 14,0</td>
<td> 480</td><td> 15,4</td>
<td rowspan="5">50% DMSO + 50% water</td><td> 0</td><td> 4,2</td>
<td> 10</td><td> 6,9</td>
<td> 30</td><td> 6,9</td>
<td> 120</td><td> 7,4</td>
<td> 480</td><td> 12,6</td>
<td rowspan="5">70% DMSO + 30% water</td><td> 0</td><td> 56,7</td>
<td> 10</td><td> 89,0</td>
<td> 30</td><td> 98,9</td>
<td> 120</td><td> 128,7</td>
<td> 480</td><td> 120,2</td>
<td rowspan="5">10% MSM + 50% DMSO + 40% water</td><td> 0</td><td> 104,2</td>
<td> 10</td><td> 116,5</td>
<td> 30</td><td> 127,9</td>
<td> 120</td><td> 128,4</td>
<td> 480</td><td> 140,4</td>
<td rowspan="5">10% MSM + 70% DMSO + 20% water</td><td> 0</td><td> 26,8</td>
<td> 10</td><td> 37,3</td>
<td> 30</td><td> 30,9</td>
<td> 120</td><td> 33,9</td>
<td> 480</td><td> 44,4</td>
In view of the various possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be construed as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. Therefore, the present inventors claim as their invention everything that is within the scope of these claims.
121
Contents102
1 sheet
Sheet 1
99 members in 16 offices
Priority claims24
| Document | Office | Kind | Date |
|---|---|---|---|
| 256935P | United States of America | – | |
| 25693509 | United States of America | P | |
| 25693509 | United States of America | P | |
| 257751P | United States of America | – | |
| 25775109 | United States of America | P | |
| 25775109 | United States of America | P | |
| 259098P | United States of America | – | |
| 25909809 | United States of America | P | |
| 25909809 | United States of America | P | |
| 294437P | United States of America | – | |
| 29443710 | United States of America | P | |
| 29443710 | United States of America | P | |
| 2010054845 | United States of America | W | |
| 2010054845 | United States of America | W | |
| 256935P | – | – | – |
| 257751P | – | – | – |
| 259098P | – | – | – |
| 294437P | – | – | – |
| PCTUS2010054845 | – | – | – |
| US20090256935P | – | – | – |
| US20090257751P | – | – | – |
| US20090259098P | – | – | – |
| US20100294437P | – | – | – |
| WO2010US54845 | – | – | – |
Members99
| Document | Office | Kind | |
|---|---|---|---|
| CA2778142A1 | Canada | A1 | |
| CA2778144A1 | Canada | A1 | |
| CA2779111A1 | Canada | A1 | |
| US2011105623A1 | United States of America | A1 | |
| WO2011053848A1 | World Intellectual Property Organization (WIPO) | A1 | |
| WO2011053854A1 | World Intellectual Property Organization (WIPO) | A1 | |
| WO2011053874A1 | World Intellectual Property Organization (WIPO) | A1 | |
| WO2011053875A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU2010281739A1 | Australia | A1 | |
| AU2010281739B2 | Australia | B2 | |
| US2011136210A1 | United States of America | A1 | |
| US2011152231A1 | United States of America | A1 | |
| AU2011204961A1 | Australia | A1 | |
| AU2010313253A1 | Australia | A1 | |
| AU2010313228A1 | Australia | A1 | |
| US2012149672A1 | United States of America | A1 | |
| IL219320A0 | Israel | A0 | |
| IL219320D0 | Israel | D0 | |
| IL219321A0 | Israel | A0 | |
| IL219321D0 | Israel | D0 | |
| IL219407A0 | Israel | A0 | |
| IL219407D0 | Israel | D0 | |
| US8217085B2 | United States of America | B2 | |
| MX2012005159A | Mexico | A | |
| MX2012005014A | Mexico | A | |
| US2012207827A1 | United States of America | A1 | |
| KR20120093993A | Republic of Korea | A | |
| US2012220667A1 | United States of America | A1 | |
| EP2493314A1 | European Patent Office (EPO) | A1 | |
| EP2493315A1 | European Patent Office (EPO) | A1 | |
| EP2493464A1 | European Patent Office (EPO) | A1 | |
| EP2494059A1 | European Patent Office (EPO) | A1 | |
| KR20120107951A | Republic of Korea | A | |
| CN102724973A | China | A | |
| US2012264726A1 | United States of America | A1 | |
| CN102834520A | China | A | |
| US2013045941A1 | United States of America | A1 | |
| JP2013509197A | Japan | A | |
| JP2013509437A | Japan | A | |
| JP2013509440A | Japan | A | |
| US2013065967A9 | United States of America | A9 | |
| EP2493464A4 | European Patent Office (EPO) | A4 | |
| EP2493314A4 | European Patent Office (EPO) | A4 | |
| EP2493315A4 | European Patent Office (EPO) | A4 | |
| US8546373B2 | United States of America | B2 | |
| EP2494059A4 | European Patent Office (EPO) | A4 | |
| US2013338130A1 | United States of America | A1 | |
| NZ600145A | New Zealand | A | |
| AU2011204961B2 | Australia | B2 | |
| KR20140015146A | Republic of Korea | A | |
| AU2010313228B2 | Australia | B2 | |
| CN102724973B | China | B | |
| NZ600134A | New Zealand | A | |
| US8841100B2 | United States of America | B2 | |
| US2014349375A1 | United States of America | A1 | |
| JP5651186B2 | Japan | B2 | |
| AU2010313253B2 | Australia | B2 | |
| SG10201500283UA | Singapore | A | |
| AU2015202432A1 | Australia | A1 | |
| IL219320A | Israel | A | |
| CN102834520B | China | B | |
| ZA201203054B | South Africa | B | |
| ZA201203083B | South Africa | B | |
| JP5947721B2 | Japan | B2 | |
| US9487749B2 | United States of America | B2 | |
| EP2494059B1 | European Patent Office (EPO) | B1 | |
| BR112012010270A2 | Brazil | A2 | |
| AU2015202432B2 | Australia | B2 | |
| DK2494059T3 | Denmark | T3 | |
| ES2616630T3This record | Spain | T3 | |
| IL219321A | Israel | A | |
| KR101763560B1 | Republic of Korea | B1 | |
| US9839609B2 | United States of America | B2 | |
| MX353059B | Mexico | B | |
| US9855212B2 | United States of America | B2 | |
| MX353712B | Mexico | B | |
| KR101826531B1 | Republic of Korea | B1 | |
| EP2493315B1 | European Patent Office (EPO) | B1 | |
| US2018092838A1 | United States of America | A1 | |
| EP2493464B1 | European Patent Office (EPO) | B1 | |
| CA2778142C | Canada | C | |
| DK2493464T3 | Denmark | T3 | |
| ES2674019T3 | Spain | T3 | |
| EP2493464B8 | European Patent Office (EPO) | B8 | |
| IL219407A | Israel | A | |
| IL219407B | Israel | B | |
| IL259683D0 | Israel | D0 | |
| US2018214373A1 | United States of America | A1 | |
| ZA201203907B | South Africa | B | |
| MX367373B | Mexico | B | |
| CA2779111C | Canada | C | |
| BR112012010272A2 | Brazil | A2 | |
| US10596109B2 | United States of America | B2 | |
| EP2493314B1 | European Patent Office (EPO) | B1 | |
| CA2778144C | Canada | C | |
| IL259683A | Israel | A | |
| IL259683B | Israel | B | |
| US2020297625A1 | United States of America | A1 | |
| US2023240984A1 | United States of America | A1 |
Numbers
- Publication
- 2616630
- Publication, DOCDB
- 2616630
- Publication, EPODOC
- ES2616630T
- Application
- 10827570
- Application, DOCDB
- 10827570
- Application, EPODOC
- ES20100827570T
Titles2
- Spanish
- Uso de metilsulfonilmetano (MSM) para modular la actividad microbiana
- English
- Use of methylsulfonylmethane (MSM) to modulate microbial activity
Classification
- CPC, 20
- C12N1/38
- A61K31/437
- A61K45/06
- A61K31/43
- A61K31/431
- C12N1/18
- C12N1/20
- C12P7/06
- C12P7/56
- A61K9/0014
- A61K31/10
- A61P31/00
- A61P31/04
- A61P31/10
- A61P31/16
- A61P43/00
- Y02E50/10
- Y02E50/30
- A01N41/10
- Y02A50/30
- IPC, 10
- C12P7 06
- C12G1 00
- C12P7 56
- C12C11 00
- C12G3 10
- C12Q1 02
- C12N1 38
- C12N1 20
- C12N1 18
- C12H6 00