Use of methylsulfonylmethane (msm) to modulate microbial activity.

USE OF METILSULFONILMETANO (MSM) PARA -

MICROBIAL

CROSS REFERENCE TO RELATED REQUESTS

This application claims priority of the US Provisional Patent Applications Serial No. 61 / 257,751 filed on November 3, 2009, Serial No. 61 / 259,098 filed on November 6, 2009, Serial No. 61 / 294,437 filed at January 12, 2010 and Serial No. 61/256, 935, filed on October 30, 2009, each of which is hereby incorporated by reference in its entirety.

FIELD OF DESCRIPTION

This description relates to the field of methylsulfonylmethane (MSM), specifically to methods of using MSM, to modify biological activity, such as to improve or inhibit microbial activity including bacterial growth.

BACKGROUND

Microorganisms (or microbes) are microscopic organisms, including bacteria, fungi, archaea, protists, plants (for example, green algae), viruses, prions, parasites, and animals such as amoebas, plankton. Depending on the context, the microorganisms can be seen as either harmful or beneficial. In some cases, microorganisms can be harmful and lead to diseases and diseases in plants, animals or humans. Additionally, in addition to causing infections or diseases, undesirable microbial growth may also occur in consumer products, such as food contamination. In other cases, the growth of microorganisms is beneficial and routinely exploited in biotechnology, diagnostic modern technologies, in chemical processes (eg fermentation), in food and beverage preparation,

COMPENDIUM

Here we describe methods to modulate microorganism activity with MSM. MSM is a sulfur organ compound with the formula (CH3) 2S02 · In particular, the surprising ability of MSM to improve or inhibit the activity of microorganisms, such as growth or survival of microorganisms, is described here, depending on the concentration of MSM that The microorganism is provided (for example, in the medium in which the organism grows). MSM at a concentration of about 0.5% to about 5% by weight of medium or by weight of moisture content of the medium, improves microbial activity while MSM at a concentration of about 6% to about 17% by weight of medium or weight of moisture content of the medium, inhibits microbial activity.

Here we describe the surprising discovery that MSM can both inhibit and improve microbial activity, depending on the concentration of MSM. For example, MSM concentrations between about 6 and about 17 weight percent of the medium (or moisture content of the medium inhibit microbial activity by reducing or otherwise impacting growth, survival rate (e.g. accelerate cell deterioration or death, such as programmed cell death), metabolism, reproduction (eg, 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 between approximately 0.

As such, methods of using MSM to modulate microbial activity, such as to improve or inhibit the activity of microorganisms, are described herein.

In some embodiments, a method for improving the fermentation efficiency of a microorganism is described. For example, the method includes contacting medium containing a microorganism capable of fermentation with MSM, wherein MSM is provided at a concentration of about 0.5% to about 5% by weight of the medium or at a concentration of about 0.5% to about 5%. % by weight of the moisture content of the medium, where MSM increases the fermentation efficiency of the microorganism compared to the fermentation efficiency in the absence of MSM.

In some embodiments, in vitro methods to enhance the growth of one or more probiotic microorganisms are described. In some examples, the methods comprise contacting one or more probiotic microorganisms with a medium capable of supporting the growth of one or more probiotic microorganisms; and provide MSM to average about 0.4% to about 5% by weight of the medium or by weight of the moisture content of the medium thereby enhancing the growth of one or more microorganisms in vitro compared to growth of one or more microorganisms in vitro in the absence of MSM.

Methods for improving 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 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 the moisture content of the medium, thereby improving the growth of the one or more microorganisms in the diagnostic test sample, in comparison with growth of one or more microorganisms in the absence of MSM.

In addition, methods for inhibiting microbial activity are described. 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 about 10% to about 16% by weight in volume, thereby inhibiting the microbial activity of H1N1 influenza.

The above and other characteristics of the description will be more apparent from the following detailed description of various modalities.

Detailed Description I. Generality of Various Modalities

Here we describe the surprising discovery that MSM can both inhibit and improve microbial activity, depending on the concentration of MSM. For example, MSM concentrations between about 6 and about 17 weight percent medium (or medium moisture content) inhibit microbial activity by reducing or otherwise impacting growth, survival rate (e.g. accelerate cell deterioration or death, such as programmed cell death), metabolism, reproduction (eg, 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 between approximately 0.

As such, methods for MSM uses to modulate microbial activity are described herein, such as to enhance or inhibit the activity of microorganisms.

In some embodiments, a method for improving the fermentation efficiency of a microorganism is described. For example, the method includes contacting medium containing a microorganism capable of fermentation with MSM, wherein MSM is provided at a concentration of about 0.5% to about 5% by weight of the medium or at a concentration of about 0.5% to about 5%. % by weight of the moisture content of the medium, wherein the MSM increases the fermentation efficiency of the microorganism compared to the fermentation efficiency in the absence of MSM. In some examples, improving the fermentation efficiency comprises at least a 50% increase in production of alcohol, carbon dioxide or acid in the presence of MSM by the microorganism, compared to alcohol or acid production in the absence of MSM. For example, improving the fermentation efficiency comprises an increase of at least 50% in the production of ethanol, methanol or a combination thereof as compared to production of ethanol, methanol or a combination thereof in the absence of MSM.

In some examples, improving the fermentation efficiency comprises an increase of at least 50% in carbon dioxide production in the presence of MSM by the microorganism, compared to production of carbon dioxide in the absence of MSM, the microorganism is yeast and the method of improving fermentation is for the production of bread.

In some examples, improving the fermentation efficiency comprises an increase of at least 50% in lactic acid production in the presence of MSM by the microorganism, compared to production of lactic acid in the absence of MSM and the method of improving fermentation It is for the production of a dairy product.

In some embodiments, the method of improving fermentation efficiency is for the production of beer, cider, wine, a biofuel, bread, dairy product or any combination thereof. In some examples, the microorganism is yeast and the method for improving fermentation is for the production of beer. In some examples, the microorganism is algae and the method to improve fermentation is for the production of biofuel.

In some modalities, the concentration of MSM is approximately 0.5%. In some examples, the medium comprises a sodium chloride concentration of less than 5% of the total moisture content.

In vi tro methods are also described to improve the growth of one or more probiotic microorganisms. 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 about 0.4% to about 5% by weight of the medium or by weight of a moisture content of the medium in this manner improving the growth of one or more microorganisms in vitro as compared to growth of the one or more microorganisms in vitro in the absence of MSM.

In some examples, the concentration of MSM is 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 ácidoophílus, Lactobacillus delbrueckii, Bacillus coagulans, Lactobacillus rhamnosus, Bifidobacterium bifidum or any combination thereof. «Λ ·» -

In some examples, the means 'liTflpience' a product containing a probiotic, such as milk, yogurt, rice yogurt, frozen yogurt, chocolate, cheese, beer, wine, vinegar, sauerkraut or any combination thereof.

Methods for improving the growth of a microorganism in a diagnostic test sample are also described. 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 about 0.4% to about 5% by weight of the medium or by weight of a moisture content of the medium, thereby improving the growth of one or more microorganisms in a diagnostic test sample compared with growth of one or more microorganisms in the absence of MSM.

In addition, methods for inhibiting microbial activity are described. 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 about 10% to about 16% by weight in volume, thereby inhibiting the microbial activity of H1N1 influenza. In some examples, the medium comprises a body fluid, a body tissue or a surface. In some examples, contacting the medium 1 1 1 - comprises spraying or rubbing with MSM the medium susceptible to microbial contamination. In some examples, the surface is a domestic surface, bedding, covers, surface or industrial equipment, blood, skin or a combination thereof. In some examples, MSM is provided in a composition, wherein the composition is free of bleach or free of alcohol or consists essentially of water. In some examples, the method further comprises sterilizing the medium after adding MSM. In some examples, the medium is free of conservatives. 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 HIN1 influenza in the absence of MSM. II. Abbreviations and Terms DMEM: Dulbecco Modified Eagle Medium DMSO: Dimethyl Sulfoxide DNA: Deoxyribonucleic Acid ELISA: Enzyme Linked Immunosorbent Assay IC50: Inhibitory Concentration 50 LAB: Lactic Acid MIC: Minimum Inhibitory Concentration MSM: Methylsulfonylmethane ΡΑ6Ξ: Gel Electrophoresis of polyacrylamide PBS:

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" refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "comprises a bacterial cell" includes simple or multiple bacterial cells and is considered equivalent to the phrase "comprising at least one bacterial cell". The term "or" refers to a single element of established alternate elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used here, "comprises" means " including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Coid Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Coid 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 & amp; Sons, 1999; Harlow & amp; Lane, Antibodies: A Laboratory Manual, Coid Spring Harbor Laboratory Press, 1990; and Harlow & amp; Lane, Using Antibodies: A Laboratory Manual, Coid 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 Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; o Vogel, A Textbook of Practical Organic Chemistry,

Including Qualitative Organic Analysis, Fourth E <^ tioi ^ f Iflew_

York: Longman, 1978. Additional terms commonly used in molecular genetics can be found in Benjamin Lewin, Genes 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 commonly used in chemistry can be found in Loudon , Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002, p. 360-361, 1084-1085; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth

Edition, Wiley-Interscience, 2001; o Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978.

Administration: To provide or give a subject a compound, such as MSM, by any effective route. Exemplary routes of administration include, but are not limited to, routes of injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal and intravenous), oral, sublingual, rectal, transdermal (such as topical), intranasal, vaginal and inhalation. A particular type of administration is topical.

Bacterial pathogen: A bacterium that causes disease (pathogenic bacteria). Examples of pathogenic bacteria for which MSM can be used to modify, 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 lariy · GamjPji'iojÍsiagtg'i · i · 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 perfringens, 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 uropathogenic E. coli) Enterococcus sp. (such as Enterococcus faecalis and Enterococcus faecíum) 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 ififrcfeicj Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm 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 acne, 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, 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 acne, 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, 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 acne, 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, (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 acne, 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, (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 acne, 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, (such as Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acne, 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, (such as Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acne, 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 enteric Salmonella, 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 pneumoniae (for example Streptococcus pneumoniae serotype resistant to chloramphenicol 4, Streptococcus pneumoniae serotype resistant to spectinomycin 6B, Streptococcus pneumoniae serotype resistant to streptomycin 9V, Streptococcus pneumoniae serotype resistant to erythromycin 14,

Streptococcus mutans, Streptococcus pyogenes, Group A streptococci, Streptococcus pyogenes, Group B streptococci, Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus, Streptococcus equismilis, Group D streptococci, Streptococcus bovis, Group F streptococci, and Streptococcus anginosus streptococci Group G), 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,

In some embodiments, MSM is used to modify, such as increasing or decreasing the biological activity of one or more of the organisms mentioned above.

Beta-lactam antibiotics: A class of antibiotic agents that contain a β-lactam nucleus in their molecular structure. Examples include the antibiotic families of penicillin, cephalosporin, monobactam and carbapenem. 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 ingredient of the substance or pharmacophore, but can be modified by the other constituents. The activity in general is dose dependent and it is not uncommon to have effects in the range from beneficial to adverse for a substance when going from low to high doses. In one example, MSM alters, as it increases or decreases the biological activity of a microorganism, such as bacteria.

Blocombustibie: A fuel derived from a metabolic product of a living organism. It is a source of renewable energy, unlike other natural resources such as oil, coal / coal and nuclear fuels. A biodiesel fuel is a processed fuel equivalent to diesel derived from biological sources that can be used in unmodified diesel engine vehicles. Biodiesels are attractive for fuels, and some other uses, because they have a low vapor pressure, are non-toxic, stable and do not deteriorate or detonate with slight heating. Chemically, biodiesels in general are defined as mono-alkyl esters of long-chain fatty acids derived from renewable lipid sources.

Bleaching: A solution of approximately 3-6% sodium hypochlorite bleach (NaClO), and oxygen, which contains hydrogen peroxide or a peroxide-releasing compound, such as sodium perborate, sodium percarbonate, sodium persulfate, pyrophosphate tetrasodium, or urea peroxide together with catalysts and activators, for example, tetraacetylethylene diamine and / or sodium nonanoyloxybenzene sulfonate. Bleaching powder is calcium 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. These conditions usually include temperature ranges, aeration levels and media selection which, when combined, allow the microorganism to grow. Exemplary media include broths or gels. To determine if the culture conditions allow production of the product, the microorganism can be cultured for 2, 4, 6, 8, 12, 24, 36, 48 or 72 hours and a sample can be obtained and analyzed. For example, the cells in the sample or the medium in which the cells grow can be tested for the presence of the desired product. When the presence of a product is tested, tests may be used,

Contact: Place in direct physical association; including in solid, liquid and gas form. The contact includes contact between one molecule and another molecule. The contact can occur in vitro with isolated cells, tissue or a solid surface (such as a domestic or industrial surface) or in vivo when administered to a subject.

Control: Samples that are considered normal (for example, function or representative activity in the absence of the variable being tested) as well as laboratory values, even if possibly arranged arbitrarily, keeping in mind that these values ​​may vary from laboratory to laboratory. A control group is virtually identical to the treatment group, except for the single variable of interest whose effect is tested, which only applies to the treatment group.

Cultivation: Maintain a cell in a medium that allows the organism to continue living. For example, the culture includes incubating a microorganism in a fermentation medium, such as a fermentation broth or a fermentation gel. A person of ordinary skill in the art will appreciate that the time, temperature and other physical conditions associated with the cultivation and the desired result of the culture.

For example, a microorganism that is grown to produce ethanol can be placed in a fermentation broth containing a source of carbohydrates, various minerals and trace elements, as well as MSM and compounds useful for inducing production, including less than 5% NaCl. .

Decrease: To reduce the quality, quantity or strength or concentration 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, the administration of MSM decreases or inhibits bacterial growth, for example by at least 2-fold, for example at least 3-fold or at least 4-fold, 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 afflicted with a bacterial infection). These decreases can be measured using the methods described herein, as well as those known to a person of ordinary skill in the art. In some embodiments, MSM is used to inhibit growth of specific microorganisms. In other embodiments, MSM is used to inhibit growth of a wide range of microorganisms in certain media or products. In some modalities, log scale reductions are achieved after the first 24 hours. as well as those known by a person with ordinary skill in the specialty. In some embodiments, MSM is used to inhibit growth of specific microorganisms. In other embodiments, MSM is used to inhibit growth of a wide range of microorganisms in certain media or products. In some modalities, log scale reductions are achieved after the first 24 hours. as well as those known by a person with ordinary skill in the specialty. In some embodiments, MSM is used to inhibit growth of specific microorganisms. In other embodiments, MSM is used to inhibit growth of a wide range of microorganisms in certain media or products. In some modalities, log scale reductions are achieved after the first 24 hours.

Dimethyl sulfoxide (DMSO): Dimethyl sulfoxide (DMSO), also known as methylsulfinylmethane or methyl sulfoxide, is a sulfur organ compound with the formula (CH3) 2SO. This colorless liquid is a polar aprotic solvent that dissolves both polar and non-polar compounds and is miscible in a wide range of organic solvents as well as in water. It has a distinctive property of penetrating the skin very easily, so that one can try it shortly after it comes into contact with the skin. DMSO is well known as a nutritional supplement and as a pharmaceutical agent. A person with skill in the relevant technique will be familiar with these uses. Various grades of DMSO are commercially available (eg, product No. 472301 from Sigma-Aldrich, Corp., St. Louis,

Improvement or increase: To increase the quality, quantity or strength or concentration of something. In one example, MSM increases or improves the activity of a microorganism, for example with respect to activity in the absence of MSM. In a particular example, MSM increases the activity of a microorganism, such as improving the growth, reproduction, proliferation, survival rate, metabolism, vitality, robustness, action and / or function of a microorganism by at least 10%, at least 20 %, at least 50%, or at least even 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 certain contexts. In some examples, MSM is used to improve the growth of specific microorganisms. In other examples, MSM is used to enhance the growth of a wide range of microorganisms in certain media or products. In some examples, improving microbial activity includes improving microbial products or microbial metabolites. For example, MSM increases or improves fermentation efficiency or culture efficiency such as 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%. These increases can be measured using the methods described here. Improving microbial activity includes improving microbial products or microbial metabolites. For example, MSM increases or improves fermentation efficiency or culture efficiency such as 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%. These increases can be measured using the methods described here. Improving microbial activity includes improving microbial products or microbial metabolites. For example, MSM increases or improves fermentation efficiency or culture efficiency such as 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%. These increases can be measured using the methods described here.

Fermentation: A process of screening energy from the oxidation of organic compounds, such as carbohydrates, and using an endogenous electron acceptor, which is usually an organic compound. During fermentation, pyruvate is metabolized to several different compounds. The homolactic fermentation is the production of lactic acid from pyruvate; alcoholic fermentation is the conversion of pyruvate into ethanol and carbon dioxide; and the 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, provided sugars are readily available for consumption. Sugars are a common fermentation substrate 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. The yeast carries out the 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 actively metabolizes carbon). A fermentation medium usually 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 that both fermentation product, such as alcohol, lactic acid, micro-organisms or other desired fermentation product, is produced in a relative manner to a control (such as in the absence of MSM) or to an amount that can be produced theoretically.

Fermentation medium: Any substance used to grow cells, such as mammalian cells and microorganisms. Means of fermentation includes any growth medium (eg, broth or gel) that supports the life of microorganisms (eg, a microorganism that actively metabolizes carbon). A fermentation medium usually 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.

Pathogen fungal: A fungus that causes disease. Examples of fungal pathogens for which MSM can be used to modify, include without limitation any one or more of (or any combination of) Trichophyton rubrum, T. mentagrophytes, Epidermophyton floccosum,

Microsporum canis, Pityrosporum orhimilare (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), Cryptococcus 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 modalities,

Incubation: A term that includes a sufficient amount of time for an agent, such as MSM, to interact with a cell or tissue.

Inhalation or Inhalation Device: A device capable of delivering a composition to a subject, for example to the lung tissue of a subject. For example, an inhalation device may be an inhaler, a nebulizer or a ventilator. Inhalation devices described herein are constructed from a material adapted to contact 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 to make direct contact with bacterial pathogens in the lung tissue of a subject. Inhalation devices are configured to generate particles of a composition that are in a range of size. In some modalities,

Inhibit Microbial Activity or Inhibit a Disease or Infection: The phrase "inhibit microbial activity" refers to reducing growth, reproduction, proliferation, survival rate, metabolism, vitality, robustness, action and / or function of microorganisms. The phrase "inhibit or treat an infection, disease or condition" refers to preventing or reducing the entire development of an infection, disease or condition, for example in a subject who is at risk for developing an infection, such as a bacterial infection. . "Treatment" refers to a therapeutic intervention that improves a sign or symptom of a pathological condition, after it has begun to develop. As aguí is used, the term "improvement", with reference to a disease, pathological condition or symptom, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example by a delayed onset of clinical symptoms of the infection / disease in a susceptible subject, a reduction in severity of some or all of the clinical symptoms of the infection / disease, a minor progress of the infection. disease, a reduction in the number of relapses of the infection / disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular infection / disease, such as a bacterial infection particular.

Medium or media: 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, living cells, host cells, diagnostic tests, and other solid, liquid, on matrix, gelatinous or gaseous environments.

Methylsulfonylmethane (MSM): A sulfur organ compound with the formula (CH3) 2S02 · MSM has been commercialized! zado__y__ sold substantially as a dietary supplement. MSM is also known as DMSO2, Dimethyl sulphone 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 water-like dissolution properties while MSM is less polar and less reactive. MSM is also a metabolite of DMSO. MSM has the following chemical structure:

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Microorganisms: A member of the prokaryotic or eukaryotic microbial species of the domains Archaea, Bacteria, and Eucarya, the latter includes yeast and filamentous fungi, protozoa, algae or higher Protista. The terms "microbial cells" and "microbes" are used interchangeably with the term "microorganism". Microbes can include wild-type, genetically engineered or modified organisms. Microorganisms include viruses, prions, parasites, fungi, molds, yeast and bacteria.

In some embodiments, MSM is used to improve the activity of a broad spectrum of microorganisms including, but not limited to, viruses, prions, parasites, fungi, mold, yeast, algae and bacteria. In other embodiments, MSM is employed to inhibit the activity of microorganisms, including but not limited to fungi, mold, yeast, bacteria and viruses.

Modular or modulation: Adjust, alter, regulate an activity, a degree or speed thereof and including an increase or decrease 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 required food, consume tissues and cells of the body and eliminate toxic waste, which makes people sick. Examples of fungal pathogens to be used according to the methods and compositions described, include without limitation any one or more of (or any combination of) Malaria (Plasmodium falciparum, P. vivax, P. malariae), Schistosomes,

Trypanosomes, Leishmania, Filaria nematodes, Trichomoniasis, Sarcosporidiasis, Taenia [T. saginata, T. solium), Leishmania, Toxoplasma gondii, Trichinellosis (Trichinella spiralis) or Coccidiosis (Eimeria species). MSM can be used to inhibit or prevent activity of one or more of the organisms mentioned above.

Pharmaceutical composition: A compound or chemical composition capable of inducing a desired therapeutic or prophylactic effect when properly 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 together with an additional compound induces the desired response (such as inducing 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.

Acceptable Pharmaceutical Carriers or Carriers: The acceptable pharmaceutical carriers (carriers) useful in this description are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), describe compositions and compositions suitable for 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 used. For example, parenteral compositions usually comprise injectable fluids including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol or the like, as a carrier. In a particular modality, the carrier is one that allows the therapeutic compound to cross the blood-brain barrier. For solid compositions (e.g., powder, pill, tablet or capsule forms), conventional non-toxic solid carriers may include for example pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may 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. Conventional non-toxic solid carriers may include for example pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may 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. Conventional non-toxic solid carriers may include for example pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may 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.

Probiotics: A nondigestible food ingredient that stimulates the growth and / or activity of bacteria in the digestive tract that are beneficial for the health of the body. Typically, probiotics are carbohydrates (such as oligosaccharides); However, products without carbohydrates are also sources of these ingredients. Probiotics can be short chain, long chain, and / or broad spectrum probiotics. Short-chain probiotics (such as oligofructose), containing 2-8 bonds per saccharide molecule, typically ferment more rapidly on the right side of the colon, providing nutrition to the bacteria in that area. Longer chain probiotics (such as inulin) contain 9-64 bonds per molecule of saccharide, and tend to ferment more slowly, nourishing bacteria predominantly in the colon on the left side. Spectrum probiotics with scope of molecular bond lengths of 2-64 bonds per molecule and nourish bacteria through the colon (such as inulin enriched with oligofructose) (OEI = Oligofructose-Enriched Inulin). In some examples, a probiotic increases the number and / or activity of bifidobacteria and lactic acid bacteria. Bifidobacteria and lactic acid bacteria (lactobacilli or LABs) are bacteria that improve digestion (including improving 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 diet sources of probiotics include soybeans, sources of inulin (such as Jerusalem artichoke, jicama and chicory root), raw oats, unrefined wheat, unrefined barley, garlic, leeks, onion, asparagus, banana and yacon. Probiotic oligosaccharides are increasingly adding foods for their health benefits. Some oligosaccharides which are used in this manner are fructooligosaccharides (FOS), xylooligosaccharides (XOS), polydextrose and galactooligosaccharides (GOS). Some monosaccharides such as tagatose are also sometimes used as probiotics. As used here, MSM is a probiotic. polydextrose and galactooligosaccharides (GOS). Some monosaccharides such as tagatose are also sometimes used as probiotics. As used here, MSM is a probiotic. polydextrose and galactooligosaccharides (GOS). Some monosaccharides such as tagatose are also sometimes used as probiotics. As used here, MSM is a probiotic.

Probiotic: A microorganism that confers a health benefit on the host, including, but not limited to, confer protection of or treatment of disease or undesirable effects. Probiotics can confer health benefits to a product, such as increasing the nutritional quality of edible products, probiotics include beneficial bacteria, 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; but certain yeasts and bacilli can also be probiotics. Probiotics are commonly consumed as part of fermented foods; such as in yogurt, soy products or as diet supplements. Live probiotic cultures are available in fermented milk products and foods fortified with probiotics. However, tablets, capsules, powders and sachets containing the bacteria in dry form by freezing are also available. Exemplary probiotic strains include, but are not limited to Bacillus coagulans GBI-30, 6086 (Ganeden Biotech), Bifidobacterium LAFTI® B94 (Institut-Rosell-Lallemand), Lactobacillus acidophilus LAFTI® IOL (Institut-Rosell-Lallemand), Lactobacillus casei LAFTI ® L26 (Institut-Rosell-Lallemand), Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium breve (Yakult), Bifidobacterium infantis 35624 (Procter &amp; Gamble), Bifidobacterium animalis subsp. lactis HN019 (Danisco), Bifidobacterium longum BB536 (Morinaga Milk Industry), Lactobacillus acidophilus DDS-1 (Nebraska Cultures),

Quantify: Determination or measurement of a quantity (such as a relative amount) of a molecule or the activity of a molecule, such as the activity of analyte present in a sample. Stem Cell: A cell that has the ability to replicate itself indefinitely and under the right conditions, or given the right signals, can differentiate in some or all of the different types of cells that make up an organism. Stem cells have the potential to develop into mature, differentiated cells such as cardiac cells, skin cells or nerve cells. The fertilized egg is a stem cell because it has the potential to generate all the cells and tissues that make up an embryo and that support its in utero development. Adult mammals include more than 200 cell types, for example, neurons, myocytes, epithelial cells, erythrocytes, monocytes, lymphocytes, osteocytes and chondrocytes. Other cells that are essential for embryonic development but are not incorporated into the body of the embryo include extra embryonic tissues, placenta and umbilical cord. All these cells are generated from a single fertilized egg. Pluripotent cells can give rise to cells derived from all three embryonic germ layers-mesoderm, endoderm and ectoderm. In this way, pluripotent cells have the potential to give rise to any type of cell. Unipotent stem cells are capable of differentiating over only one lineage. Embryonic stem cells are pluripotent cells derived from the blastocyst. Adult stem cells are undifferentiated cells that are found in a differentiated tissue that can replicate and become specialized to give all types of specialized cells of the tissue from which they originate. Adult stem cells are capable of self-renewal for the lifetime of the organism. Sources of adult stem cells have been found in bone marrow, bloodstream, cornea, retina, dental pulp, liver, skin, gastrointestinal tract and pancreas. MSM is used here to increase the efficiency, stability and / or viability of stem cell culture. Sources of adult stem cells have been found in bone marrow, bloodstream, cornea, retina, dental pulp, liver, skin, gastrointestinal tract and pancreas. MSM is used here to increase the efficiency, stability and / or viability of stem cell culture. Sources of adult stem cells have been found in bone marrow, bloodstream, cornea, retina, dental pulp, liver, skin, gastrointestinal tract and pancreas. MSM is used here to increase the efficiency, stability and / or viability of stem cell culture.

Sterilization: A gnft .. process eliminates all forms of life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medicine, or in a compound such as biological culture medium. The sterilization can be achieved by methods known to a person with ordinary skill in the art, including applying the appropriate combinations of heat, chemicals, radiation, high pressure and filtration.

Subject: Live multicellular vertebrate organisms, a category that includes humans and non-human mammals.

Symptoms and signs: Any subjective evidence of illness or a condition in a subject, for example, evidence as perceived by the subject; a notable change in a condition of the subject indicative of some bodily or mental state. A "sign" is any abnormality indicative of disease, which is discovered when examining or evaluating a subject. A sign is usually an objective indication of disease. Signs include, but are not limited to, any measurable parameters such as tests for detecting 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%, at least 50%,

Therapeutically effective amount or concentration:

An amount of a composition that alone, or together with one or more additional therapeutic agents is sufficient to achieve a desired effect in a subject or in a cell, which is treated with the agent. The effective amount of the agent will depend on several factors, including but not limited to the subject or cells being treated, and the manner of administration of the therapeutic composition. In one example, an effective therapeutic amount or concentration is one that is sufficient to prevent advancement, delay progress or cause regression of the 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 do not have to be completely eliminated by the composition to be effective. For example, a composition can decrease the sign or symptom by a desired amount, for example by at least 20%, by at least 50%, by at least 80%, by at least 90%, by at least 95%, by 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 microorganism activity (such as bacterial growth) by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at less 80%, at least 90%, at least 95%, at least 98% or even at least 100%, compared to the activity of microorganism in the absence of MSM.

An effective therapeutic amount of a described pharmaceutical composition can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective therapeutic amount may depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. An effective therapeutic amount of an agent can be measured as the concentration (moles per liter or molar-M) of the agent in the blood (in vivo) or a buffer (in vitro) that produces the desired effect (s). Alternatively, an effective therapeutic amount of the agent can be measured as the amount administered to a subject by the subject's body weight, eg, mg agent / kg body weight. Untreated cells: A cell that has not come in contact with the desired agent, such as MSM. In one example, an untreated cell is a cell that receives the vehicle in which MSM is delivered.

Virus: A microscopic infectious organism that reproduces inside living cells. A virus consists essentially of a nucleic acid nucleus surrounded by a seeding in protein plates, and has the ability to replicate itself only within a living cell. "Viral Replication" is the production of additional viruses by the occurrence of at least one viral life cycle. A virus can disrupt the normal functions of host cells, causing the cell to behave in a way determined by the virus. For example, a viral infection can result in a cell that produces a cytokine, or that responds 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 according to the methods and compositions described, 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, Bunyavirus (such as Crimean-Congo haemorrhagic fever virus and Hantavirus), Barnavirus, Birnavirus, Bornavirus (such as virus) of Boma disease), Bromovirus, Calicivirus, Chrysovirus, Coronavirus (such as Coronavirus and SARS), Cistovirus, Closterovirus, Comovirus, Dicistrovirus, Flavirus (such as yellow fever virus, West Nile virus, Hepatitis C virus and Dengue Fever virus), Filovirus (such as Ebola virus and Marburg virus), Flexivirus, Hepevirus (such as Hepatitis E virus),

Measles and Mumps), Picornavirus (such as Polio virus, common cold virus and Hepatitis A virus), Potivirus, Poxvirus (such as Variola and Bovine Smallpox), Sequivirus, Reovirus (such as Rotavirus), Rhabdovirus (such as Rabies virus), Rhabdovirus (such as Vesicular stomatitis virus, Tetravirus, Togavirus (such as Rubella virus and Ross River virus), Tombusvirus, Totivirus, Tymovirus, and Norovirus among others.

In some embodiments, MSM is employed to inhibit a biological activity of one or more of the viruses mentioned above.

Yeast: A eukaryotic microorganism classified in the Kingdom of Fungi, with approximately 1,500 described species. Most reproduce asexually by budding, although a few breed by binary fission. Yeasts in general are unicellular, although some species can become multicellular through the formation of a string of connected budding cells known as pseudohifa, or false hyphae. Exemplary yeasts which can be employed in the methods and compositions described include but are not limited to Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Pichia, Cryptococcus, Zygosaccharomyces, Torulopsis, Hansenula, and Debaryomyces.

: i. MSM compositions

Here, MSM compositions are described for use in modulating microbial activity, such as improving or decreasing microbial activity. In some embodiments, an MSM composition for use in improving microbial activity includes about 0.02% to about 5% MSM by weight of the medium (such as culture medium) or by weight of the moisture content of the medium (such as culture medium). ), such as from about 0.04% to about 4%, about 1% to about 3%, including about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, approximately 0.1%, approximately 3%, approximately 0.5%, approximately 1%, approximately 2%, approximately 2.5%, approximately 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, for example water in a product. By way of example, a composition with 5% MSM by weight of the medium, will contain 5 grams of MSM per 100 grams of medium or a composition with 5% of MSM by weight of the moisture content of the medium will contain 5 grams of MSM per 100. grams or the polar solvent in the middle, excluding solids.

In some embodiments, the described nnrnnnsns.innfis include a medium capable of supporting growth of a microorganism, a microorganism and MSM. In some examples, one medium includes one or more of the following: products containing probiotics, 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, edible or not, that benefit from improved microbial activity.

In some embodiments, improved microbial activity includes improving the fermentation of a microorganism. Thus, in some particular examples, a composition includes a medium capable of supporting growth of fermentative microorganisms, a fermentative 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%, 0.5% to about 3%, 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%, approximately ? r anrnyiTnaHamantfl. 3.0%, approximately 4% or approximately 4.5% of MSM by weight of medium or weight of the moisture content of the medium, wherein the concentration of MSM is effective to improve the fermentation of the microorganism. In some embodiments, the compositions described are used to produce a fermented beverage, such as beer, cider and / or wine. In some embodiments, a composition for improving fermentation efficiency includes MSM added to packages of yeast to generate fast-activating yeast for home or commercial use. The compositions described are used to produce a fermented beverage, such as beer, cider and / or wine. In some embodiments, a composition for improving fermentation efficiency includes MSM added to packages of yeast to generate fast-activating yeast for home or commercial use. The compositions described are used to produce a fermented beverage, such as beer, cider and / or wine. In some embodiments, a composition for improving fermentation efficiency includes MSM added to packages of yeast to generate fast-activating yeast for home or commercial use.

In some embodiments, improving microbial activity includes improving the growth of probiotics. Thus, in some examples, a composition for improving the growth of probiotics includes a medium capable of supporting the growth of probiotics and MSM at a concentration of about 0.04% to about 5% by weight of the medium or by weight of the moisture content of the medium, where the concentration of MSM is effective to improve the activity (for example, growth) of the probiotics. In addition, a composition for improving the growth of probiotics includes about 0.04% to about 5% MSM, such as about 0.1% to about 4%, 0.5% to about 3%, about 1% to about 2%, including about 0.04% , a ^ pmY ^ m ^ mpntP ··, n, ^ r- approximately 0.06%,

In some embodiments, improving microbial activity includes improving the microbial production of biofuel. Thus, in some examples, a composition for improving microbial production of biofuel includes a medium capable of supporting the growth of algae, algae capable of producing a biofuel and MSM at a concentration of about 0.4% to about 5%, such as about 0.1% to about 4%, 0.5% to about 3%, 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 %, approximately 0.3%, approximately 0.5%, approximately 0.7%, approximately 1%, approximately 1.5%, approximately 2.0%, approximately 2.5%, about 3.0%, about 4%, or about 4.5% of MSM by weight of medium or weight of the moisture content of the medium, wherein the concentration of MSM is effective to improve biofuel production from the algae. In other examples, a composition includes algae and MSM, and optionally other ingredients to improve the growth of algae. In several modalities, the composition is useful to improve algae activity for biofuel, algae farms, aquaculture, medicines, etc. In one embodiment, the method comprises exposing algae to MSM, for example, at a concentration of about 0.04% to about 5% by weight of medium or by weight of the moisture content of the medium. where the concentration of MSM is effective to improve the production of biofuel from algae. In other examples, a composition includes algae and MSM, and optionally other ingredients to improve the growth of algae. In several modalities, the composition is useful to improve algae activity for biofuel, algae farms, aquaculture, medicines, etc. In one embodiment, the method comprises exposing algae to MSM, for example, at a concentration of about 0.04% to about 5% by weight of medium or by weight of the moisture content of the medium. where the concentration of MSM is effective to improve the production of biofuel from algae. In other examples, a composition includes algae and MSM, and optionally other ingredients to improve the growth of algae. In several modalities, the composition is useful to improve algae activity for biofuel, algae farms, aquaculture, medicines, etc. In one embodiment, the method comprises exposing algae to MSM, for example, at a concentration of about 0.04% to about 5% by weight of medium or by weight of the moisture content of the medium. The composition is useful to improve algae activity for biofuel, algae farms, aquaculture, medicines, etc. In one embodiment, the method comprises exposing algae to MSM, for example, at a concentration of about 0.04% to about 5% by weight of medium or by weight of the moisture content of the medium. The composition is useful to improve algae activity for biofuel, algae farms, aquaculture, medicines, etc. In one embodiment, the method comprises exposing algae to MSM, for example, at a concentration of about 0.04% to about 5% by weight of medium or by weight of the moisture content of the medium.

MSM compositions are described for inhibiting microbial activity. In some embodiments, an MSM composition for inhibiting microbial activity includes about 6% to about 17%, such as about 7% to about 15%, about 10% to about 12%, such as about 6%, about 7%, about 8%, approximately 9%, approximately 10%, approximately 11%, approximately 13%, approximately 14%, approximately 15%, or approximately 16% of MSM by weight of medium or weight of the moisture content of the medium, wherein the MSM concentration 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 compositions described including MSM for modulating microbial activity has a sodium chloride concentration of less than 5% of total moisture content of the medium, such as about 1% to about 3% sodium chloride, including 0% , 0.1%, 0.3%, 0.5%, 0.75%, 1%, 2%, 2.5%, 3% or 4%. In some examples, a described composition of MSM is free of preservatives. For example, MSM is added to a food, cosmetic or beverage product that requires or desires a list of all-natural ingredients. In some embodiments, an MSM composition consists or consists essentially of MSM and all natural non-toxic ingredients. In other examples, a disclosed composition of MSM includes one or more additional preservatives. Conservatives include,

In several modalities, MSM is used to extend or prolong the shelf life of products and is capable of reducing microbial activity by at least 2-, 3-, 4-, 5-, 10-, 25-, 50-, 100- , 1000-fold in compaipaoa-ÓM em pmüewrtwwy- without MSM or in comparison with products with less effective antimicrobial agents. In other embodiments, MSM is able to achieve comparable levels of antimicrobial activity compared to agents that produce undesirable side effects. Thus, in one embodiment, MSM can be used in place of an unwanted conservative. In various embodiments, compositions include formulations free of preservatives or with reduced preservatives comprising MSM.

In several modalities, MSM is used in products (for example, 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. In this way, a pH that is optimal for the product can be selected. In various embodiments, products 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 chemical products, 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 MSA aaidlUlltibles, for example to prolong the inhibitory or stimulatory actions of MSM.

In certain embodiments, the addition of MSM is an effective antimicrobial agent. For example, in one embodiment, a composition comprising MSM has the same or improved antimicrobial effect as compared to a formulation without MSM. In other modalities, MSM serves as an antibacterial agent. In certain modalities, MSM is used as a substitute for a chemical food preservative. In still other embodiments, MSM can be used in combination with a preservative. In certain of these modalities, the use of MSM reduces the amount of, or completely replaces, traditional conservatives. In some modalities, MSM can increase the shelf life of a product, including products that traditionally would not have a preservative. In still other embodiments, MSM serves as a virucide, fungicide and / or bacteriocide. In additional embodiments, MSM is a bacteriostatic. In some embodiments, MSM is a broad spectrum inhibitor of microbial activity. In other modalities, MSM selectively exterminates a certain kingdom, genus or species. In some embodiments, MSM selectively inhibits aerobic bacteria. In other embodiments, MSM selectively inhibits anaerobic bacteria. In some embodiments, MSM selectively inhibits baa? gr · ^ ™ -positives. In other embodiments, MSM selectively inhibits gram-negative bacteria. MSM selectively inhibits anaerobic bacteria. In some embodiments, MSM selectively inhibits baa? gr · ^ ™ -positives. In other embodiments, MSM selectively inhibits gram-negative bacteria. MSM selectively inhibits anaerobic bacteria. In some embodiments, MSM selectively inhibits baa? gr · ^ ™ -positives. In other embodiments, MSM selectively inhibits gram-negative bacteria.

In several modalities, MSM is used to inhibit the growth of microorganisms, including those found in cosmetics, beauty and health aids, parenterals, topically used products and oral products. In several preferred embodiments, MSM is employed to inhibit the growth of microorganisms in products packaged in single or multiple dose containers. MSM formulations according to several of the embodiments described herein are in any convenient form, including but not limited to, powder, cream, liquid, paste, solid or gel form.

In several modalities, MSM is added to cosmetic products susceptible to microbial contamination. Cosmetics may include, but are not limited to lipstick, lip gloss, lip liner, lip volume booster, lip balm, lip resurfacing and lip resurfacing, foundation, powder, rouge, blush, bronzer, mask, eyeliner of eyes, eyeshadow, mineral powder for eyes, pencils for glare of eyes, pencils for eyebrows, enamel, concealer, skin care products, creams, lotions, serums, moisturizer, sunscreen, products for repair of the skin (for example, for acne, sunburn, wrinkles, dark circles), and sunscreen. '

In several modalities, MSM is added to a cosmetic cream matrix susceptible to microbial contamination. In some of these modalities, the cream includes jojoba, aloe vera, cocoa butter, shea butter, coconut oil, or combinations thereof.

In several modalities, MSM is added to personal care products susceptible to microbial contamination. These products include products used for daily moisturization needs, products to treat psoriasis or eczema, products to treat dry or irritated skin, products to treat sun and wind burns, products before and after shaving, oils or creams for massage , personal lubricants, products for acne treatment and exfoliants or emollients. According to other modalities, MSM is added to products to soften the skin on the hands or feet (such as calluses), skin care products after swimming, make-up remover, lotion for children's skin, and cream for rash from diapers. In some modalities,

In several modalities, MSM is added to medicinal products or equipment susceptible to microbial contamination.

Medicinal products include, but not -r.-imi t- aήη &lt;=. to treatment and prevention of catarrh or influenza, preventive and treatment for allergy, nasal irrigant, medicinal drops, eye drops, inhalants, treatments for athlete's foot, herpes and canker medicine or fires, creams for burns, ointments for cuts and infections , and sprays or bactericidal, fungicidal and virucidal lotions. In some modalities, MSM is used to inhibit microbial activity in inhalers, nebulizers, ventilators, catheters, syringes, tubes for intubation, equipment in hospital wards, furniture and surfaces, diagnostic equipment, fabrics, bedding and covers for patients. In several modalities, 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 blood, which can be particularly helpful for patients with sepsis. In another embodiment, MSM is injected into a patient to inhibit microbial activity locally or systemically. In other embodiments, a composition that includes MSM is applied topically to a microbial infection present on a dermal surface.

In some modalities, MSM products are used nasally. In other embodiments, these products are used orally and / or as a vapor. Still in other modalities, the product is a drop for use eyes rsgstJadám..u .... Qtro. ocular medicinal product.

In several modalities, MSM is added to medicinal products used to prevent or treat fungal infections. In some of these modalities, the product is used to prevent or treat athlete's foot. In some modalities, the product is used topically. In some of these modalities, the product is a cream, ointment, spray, gel or powder. In other modalities, the product is used orally.

In several modalities, MSM inhibits the activity of mycotoxins, toxic metabolites produced by an organism of the fungal kingdom, including fungi, molds and yeasts. Products that comprise MSM are also used to decontaminate surfaces and equipment that are susceptible to contamination by these organisms and / or metabolites. In some embodiments, MSM inhibits the activity of fungal kingdom organisms (eg, fungi, molds, and yeasts). In still other embodiments, MSM inhibits microbial toxins directly and / or indirectly by inhibiting the activity of microbes. In one embodiment, MSM inhibits the formation and / or detachment of microbial metabolites.

In several modalities, MSM is added to products used to prevent or treat viral infections. In one embodiment anti-viral nasal sprays or nebulizations comprising MSM are provided. Products that comprise MSM are also useful for decontaminating surfaces and equipment that are susceptible to viral contamination. In one embodiment, MSM is used to inhibit influenza virus, including H1N1, either in a biological tissue or on an external surface. In some modalities, MSM is used to inhibit human immunodeficiency virus, herpes simplex virus, papilloma virus, influenza virus, influenza, hepatitis, and other viruses.

In some modalities, MSM inhibits algae. In some modalities, MSM inhibits algal blooms. In some embodiments, MSM inhibits undesirable phytoplankton activity. In other modalities, MSM inhibits macroalgae species. In other modalities, MSM inhibits dinoflagellates of the genus Alexandrium and Karenia. In several modalities, MSM inhibits the toxic metabolites (including by-products) of algae.

In some modalities, MSM is added to medicinal products used to treat a burn, cut or wound. Injuries or injuries may include, but are not limited to, lacerations, split lacerations, excessive stretching, crushing compression, lacerations by cutting, tearing, incisions, incisional injuries or wounds, abrasions, puncture wounds, penetration wounds. In some modalities, MSM is incorporated into a bandage used to cover a wound. In other modalities, MSM is added to a cream or ointment. In some of these modalities, the product formulated with MSM acts as an antiseptic.

Microflora of the skin (bacteria, fungi, viruses, phages, archaebacteria) play a significant role in common dermatological conditions, such as atopic dermatitis (a common form of eczema). Typically, a specific microbe colonizes the skin to unbalance the balance of commensal microflora, or microbes release toxic substances or invade cells to induce an inflammatory response directly. In this way, in some modalities, MSM is incorporated into a topical product that inhibits the growth of this microflora. Subcutaneous administration of MSM is provided in other modalities.

In other modalities, MSM is added to optical products susceptible to microbial contamination and / or to optical products to improve its antimicrobial activity. Optical products may include solutions for cleaning or disinfecting contact lenses. In some of these modalities, MSM is incorporated into various products applied to contact lenses such as a solution for contact lens storage. In some modalities, MSM is added to eye drops used in conjunction with contact lenses. In other modalities, MSM is added to chemical solutions used in my ocular diagnosis pnt-na, such as solution for pupil dilation of multiple uses.

In several modalities, MSM is added to oral products susceptible to microbial contamination and / or to oral products to improve its antimicrobial activity. In some modalities, these products are used for cleaning teeth. In some modalities, MSM is incorporated into toothpaste or gel for teeth. In some modalities, MSM is incorporated into or coated over the bristles of a toothbrush. In other embodiments, MSM formulations are incorporated into or used for plating dental floss. In other modalities, the product is used to cleanse the tongue. In other modalities, the product is a mouth rinse, irrigant or mouth rinse for professional or home dental use. Still in other modalities, the product is a chewing gum or candy or sweet. In some modalities,

In some modalities, MSM is added to foods that contain probiotic organisms, such as milk, yogurt, rice yogurt, frozen yogurt, kefir, juice, canned or pickled vegetables, cabbage or fermented cabbage, fermented bean paste, pickled olives , chocolate, cheeses and other dairy products, and certain cereals. In some modalities, MSM is added to products that are diet supplements, including but not limited to, probiotic pills, capsules and liquids. In some of these modalities, MSM is added to a supplement for human ingestion. In other modalities, 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 modalities, MSM is added to a product that is formulated as a solid or liquid.

In several embodiments, MSM is added to a food product that is susceptible to microbial infection. In some embodiments, MSM can be mixed, intermixed, formulated or otherwise incorporated into a food product. In other embodiments, MSM is applied to the surface of a food product. For example, in some embodiments, MSM can be sprayed onto a food product. These food products may include, but are not limited to, fruits, vegetables, fish and meat products. In some modalities, MSM is used in processing or packaging facilities to prolong the shelf life of food products. In several embodiments, the addition of MSM (example, κ ° »» about 25%) increases the deterioration time of ingerid products. For example, MSM can be baked or added to breads, pasta, or dough to increase the shelf life of edible products by approximately 10% to 100% (Example, 20%, 30%, 40%, 50%, 75%, 150%, 200% or more). For example, in one modality, if the storage life of an edible product is 10 days, the addition of MSM will increase the shelf life to at least 11 days in some modes (for example, 11 days, 14 days, 15 days , 20 days or 25 days). As an additional example, in another embodiment, if an edible product has a storage life of 14 days at room temperature and / or 30 days in the refrigerator and / or 3 months in the freezer, the addition of MSM will increase shelf life. 30 days at room temperature and / or 60 days in the refrigerator and / or 6 months in the freezer. In additional modalities, the use of MSM will allow the shipment and / or storage of an edible product at room temperature, where the product will otherwise have to be shipped and / or stored at colder temperatures. Still in other modalities, the use of MSM will obviate the need for sterilization of edible products.

In some examples, any of the described compositions of MSM consists essentially of water. For example, MSM is particularly effective when combined with water or other liquid components. In some examples, a described composition of MSM is free of bleach, alcohol free or a combination thereof. In various embodiments, a composition for modulating microbial activity includes a compound related to MSM instead of or in addition to MSM. Related compounds include, but are not limited to, DMSO and dimethylsulfoxide (DMS). MSM employee according to any of the modalities that are arranged here can be isolated, purified or processed. MSM that is designated as Generally Recognized As Safe (GRAS = Generally Recognized As Safe) is used for several modalities described here.

In some embodiments, MSM is combined with one or more of the following ingredients (or derivatives, metabolites, precursors, oils, extracts, esters, acids, salts, and their related compounds): abietic acid, acacia, Acacia Senegal gum, acai extract, acetic acid, acetone, acetyl glucosamine, acmella oleracea extract, strict adenofora, marginata (marine plant), albumen, alcohol, aldenin, alfalfa, algae extract, alkyl guanine transferase, alkylamides, allantoin, aluminum hydroxide, almonds, aloe vera, alpha lipid alcohol, aluminum benzoate, aluminum chloride, amino acids, aminopropan sulfonic acid 3, ammonium glycolate, ammonium lauryl sulfate, anemarrhenae root extract asphodeloides, aniz oil, antioxidants, apigenin, apricot or damask, apricot seed,arachidonic acid, arbutin, argan oil, argania spinosa leaf extract, arginine, argirelin, arnica extract, dracunculus artemisia oil (tarragon), ascorbic acid, ascorbyl palmitate, ascorbyl tetraisopalmitate, aspergillus ferment, aspidosperma quebracho, astaxanthin , atelocollagen, oat grain extract (avena sativa), avobenzone, azelic acid, azuki fritters, mint balm extract, balsam Peru, bamboo trunks or extract, barley extract (hordeum vulgare), barium sulfate, barley, basil, bee pollen, beeswax, bentonite, benzoyl peroxide, root extract beta vulgaris (beet), beta carotene, cranberry, biotin, bismuth oxychloride, vejigoso sargassum extract, borage oil, boric acid, rust boric, liquid bovine placenta, brewer's yeast, bronopol,butyl acetate, butyl stearate, butylated hydroxyanisole, butylated hydroxytoluene, butylated glycol, butylparaben, butyrospermum parkii, triglyceride of cl8-36 acid, caffeine, calamine, calcium, calendula extract, carnauba wax, extract of camellia oleifera leaves, hnia extract .q camelia sinensis, camphor, oil of canaga odorata flowers (ylang ylang), candelilla wax, canola sterols, caprylic acid, caprylic / capric triglyceride, capryl glycol, capsicum oleoresin, caramel, carmine, carotenoids, caragenine, carrot oil, carrot seed oil, carthamus tinctorius (safflower) seed oil, castor oil, cellulose, gotu kola, calendula officinalis, alba wax, carnauba wax, ceramide, cerebrosides, cerium ammonium ferrocyanide, cetearet-3 , cetearyl alcohol, cetearyl glucoside,cetearil olivato, cetil alcohol, cetil lactato, chamomile oil, chamomilla recutita flower extract (feverfew), chestnuts, chestnut extract, chloroxylenol, chlorphenesin, cholesterol, choline, chondrus crispus (Irish moss), green chromium hydroxide, oxide of green chrome, cinnamon alcohol, citric acid, citronellol, citrus, citrus oil nobilis (green tangerine), clove powder, clove flower extract, glyceryl coconate, cocamidopropyl betaine, cocoa, cocoa butter, caprylate / caprate, oil coconut, coconut wax, cod liver oil, coenzyme qlO, collagen, comfrey extract, echinacea extract, cerifene wax (carnauba), copper, coriander, coriander oil (coriandrum sativum), corn starch, corn flower extract, creatine, crithmum maritimum extract, cucumber, cyclomethicone,cyclopentasiloxane, dantoin 685, decyl glucoside, deionized water, diazolidinyl urea, dehydrated dicalcium phosphate, dicapryl carbonate, diethanolamine, dilaurate, dimethicone, dimethylaminoethanol, wild potato root extract (dioscorea villosa), dipotassium glycyrrhizinate, dihydryl biphenyl disulfonate disodium, ed disodium , hydantoin dmdm, echinacea extract (echinacea angustifolia), edta, rose eijitsu, oleifera elaeis, elastin, elderflower, emollients, enzymes, alpandinum extract (epilobium fleischeri), horsetail leaf extract (equisetum hiemale), erucate, essential fatty acids, essential oils, ethanol, ethoxydiglycol, ethyl acetate, ethylene / acrylic acid copolymer, ethylhexyl palmitate, ethylhexylglycerin, ethylparaben, eucalyptus extract, eukarion, fruit extract euterpe oleracea,evening primrose oil, exfoliants, fatty acids, fatty alcohols, fennel oil, ferric oxide, flavanoids, flavonolignano, fish oils, flax, floralozone, fluoride, formaldehyde, fruit acids, fruit extract, fruit extracts, gaba, acid gamma linolenic, gelatin, geraniol, geranium oil, red seaweed (gigartina papillata), ginger, ginger oil, ginko biloba oil, ginseng, glucosamine, glucose oxidase, sugar glucose, glyceret, glyceret-26, glycerin, glycerol, glycerol stearate, hydrogenated glyceryl rosinate, glyceryl oleate, glyceryl stearate, glycol distearate, glycolic acid ^^ gold ^^ gold seal extract, grape seeds, grapeseed oil, grapefruit, grapefruit oil, grapefruit seed extract , green tea, gums, hazelnut oil,cross-linked polymer of hdi / trimethylol hexylactone, hemp seed oil, hexamidine, hexylene glycol, homosalate, honey, hordeum distychum extract, hordihydroguaracetic acid, hormones, humectant, humulus lupulus extract, hyaluronic acid, goldenseal extract (hydrastis) canadensis), hydrocortisone, hydrocotyl extract, hydrogenated castor oil laurate, hydrogenated polyisobutene, hydrogenated polyisobulene, hydrolyzed animal protein, hydrolyzed keratin, hydrolyzed rhizobia gum, hydrolyzed soy protein, hydrolyzed wheat protein, hydroxy acids, hydroxyethylcellulose, hydroxyethyl -cellulose, hydroxyisohexyl 3-cyclohexane carboxaldehyde, hydroxypropyl cellulose, hydroxypropyltrimonium honey, hydroquinone, hydroxystearate, hypericum extract, idebenone, imidazolidinyl urea, iodine, Irish moss,oxides of iron, isobutylparaben, isododecane, isohexadecane, isononil isononanoate, isopentildiol, isopropyl alcohol, isopropyl lanolate, isopropyl linoleate, isopropyl myristate, isostearate, isostearic acid, ivy extract, jasmine oil, jojoba butter, jojoba oil, extract of juniper

Ui u · raoviRiMt; or juniper, juniper oil, kaolin, queraff®, FS, 'JAt · sprays, quinerasa, kinetina, kojic acid, oil-cteuua-UQE do> lQ' Ineti ^ * »o of kukui, lactic acid, lactoperoxidase, skin extract of lion or alderilla, extract of seaweed (laminaria digitata), lanolin, wood extract larix sibirica, lauramida, laurato, lauril ether, lauryl alcohol, lauryl glucoside, lavender, lavender oil, lecithin, lemon oil, licorice, oil of lime, limonene, lindeno extract, linoleic acid, linolenic acid, liposomes, carob tree, lycium barbarum fruit extract, lycium barbarum fruit extract (goji berry), lycopene, macadamia nut oil, matcha, magnesium aluminum silicate, ascorbyl magnesium phosphate, magnesium myristate, magnesium stearate, magnesium sulfate (epson salts), fruit extract (acerola), malpighia punicifolia, manganese violet, mango butter, calendula, altea chocolate extract, matcha tea powder, matricaria oil, mea, meadow queen, melaleuca oil, melon oil, organic mint extract (peppermint), menthol, methyl acetate, methyl ethyl ketone, methyldihydrojasmonate, methylparaben, mica, microdermabrasion compounds, milk protein, minerals, mineral oil, mipa, monoethanolamine, monostearate, montmorillonite (green clay), artemisia or artemega extract (artesemia vulgaris), blackberry, mulberry root extract (morus nigra), murumuru, fungi, myristate, myristate, myristic acid, miristil miriotia.tioy arrayán green (myrtus comunis), n-acetyl glucosamine, nephrite powder, neroli oil,nettle leaf, neuropeptides, niacin, nitrosaraines, nonyl nonoxinol-150, nucleic acids, nutmeg powder, nuts, oats, oatmeal, oat cereals, linalool oil, ocimum basilicum (basil linalool), octinoxate, octosalate, oleate , oleic acid, oleyl alcohol, oligopeptides, oligosaccharides, olive fruit extract, olive oil, omega-3, orange peel oil, orthoboric acid, oxybenzone, ozokerite, padina pavonica talus extract, palm oil, palmitate, palmitic acid, palmitoil, panthenol, panthenol, para-aminobenzoic acid, paraben, paraffin, passion fruit extract incarnata, passion fruit, patchouli, peach seeds, peat extract, pectin, spike, mint, peppermint oil, peptides , petrolatum, phellodendron amurense bark extract, phenoxyethanol, phenyl trimethicone,phenylethyl resorcinol, phosphoric acid, phytochemicals, pine extract, pineapple extract, leaf extract of Plantago lanceolata, banana leaf extract, pollen extract, root extract Poligonum cuspidatum, polypeptides, polysaccharides, polysilicone, polysorbate, polysorbate, polyvinylpyrrolidone, progesterone, propylene glycol, propylheptyl caprylate, propylparaben, pumpkin seed extract, pomegranate extract (Punic granatum), Punic granatum / punica extract "" "grana tum, Pycnogenol, Quaternium-15, cortisol extract - keel ja saponaria (soap), quillia extract, bookratol, retinoic acid, retinoids, retinol, retinol palmate, fruit extract Ribes rubrum, rice, rice bran wax, castor oil, rose oil, rosehip , rosemary, rosemary oil,rose water, royal jelly, fruit extract Rubus villosus, Saccharum officinarum (sugar cane), salicilic acid, sage, sandalwood oil, saponins, sassafras, saw palmetto, sarmentosus saxifrage extract, Sclareolide, extract of Scutellaria baicalensis, seaweed, Secale cereale seed extract (rye), water moss extract (Selaginella tamariscin), selenium, sesame oil, sesquioleate, hundred knuckle tea herb, shea butter, silibinin, silica, silicon, sirtuin, alginate sodium, sodium ascorbate, sodium bisulfate, sodium borate, sodium carbonate, sodium chloride, sodium citrate, sodium dehydroacetate, ethyl paraben, sodium glycyrrhetinate, sodium hyaluronate, sodium lactobionate, sodium lauryl sulfate, methyl paraben sodium, polystyrene sodium sulfonate,sodium propylparaben, sodium stearate, sodium thioglycolate, sodium acrylodimethyl taurate, sorbitan isostearate, olive, sorbitan sesquioleate, sorbitan stearate, sorbitol, sorbitol, soybean, soy wax, soybean oil, spearmint oil, squalane , herb of <= ^ η P ^ hi-o / san Juan, stearate, stem cells, sucrose stearate, sugarcane extract, sulfate, sunflower oil, sweet almond oil, comfrey leaf extract (Symphytum officinale ), leaf extract of Symphytum officinale, synthetic fluorflolopitae, Tamarindus indica seed extract, tea tree oil, thyme extract, tin oxide, titanium dioxide, titanium dioxide, tocopherol, tocopherol acetate, tocopherol acetate , toluene, tomato, tragacanth, tretinoin, tribehenin, triclosan, tridecyl trimellitate,triethanolamine, trihydroxystearin, triisostearyl citrate, triisostearate trimethylolpropane, trimyristate, trimethylsiloxysilicate, tripeptide, turmeric, tyrosine, ubiquinone, grocery, undecylenyl phenylalanine, urea, uridine, fruit extract Vaccinium macrocarpon, vegetable glycerin, vetiver oil, vitamin A, vitamin B1-B12, vitamin C, vitamin C asters, vitamin D, vitamin E, vitamin K, vitamins, nutshell powder, water, wheat germ oil, whey protein (proteinum lactis), birch bark extract white, willow bark, wintergreen oil, magic hazel or witch hazel, xanthan gum, xanthan gum, yarrow extract, yeast, yerba mate, cassava, zinc oxide, zinc stearate.trimethylsiloxysilicate, tripeptide, turmeric, tyrosine, ubiquinone, grocery, undecylenyl phenylalanine, urea, uridine, fruit extract Vaccinium macrocarpon, vegetable glycerin, vetiver oil, vitamin A, vitamin B1-B12, vitamin C, vitamin C aster, vitamin D, vitamin E, vitamin K, vitamins, nutshell powder, water, wheat germ oil, whey protein (proteinum lactis), white birch bark extract, willow bark, wintergreen oil, magic hazelnut or Witch hazel, xanthan gum, xanthan gum, yarrow extract, yeast, yerba mate, cassava, zinc oxide, zinc stearate.trimethylsiloxysilicate, tripeptide, turmeric, tyrosine, ubiquinone, grocery, undecylenyl phenylalanine, urea, uridine, fruit extract Vaccinium macrocarpon, vegetable glycerin, vetiver oil, vitamin A, vitamin B1-B12, vitamin C, vitamin C aster, vitamin D, vitamin E, vitamin K, vitamins, nutshell powder, water, wheat germ oil, whey protein (proteinum lactis), white birch bark extract, willow bark, wintergreen oil, magic hazelnut or Witch hazel, xanthan gum, xanthan gum, yarrow extract, yeast, yerba mate, cassava, zinc oxide, zinc stearate.Vitamin B1-B12, Vitamin C, Vitamin C Aster, Vitamin D, Vitamin E, Vitamin K, Vitamins, Nutshell Powder, Water, Wheat Germ Oil, Whey Protein (Protein Lactis), Bark Extract white birch, willow bark, wintergreen oil, magic hazel or witch hazel, xanthan gum, xanthan gum, yarrow extract, yeast, yerba mate, yucca, zinc oxide, zinc stearate.Vitamin B1-B12, Vitamin C, Vitamin C Aster, Vitamin D, Vitamin E, Vitamin K, Vitamins, Nutshell Powder, Water, Wheat Germ Oil, Whey Protein (Protein Lactis), Bark Extract white birch, willow bark, wintergreen oil, magic hazel or witch hazel, xanthan gum, xanthan gum, yarrow extract, yeast, yerba mate, yucca, zinc oxide, zinc stearate.

In some embodiments, MSM or the combination consists of, essentially consisting of or consist of, the combination with one, two, three, four, five or more of the ingredients identified above. In several embodiments, MSM inhibits microbial activity in the formulation. In other embodiments, MSM offers the same or better anti-microbial effect when used to replace a preservative in the formulation (some of which were previously identified). In certain embodiments, MSM offers the same or better antimicrobial effect when used with a reduced amount of preservative. In still other embodiments, the addition of MSM to a formulation having a preservative improves the effects of the preservative. The ingredients identified here can be used with MSM in a cosmetic formulation (eg, oral, injectable,

In some embodiments, the composition includes MSM, but is free from one or more of the following compounds: sulfates, GMOs, synthetic fragrances, synthetic dyes, formaldehyde, potassium sorbate, methyl paraben, methylchloroisothiazolinone, cocamidopropyl betaine, parabens, decyl polyglucose, polyaminopropyl biguanide, phenoxyethanol, sodium laureth sulfate, tetrasodium EDTA, decyl glucoside, polyethylene glycol, propylene glycol, phthalate ^ Sn ^ "In some modalities, the use of MSM allows the formulation of the formulation to be free. of any synthetic ingredient Still in other embodiments, the use of MSM allows the manufacture of a formulation that is free of any ingredient that causes allergy, immunosuppressant and / or inflammatory.

In several modalities, 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 multiple purpose function, according to some modalities. For example, not only does MSM inhibit the growth of undesirable microorganisms, MSM also beneficially affects the product to which it is added in various modalities (for example, MSM serves as an antioxidant, regenerative compound, anti-wrinkle compound, humectant, skin lightener , softening, circulation stimulating, neutralizing, reparative, hair / nail enhancer, healing catalyst, coating agent, etc., or combinations of two or more thereof). In several modalities, the anti-microbial properties of MSM increase the shelf life, half-life, efficacy and / or stability of the formulation (or the specific ingredient identified here). The use of MSM can be particularly beneficial in some embodiments, for cosmetic or other formulations that are shared by more than one person (for example, cosmetics employed by make-up artists or in cosmetics counters).

Cosmetics may include, but are not limited to lipstick, lip gloss, lip liner, lip booster, lip balm, lip and lip conditioner, foundation, powder, rouge, blush, sunscreen, mask, eyeliner, eye shadow, mineral eye powder, eye shine pencils, eyebrow pencils, enamel, concealer, skin care products (eg, microdermabrasion products, soothing gels) creams, lotions , serums, moisturizer, sunscreen, products for skin repair (for example, for acne, sunburn, wrinkles, dark circles), and scrubbers. Formulations for face, hair and body (for example, shampoo, soaps, conditioners, sprays, gels, serums, restorative treatments, deodorants, etc. ) are provided in several modalities. Cosmetics, such as cosmeto-ceutical and nutraceutical products, are provided in various embodiments of the invention. Dermal fillers and other dermatological products (such as hyaluronic acid, waglerin 1, acetyl hexapeptide-8, palmitoyl tetrapeptide-7, palmitoyl oligopeptide, liposomes, collagen, calcium hydroxyl-apatite, poly-lactic acid and botulinum toxin) are provided in several modalities. Formulations anti-wrinkle, anti-acne, anti-aging, exfoliating, moisturizing and anti-stretching, fragrances, mineral makeup, and primers, are provided in various modalities. Dermal gels, for cosmetic and medical use (for example, which inhibit or prevent microbial infection and / or wound healing) are provided in some embodiments. such as cosmeto-cerutic and nutra-cerutic products, are provided in various embodiments of the invention. Dermal fillers and other dermatological products (such as hyaluronic acid, waglerin 1, acetyl hexapeptide-8, palmitoyl tetrapeptide-7, palmitoyl oligopeptide, liposomes, collagen, calcium hydroxyl-apatite, poly-lactic acid and botulinum toxin) are provided in several modalities. Formulations anti-wrinkle, anti-acne, anti-aging, exfoliating, moisturizing and anti-stretching, fragrances, mineral makeup, and primers, are provided in various modalities. Dermal gels, for cosmetic and medical use (for example, which inhibit or prevent microbial infection and / or wound healing) are provided in some embodiments. such as cosmeto-cerutic and nutra-cerutic products, are provided in various embodiments of the invention. Dermal fillers and other dermatological products (such as hyaluronic acid, waglerin 1, acetyl hexapeptide-8, palmitoyl tetrapeptide-7, palmitoyl oligopeptide, liposomes, collagen, calcium hydroxyl-apatite, poly-lactic acid and botulinum toxin) are provided in several modalities. Formulations anti-wrinkle, anti-acne, anti-aging, exfoliating, moisturizing and anti-stretching, fragrances, mineral makeup, and primers, are provided in various modalities. Dermal gels, for cosmetic and medical use (for example, which inhibit or prevent microbial infection and / or wound healing) are provided in some embodiments. Dermal fillers and other dermatological products (such as hyaluronic acid, waglerin 1, acetyl hexapeptide-8, palmitoyl tetrapeptide-7, palmitoyl oligopeptide, liposomes, collagen, calcium hydroxyl-apatite, poly-lactic acid and botulinum toxin) are provided in several modalities. Formulations anti-wrinkle, anti-acne, anti-aging, exfoliating, moisturizing and anti-stretching, fragrances, mineral makeup, and primers, are provided in various modalities. Dermal gels, for cosmetic and medical use (for example, which inhibit or prevent microbial infection and / or wound healing) are provided in some embodiments. Dermal fillers and other dermatological products (such as hyaluronic acid, waglerin 1, acetyl hexapeptide-8, palmitoyl tetrapeptide-7, palmitoyl oligopeptide, liposomes, collagen, calcium hydroxyl-apatite, poly-lactic acid and botulinum toxin) are provided in several modalities. Formulations anti-wrinkle, anti-acne, anti-aging, exfoliating, moisturizing and anti-stretching, fragrances, mineral makeup, and primers, are provided in various modalities. Dermal gels, for cosmetic and medical use (for example, which inhibit or prevent microbial infection and / or wound healing) are provided in some embodiments. poly-lactic acid and botulinum toxin) are provided in various modalities. Formulations anti-wrinkle, anti-acne, anti-aging, exfoliating, moisturizing and anti-stretching, fragrances, mineral makeup, and primers, are provided in various modalities. Dermal gels, for cosmetic and medical use (for example, which inhibit or prevent microbial infection and / or wound healing) are provided in some embodiments. poly-lactic acid and botulinum toxin) are provided in various modalities. Formulations anti-wrinkle, anti-acne, anti-aging, exfoliating, moisturizing and anti-stretching, fragrances, mineral makeup, and primers, are provided in various modalities. Dermal gels, for cosmetic and medical use (for example, which inhibit or prevent microbial infection and / or wound healing) are provided in some embodiments.

In various modalities, products containing MSM can be shipped and / or stored under conditions of high temperature and high humidity, which would otherwise be favorable for microbial activity.

In some embodiments, products that include MSM are packaged in containers adapted for multi-use applications, and exposure to external microorganisms, such as from air or contact with a body part (eg, fingers). The use of MSM is particularly beneficial in several modalities, because it increases the shelf life of these products. In some embodiments, products that comprise MSM are also packaged in sealed containers for single use. In one embodiment, a single-use product (such as a seasoning package, dressing package, or dressing, a travel cosmetic package, etc.) will have a longer shelf life and / or will not require further refrigeration if MSM It is used in conjunction with the product and / or packaging.

In some modalities, MSM is incorporated directly into packaging materials to, for example, improve storage life. For example, MSM can be incorporated into food storage bags to inhibit microbial growth. In other modalities, MSM can be incorporated in containers and / or caps to improve the shelf life of food, cosmetics and other products by inhibiting unwanted microbial growth. Still in other modalities, MSM can be incorporated into sowing products in plates such as plastic wrap and plastic wrap.

In various embodiments, a composition for inhibiting microbial activity in a topical cream or ointment includes MSM, wherein MSM is configured to affect microbial contamination by inhibiting microbial activity. MSM is provided in a concentration of at least 5% according to one modality (eg, 5-10%, 10-16%, 16-20%, 20-30%, 30-40%, 40-50%, 50-75% or higher, and their overlapping intervals). In some examples, the composition is a preservative-free cream. In one embodiment, MSM inhibits microbial activity by at least 50% in the cream at room temperature.

In some embodiments, pharmaceutical compositions include MSM, DMSO, and / or anti-microbial agents or combinations thereof, which are formulated for use in human or veterinary medicine.

For example, the pharmaceutical compositions provided include about 0.01% MSM by weight to about 20% MSM by weight. In some embodiments, a pharmaceutical composition containing between about 0.01% to about 5% MSM by weight. Other embodiments contain between 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 modalities include approximately 10-16% MSM,

Additional exemplary anti-microbial agents which may be included in a described composition, include but are not limited to penicillin derivatives, cephalosporins, penems, monobactams, carbapenems, Beta-lactamase inhibitors and combinations thereof. Examples of penicillin derivatives include but are not limited to, aminopenicillins (e.g., amoxicillin, ampicillin, and epicillin); carboxypenicillins (for example, carbenicillin, ticarcillin, and temocillin); ureidopenicillins (for example, azlocillin, piperacillin and mezlocillin); mecillinam, sulbenicillin, benzathine penicillin penicillin G (benzylpenicillin), penicillin V (phenoxymethyl penicillin), Penicillin 0 (alilmercaptometilpenicilinico), procaine penicillin, oxacillin, methicillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, co-amoxiclav (amoxacillin plus clavulanic acid), and piperacillion. Examples of cephalosporins include, but are not limited to, cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefamandole, cefotetan, cefoxitin, cefranide, ceftriaxone, cefotaxime, cefpodoxime proxetil, ceftazidime, cefepime, cefoperazone, ceftizoxime, cefixime, and cephyriroma. 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 4,4-acid dioxide ([2S- (2-alpha, 3beta, 5alpha)] - 3-Methyl-7-oxo-3- (1H - 1.2.3- triazol-l-ylmethyl) -4-thia-l-azabicyclo [3.2.

Many antibiotics have an established minimum inhibitory concentration (MIC = Minimum Inhibitory Concentration) in which they are effective in reducing or killing certain bacteria. In some embodiments, a disclosed pharmaceutical composition includes an amount of Beta-lactam antibiotic equal to about 0.001 to 100 MICs for the particular bacterial pathogens described herein. In some embodiments, 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 approximately 90-100 MIC of a beta-lactam antibiotic. In some embodiments, the pharmaceutical composition comprises approximately 0.001, 0.01, 0.1, 0.5 or 1 MIC of a beta-lactam antibiotic.

Pharmaceutical compositions provided herein also include combinations of MSM and anti-microbial compounds, for example a combination of MSM and a beta-lactam antibiotic. In some embodiments, pharmaceutical compositions provided herein include 10-16% MSM and an amount of Beta-lactam antibiotic equal to 1 MIC for a bacterial pathogen with which the composition will contact.

A person skilled in the art will know the MIC of an antibiotic for a particular bacterial pathogen, or the skilled person 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 antibiotic. Particular bacterial pathogens are described here, for example the use of the Etest® antibiotic test system (bioMérieux, Durham, NC).

The dosage form of the pharmaceutical composition will be influenced by the selected administration mode. For example, in addition to injectable fluids, formulations for inhalation, topical, ophthalmic, peritoneal and oral can be employed. Preparations for inhalation may include aerosols, particles and the like. In general, the goal for the particular size for inhalation is approximately 1 μm or less in order for the pharmaceutical product to reach the alveolar region of the lung for absorption.

Pharmaceutical compositions that include MSM, DMSO, an antimicrobial agent or therapeutic 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 active ingredients, can be formulated with an appropriate solid or liquid carrier, depending on the particular mode of selected administration. Oral formulations can be liquid (for example, syrups, solutions or suspensions), or solid (for example, powders, pills, tablets or capsules). For solid compositions, conventional non-toxic solid carriers may include pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. Current methods for preparing these dosage forms are known, or will be apparent, to those skilled in the art.

For oral administration, the pharmaceutical compositions can take the form, for example, of tablets or capsules prepared by conventional means with acceptable pharmaceutical excipients such as binding agents (for example, pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration can take the form, for example, of solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. These liquid preparations can be made by conventional means with acceptable pharmaceutical additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring agents, colorants and sweeteners as appropriate. These liquid preparations can be made by conventional means with acceptable pharmaceutical additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring agents, colorants and sweeteners as appropriate. These liquid preparations can be made by conventional means with acceptable pharmaceutical additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring agents, colorants and sweeteners as appropriate. lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring agents, colorants and sweeteners as appropriate. lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring agents, colorants and sweeteners as appropriate.

For administration by inhalation, the compounds to be used in accordance with the present disclosure are conveniently supplied in the form of an aerosol spray presentation from pressure packs or a nebulizer, with the use of a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other convenient gas. In the case of a pressurized aerosol, the dose unit can be determined by providing a valve to supply a metered amount. Capsules and cartridges for use in an inhaler or insufflator can be formulated containing a powder mixture of the compound and a convenient powder base such as lactose or starch.

For topical administration, the compounds, for example, can be mixed with a liquid delivery agent for local administration. Therapeutically, therapeutically (such as DMSO, MSM and / or other therapeutic compounds as described herein) are easily soluble or suspended in water, and as such, this would be useful for delivery since the Water does not cause adverse effects in biological tissues. This allows sufficiently high doses to be administered locally or systemically, without secondary toxicity of 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 selected. The acceptable pharmaceutical carriers and excipients useful in this description are conventional. For example, parenteral formulations usually comprise injectable fluids which are pharmaceutically and physiologically acceptable fluid carriers, such as water, physiological saline, other solutions of balanced salt, aqueous dextrose, glycerol or the like. Excipients that may be included are, for example, proteins, such as plasma preparations or human serum albumin. 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. Current methods for preparing these dosage forms are known or will be apparent to those skilled in the art.

Pharmaceutical compositions that include an effective therapeutic amount of MSM, in some embodiments, will be formulated in unit dosage forms, suitable for individual administration of precise doses. The amount of MSM administered will depend on the subject to be treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing physician. Within these limits, the formulation to be administered will contain an amount of the active ingredient (s) in effective amounts to achieve the desired effect of the subject being treated.

Preparations for administration can conveniently be formulated to provide controlled release of the therapeutic agent or agents (eg, DMSO, MSM, Beta-lactam antibiotic and so forth). For example, the pharmaceutical compositions may be in the form of particles comprising a biodegradable polymer and / or a bioadhesive polymer and / or polysaccharide gelling agent, a. amphiphilic polymer, an agent that modifies the interphase properties of particles and an active pharmacological substance. These compositions exhibited certain biocompatibility characteristics that allow for an active substance to be released from the active substance. See, for example, U.S. Patent No. 5,700,486.

Polymers can be used for controlled release. Various degradable and non-degradable polymer matrices for use in controlled drug or drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537, 1993). For example, the block copolymer, polaxamer 407 exists as a viscous liquid, however mobile at low temperatures but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of interleukin-2 and recombinant urease (Johnston et al., Pharm. Res. 9: 425, 1992; Pee, J. Parent. Sci. Tech. 44 (2): 58, 1990).

Alternately, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm, 112: 215, 1994). In yet another aspect, liposomes are employed for controlled release as well as drug targets of lipid-encapsulated compounds (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA, 1993). Numerous additional systems for controlled delivery of therapeutic proteins are known (e.g., U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; US No. 4,837,028; U.S. Patent No. 4,957,735; and U.S. Patent No. 5,019,369; U.S. Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S. Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S. Patent No. 5,004,697; U.S. Patent No. 4,902,505; U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No. 5,254,342; and U.S. Patent No. 5,534,496).

In various embodiments, pharmaceutical compositions include DIVISO and / or MSM, and a therapeutic agent for treating an infectious disease, such as H1N1, herpes simplex virus, or HIV (HIV). In some embodiments, compositions that include DMSO and / or MSM are provided as an inhalant to treat an infectious disease. In some embodiments, pharmaceutical compositions for treating an 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 embodiments, the compositions are consumed orally to treat the infectious disease, while in some other embodiments, the compositions are applied topically. In a particular modality,

In some embodiments, pharmaceutical compositions that include DIVISO and / or MSM allow antibiotics (or other therapeutic agents) to penetrate lung tissue infected with an infectious disease. In one embodiment, these compositions include DMSO and / or MSM: (i) they allow antibiotics to reach deeper levels of infected tissue; (ii) they allow direct contact of infected tissue; (iii) they extend the time of exposure of the antibiotic to the infected tissue; and / or (iv) decrease the time to achieve a desired antibiotic effect. In one embodiment, 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 embodiments, pharmaceutical compositions including DMSO and / or MSM formulations, which others include antiparasitic agents effective to treat infections caused by parasites, such as nematodes, cestodes, trematodes, protozoa or amoebae.

In some embodiments, pharmaceutical compositions that include DMSO and / or MSM formulations also include antifungal agents that are effective in treating fungal infections, such as those that occur in a colibrillar, candidiasis, and Cryptococcus ργ • "* · ίί Ίγιγ γ τ-ρή ñ ~ (cryptococcal meningitis, for example).

In some embodiments, pharmaceutical compositions that include DMSO and / or MSM formulations further include antiviral agents that are effective in treating viral infections. In some modalities, specific classes of antiviral agents are used, they are used to treat infections caused by a particular type of virus. In some modalities, agents target HIV (HIV), herpes virus, hepatitis B or C virus, and influenza virus, such as H1N1.

In various embodiments, DMSO and / or MSM compositions include antibiotics that are effective in treating bacterial infections, for example by inhibiting bacterial growth, metabolism, proliferation, activity and / or function. In some modalities, bacteriostatic antibiotics are used, while in other modalities, bactericidal antibiotics are used. In still other embodiments, both bacteriostatic and bactericidal antibiotics are incorporated into a single formulation comprising DMSO and / or MSM. In some embodiments, antibiotics of one or more classes are incorporated into a composition including DMSO and / or MSM. In certain embodiments, a composition includes one or more than one: aminoglycoside, antamicine, carbacephem, carbapenem, cef alosporin (Ia, 4th or 5th generation), glycopeptides, macrolide, monobactam, penicillin, polypeptide,

In some embodiments, specific diseases are targeted by incorporating specific antibiotics into a described composition that includes DMSO and / or MSM. For example, macrolides, such as azithromycin or erythromycin, are incorporated into formulations used to treat respiratory or mycoplasma infections. Also, penicillins, such as amoxicillin or oxacillin, are incorporated into formulations used to treat a wide range of streptococcal infections.

Still in other embodiments, microorganisms that cause specific disease are targeted by the specific antibiotics incorporated in a formulation comprising DMSO and / or MSM. For example, aminoglycosides, such as neomycin, are incorporated into formulations used to treat infections of Escherichia coli. In several embodiments, antibiotics typically employed to combat microbial infections are employed. In certain embodiments, antibiotics including, but not limited to, isoniazid, rifampicin, pyrazinamide and ethambutol are incorporated into formulations comprising one or more of DMSO and MSM, and are used to treat an infectious disease, including an infectious disease resistant to drug.

In various embodiments, compositions are provided including DMSO, MSM and one or more of the following therapeutic agents: rifampicin, isoniazid, pyrazinamide and ethambutol. In other embodiments, compositions that include DMSO and at least one of rifampicin, isoniazid, pyrazinamide and ethambutol are provided. In further embodiments, compositions are provided which include MSM and at least one of rifampicin, isoniazid, pyrazinamide and ethambutol. In various embodiments, compositions that include DMSO and / or MSM in combination with rifampicin, isoniazid, pyrazinamide and ethambutol are provided to treat an infectious disease, including a drug-resistant infectious disease.

In some embodiments, rifampicin is provided in a total daily dose in the range of about 400 mg to about 800 mg per day. In some embodiments, rifampicin is provided in a total daily dose range of about 500 mg to about 700 mg per day, while still in other embodiments, it is provided in a total daily dose range of from about 550 to about 650 mg per day , including 560, 570, 580, 590, 600, 610, 620, 630 and 640 mg per day.

In some embodiments, it is possible to pre-stimulate a total daily dose range of about 100 mg to about 500 mg per day. In some embodiments, isoniazid is provided in a total daily dose range of about 200 mg to about 400 mg per day, while still in other embodiments, it is provided in a total daily dose range of about 250 mg to about 350 mg per day. day, including 260, 270, 280, 290, 300, 310, 320, 330 and 340 mg per day.

In some embodiments, pyrazinamide is provided in a total daily dose range from about 1.0 to about 4.0 g per day. In some embodiments, pyrazinamide is provided in a total daily dose range of from about 2.0 to about 3.0 g per day, while still in other modalities, it is provided in a total daily dose range of approximately 2.0 to 2.5 g per day, including 2.1, 2.2, 2.3 and 2.4 g.

In some embodiments, ethambutol is provided in a total daily dose range of about 0.5 to about 2.5 g per day. In some embodiments, ethambutol is provided in a total daily dose range of about 1.0 to 2.0 g per day, while still in other modalities, it is provided in a total daily dose range of approximately 1.0 a • • • • ••• rr * '-, + · - *. -> · ♦ · - · ---- approximately 1.5 g per day, including 1.1, 1.2, 1.3 and 1.4 g.

In some embodiments, pharmaceutical compositions including DMSO and / or MSM are used to pre-treat a patient suffering from an infectious disease, such as H1N1. In some embodiments, the doses of DMSO and / or MSM used for pretreatment of patients are in the range of about 10% to 50% weight by volume. In some embodiments, the dose of pretreatment 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 embodiments, approximately 50% to approximately 100% DMSO and / or MSM are employed. In several modalities,

In some embodiments, a pharmaceutical composition is prepared wherein antimicrobials are dissolved in DMSO and / or MSM prior to administration. This is particularly advantageous in certain embodiments because the antimicrobial and DMSO (and optionally MSM) can be administered to a subject by inhalation. Inhalants, according to some embodiments, provide direct access of DMSO and / or MSM to infected lung tissue to sensitize bacterial cells to the antibiotic.

In one embodiment, an inhalant is provided to target the site of infection (eg, lungs) of various infectious diseases. In some of these embodiments, the inhalant device comprises a nebulizer. In other modalities, an inhaler is used. In some embodiments, a pressure metered dose inhaler is employed, and the formulation is inhaled in the form of a liquid aerosol. In other embodiments, dry powder inhalers are employed, and the formulation is inhaled into a powdered aerosol form. In various embodiments, oral, intravenous, intramuscular or subcutaneous administration is employed in addition to or in place of inhalant therapy.

The ability to administer antimicrobial agents as an inhalant (e.g., in a powdered aerosol form) with DMSO and / or MSM is especially advantageous in some embodiments, because it allows for increased storage stability and prior packaged doses. This is particularly helpful for individuals in underdeveloped or developing nations who do not have regular access to health care facilities. Complete courses of treatment can be provided to an affected subject in a single visit to a health care practitioner without need for a stay in hospital or repeated visits. In various embodiments, the forms described herein are suitable for self-administration (e.g.

In certain embodiments, the total volume of inhaled DMSO and / or MSM is approximately 2-8 mL. In some embodiments, the total volume of inhaled DMSO and / or MSM is from about 2 mL to about 4 mL. In some embodiments, the total volume of inhaled DMSO and / or MSM is from about 6 mL to about 8 mL. In still other embodiments, the total volume of DMSO and / p MSM inhaled is from about 3 mL to about 7 mL, including 4, 5 and 6 mL. Thus, in some embodiments, the concentration of DMSO administered by inhalation is in the range of about 65% to about 95%, including 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92 , 93 and 94%.

In several modalities, MSM is included with the DMSO and inhaled antimicrobial compounds. In certain embodiments, the amount of MSM inhaled is in the range of about 0.01% by weight to about 70% by weight of the inhalant. In other embodiments, the inhaled formulation contains between about 0.01% and 10% MSM by weight. Other embodiments contain between about 10 and 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% of MSM. Still other embodiments 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% of MSM or about 65-70% of MSM. Thus, in some embodiments of the inhaled formulation containing MSM, the concentration of DMSO administered is in the range of 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 various embodiments, the use of MSM reduces the amount of DMSO required to achieve a comparable effect and / or improves the efficacy of DMSO by at least 10%, 25%, 50%, 100%, 2-fold, 3-fold, 5-times, 10-times, 50-times or 100-times. In other embodiments, the use of MSM reduces the amount of a therapeutic agent required to achieve a comparable effect and / or improves the efficacy of the therapeutic agent by at least 10%, 25%, 50%, 100%, 2-fold, 3 - times, 5-times, 10-times, 50-times or 100-times. In additional embodiments, the use of DMSO reduces the amount of a therapeutic agent required to achieve a comparable effect and / or improves the efficacy of the therapeutic agent by at least 10%, 25%, 50%, 100%, 2-fold,. .Mb · times, 50-fold or 100-fold. In still other modalities,

In various embodiments, a pre-treatment formulation including DMSO, alone or in combination with MSM, is administered to a subject in intravenous, intramuscular, topical or oral form to improve the effects of an inhalant therapy comprising DMSO and / or MSM with agents therapeutic, such as antibiotics. Previous treatment with DMSO, alone or in combination with MSM, improves the therapeutic effects of the inhalant in at least 10%, 25%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 50-fold or 100-fold.

In various embodiments, subjects having an infectious disease 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 embodiments, the formulation additionally includes other therapeutic agents, carriers or excipients. In one embodiment, the formulation further includes arginine, vitamin D, antioxidants, macrolides, linezolid, thioacetazone, thioridazine or combinations thereof. ~ 'DMSO easily dissociates the integrity of many materials (particularly plastics and polymers used in the manufacture of disposable medical equipment). Accordingly, various embodiments of the invention comprise devices to facilitate the storage and administration of DMSO. In some modalities, DMSO is stored in glass bottles and administered through non-reactive tubing. In other modalities, inhaling devices are specially designed to be resistant to DMSO. In some embodiments, portions of the inhalation devices are disposable or replaceable. According to various embodiments, formulations comprising DMSO are manufactured, stored and / or administered using materials and devices described in US Patent Application Serial No. 12/066, 480, which is the entry into the National Phase of International Application No .: PCT / US06 / 35499, filed on September 11, 2006, which is hereby incorporated by reference in its entirety. Inhalant devices are specially designed to be resistant to DMSO. In some embodiments, portions of the inhalation devices are disposable or replaceable. According to various embodiments, formulations comprising DMSO are manufactured, stored and / or administered using materials and devices described in US Patent Application Serial No. 12/066, 480, which is the entry into the National Phase of International Application No .: PCT / US06 / 35499, filed on September 11, 2006, which is hereby incorporated by reference in its entirety. Inhalant devices are specially designed to be resistant to DMSO. In some embodiments, portions of the inhalation devices are disposable or replaceable. According to various embodiments, formulations comprising DMSO are manufactured, stored and / or administered using materials and devices described in US Patent Application Serial No. 12/066, 480, which is the entry into the National Phase of International Application No .: PCT / US06 / 35499, filed on September 11, 2006, which is hereby incorporated by reference in its entirety.

In certain embodiments, the delivery device provides droplets or particles of the inhaled formulation, of a size capable of reaching the bronchioles of the patient's lungs. In some embodiments, the delivery device is synchronized with a patient's breathing rate to bring the formulation to the bronchioles. Inhalant therapy according to one modality, allows more direct administration of the inhaled formulation to infected lung target tissues. Direct shipping is advantageous in some embodiments because it allows reduction of the amount of antimicrobial compounds incorporated in the formulation while maintaining or improving the efficacy of the formulation against infectious microorganisms. In other modalities, Direct administration increases the efficacy of a given antimicrobial regimen against one or more drug-resistant strains of microorganisms. Direct delivery or direct targeting, in accordance with other modalities, minimizes side effects by minimizing contact with non-target or non-target tissue.

The small size of droplets or particles that are provided according to some modalities reduces the volume of DMSO and / or MSM that is administered compared to traditional fan therapy. For example, in one embodiment, the use of an inhalant device (e.g., nebulizer) will be effective with about 6 mg to about 25 mg of DMSO and / or MSM daily, compared to 50-100 mg daily when administered to through certain other routes. Reducing DMSO is beneficial in some modalities because it reduces undesirable side effects and odors. In ntrag m ^ j1iiiinp) higher amounts of DMSO are used and tolerated.

In several embodiments, the addition of MSM unexpectedly reduces the unpleasant odor that is normally experienced with the use of DMSO. For example, in certain embodiments, the DMSO and MSM formulations do not produce perceptible odor after use. In some other embodiments that have DMSO concentrations approaching or exceeding 50%, the MSM combination in the formulation reduces or eliminates the DMSO-based odor. This result is unexpected, since the use of DMSO is usually associated with a strong unpleasant odor.

In some embodiments, 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 throat. , since the droplets or particles travel deeper into the lungs of the patient. In some embodiments, the depth of travel of the droplets or particles increases the concentration of the antibiotics dissolved in the lungs of the patient.

In various embodiments, compositions of DMSO and / or MSM are combined with therapeutic agents (such as antibiotics) and are provided as an aerosol to deliver locally active drugs to the respiratory system to treat a respiratory disease. In one embodiment, the lower airways are contacted (or brought into contact exclusively) with the composition. In other embodiments, the composition is used to systemically treat diseases. For systemically active drugs, the aerosol particles are sized to reach the alveolar surface in peripheral areas of the lung.

In some embodiments, 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 embodiment, inhalation supply provides a large area of ​​lung absorption. For locally acting drugs, the onset of action is immediate in some modalities. Inhaled formulations systemically active, according to some modalities, quickly reach the bloodstream. Inhalation therapy provides a therapeutic effect within approximately 1-90 minutes in some modalities. In one embodiment, DMSO and / or MSM improve the bioavailability of the therapeutic agent. In a further embodiment, DMSO and / or MSM reduces degradation of the therapeutic agent. In another modality, The aerosol formulations described herein reduce gastrointestinal side effects or skin irritation that may occur with oral or topical treatment. --P-

In several embodiments, the inhalant particles are sized to minimize the deposition of these particles by inertial impact in the upper respiratory tract without reaching the site of action. In various embodiments, the particles are sized to minimize deposit in the mouth and throat, thereby minimizing swallowing / swallowing and ingestion and undesired systemic or local side effects. In various embodiments, the particles are less than 2, 5 or 10 μιη. In one embodiment, the particles are approximately 3-5 μm and are transported to the smaller bifurcations and airways of the bronchi and bronchioles. In another embodiment, the particles are less than 3 pm and follow the air flow to the alveoli. In several modalities, the use of DMSO and / or MSM makes it possible to optimize the particle size of the therapeutic agent. In this way, diseases such as an infectious disease can be treated more effectively. Furthermore, in several modalities, the use of DMSO and / or MSM sensitizes antibiotics to drug-resistant microorganisms.

In various embodiments, DMSO and / or MSM form a solution, mixture, emulsion, suspension or other convenient combination with the therapeutic agent. In a modality, homogenization, sonication, ρ »· Α ^ Γ, ί · -1η, ·; ^ 4- ^ Λΐα - ,. £] · ιιΐΓίη Ha aH- ^ shear or cut, or other mechanical methods are used to combine the therapeutic agent with DMSO and / or MSM. In other embodiments, the therapeutic agent readily dissolves in DMSO. Unlike other strong solvents, DMSO is not harmful to lung tissue. Thus, DMSO is especially advantageous in some embodiments because it can both dissolve the therapeutic agent and deliver the agent without damage to the lung tissue. In some embodiments, DMSO dissolves at least 50%, 75%, 90%, 95% or 99% of the therapeutic agent, and in one embodiment,

In some embodiments, sprays, gels or fabrics comprising DMSO, alone or in combination with MSM, and antibacterial agents, are provided to disinfect medical equipment, surfaces and the body, to minimize the spread of infectious disease.

In various embodiments, a pharmaceutical composition comprising DMSO and / or MSM and antimicrobial agents is used as a treatment for an infectious disease.

epidemic typhus, infectious erythema, exanthem subitum, f asciolopsiasis, fa.sr.i ni os is. __ i η, Βοτηη, ί g,. Jami .1 i ar fatal (FFI = Fatal Familial Insomnia), filariasis, food poisoning, free-living amoebic infection, Fusobacterium infection, gas gangrene (Clostridial myonecrosis), geotrichosis, Gerstmann-Stráussler-Scheinker syndrome (GSS) = Gerstmann-Straussler-Scheinker), giardiasis, amormado, gnatostomiasis, gonorrhea, inguinal granuloma (Donovanosis), Group A streptococcal infection, Group B streptococcal infection, Haemophilus influenzae infection, hand, foot and mouth disease (HFMD = Hand, Foot and Mouth Disease), Hantavirus, Helicobacter pylori infection, hemolytic-uremic syndrome (HUS = Hemolytic-Uremic Syndrome),

In certain embodiments, the formulations described herein are also effective in treating one or more of the following infectious diseases: Epstein-Barr virus, infectious mononucleosis (monkey), influenza (influenza),

Isosporiasis, Kawasaki disease, Kingella kingae infestana, Kuru, Lassa fever, legionellosis, leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease, lymphatic filariasis, lymphocytic choriomeningitis, malaria, Marburg haemorrhagic fever (MHF), measles, melioidosis ( Whitmore's disease), meningitis, meningococcal disease, Metagonimiasis, microsporidia microsporidiosis, molluscum contagiosum (MC), mumps, murine typhus, Mycoplasma pneumonia, mycetoma, myiasis, neonatal conjunctivitis, onchocerciasis (river blindness), paracoccidioidomycosis (South American blastomycosis) , paragonimiasis, pasteurellosis, pediculosis capitis (head lice), Pediculosis corporis (body louse), Pediculosis pubis (crabs, pubic lice), pelvic inflammatory disease (PID), whooping cough (pertussis), plague,pneumococcal infection, Pneumocystis carinii pneumonia (PCP), pneumonia, poliomyelitis, poliovirus, primary amebic meningoencephalitis (MAP), progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite fever, respiratory syncytial virus, Rinosporidiosis, rhinovirus infection , Rickettsial infection, rickettsial disease, Rift Valley fever (FVR), Rocky Mountain spotted fever FMR), rotavirus infection, rubella, salmonellosis, Severe Acute Respiratory Syndrome (SARS), scabies, schistosomiasis, sepsis, shigellosis, herpes (herpes zoster), smallpox, sporotrichosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, syphilis, taeniasis, tetanus (trismus), tinea barbae (folliculitis), tinea capitis (tinea capitis), body ringworm ,ringworm of the body), tinea cruris (inguinal ringworm), manuum ringworm of the hand), ringworm, tinea pedis (athlete's foot), Tinea unguium (onychomycosis), tinea versicolor (pityriasis versicolor), toxocariasis (ocular larva migrans (OLM) )), toxocariasis (visceral migrans larvae (VLM)), toxoplasmosis, trichinosis, trichomoniasis, trichuriasis (T. trichiura infection), tularemia, urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever , White Stone, Yersiniosis, yellow fever, and Zygomycosis.trichinosis, trichomoniasis, trichuriasis (infection by T. trichiura), tularemia, infection by ureplasma urealyticum, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, White Stone, Yersiniosis, yellow fever, and Zygomycosis.trichinosis, trichomoniasis, trichuriasis (infection by T. trichiura), tularemia, infection by ureplasma urealyticum, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, White Stone, Yersiniosis, yellow fever, and Zygomycosis.

In various embodiments, the compositions described herein are particularly effective in treating one or more infectious diseases that are resistant to drug therapies. In addition to those infectious diseases cited above, which may already be or may become resistant to drugs in the future, certain modalities are effective to treat, among others, resistance to drugs of: measles, tetanus, malaria, upper respiratory infections and lower, hepatitis, typhoid fever, infection of Staphylococcus aureus intermediate vancomycin / glycopeptide, enterococci r ^ «? i - vancomycin, methicillin-resistant Staphylococcus aureus (MRSA = Methicillin-Resistant Staphylococcus Aureus) and streptococcus pneumoniae.

In some embodiments, the treatment of an infectious disease comprises the pretreatment of a patient with DIVISO, followed by the administration of a pharmaceutical composition comprising DMSO and antimicrobial agents. In other embodiments, the treatment of an infectious disease comprises the pretreatment of a patient with DMSO, followed by the administration of a formulation comprising DMSO, MSM, and antimicrobial agents. In some embodiments, pretreatment with DMSO is administered intravenously by a rapid drip IV catheter. In other embodiments, the DMSO is delivered with a bolus IV injection. Still in another modality, the previous treatment with DMSO is not carried out. Additional treatment compositions additionally include MSM,

In various embodiments, compositions that include DMSO and anti-microbial agents, or DMSO, MSM and antimicrobial agents are administered orally, intravenously, intramuscularly or subcutaneously. However, as the site of infection of several infectious diseases are the lungs in some modalities, the formulations -aH ™ -; no other pnr __ inhalation. In some of these embodiments, the inhalant medium comprises a nebulizer. In other modalities, an inhaler is employed.

In various embodiments, the subjects are pretreated with DMSO using intravenous DMSO by rapid drip within, for example, a period of ten minutes. In one embodiment, DMSO will be provided in glass bottles with proprietary non-reactive tubing. The subjects will then receive antibiotics dissolved in DMSO at a dose of 3 mL through an inhaler or oral spray, three times a day with meals. In one embodiment, pretreatment with DMSO 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 embodiment, 56 mg of DMSO in 200 mL of 5% dextrose and water are provided. In one embodiment, the following antibiotics are provided: rifampicin, isoniazid, pyrazinamide, and ethambutol. In one modality, Approximately 600 mg of rifampicin, 300 mg of isoniazid, 2.4 g of pyrazinamide, and 1.2 g of ethambutol are administered per day, through an inhaler / nebulizer or oral spray supplied in a dose of 3 mL, three times a day. In one embodiment, antibiotics are combined with DMSO for delivery by inhalation, with or without prior treatment with DMSO. Previous treatment with MSM is also provided in several modalities. Preliminary intravenous treatment of DIVISO, MDM or the combination of the two is provided in some modalities. In some examples, pretreatment formulations include therapeutic agents. In one embodiment, antibiotics are combined with DMSO for delivery by inhalation, with or without prior treatment with DMSO. Previous treatment with MSM is also provided in several modalities. Preliminary intravenous treatment of DIVISO, MDM or the combination of the two is provided in some modalities. In some examples, pretreatment formulations include therapeutic agents. In one embodiment, antibiotics are combined with DMSO for delivery by inhalation, with or without prior treatment with DMSO. Previous treatment with MSM is also provided in several modalities. Preliminary intravenous treatment of DIVISO, MDM or the combination of the two is provided in some modalities. In some examples, pretreatment formulations include therapeutic agents.

In several modalities, therapeutic effects are obtained within two weeks of treatment, within two months of treatment, and / or within six months of treatments. Other therapeutic windows are also provided.

In some modalities, patients previously treated with DMSO show additional improvement than those treated with DMSO inhalant and antibiotics without prior intravenous DMSO treatment. In some modalities, patients treated with DMSO with DMSO inhalant and antibiotics show further improvement than those treated with antibiotics alone. In several embodiments, the addition of MSM to the formulation improves the therapeutic effects or reduces side effects. In one modality, MSM is used only as pre-treatment.

In various embodiments, the compositions described herein are employed not only to treat undesirable symptoms and diseases, but may also act as a preventive agent. For example, a formulation can be taken on a regular basis to prevent the onset of a disease. In one modality, in a r-jesnn subject (for example, family members or subjects who are exposed to patients who have an infectious disease) they are given lower doses of DMSO and / or MSM and antibiotics to prevent the onset of the illness.

IV. Methods of Use of MSM

Here methods are described for using any of the described 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 improving microbial activity are described as including methods for improving microbial growth, fermentation efficiency, culture efficiency, microbial survival or any combination thereof. Methods for inhibiting microbial activity are also described, which include methods for inhibiting microbial growth (such as bacterial growth) or infection. In some modalities, MSM selectively improves the activity (for example, growth) of a microorganism (such as a probiotic microorganism) and inhibits the activity of undesirable microbes (such as undesirable fungal or bacterial activity). A. Methods for Improving Microbial Activity

Methods for improving microbial activity are described. In one embodiment, a method for improving the activity of a microorganism includes providing microorganisms, a medium capable of supporting the growth of microorganisms, and MSM in an amount sufficient to improve activity (eg, fermentation efficiency, growth, culture efficiency). and / or microbial survival) of the microorganisms and contacting the MSM with the medium, thereby improving the growth of the microorganisms in the medium. It is contemplated that MSM can be added to the medium before, concurrently with or after the medium comes in contact with the microorganisms. In a particular embodiment, MSM is provided at a concentration of approximately 0. 04% up 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 about 0.5% to about 5% MSM) is used to improve microbial growth. For example, MSM is employed to improve the fermentation efficiency, such as to improve the fermentation efficiency associated with the production of beer, cider, wine, a biofuel, dairy product or any combination thereof. In several examples, MSM improves the production of a certain food or beverage processing process, which is based on microorganisms, such as brewing, winemaking, baking, marinating, processing dairy products and the like. In additional examples, MSM is used to improve the growth of one or more probiotic microorganisms or a microorganism in a diagnostic test sample. In still further examples, MSM is employed to improve the efficiency of culture and / or survival of microorganisms.

i. Methods for Improving Fermentation Efficiency of Microorganism with MSM

In several modalities, MSM is used to facilitate the production of energy. Thus, methods for improving energy production are described herein, including methods for improving fermentation efficiency of microorganisms. For example, microorganisms can be used in a fermentation process to produce ethanol and in biogas reactors to produce methane. Fermentation is a process that generates energy with which organic or synthetic molecules are degraded through metabolism by microorganisms. Some forms of microorganisms, such as bacteria or yeast, can be used to convert various forms of agricultural and urban waste into useful fuels. Microorganisms can be used as living microbial fuel cells. In some modalities, MSM improves bacterial growth and metabolism. In some modalities, MSM improves the production of bacterial energy. In some modalities, MSM improves the development and metabolism of 1 &lt;5 VdUlfLct. In some modalities, MSM improves the production of yeast energy.

In several embodiments, MSM is used to activate or improve one or more of the following: (i) fermentation of ethanol or other anaerobic respiration employed primarily by yeast when oxygen is not present in sufficient quantity for normal cellular respiration; (ii) production of fermentative hydrogen; (iii) industrial fermentation or other decomposition and re-assembly of biochemicals for the industry; (iv) the conversion of carbohydrates into alcohols or acids under anaerobic conditions used for food preparation (eg, breads, dairy products, beans, vinegar, sour cabbage, quinchi, fish and tofu); (v) fermentation for making brandy, whiskey, vodka, beer, wine or cider, (vi) fermentation to produce glucosamine;

In one embodiment, a method for improving the efficiency of fermentation of a microorganism includes bringing average contact containing a microorganism capable of fermenting with MSM, wherein the MSM is provided at a concentration of about 0.04% to about 5% by weight of medium or at a concentration of about 0.04% to about 5% by weight of the moisture content

• S ···· "I ** · '···· Μ'> 1 I I of the medium, wherein the concentration of MSM increases the efficiency of fermentation of the microorganism compared to the efficiency of fermentation in the absence of MSM .

In one embodiment, the improved fermentation efficiency is indicated by an increase of at least 10%, such as approximately an increase from 20% to 80%, approximately an increase from 30% to 50%, includingapproximately an increase of 10%, approximately to 20%, approximately to 30%, approximately to 40%, approximately to 50%, approximately to 60%, approximately to 70%, approximately to 80%, approximately to 90%, approximately to 100 %, approximately 150%, approximately 200%, approximately a 300% increase in alcohol, carbon dioxide or acid production in the presence of MSM by the microorganism compared to production of alcohol, carbon dioxide or acid in the absence of MSM. For example, the method to improve fermentation efficiency is for the production of beer, cider, wine, biofuel, bread, dairy products or any combination thereof. In some examples, improving the fermentation efficiency includes an increase of at least 10%, such as approximately an increase from 20% to 80%, approximately an increase from 30% to 50%, including about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% approximately 90%, approximately 100%, approximately 150%, approximately 200%, approximately 300% in production of ethanol, methanol or a combination thereof as compared to the production of ethanol, methanol or a combination thereof. same in the absence of MSM. In a particular example, the microorganism is yeast and the method for improving the fermentation is for the production of beer. In another example, the microorganism is algae and the method to improve the fermentation is for the production of a biofuel. about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, approximately 200%, approximately 300% in production of ethanol, methanol or a combination thereof as compared to production of ethanol, methanol or a combination thereof in the absence of MSM. In a particular example, the microorganism is yeast and the method for improving the fermentation is for the production of beer. In another example, the microorganism is algae and the method to improve the fermentation is for the production of a biofuel. about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, approximately 200%, approximately 300% in production of ethanol, methanol or a combination thereof as compared to production of ethanol, methanol or a combination thereof in the absence of MSM. In a particular example, the microorganism is yeast and the method for improving the fermentation is for the production of beer. In another example, the microorganism is algae and the method to improve the fermentation is for the production of a biofuel. about 90%, about 100%, about 150%, about 200%, about 300%, production of ethanol, methanol or a combination thereof compared to production of ethanol, methanol or a combination thereof in the absence of MSM. In a particular example, the microorganism is yeast and the method for improving the fermentation is for the production of beer. In another example, the microorganism is algae and the method to improve the fermentation is for the production of a biofuel. about 90%, about 100%, about 150%, about 200%, about 300%, production of ethanol, methanol or a combination thereof compared to production of ethanol, methanol or a combination thereof in the absence of MSM. In a particular example, the microorganism is yeast and the method for improving the fermentation is for the production of beer. In another example, the microorganism is algae and the method to improve the fermentation is for the production of a biofuel. In a particular example, the microorganism is yeast and the method for improving the fermentation is for the production of beer. In another example, the microorganism is algae and the method to improve the fermentation is for the production of a biofuel. In a particular example, the microorganism is yeast and the method for improving the fermentation is for the production of beer. In another example, the microorganism is algae and the method to improve the fermentation is for the production of a biofuel.

In some embodiments, improving the fermentation efficiency includes an increase of at least 10%, such as approximately an increase from 20% to 80%, approximately an increase from 30% to 50%, including approximately an increase of 10%, approximately an increase of 20%, approximately an increase of 30%, approximately to 40%, approximately to 50%, approximately to 60%, approximately to 70%, approximately to 80%, approximately to 90%, approximately to 100%, approximately 150%, approximately 200%, approximately 300% in carbon dioxide production in the presence of MSM by the microorganism compared to carbon dioxide production in the absence of MSM. In a particular example, the microorganism is yeast and the method for improving 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 improving the fermentation efficiency include an increase of at least 10%, such as approximately an increase from 20% to 80%, approximately an increase from 30% to 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%, approximately 200%, approximately 300% in lactic acid production in the presence of MSM by the microorganism compared to lactic acid production in the absence of MSM.

In some embodiments, the concentration of effective MSM to improve the fermentation efficiency is about 0.04% to about 5%, such as about 0.1% to about 4%, 0.5% to about 3%, about 1% to about 2%, which includes about 0.04%, 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 modalities,

In some examples, the means for the method of improving the efficiency of a microorganism includes a concentration of sodium chloride at least 5% of the total moisture content of the medium, such as about 1% to about 3% sodium chloride. , including 0%, 0.1%, 0.3%, 0.5%, 0.75%, 1%, 2%, 2.5%, 3% or 4%.

In a certain modality, MSM is used for beer production. 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 activation of the yeast culture, improve fermentation, reduce potential environmental contamination (such as undesirable microorganisms transported by air) or a combination thereof. For example, an increase in the efficiency to activate the yeast (such as an increase in the efficiency of the start-up process), an increase in efficiency of the fermentation process or its combination, is indicated by an increase of at least 10%, such as approximately an increase from 20% to 80%, approximately an increase from 30% to 50%,

In several modalities, MSM is used to improve the activity of algae, including the fermentation process associated with the generation of biofuel from the use of algae. In one modality, this is particularly beneficial for the cultivation of algae (algal farms), to make or process vegetable oil, biofuel, 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 higher. MSM can be particularly advantageous because by improving the activity of algae (such as algae growth), The production of biofuels can be scaled, economically competitive and / or commercially viable. In one modality, MSM improves the process by which the algae product is harvested and converted into a biofuel. In other modalities, MSM improves the process by which the carbohydrate content of the algae is fermented into bioethanol and biobutanol. In some modalities, MSM improves the algae process by (i) increasing the yield of algae, (ii) forming more robust algal colonies, (iii) shortening the time for harvest, (iv) shortening the fermentation time, (v) ) improve the fermentation and / or otherwise support or improve the growth, reproduction, proliferation, survival rate, metabolism, vitality, robustness, action and / or function of the algae. Seaweed, include but are not limited to, Botryococcus braunii, Chlorella, Dunaliella tertiolecta, Gracilaria, Pleurochrysis carterae, and Sargassum, are improved by MSM according to several modalities. ii. Methods to Improve Microbial Growth with

MSM

In some embodiments, the addition of MSM is particularly advantageous because MSM promotes the growth of certain microorganisms (e.g., probiotics). In some embodiments, microorganisms developed with a medium composition comprising MSM have a higher growth rate curve compared to a comparable composition without MSM. In some embodiments, microorganisms developed with a composition comprising MSM have an increased total population density as compared to a comparable composition without MSM. In certain embodiments, MSM significantly improves the simultaneous growth of one or more microorganisms. In some embodiments, medium supplemented with an MSM composition to improve microbial activity (such as a concentration range of about 0.

Some microorganisms are anaerobic organisms (anaerobes). Anaerobes do not require oxygen for growth. Anaerobes can be used for fermentation and / or culture. In some modalities, MSM has a positive impact on anaerobes, such as Bifidobacterium, among others. In some of these modalities, MSM has a greater positive impact on the growth of anaerobes than other microorganisms. In other modalities, MSM has a greater positive impact on growth of aerobic bacteria compared to other microorganisms. Still in other modal ^ da ^ te-s ^ 1 os. laarnhi n «and, anaerobes are both positively impacted by the presence of MSM.

Bacteria can generally be classified as gram-positive or gram-negative, depending on the structure of your 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, green bacteria sulfur and greens that are not sulfur. Enteric bacteria are Gram-negative in the form of rods; most occur normally or pathogenically in the intestines of humans and other animals. In some modalities, MSM has a positive impact on the growth of gram positive bacteria. In other modalities, MSM has a positive impact on the growth of gram negative bacteria. In some of these modalities, MSM has a greater positive impact on gram-negative bacteria than on gram-positive bacteria. In other modalities, MSM has a greater positive impact on gram-positive bacteria than on gram-negative bacteria. Still in other modalities, MSM has a positive impact on both gram-negative and gram-positive bacteria.

Probiotics include live microorganisms that are considered healthy for the host organism. Lactic acid bacteria (LAB = Lactic Acid Bacteria) and bifidobacteria are common types of microbes used as probiotics. Certain yeasts and bacilli are also used. In several modalities, MSM is used to improve the survival or growth of at least one probiotic. Survival effect of probiotic organisms can be measured in three points according to some modalities: survival, colonization and production of lactic acid. 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, do not provide benefit. In this way in some modalities, MSM positively affects probiotic survival. In certain modalities, MSM improves initial survival when bacteria are exposed to a new environment. Thus, in these embodiments, a product comprising a probiotic and MSM establishes a larger or more healthy population (or both) of probiotic bacteria in the intestine compared to probiotic products only. In certain modalities, MSM improves the long-term survival of probiotics. Thus, in these modalities, a product comprising a probiotic and MSM establishes a longer duration, and based on growth, a greater population of probiotic bacteria in the intestine compared to probiotic products alone. Of those probiotic bacteria that reach the intestine alive, those that colonize, (multiply in) the intestine in general provide benefit. In this way, in several modalities, MSM improves the speed and frequency of probiotic multiplication. Still in other modalities, MSM increases the production of lactic acid.

In some modalities, MSM has a positive impact on probiotic growth. In some modalities, MSM has a positive impact on the microbial flora of the gastrointestinal tract. In some of these modalities, MSM has a positive impact on intestinal health. In some embodiments, foods containing probiotics are supplemented with MSM, and the resulting probiotic levels achieved in the gastrointestinal tract are greater than after ingestion of the probiotic-containing food alone. In some of these modalities, the addition of MSM results in a higher level of probiotic organism in a shorter time frame with ingestion of food containing probiotic alone. In some modalities, probiotics require 24 to 48 hours before effects are observed,

Bacterial growth typically has an initial latent phase where the bacteria adjust to the environment, before entering the log phase, where the cells double. After the log phase, there is a stationary phase. During the stationary phase, the rate of growth slows down as a result of nutrient depletion and accumulation of metabolic by-products. This phase is reached as microbes begin to deplete the resources that are available to them. This phase is a relatively constant value since the rate of microbial growth is equal to the rate of microbial death. In the death phase, bacteria typically deplete nutrients and population numbers fall.

In some modalities, MSM impacts the latency phase, log phase, stationary phase, death phase or any combination thereof. In certain embodiments, MSM shortens the latency phase, such that bacteria, such as probiotic bacteria, start the log phase at a previous time. In several modalities, MSM prolongs the stationary phase. In certain modalities, the mortality, index or mortality rate is slowed down in the presence of MSM. Certain embodiments of the description as described herein positively affect one or more, and in certain embodiments all phases of the growth of probiotic bacteria.

In some modalities, MSM impacts the metabolism of microbes (eg, probiotics), in the later stage. During the latency phase, the ..... mic-r-obios mature (grow in size) and are not yet able to divide (in this way without growth in number). During the latency phase of the microbial growth cycle, the synthesis of RNA, enzymes and other molecules occurs. In some modalities, MSM decreases the duration of the latency phase by accelerating the maturation (and adaptation of microorganisms to environmental stressors) of the microorganisms, thus allowing microbial division rather than in MSM-free medium.

In some embodiments, the MSM supplement results in an increase in the log phase of microbial growth (e.g., probiotics). The exponential phase (sometimes called the log phase) of growth is a period characterized by cellular duplication. The number of new microbes that appear per unit of time is proportional to the present population. If the growth is not limited, the duplication will continue at a constant speed so that both the number of cells and the rate of population increase doubles with each consecutive period of time. The exponential growth can not continue indefinitely, however, because the medium is soon depleted of nutrients and enriched with waste. In some modalities, MSM increases the total duration of the exponential phase. In other modalities, the presence of MSM in the medium of f-rorimiont-n leads to the microbial on the exponential phase more rapidly than the microorganisms in MSM-free medium. The initial growth environment with medium supplemented with MSM can lead to multiplication and cell survival.

In several modalities, MSM affects the stationary phase of microbial growth (eg, probiotic). In one example, the media supplement MSM extends the stationary phase by microbes in comparison to MSM-free medium.

In some modalities, MSM improve probiotic growth, which in turn attests and takes nutrients from unwanted microbes. In other modalities, MSM improves the probiotic activity, which in turn improves the production of lactic and acetic acids to reduce the ambient pH and inhibit the activity of undesirable bacteria. In additional modalities, MSM improves probiotic activity, which in turn stimulates the production of imunomodulatory agents (e.g., cytokines), thereby improving the immune response. In certain modalities, MSM improves probiotic activity, which in turn improves bactericidal activity with respect to undesirable microbial contamination. In one modality, MSM improves probiotic growth at a higher rate than undesirable microbes,

Without being bound by a particular theory, in several modalities, 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 better able to adapt and / or recover from environmental changes. In some embodiments, MSM serves as a substrate or cofactor for microbial metabolism and / or anaplastic biochemical pathways. In some modalities, MSM positively impacts the latency growth phase. In some modalities, MSM increases the log phase of microbial growth. In still further modalities, MSM increases the duration of the stationary phase of microbial growth. In some modalities, MSM decreases the rate of population decline of certain microbes. In certain modalities, MSM provides a selective or semi-selective growth environment, such that certain microbes grow more rapidly (or to reach a larger population size, or both) compared to other microbial species. In certain modalities, MSM impacts the metabolic activity of microorganisms, while in other modalities, MSM creates an environment that leads more to microbial growth.

As such, methods of microbial growth are provided. In some embodiments, methods for improving microbial growth include in vitro methods to enhance the growth of one or more microorganisms. In one example, in vitro methods to enhance 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 about 0.4% up to about 5% by weight of the medium or by weight of moisture content of the medium in this manner improving the growth of one or more microorganisms in vitro compared to growth of the one or more microorganisms in vitro in the absence of MSM. It is contemplated that similar methods can be employed 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 the weight of the microorganism or number of cells such as an increase of at least 10%, such as approximately an increase from 20% to 80%, approximately an increase of 30% at 50%, including about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, approximately to 100%, approximately to 150%, approximately to 200%, approximately to 300% of increase in compari- tion ^ cor ^^ a ,. control (such as the weight of the microorganism or number of cells in the absence of MSM). Increases in the growth of microorganism can be detected by methods known to those skilled in the art including those described in the Examples. to. Methods to Improve Growth of a

Probiotic microorganism

Methods for improving growth of one or more probiotic microorganisms are described. For example, methods for improving 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 about 0.4% to about 5% by weight of the medium or by weight of moisture content of the medium, thereby improving the growth of one or more microorganisms compared to growth of one or more microorganisms in the absence of MSM. In one example, the concentration of MSM is about 1% to about 3% by 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%,

In some examples, the means for improving microbial growth, such as probiotic growth, includes a product containing probiotic, such as milk, yogurt, rice yogurt, frozen yogurt, chocolate, cheese, beer, wine, vinegar, sour cabbage or any combination of them.

It is contemplated that the method can be used to enhance the growth of any probiotic microorganism, including, but not limited to Lactobacillus acidophilus, Lactobacillus delbrueckii, Bacillus coagulaos, Lactobacillus rhamnosus, Bifidobacterium bifidum or any combination thereof. In one embodiment, the methods described are used to improve the activity of the bacterium Lactobacillus rhamnosus. In other embodiments, the methods described are used to improve the activity of species within the genus Lactobacillus. For example, a method for improving the activity (eg, growth) of Lactobacillus acidophilus includes contacting

Lactobacillus acidophilus with a medium capable of supporting the growth of Lactobacillus acidophilus; and provide MSM to the medium with approximately less than 1% (such as 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 improving the growth of Lactobacillus acidophilus compared to growth of Lactobacillus acidophilus in the absence of MSM.

In other embodiments, the methods described are used to improve the activity of Bifidobacterium bifidum. For example, a method for improving the activity (e.g., 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 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 in this way improving the growth of Bifidobacterium bifidum compared to the growth of Bifidobacterium bifidum in the absence of MSM.

Increases in growth of probiotic microorganism can be detected by methods known to those skilled in the art including those described in the Examples. b. Methods for Improving the Growth of a Microorganism in a Diagnostic Test Sample or Industrial Test Sample Methods for improving the growth of a microorganism in a diagnostic test sample or industrial test sample are described. In one embodiment, a method for improving the growth of a microorganism in a diagnostic test sample is provided. In one example, the method includes contacting a diagnostic test sample (eg, blood, tissue, scrapes, mrpnralps fluids and metabolic products, and the like) comprising one or more microorganisms with a medium capable of supporting the growth of the one or more microorganisms; and providing MSM to the medium at a sufficient concentration to enhance microbial growth, thereby improving the growth of the one or more microorganisms in the diagnostic test sample compared to growth of the one or more microorganisms in the absence of MSM.

In some embodiments, a method for improving the growth of a microorganism in an industrial test sample is provided. In one example, the method includes contacting an industrial test sample (eg, water sample, domestic bacterial or mold sample and other like samples) comprising one or more microorganisms with a medium capable of supporting the growth of the one or more microorganisms; and providing MSM to the medium at a sufficient concentration to enhance microbial growth, thereby improving the growth of one or more microorganisms in the industrial test sample as compared to growth of the one or more microorganisms in the absence of MSM.

In various embodiments, MSM is provided in a composition to facilitate diagnostic assays or assays of industrial test samples, such as at a concentration of about 0.04% to about 5% by weight of the sample or by weight of the moisture content of the sample. sample. In some embodiments, MSM is provided in a composition to facilitate diagnostic assays or assays of industrial test samples such as any of the MSM compositions capable of improving the microbial activity described in Section III. In certain embodiments, MSM is added directly to the diagnostic or industrial test sample comprising microorganisms.

According to various modalities described herein, MSM can shorten the detection and / or analysis time by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. According to several modalities described herein, MSM can improve 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 weight of the microorganism or its number of cells, including an increase of at least 10%, such as approximately an increase from 20% to 80%, an increase of approximately 30% to 50%, including approximately an increase of 10%, approximately an increase of 20%, approximately an increase of 30%, approximately an increase of 40%, approximately an increase of 50%, approximately an increase of 60%, approximately an increase of 70%, approximately an increase of 80%, approximately an increase of 90%, approximately an increase of 100%, approximately an increase of 150%, approximately an increase of 200%, approximately an increase of 300% compared to a control (such as the weight of the microorganism or number of cells in the absence of MSM). Increases in the growth of microorganisms can be detected by methods known to those skilled in the art, including those described in the Examples. approximately an increase of 300% compared to a control (such as the weight of the microorganism or number of cells in the absence of MSM). Increases in the growth of microorganisms can be detected by methods known to those skilled in the art, including those described in the Examples. approximately an increase of 300% compared to a control (such as the weight of the microorganism or number of cells in the absence of MSM). Increases in the growth of microorganisms can be detected by methods known to those skilled in the art, including those described in the Examples.

In several modalities, MSM is used in conjunction with medical screen tests and rapid diagnostic tests, such as in urine or blood samples. In many cases, diagnostic tests are performed to identify possible microbial infections. Several groups of microorganisms, including bacteria, viruses, mold and yeast, can cause infections. If a microorganism is found, more tests are done to determine which antibiotics can be effective in treating the infection. To diagnose these infections as soon as possible, in some modalities, MSM is used to supplement the growth medium used in diagnostic tests to increase the growth rate of microorganisms in the patient's sample, thereby improving the detection time of the proof. In some modalities, MSM can improve the detection sensitivity of a diagnostic test. In some modalities, the diagnostic test is a urine test. In some modalities, the diagnostic test is a blood test. In other embodiments, other patient samples may be developed or cultured for diagnostic purposes, such as sputum, saliva, skin scrapes, dental scrapes, vaginal or cervical specimens, and the like. In one embodiment, MSM is used to provide a rapid streptococcal test. For example, a sample of body fluid (the diagnostic test sample) is added to a test tube or culture dish (the medium). The medium supports the cultivation of any microbes that may exist in the body fluid. By providing a medium that is pre-dosed with MSM or by adding MSM before or after adding the body fluid to the test tube or culture dish, the microbes in the body fluid (or its test products or metabolites) will increase and will be easier to rehearse. In this way, the diagnosis is facilitated.

In several modalities, the use of MSM facilitates the medical diagnosis of viral infections by supporting the growth of viruses for diagnostic testing. Viruses include, but are not limited to, human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza, 1INDU # ^ a '&amp; 5sfc ¥: s ^ p and other viruses. Similarly, the diagnosis - ··· méd-ieo- «de-ntg.üya., - infections, such as those caused by bacteria, fungi, yeast and parasites is also facilitated by MSM according to various modalities. The use of MSM facilitates the development of vaccines in one modality.

In several modalities, MSM is used to improve the detection of microbes in a commercial or industrial test. Microorganisms are a common water pollutant. Many water safety test equipment evaluates the quality of drinking water through testing methods of the Environmental Protection Agency (EPA =

Environmental Protection Agency) to test among other things, for the presence of bacteria. Mold found in home, office and school environments has been linked to lung disorders and allergic symptoms. However, some tests used to detect bacteria or mold may be time consuming for analysis while some tests additionally detect only viable (living) organisms. In this way, in several modalities, MSM is used to supplement growth media used in commercial detection tests. In some modalities, medium supplemented with MSM improves the detection time of the tests. In some modalities, medium supplemented with MSM improves the detection sensitivity of these tests. In certain modalities, MSM restores environmentally stressed bacteria that were previously not viable. In still further embodiments, diagnostic test kits comprising medium supplemented with microorganism-specific MSM are used to improve the detection time or sensitivity of a test aimed at detecting a particular microorganism. In other embodiments, MSM is used to supplement a broad spectrum growth medium, such that a variety of microorganisms are detected more rapidly or with increased sensitivity.

iii. Methods to Improve Survival of Microorganisms and Cells with MSM

Methods for improving the survival of microorganisms (including, but not limited to probiotic microorganisms) or cells (such as stem cells or recombinant cells) are disclosed. For example, methods for improving the survival of microorganisms or cells, such as cells in culture, include contacting one or more microorganisms or select cells with MSM at about 0.4% to about 5% by weight of the medium or by weight of a content of moisture of the medium, thereby improving the survival of one or more microorganisms or collection of cells compared to the survival of one or more microorganisms or collection of cells in the absence of MSM. In one example, the concentration of MSM is about 1% to about 3% of the weight of the medium or the moisture content of the medium.

According to several modalities, MSM improves the initial survival of microorganisms (including, but not limited to, probiotic microorganisms). In one modality, MSM improves the long-term survival of microorganisms. In one embodiment, MSM extends the stationary phase of a growth curve of the microorganisms.

In several modalities, MSM extends the shelf life of a product by extending the life span of beneficial bacteria compared to products without MSM. For example, a product containing probiotic can have a shelf life of several weeks after that time the probiotic organism begins to decline in health and / or population. However, in some embodiments, the addition of MSM to a probiotic-containing product increases the length of product packaging time to the decline in health and / or probiotic population. In these modalities, the probiotic product is functional (in terms of supplying a population of healthy and active probiotics to the gastrointestinal tract (GI) of the consumer), for a longer period of time after packing.

In several embodiments, the addition of MSM increases the time to deterioration of ingestible products by supporting or enhancing the activity of beneficial microbes, with a resulting decrease in the activity of undesirable microbes. For example, MSM can increase the shelf life of edible products, such as a probiotic product, by approximately 10% to 100% (eg, 20%, 30%, 40%, 50%, 75%, 150%, 200 % or more) . For example, in one mode, if the storage life of an edible product is 10 days, the addition of MSM will increase the shelf life to at least 11 days in some modes (for example, 11 days, 14 days, 15 days , 20 days or 25 days). As an additional example, in another modality, if an edible product has a storage life of 14 days at room temperature and / or 30 days in the refrigerator and / or 3 months in the freezer, the addition of MSM will increase the 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 improves the activity of beneficial microbes and inhibits (either directly or indirectly) the activity of undesirable bacteria, thereby reducing or eliminating the need for sterilization (eg, by irradiation, filtration). , heat, chemical products, 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 agents 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 improving gene therapy are described wherein one or more processes associated with gene therapy are improved or increased by treating the recombinant cells or microorganisms with a concentration of MSM (such as a concentration of about 0. 04% to approximately 5% of MSM) capable of improving one or more gene therapy processes (such as expression, growth or survival of recombinant cells or microorganisms), thereby increasing the effectiveness of gene therapy. iv. Methods to Improve Crop Efficiency with

MSM

Here we describe methods to improve crop efficiency with MSM. In one embodiment, methods for improving the efficiency of culture of antibiotics, steroids, cells (for example, recombinant and wild type), microorganisms and fertilizers. For example, in several modalities, MSM is used to supplement culture medium used for the growth or propagation of microbial organisms. In several modalities, medium supplemented with MSM improves culture efficiency by improving cell growth.

In some embodiments, methods to improve crop efficiency include improving / promoting microbial activity in environmental and industrial fields. Microorganisms participate in elementary cycles such as the carbon cycle and nitrogen cycle, as well as fulfill other vital functions in virtually all ecosystems, such as recycling waste products and / or the remains of other organisms through decomposition. In this way, in some modalities, the use of MSM can improve waste decomposition and waste management. Many biological oxidation processes for industrial wastewater treatment have in common the use of oxygen (or air) and microbial action. Especially cultivated microbes are used in the biological treatment of sewage and industrial waste effluents, a process known as bioaugmentation. Bioaugmentation is used to ensure that microorganisms in situ can degrade contaminants. In some modalities, MSM improves the degradation of certain microorganisms of contaminants. In some modalities, MSM is added to gardening products, such as soil, fertilizers and compost bins, to improve the activity of beneficial microorganisms. As such, MSM is used to increase the efficiency of fertilizers and compost reactions. fertilizers and compost bins, to improve the activity of beneficial microorganisms. As such, MSM is used to increase the efficiency of fertilizers and compost reactions. fertilizers and compost bins, to improve the activity of beneficial microorganisms. As such, MSM is used to increase the efficiency of fertilizers and compost reactions.

In one embodiment, a method to improve the efficiency of a fertilizer includes applying MSM to the medium in an amount sufficient to improve the activity of a fertilizer, thereby improving the activity of the fertilizer. In a particular embodiment, MSM is dissolved in a solution at a final concentration of about 0.04% to about 5%. This solution is then sprayed on a plant surface either before, after or simultaneously with the fertilizer. An increase in fertilizer efficiency is indicated by an Hp increase to at least 10%, such as approximately an increase from 20% to 80%, approximately an increase from 30% to 50%, including approximately an increase of 10%, approximately one increase of 20%, approximately an increase of 30%, approximately an increase of 40%, approximately an increase of 50%,

In another embodiment, a method for improving the composting efficiency is described. This method includes applying MSM to the compost in an amount sufficient to improve 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 about 0.04% to about 5%. This solution is then applied to the compost (such as by pouring or spraying the solution) and allowing sufficient time to improve the composting efficiency. An increase in compost efficiency is indicated by increase of at least 10%, such as approximately an increase from 20% to 80%, approximately an increase from 30% to 50%, including approximately an increase of 10%, approximately an increase of 20%, approximately an increase of 30%, approximately an increase of 40%, approximately an increase of 50%, approximately an increase of 60%, approximately an increase of 70%, approximately an increase of 80%, approximately an increase of 90%, approximately an increase of 100%, approximately an increase of 150%, approximately an increase of 200%, approximately a 300% increase in nitrate levels compared to a control (such as nitrate levels in the absence of MSM). In other examples, an increase in compost efficiency is indicated by an increase of at least 10%, such as approximately an increase from 20% to 80%, approximately an increase from 30% to 50%, including approximately an increase of 10%. %, approximately an increase of 20%, approximately an increase of 30%, approximately an increase of 40%, approximately an increase of 50%, approximately an increase of 60%, approximately an increase of 70%, approximately an increase of 80%, approximately an increase of 90%, approximately an increase of 100%, approximately an increase of 150%, approximately an increase of 200%, approximately a 300% increase in the amount of time that organic matter decomposition occurs compared to a control (such as decomposition rate in the absence of MSM). __ B. Methods to Inhibit Microbial Activity approximately a 300% increase in the amount of time that organic matter decomposition occurs compared to a control (such as decomposition rate in the absence of MSM). __ B. Methods to Inhibit Microbial Activity approximately a 300% increase in the amount of time that organic matter decomposition occurs 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 described. In one embodiment, a method for inhibiting microbial activity includes selecting a medium that is susceptible to contamination; and contacting the medium with MSM at a concentration of about 6% to about 16% by weight in volume, thereby inhibiting microbial activity as compared to microbial activity in a control (such as microbial activity in the absence of MSM) . By an increase of at least 10%, such as approximately a decrease from approximately 20% to 80%, a decrease from approximately 30% to 50%, including a decrease of approximately 10%, a decrease of approximately 20%, a decrease of approximately 30%, a decrease of approximately 40%, a decrease of approximately 50%,

In some embodiments, a method for inhibiting microbial activity includes selecting a medium that is susceptible to bacterial contamination and contacting the medium with MSM at a concentration of about 6% to about 16% by weight by volume, thereby inhibiting bacterial activity. . In some embodiments, a method for 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, parainfluenza virus, influenza, hepatitis, or other similar viruses); and contacting the medium with MSM at a concentration of about 6% to about 16% by weight in volume, thereby inhibiting viral activity.

In a particular example, a method for inhibiting microbial activity includes selecting a medium that is susceptible to H1N1 influenza contamination; and contacting the medium with MSM at a concentration of about 10% to about 16% by weight per volume, thereby inhibiting the microbial activity of H1N1 influenza. In some embodiments, MSM inhibits microbial activity by reducing the growth rate of H1N1 influenza by at least 10%, such as by approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60. %, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 150%, approximately 200%,

In various embodiments, the methods include MSM at about 8% (by weight) or greater, of a total product weight or moisture content. In certain embodiments, MSM is an effective antimicrobial agent when used at concentrations between about 5% and about 16%. In certain embodiments, MSM is an effective antimicrobial agent when used at concentrations (based on the total weight or moisture content of a product) between about 9% and about 16%, between about 10% and about 16%, between about 12% and approximately 16%, between approximately 9% and approximately 13%, and between approximately 10% and approximately 12%. In certain embodiments, MSM is an effective antimicrobial agent when used at concentrations between about 5% and about 16%, including 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% and 15%. In several modalities described herein, the MSM percentages are based on the moisture content of a product. In some modalities, MSM is particularly effective when combined with water or other liquid components. In various embodiments, the percentages of MSM herein are based on the amount of a polar solvent in a product or other medium.

In some embodiments, the methods described to inhibit microbial activity include inhibition of growth of specific microorganisms. In some examples, the methods include inhibiting growth of a wide range of microorganisms in certain media or products. In some modalities, reductions in log scale are achieved after the first 24 hours. In some other modalities, significant log scale reductions are evident within 24-48 hours. In some embodiments, the disclosed methods include MSM formulations that produce reduction at microbial (e.g., bacterial) levels in the range of about 0.5 log to about 5 log or more within two weeks. In some modalities, The methods described to inhibit microbial activity result in a log reduction between approximately a 1 log reduction and approximately 3 log or more reductions. In other embodiments, the methods described to inhibit microbial activity inhibit lethally the growth of certain microorganisms. In one embodiment, a method that uses an MSM formulation between about 12% and about 16% fatally kill certain microbes (e.g., bacteria) within about 48 hours. In another embodiment, a formulation comprising MSM between about 8% and about 12% lethally kill certain microbes (e.g., bacteria) within about three to seven days. In other embodiments, the methods employ an MSM formulation between about 5% and about 8%, in combination with a reduced amount of a conventional preservative, which lethally kill certain microbes (eg, bacteria) within approximately 48 hours. With higher concentrations of preservative, MSM levels can be further reduced.

In some embodiments, the methods described for inhibiting microbial activity with MSM (such as with approximately 6% to approximately 16% MSM) impact the metabolism of microbes in the latency phase. For example, the described method increases the duration of the latency phase. An alteration, such as an increase in the latency phase, can be detected by methods known to those skilled in the art including those described in the Examples.

In some modalities, the supplement with MSM results in a decrease in the log phase of the growth of microbes. The exponential phase (sometimes called the log phase) of growth is a period characterized by cellular duplication. The number of new microbes that appear per unit of time is proportional to the present population. If growth is not limited, the duplication will continue at a constant rate so that both the number of cells and the rate of population increase doubles with each consecutive period of time. Exponential growth can not continue indefinitely, however, because the environment is soon depleted of nutrients and enriched with wastes. In some modalities, MSM decreases the total duration of the exponential phase. In other modalities,

In various embodiments, the methods described to inhibit microbial activity include modulating the stationary phase of microbial growth. During the stationary phase, the rate of growth slows down as a result of nutrient depletion and accumulation of metabolic byproducts. This phase is reached as the microbes begin to deplete the resources that are available to them. This phase is a relatively constant value since the rate of microbial growth is equal to the rate of microbial death. The MSM supplement of medium at certain concentrations shortens the stationary phase for microbes in one embodiment.

It is contemplated that a medium includes ~ r y-η- | -jnr - environment that contains or is suitable for supporting contamination including but not limited to cosmetics, broths, agar, crops, foods, beverages, cell suspensions, 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 rubbing the medium susceptible to microbial contamination with an MSM composition / formulation. For example, a surface may include any surface susceptible to contamination including, but not limited to a domestic surface, an industrial surface (such as surfaces in public restrooms, door handles, floors, walls, hand rails, shopping carts and the like ), beds, covers, equipment or industrial surfaces, blood, skin or a combination thereof. For example, a domestic surface may include a door handle, door knob, a canister

• • • Litter, a counter, floor, toilet seat, or any surface that is commonly touched or "" '' exposed to possible contaminants.

Accidental microbial growth can occur in many cosmetic products, beauty and health aids, topical products, and oral products. Acute or continuous use of products with microbial contamination can lead to adverse health effects for the user. Contamination can occur, for example during manufacturing, packaging or repetitive use by a consumer including repeated opening and closing of containers, hand contact, skin or mucous membranes or repeated administration / withdrawal of individual doses. In the absence of antimicrobial properties, these products may allow the accidental growth of many different and potentially harmful microorganisms.

Antimicrobial preservatives can be added to products to protect them from microbial growth. Common general use of antimicrobial preservatives includes calcium propionate, sodium nitrate, sodium nitrite, sulfites (sulfur dioxide, sodium bisulfite, hydrogen potassium sulfite, etc.) and disodium EDTA. Cosmetic preservatives include formaldehyde, potassium sorbate, methylparaben and methylchloroisothiazolinone.

In many cases, preservatives should be added in a minimum effective concentration, since adverse reactions can occur at certain concentrations or doses. In this way, while preservatives can inhibit microbial growth, they also have the potential to cause chemical burns and / or irritate mucous membranes and skin. Some modern synthetic conservatives have become controversial because they have been shown to cause respiratory or other health problems. In addition to certain preservatives for commercial products may present unique complications with solubility, pH limits, deactivation by some polyethylene glycol (PEG) compounds, and a change in the color, consistency or fragrance of a product.

Methods for inhibiting microbial activity in a consumer product are also described. In one embodiment, 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. MSM is provided in a concentration of at least 10% according to one modality (eg, 10-16%, 16-20%, 20-30%, 30-40%, 40-50%, 50-75% or superior and intervals or overlapping ranges thereof). The medium is free of "conservatives in some modalities. -

In some embodiments, methods are provided for inhibiting microbial activity in a cosmetic cream at room temperature. In one embodiment, 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. MSM is added at a concentration of at least 5% according to one modality (eg, 5-10%, 10-16%, 16-20%, 20-30%, 30-40%, 40-50%, 50-75% or higher, and their ranges or overlapping ranges). The medium is free of conservatives in some modalities. The medium includes a cosmetic cream in some modalities. In one example, MSM inhibits microbial activity by at least 50% in the 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 of diagnosis, and other solid, liquid, matrix, gelatinous or gaseous environments. For example, in one embodiment, the medium includes an optical product or a product for oral hygiene or health. The medium may also include a body tissue or fluid, such as blood. In one example, the medium is sterilized before adding MSM. and / or after adding MSM. In other examples, sterilization is not required. 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 bacteria and / or gram negative bacteria, fungi, parasites, yeast, mold, viruses or combinations thereof (eg, bacteria and mold, or other combinations). In several modalities, microbial contamination is caused by one or more of the following genera: Candida, Aspergillus, Escherichia, Pseudomonas, Staphylococcus and Streptococcus, or their combinations. In another embodiment, the microbial contamination is caused by an infectious disease including any of the infectious diseases described herein.

In various embodiments, methods for treating an infectious disease are described, including but not limited to H1N1, herpes simplex virus or HIV (HIV). In one embodiment, the method includes administering an effective therapeutic amount of a therapeutic agent and DMSO alone, MSM alone or a combination of DMSO and MSM. The concentration of DMSO and / or MSM is in the range of about 6% to about 17% in a composition.

In some modalities, MSM inhibits the activity, ...... Tl, microbial by reducing the growth rate of one or more microbes by more than 50%, which in turn increases the storage life of the medium. It is contemplated that MSM may confer a therapeutic and / or aesthetic benefit. In some modalities, the therapeutic or aesthetic benefit is not related to microbial inhibition.

In some embodiments, the methods described for inhibition of microbial activity, inhibit microbial activity at temperatures that lead to microbial activity, including 20-25 ° C, 25-30 ° C, 30-40 ° C, 40-50 ° C and above (and its overlapping ranges). In some embodiments, MSM inhibits microbial activity at moisture levels favorable for microbial activity, including 50% -60%, 60-70%, 70-80%, 80-95%, and above (and overlapping ranges or ranges of same). MSM is particularly advantageous in several embodiments, because it can be used at higher concentrations than other preservatives, which when used even in low concentrations can cause adverse effects. For example, conservatives have been implicated in atopic dermatitis, rashes, flushing, abdominal pain, nausea, asthma, rhinitis, muscle pains, joint pains, fatigue, numbness or numbness, migraines, hyperactivity disorder and attention deficit, -drops and arrhythmias. In contrast, MSM is not known to cause these effects in concentrations that are provided in accordance with the present preferred embodiments. Furthermore, MSM has a dual function according to some modalities. Not only does MSM inhibit the growth of undesirable microorganisms, MSM also beneficially affects the product to which it is added in various modalities. MSM is not known to cause these effects in concentrations that are provided in accordance with the present preferred embodiments. Furthermore, MSM has a dual function according to some modalities. Not only does MSM inhibit the growth of undesirable microorganisms, MSM also beneficially affects the product to which it is added in various modalities. MSM is not known to cause these effects in concentrations that are provided in accordance with the present preferred embodiments. Furthermore, MSM has a dual function according to some modalities. Not only does MSM inhibit the growth of undesirable microorganisms, MSM also beneficially affects the product to which it is added in various modalities.

In some embodiments, the methods described to inhibit microbial activity not only inhibit microbial activity, but provide one or more other beneficial effects, including but not limited to, reduction of muscle cramps, skin irritation, pain reduction, joint lubrication, reduction of inflammation, treatment of rheumatoid arthritis and osteoarthritis, cardiovascular improvements, lubrication of the skin, improved wound healing and improved scalp, hair, cuticle and nails.

In some embodiments, the methods described to inhibit microbial activity are employed to avoid or minimize the formation of new microbes. In other modalities, the methods are used to exterminate or reduce existing microbes. In one embodiment, MSM can convert an otherwise unusable contaminated product into a usable product.

According to several modalities, the methods instantaneously inhibit the microbial activity. In other modalities, the methods inhibit microbial activity 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 and more.

In various embodiments, MSM is added to cleaning agents to improve antimicrobial activity (e.g., to inhibit microorganism activity). In some modalities, MSM is added to a soap formulation. In some modalities, the product is a dry soap whereas in other modalities, the product is a liquid soap. In some embodiments, MSM is added to a gel formulation to give a disinfectant. For example, methods for inhibiting microbial activity include methods for disinfecting a surface, such as the body, equipment, floors, materials, walls, etc. In certain embodiments, the resulting disinfectant is an instant disinfectant. In other modalities, the disinfectant acts in a non-instantaneous way (for example, it is effective over time). In some modalities, the disinfectant is applied to the body. Still in other modalities, 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 are not limited to household surfaces, vehicles, computers, clothing and toys.

In some additional embodiments, methods for inhibiting microbial activity include spraying or incorporating MSM (eg, from about 5% to about 50% in face masks or filters.) Filters may include, but are not limited to, air conditioning filters, filters, and filters. Air, water filters, and recycled air environments, such as aircraft, can especially benefit from MSM filtration systems, wastewater treatment and water filtration plants can also incorporate MSM to inhibit microbial activity. It provides to reduce microbial contamination in flowers arrangement and in gardening products (such as fertilizers and soil or substrates).

In some embodiments, methods of inhibiting microbial activity include inhibiting microbial activity of a microorganism present in animal feed and to prevent microbial growth during storage or processing of the food. Types of foods for animals include, but are not limited to, compound feed, feed or fodder. The animal feed may consist of raw materials and / or additives. The raw food can be provided as straw or grains. Alternatively, the raw material can be manufactured and served, peeled or chopped or textured. In some modalities, MSM is applied to animal feed to reduce mold growth. In other modalities, MSM is applied to animal feed to reduce fungal growth. In some modalities, MSM is applied to raw food materials and is thus incorporated into a particular food product. In additional modalities, the product is applied to the food during or after manufacture. In some modalities, the product is applied for food, for long-term storage.

V. Methods to Produce Products that include MSM

Here are described methods for producing products that include MSM. In some embodiments, MSM is incorporated into a step that will reduce the crystallization of MSM. In one embodiment, MSM is incorporated into a product before emulsifying the product. In another embodiment, MSM is encapsulated (for example, in a polymer lipid material or other) before addition to a product. MSM micro encapsulated, according to some modalities, can be designed for MSM dose release or over time. Still in other modalities, MSM is combined with the aqueous portion of a product before mixing the wet and dry ingredients. In one embodiment, MSM in the form of dry powder 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 higher). In some embodiments, MSM is not materially affected by heat, and can be added before heating. Solutions having a temperature greater than about 35 ° C support MSM concentrations greater than 50% in some embodiments. In several modalities, MSM does not substantially impact the pH of the product to which it is added. In one embodiment, hygroscopic solid products and other products with low moisture content comprise MSM in a range of about 15% or more. Methods for making a product having a reduced conservative concentration are also described herein. In one modality, 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. MSM is added in a concentration of at least 5% to approximately 20% according to one modality (eg, 5-8%, 8-12%, 12-15%, 15-20%, and their overlapping intervals). In one embodiment, MSM and conservative inhibit microbial growth in at least 50% in the medium at room temperature, and MSM supplement or improves preservative to inhibit microbial growth, thus reducing the concentxa ^ ^ clfiX- conservative LjólL required to inhibit microbial growth. where MSM affects microbial contamination by inhibiting microbial growth. MSM is added in a concentration of at least 5% to approximately 20% according to one modality (eg, 5-8%, 8-12%, 12-15%, 15-20%, and their overlapping intervals). In one embodiment, MSM and conservative inhibit microbial growth in at least 50% in the medium at room temperature, and MSM supplement or improves preservative to inhibit microbial growth, thus reducing the concentxa ^ ^ clfiX- conservative LjólL required to inhibit microbial growth. where MSM affects microbial contamination by inhibiting microbial growth. MSM is added in a concentration of at least 5% to approximately 20% according to one modality (eg, 5-8%, 8-12%, 12-15%, 15-20%, and their overlapping intervals). In one embodiment, MSM and conservative inhibit microbial growth in at least 50% in the medium at room temperature, and MSM supplement or improves preservative to inhibit microbial growth, thus reducing the concentxa ^ ^ clfiX- conservative LjólL required to inhibit microbial growth.

In one embodiment, the medium is emulsified or otherwise mixed. In one embodiment, MSM is added to the medium before emulsification (or other mixing).

The following examples are provided to illustrate certain characteristics and / or particular modalities. These examples should not be considered as limiting the description to the particular characteristics or modalities described. EXAMPLES Example 1

MSM-based Modulation of Microbial Activity

This example describes MSM-based modulation of microbial activity, such as improving or inhibiting microbial growth depending on the concentration of MSM.

Comparative microbial growth studies were performed in medium 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 triptych soy broth (TSB = Tryptic Soy Broth) and except for Candida and Aspergillus, all were successfully transferred to fresh medium each day for 4 consecutive days before inoculation to maintain organisms in an exponential growth phase. Candida and Aspergillus had 48-58 hours of growth in TSB before inoculation in a half-trial. Aspergillus was also developed on multiple potato dextrose agar plates (PDA = Potato Dextrose Agar) for 48-58 hours. The Aspergillus inoculum was prepared by taking a surface rinse with TSB from the PDA plates with an Aspergillus lawn, and then adding it to the culture for 48-58 hours until turbidity. For each test microorganism, aliquots of 90 mL of TSB were prepared either 10% or 0% MSM. Once each set of test media was applied to plates for sterility, they were inoculated at a level of 5 and &lt;1 inoculum per 10 mLs of broth (1: 2000 inoculum dilution) with each respective microorganism. Bacterial organisms were incubated at 30 ° C + 2 ° C and incubated in fungal organisms at 25 ° C ± 2 ° C. and then it is added to the culture for 48-58 hours until turbidity. For each test microorganism, aliquots of 90 mL of TSB were prepared either 10% or 0% MSM. Once each set of test media was applied to plates for sterility, they were inoculated at a level of 5 and &lt;1 inoculum per 10 mLs of broth (1: 2000 inoculum dilution) with each respective microorganism. Bacterial organisms were incubated at 30 ° C + 2 ° C and incubated in fungal organisms at 25 ° C ± 2 ° C. and then it is added to the culture for 48-58 hours until turbidity. For each test microorganism, aliquots of 90 mL of TSB were prepared either 10% or 0% MSM. Once each set of test media was applied to plates for sterility, they were inoculated at a level of 5 and &lt;1 inoculum per 10 mLs of broth (1: 2000 inoculum dilution) with each respective microorganism. Bacterial organisms were incubated at 30 ° C + 2 ° C and incubated in fungal organisms at 25 ° C ± 2 ° C.

Fungal organisms were planted daily in PDA on days 0 to day 7 every 24 hours. Preparation and sowing in plates were performed at room temperature. Fungal plates were incubated at 25 ° C + 2 ° C for at least 3 days. Test samples were plated in triplicate plates on each test date and the averages are reported. The data expressed as colony forming units recovered by one milliliter (cfu / mL).

After these points in time, the divergence in growth curves continued through the study for Candida. These data indicate that a concentration of 10% of MSM provides a significant negative effect on the growth of various fungal organisms over time.

Table 1-1 (a). Effect of MSM on Growth of Aspergillus niger.

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Table 1-1 (b). Effect of MSM on Growth of Candida alhicans.

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The effect of growing MSM on Staphylococcus aureus is illustrated in Table 1-2. A difference in viability of the presence of higher concentrations of MSM was observed. In particular, 10% MSM appeared both to slow down the growth rate and the maximum population size of Staphylococcus aureus.

Table 1-2. Effect of MSM on Growth of Staphylococcus aureus.

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The effect of MSM on growth of Pseudomonas aeruginosa is illustrated in Table 1-3. Ten percent supplementation of MSM medium resulted in a substantial divergence in the viability of Pseudomonas aeruginosa over time. For example, medium supplemented with 10% MSM resulted in a population reduction that lasted the first 4 days of the study, but did not persist after that time. Table 1-3. Effect of 10% MSM on Growth of Pseudomonas aeruginosa.

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The effect of MSM on growth of Pseudomonas aeruginosa is illustrated in Table 1-4. Ten percent supplement of MSM medium resulted in substantially less growth of E. coli over time.

Table 1-4. Effect of 10% MSM on Growth of Escherichia coli.

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The effect of MSM on growth of Salmonella cholerasuis is illustrated in Table 1-5. Medium supplemented with 10% MSM reduced the growth of Salmonella cholerasuis for most points at study time.

Table 1-5. Effect of 10% MSM on Growth of Salmonella cholerasuis.

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These studies indicate that certain concentrations of MSM inhibit growth, including growth of Aspergillus niger, Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa and E. coli.

Example 2

Antimicrobial Effectiveness Test of Supplemented Medium

with MSM

This example describes the test results of antimicrobial effectiveness of medium supplemented with MSM.

Compounds or formulated products that have antimicrobial activity can be evaluated with the Antimicrobial Effectiveness Test (AET = Antimicrobial Effectiveness Test) of the US Pharmacopoeia (USP = United States Pharmacopeia). AET 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 simulate contamination. The product is maintained · - · jskmí-iiíi utos, 'cdlf' 'weekly analysis of microorganism levels. Depending on the route of administration of a product, the satisfaction of the ΔΕΤ generally requires a reduction of 1 to 3 log in bacteria of initial levels, which should occur in one to two weeks, No major increase in bacteria after two weeks. For yeast and mold, no increase in the initial inoculum level is allowed. Successful completion of the AET criteria demonstrates that a product, optionally supplemented with an antimicrobial compound under evaluation, can support an inoculum of up to one million micrograms per gram of product without becoming contaminated. TEA demonstrates the effectiveness of a preservative system in a product and / or can be used as part of a stability study to determine if a conservative system affected the shelf life of a product. optionally supplemented with an antimicrobial compound under evaluation, can support an inoculum of up to one million micrograms per gram of product without becoming contaminated. TEA demonstrates the effectiveness of a preservative system in a product and / or can be used as part of a stability study to determine if a conservative system affected the shelf life of a product. optionally supplemented with an antimicrobial compound under evaluation, can support an inoculum of up to one million micrograms per gram of product without becoming contaminated. TEA demonstrates the effectiveness of a preservative system in a product and / or can be used as part of a stability study to determine if a conservative system affected the shelf life of a product.

The TEA was performed by adding the specified microorganisms directly to test medium supplemented with MSM at concentrations that simulate microbial contamination. Active, fresh cultures standardized at a concentration between 100,000 to 1,000,000 cells per mL of the test product were added to the medium supplemented with MSM. Inocula were performed using Candida albicans, Aspergillus niger, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Soy broth Triptych (TSB = Tryptic Soy Broth) 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 maintained for one month, during which time the aggregated microorganisms were enumerated weekly to determine if they are growing, dying or remain close to the initial inoculation level. 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 to 2-8. The acceptance criteria for antimicrobial effectiveness are described in detail in the USP, incorporated herein by reference.

Table 2-1. AET test results for 1: 1 dilution of MSM.

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Table 2-2. Reduction Log of Inoculation of Initial Microorganism with dilution 1: 1 of MSM.

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Table 2-3. AET test results for 1: 5 dilution of MSM.

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Table 2-4. Reduction Log of Inoculation of Initial Microorganism with dilution 1: 5 of MSM.

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Table 2-5. Test results ΔΕΤ for 1:10 dilution of MSM.

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Cont.

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Table 2-6. Reduction Log of the Inoculation of Initial Microorganism with dilution 1:10 of MSM.

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Table 2-7. AET test results for 1: 100 dilution of MSM.

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* - Colonies too numerous to count (TNTC = Colonies Too Numerous To Count)

Table 2-8. AET test results for 1: 1000 dilution of MSM.

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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 microorganisms killed in medium and not simply have a static effect on the increase. Based on the culture populations on day 5, bactericidal effects were unexpected, since the culture populations were stable or showed signs of increased growth. However, in 14 days, the reduction in initial inoculation levels was observed and in 20 days, a total extermination of all microorganisms was observed using at least 10% MSM. These results were confirmed by the addition of a 90 mL target of TSB with 10 mLs of the dilution matrix tested (for example, the medium that is considered to contain the most living microorganisms.) None of the microbiologicals. It can be cultivated and no contamination was observed. These results showed that MSM, at certain concentrations, is bactericidal for these organisms.

Example 3

Bactericidal Effects of Sterile and Non-Sterile MSM in Escherichia coll

This example describes the bactericidal effects of sterile and non-sterile MSM on E. coli growth.

The USP <51> AET test methodology as described in Example 2, was used as the basis for evaluating the lethality to Escherichia coli (ATCC strain 8739) of various concentrations of MSM in the range of 5 to 16% in TSB or saline. USP <51> AET is a compendial antimicrobial effectiveness test method (United States

Pharmacopoeia) to determine if a conservator is effective based on a verified and validated methodology. The parameters of the AET were described above. In this study, after the designated incubation period, cultures were evaluated visually and then scored and developed on selective MacConkey agar for qualitative analyzes of the effects of the various concentrations of MSM. This study also evaluated the effect of pre-sterilizing MSM (by autoclaving with steam at 121 ° C for 15 minutes) before the preparation of the medium. The test medium was prepared by weighing an appropriate amount of MSM and adding to 25 mL of TSB or saline medium. Media compositions were coded as provided in Table 3-1.

Table 3-1. Compositions of tested media against E. coli.

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All tubes except negative controls were added with 250 μΐ of a 1.2xl08 culture, which produces an initial E. coli population density of 1.2xl06 / mL. The tubes were incubated at 25 ° C. At 24 hours, visible signs of growth were observed in 5-9% of MSM in TSB / TSBA medium (see Table 3-2 below). In contrast, no growth signs were seen in the tubes with 10-16% MSM in TSB / TSBA medium (see Table 3-2 below). The saline tubes do not show any signs of growth. When scratched in MacConkey medium, it resulted in heavy growth in any medium "containing 5-9% MSM, while fewer colonies were detected in the 10% MSM striped plates in TSB / TSBA medium. of the streak of 11-16% of MSM in TSB / TSBA medium.

Table 3-2. Growth profile of E. coli in medium containing MSM after 24 hours.

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As shown in Table 3-3, at 48 hours signs of heavy growth were observed in 5-10% of MSM in TSB / TSBA culture tubes. Apparent growth was observed in 11% of MSM in TSB / TSBA culture tubes. Similar to the point in time of 24 hours, few to no observable signs of growth were noted in 12-15% of MSM in the TSB / TSBA culture tubes. After scratching, heavy bacterial growth occurred in all media containing 5-10% MSM. 11% of MSM in TSB / TSBA allowed moderate growth, while the same concentration of MSM added to saline allowed strong growth. At concentrations of 12-16% MSM in TSB / TSBA, little to no growth was detected in the plates. Moderate growth was observed from similar concentrations of MSM in saline medium.

Table 3-3. Growth profile of E. coli in medium containing MSM after 48 hours.

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After 72 hours of culture, signs of heavy growth were observed in 5-10% of MSM in TSB / TSBA culture tubes (Table 3-4). Apparent growth was observed in 11% of MSM in TSB / TSBA culture tubes. Similar to the point in time of 24 hours, few to no observable signs of growth were observed in 12-15% MSM in TSB / TSBA culture tubes. After streaking, strong bacterial growth occurred in all media containing 5-10% of MSM. 11% of MSM in TSB / TSBA allowed moderate growth, while the same concentration of MSM added to saline allowed strong growth. At concentrations of 12-16% MSM in TSB / TSBA, little to no growth was detected in the plates. Moderate growth was observed from similar concentrations of MSM in saline medium.

Table 3-4. Growth profile of E. contains MSM after 72 hours. -------

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These results show that MSM concentrations of approximately 10-16% are effective to kill bacteria at certain points in time. At 24 hours, 10% MSM reduced the viable bacterial population, whereas 48-72 hours were higher effective concentrations to exterminate the majority of the bacterial population. Concentrations of 10-16% MSM in TSB / TSBA were more effective than the same concentration in a saline-based medium. The data also suggest that steam sterilization does not inherently impact the effectiveness of MSM.

Example 4

Comparison of Bactericidal Effectiveness of MSM in Saline-based Medium or Tryptic Soy Broth (TSB)

This example compares the bactericidal effectiveness of MSM in saline and medium based on TSB.

As presented in Examples 2 and 3, the methodology of the USP <51> AET test was used as the basis to evaluate the lethality to E. coli of various concentrations of MSM (in flakes or microbeads) in the range of 5 to 16% in TSB or saline. Each composition of medium was inoculated with 1.25xl06 / mL of E. coli and then cultured for seven days at 35 ° C. At the end of the incubation period, cultures were evaluated visually and then developed on tryptho-soy agar at concentrations diluted in series to ensure bacterial growth (if any) at a density that was capable of being quantified. Coated cultures were grown for 24 hours at 35 ° C before analysis.

Media compositions were coded as shown in Table 4-1 and the results of these studies are given in Table 4-2.

Table 4-1. Compositions of Medium

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Table 4-2. Log growth of E. coli in different medium with different concentrations of MSM.

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Concentrations in the range of 11-16% MSM has a negative effect of growth of E. coli in culture for 7 days. As the concentration of MSM increases over 10% in either the FTSB or PTSB media, the growth of E. coli is reduced. Both forms of MSM showed efficacy to inhibit bacterial growth.

Example 5

Effect of Sodium Chloride Free Medium on Bactericidal Effect of MSM

This example shows the effect of sodium chloride-free medium on bactericidal effects of MSM.

A study using Müller-Hinton broth medium, which does not contain NaCl, was carried out. 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 in concentrations in the range of 5-16%. After inoculating each type of medium containing MSM with 1.9 x 107 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 7 days. Aliquots were grown on tripto-soy agar at concentrations diluted in series to ensure bacterial growth (if any) at a density that was capable of being quantified. Coated cultures were grown for 24 hours at 35 ° C before analysis. Media compositions were coded as provided in Table 5-1.

Table 5-1. Compositions of Medium

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After 24 hours in culture, an approximate 1 log reduction of the initial inoculum was detected in all media having MSM concentrations greater than 13% (Table 5-2). In addition, at 12% MSM, all medium compositions reduce the growth of E. coli, except for the composition of PMHS. At 11% MSM, only the FMH medium reduced the growth of E. coli.

Table 5-2. Log growth of E. coli in Müller-Hinton medium supplemented with MSM or Müller-Hinton (plus NaCl) after 24 hours

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After 48 hours in culture, media compositions with MSM concentrations greater than 13% reduce the growth of E. coli by 1-2 logs. Certain concentrations of MSM are effective in reducing bacterial growth, which is surprising because other concentrations of MSM are effective in supporting increased bacterial activity.

Table 5-3. Log growth of E. coli in Müller-Hinton medium supplemented with MSM or Müller-Hinton (plus NaCl) after 48 hours

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After 7 days in culture, the composition of the medium containing as low as 12% MSM substantially inhibited the growth of E. coli (Table 5-4). FMHS medium was more effective at 12% MSM, resulting in a 3 log reduction in E. coli.

Table 5-4. Log growth of E. coli in Müller-Hinton medium supplemented with MSM or Müller-Hinton (plus NaCl) after 7 days

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Example 6

Bactericidal Effect of MSM in Medium with Low Protein and Free of Sodium Chloride

This example shows the bactericidal effect of MSM in low protein medium and free of sodium chloride.

Lactose broth, free of both NaCl and proteins, was used as the medium in this experiment. MSM was added to lactose broth at concentrations in the range of 5-16%. A duplicate set of medium containing MSM is supplemented with DMSO at a final concentration of 1%. Each medium composition was initially inoculated with 6.75 x 106 cfu / mL of E. coli. The cultures were incubated at 25 ° C for seven days. Aliquots of each culture were taken after 24 hours of culture and at the end of seven days of culture. Aliquots were serially diluted (with Modified Letheen diluent) and seeded onto plates on trypto-soy agar. The coated cultures were grown for 24 hours at 35 ° C and then analyzed. Media compositions were coded as shown in Table 6-1.

Table 6-1. Compositions of Medium

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Lactose broth containing 11-16% MSM reduces bacterial growth from approximately 1 log (16% MSM) to a maximum of approximately 2.2 logs (11% MSM) as shown in Table 6-2. Inhibition of bacterial growth is reduced by 1 log or more from 9-16% MSM. Table 6-2. Growth log of 24-hour E. coli in MSM-lactose broth with or without DMSO

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After 7 days of culture, a more defined pattern of inhibition of bacterial growth was evident (Table 6-3). 10% MSM in lactose broth maintained the E. coli population approximately equivalent to the initial inoculum.

Table 6-3. 7-day growth log of E. coli in lactose broth-MSM with or without DMSO

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Example 7

Evaluation of 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 in a cosmetic matrix was performed. The cosmetic matrix was a base cream (jojoba) that is used in many cosmetic products. MSM was incorporated into the cream at concentrations in the range of 5-16% MSM. Each of these concentrations was then added with E. coli at a level of 4.6xl05cfu / 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. Growth log for 48 hours of E. coli in cosmetic matrix containing MSM

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These data indicate that bacteria growing in a cosmetic cream are particularly sensitive to MSM. Surprisingly, lower concentrations of MSM (for example, the 5-9% concentration range) substantially inhibits bacterial growth in this study. In this way, in several modalities, MSM in concentrations greater than 5% is used to inhibit microbial activity.

Example 8

Evaluation of 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 preservative over a period of 28 days.

To evaluate the ability of MSM to function as a long-term antimicrobial on a cosmetic basis, 10% MSM is incorporated into a cosmetic cream matrix added with E. cali, which is evaluated for a period of 28 days using the protocol USP <51> AET. The cosmetic cream matrix in which the MSM is incorporated was free of preservatives. An additional cream, with a preservative, was also added with E. coli and evaluated. The results of these studies are illustrated in Tables 8-1 to 8-4.

Table 8-1. Effect of 10% MSM on Microbial Growth in an Unconservative Cosmetic Cream

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Table 8-2. Reduction of Initial Inoculum Log

Microorganism with 10% MSM in a MSM Dilution of Cosmetic Cream of Jojoba Free of Preservative

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Table 8-3. Microbial Growth in a Cosmetic Cream that Contains a Preservative

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Table 8-4. Reduction Log of Inoculation of Initial Microorganism in Jojoba Cosmetic Cream that Contains a Preservative

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These studies show that a cosmetic cream base containing MSM is effective to substantially inhibit microbial growth over a period of 28 days. In addition, these studies illustrate that under certain conditions, MSM is a more efficient antimicrobial agent than a standard cosmetic preservative. For example, 10% MSM reduces the microbial load to a degree greater than 48 hours compared to a cream containing preservative. Furthermore, S. aureus is reduced to almost undetectable levels at 48 hours in the cream containing MSM. In contrast, the cream containing preservative showed a modest bacterial population of 3 x 104 bacteria after 48 hours. Despite a less robust initial phase,

Example 9

Antimicrobial Activity Evaluation of MSM in Two Conservative-Free Cosmetic Compositions

This example describes MSM antimicrobial activity in two preservative-free cosmetic compositions.

As described in Example 8, above, 10% MSM is incorporated into the cosmetic matrices, which were added with various initial inocula of microbes. According to the USP <51> AET test, these aggregate microbial cultures were incubated for 28 days, with samples withdrawn at 48 hours, 7 days, 14 days and 28 days for sTeriSra "''" in ^ pTacaTT subsequent colony count . The results of these studies are shown in Tables 9-1 through 9-4 below. Table 9-1. Effect of 10% MSM on Microbial Growth in Conservative Free Cosmetic Composition # 1

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Table 9-2. Reduction Log of Inoculation of Initial Microorganism with a Cosmetic Composition Free of Conservative to 10% of MSM # 1

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Table 9-3. Effect of 10% MSM on Microbial Growth in Conservative Free Cosmetic Composition # 2

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Table 9-4. Reduction Log of Inoculation of Initial Microorganism with Composition Free Cosmetic of Conservative to 10% MSM # 2

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These studies show that MSM exhibited effective antimicrobial properties in the absence of a preservative.

Example 10

Selected concentrations of MSM Support Activity

Microbial

This example shows that select concentrations of MSM support microbial activity.

Parallel growth studies reinforced with MSM at a concentration of 0, 0.04, 0.1, 0.2, 0.4, and 1% MSM, were compared with the growth curve of various microorganisms at a concentration of 0% MSM in the control sample. Each organism (Lactobacillus rhamnosus,

Lactobacillus acidophilus, and Bifidobacterium bifidum) was grown in bacterial growth medium MRS (broth) and plated on MRS agar at different time intervals. The results are expressed in colony forming units per milliliter (cfu / mL).

For each test organism, aliquots of 100 mL of MRS broth were prepared with the respective concentration of MSM as established. Initially, a test solution of 1%, 0.4%, and 0.2% MSM (+/- 0.01%) was 0.45 g of MSM in 110 g of MRS broth and 0.20 g of MSM in 100 g, respectively. The test concentrations of 0.1% and 0.04% were prepared by making a 1:10 dilution of the 1% and 0.4% test solutions. Once each set of test media is coated for sterility, they were inoculated at a level of 100 μΐ inoculum per 100 go mLs of test broth (1: 1000 dilutions of inoculum) with each respective microorganism. All bacterial organisms were incubated at 35 ° C +/- 2 ° C for the duration of the study.

All samples were plated on MRS agar at times 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. The test samples were plated in triplicate plates on each test date and the averages are reported. The results of these studies are given in Tables 10-1 to 10-3.

For samples of Lactobacillus rhamnosus (Table ΙΟΙ), within the first 12 hours all the MSM samples recovered at least 12% or more than the control at 0%. The concentrations of 0.2% and 1% were 41% and 47% higher respectively within the first 12 hours. All the test values ​​were in line at 24 hours before leveling, the cultures were highly turbid, the organism was directed to stationary phase. However, after leveling slightly at 36 and 48 hours, the counts in the MSM samples continued to increase while the 0% control started to fall.

Table 10-1. Growth of Lactobacillus rhamnosus

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These studies suggest that MSM concentrations of about 0.1% to about 1% improve the growth / function of Lactobacillus rhamnosus, with microbial "*" levels in the range of 11% to 41% higher than at the end of 72 hours.

For samples of Lactobacillus acidophilus (Table 10-2), the concentrations of 0.04% and 0.1% MSM were the first in growth of production, followed by 0.2% and 0.4% MSM at 36 hours and the sample of 1% MSM for 48 hours . Control growth at 0% was not recovered, suggesting that MSM had a positive impact on recovery. Lower concentrations of MSM revealed a shorter recovery time than higher concentrations of MSM. Samples of 0.04% and 0.4% MSM resulted in high growth levels for Lactobacillus acidophilus. These studies illustrate that MSM affects microbial metabolism in a way that promotes microbial adaptability and recovery.

Table 10-2. Growth of Lactobacillus acidophilus

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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 sowing intervals on plates for Bifidobacterium test samples and sowing events on plates.

For 48 hours, samples of 0.04% and 0.2% MSM were 1 log higher than the control at 0% MSM. The sample of 0.2% MSM had the highest growth level for Bifidobacterium bifidum, followed by the sample of 0.04% MSM.

As observed with Lactobacillus rhamnosus (Table 10-2), samples from 0.1% to 1% MSM continued to grow while the control was directed to a phase of descending stationary growth (Table 10-3). Increase Ho i and -9 log of Bifidobacterium were observed with concentrations of 0.04% and 0.2% MSM, respectively (Table 10-3).

Table 10-3. Growth of Bifidobacterium bifidum

<img img-format="tif" img-content="drawing" file="MX353059BD02101.tif" id="idf0056" />

The general growth characteristics of probiotic organisms in medium supplemented with MSM and free of MSM are also tested by observation. Colony size of Bacillus coagulans developed in 0% medium and medium containing 5% MSM are compared (see Example 13 for detailed description). "

Example 11

Evaluation of the Influence of MSM on Storage Life

This example describes the effect of MSM on the storage life of milk. MSM as an additive has been shown to increase the growth and recovery of beneficial microorganisms in a product. This example examines whether MSM modifies microorganisms that affect the shelf-life stability of a product based on microbial counts. Milk that has a relatively short storage life was used as the product to evaluate in this study. Milk with fat concentrations are analyzed to study the effects of how the concentration of solids in the product can affect MSM. The standard storage life for milk is 18 to 21 days, the study is carried out at 28 days. Storage life study was carried out on milk products reinforced with MSM at 0.0%, 0.5%, 1.0%, 2.5%, 5.0% and 10%. The time intervals for sowing in plates of solutions in days were on days 0, 7, 14, 21, 24 and 28. The influences of the percent of solids in concentrations of MSM to the following by hundreds were evaluated: 0.0%, 1.0%, 2%, 10.5% and 40%. The growth curves of colony-forming units recovered per milliliter (cfu / mL) of my croraans were compared between the 0% MSM concentrations as a control sample. MSM raw material powder was supplied by Bergstrom Nutrition with certificate of analysis. The powder was the microgranule formula, lot # 0806809, expiration date 10/31/13. All media, water and powder of MSM raw material were checked for sterility before the study. The MSM concentrations of the work were prepared from a single MSM solution at 10. 0% and were diluted according to bottled milk to obtain the final desired concentration of MSM. The product was supplied by a local milk processing plant. Samples were collected and the study started on the day of processing. The product included two bottles of each type of product for each concentration of MSM for each day of analysis. The total of bottles for one type of product were 72 for the entire experiment. All bottles of the same type of product came from a production lot. The product included two bottles of each type of product for each concentration of MSM for each day of analysis. The total of bottles for one type of product were 72 for the entire experiment. All bottles of the same type of product came from a production lot. The product included two bottles of each type of product for each concentration of MSM for each day of analysis. The total of bottles for one type of product were 72 for the entire experiment. All bottles of the same type of product came from a production lot.

The microorganisms analyzed were the normal flora found in the product after processing. The product samples were kept at 4 ° C for the duration of the study. All the preparation and sowing in plates was carried out at room temperature. Each concentration of MSM was duplicated. Each dilution was plated in duplicate plates for each time interval sampled.

To capture the appropriate colonies per milliliter, each organism in each time interval was plated in three dilutions. All coatings were incubated at 37 ° C ± 0.5 ° C for 48 hours before the examination. The appropriate dilution plate was used for numbering and average for reporting. The appropriate plate for enumeration contains between 25 and 250 cfu / mL.

The MSM raw material sample and all media prepared with MSM were tested for background levels of microorganisms on Tryptic Soy agar. The MSM raw material was &lt;10 cfu / g and all test media were negative in all cases before inoculation. All time intervals for plating include negative control plates during casting for quality control purposes. All negative control plates were absent for growth of microorganisms. The results of these studies are shown in Tables 11.1 -15.1 below.

Table 11.1. Growth Log of Fat-free Milk

Cone. Time (days)

of MSM

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<img img-format="tif" img-content="drawing" file="MX353059BD02141.tif" id="idf0058" />

Table 12.1. 1% Milk Fat Log Growth

Conc. Time (days) of MSM

<img img-format="tif" img-content="drawing" file="MX353059BD02142.tif" id="idf0059" />

Table 13.1. Growth Log of 2% Milk Fat

Conc. Time (days) of MSM

<img img-format="tif" img-content="drawing" file="MX353059BD02143.tif" id="idf0060" />

Table 14.1. Milk Fat Log Growth aT * T $ A.L5% * iSa ~ ·

Conc. Time (days) ___

of MSM

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Table 15.1. 40% Milk Fat Log Growth

Conc. Time (days)

of MSM

<img img-format="tif" img-content="drawing" file="MX353059BD02152.tif" id="idf0062" />

When all the milk without MSM is evaluated, there was a peak in the counts on day 21. This is a typical standard peak with the milk products. The increase in normal flora reaches a point 2 log and there is an onset of product degradation. At 4 logs, the storage life of the product is questionable and the sensory factors make the product undesirable.

The evaluation study takes into consideration the nature of the product used. The product is taken from a day of batch production. Microbial counts for a single batch of milk products can vary by 0.5 to 1.5 logs. Seeing day 0 of growth, the intervals for each product are within 1.5 logs each other.

On day 7, there was a slight increase in microbial counts for control and MSM concentrations. There was a microbial count that was higher than the others for each sample, except for the 10.5% milk fat product. The microbial counts of the product at 10.5% were all within 0.75 logs each other, (control and concentrations of MSM). The non-fat milk products and 40% had an increase in the control sample. While the 1% milk products had a peak in the 5% MSM sample and the 2% milk product had a peak in the 1% MSM sample.

On Day 14, the products show normal microbial counts and growth rates. There is no abnormal growth in the products. Products with lower milk fat are within expected microbial load variabilities, when comparing white with MSM concentrations. The milk product at 40% shows a lower microbial count for cJüWCTTCi "&lt;aC 1 to 5.00% and 2.50% while the target and the other MSM concentrations are all within 0.40 logs in microbial counts. 10.5% shows that the target is 1 log higher than the concentrations of MSM with the MSM at 1.00% and 10.0% which are the only two with a microbial count.

On Day 21, the microbial counts of products are separated from the white and MSM concentrations. The 1% milk products without fat indicated that MSM in the higher concentrations (5.0% and 10.0%) slowed the growth of the normal flora. While microbial control counts and lower MSM concentrations increased to 4 logs. In the 2% milk product, the concentration of MSM at 10.0% and the concentration of 2.50% of MSM slowed the growth rate of normal flora. The microbial counts of MSM at 0.50%, 1.00%, 5.00% and the target control were 4 logs. The milk product at 10.5% showed MSM at 5.00% at 0.35 logs, slowing down the growth rate compared to the control, which was at 4.43 logs. The concentration of MSM at 0.50% was 4.36 logs. MSM at 1.00% was at 3.30 logs, MSM at 2.50% went to 2.34 logs and MSM at 10.0% went to 2.14 logs. With the increase in MSM concentrations, microbial counts decreased, except for MSM at 5.00%. The microbial load of the milk product at 40% was relatively equal in the count for the control, MSM at 0.5%, 1.0% and 10%. The concentration of MSM at 2.5% was two logs lower than the control at 2.16 logs, while MSM at 5.0% was lower at 3.22 logs.

On Day 24, the control and the MSM at 1.0% for the non-fat milk product fell around 1.3 logs, while the MSM at 0.50% maintained microbial counts. Microbial counts of MSM at 2.50% dropped, while MSM increased to 5.0%. There was no growth observed in the MSM at 10.0%. The 1% milk product had a microbial count decreased in the MSM to 0.50%, increased in the MSM to 1.00%, and without alteration in the control and 2.5% MSM. The MSM at 5.0% increased and at MSM at 10.0% it decreased. These two concentrations of superior MSM maintained a low microbial count. For the 2% milk product, all concentrations of MSM and control continued to increase in the microbial load. The 10% MSM continued to delay in the microbial count. The milk product at 10. 5% showed a decrease in control and MSM concentrations: 0.5%, 1.0%, and 10.0%. The MSM at 2.5% and the MSM at 5.0% continued to grow. The milk product at 40% showed a slight decrease in growth for the control, MSM at 5.0% and the two lowest concentrations of MSM. Microbial counts of MSM at 2.5% increased to 24 hours, while MSM at 10.0% had a significant decrease in microbial growth. MSM at 5.0% and MSM at 10.0% were counts close to the microbial loads! initial day 0 while MSM at 10.0% had a significant decrease in microbial growth. MSM at 5.0% and MSM at 10.0% were counts close to the microbial loads! initial day 0 while MSM at 10.0% had a significant decrease in microbial growth. MSM at 5.0% and MSM at 10.0% were counts close to the microbial loads! initial day 0

On Day 28, non-fat milk products were greater than the microbial load of 2 logs. The concentration of MSM at 0.5% was 3.79 logs. The highest concentrations of MSM, 2.50%, 5.0% and 10.0% did not have growth for day 28. The milk product at 1% showed an increase for the control, MSM at 0.5% and 1.0%. The MSM sample at 2.5% had a decrease in microbial load, while the MSM at 5.0% and 10.0% had no growth. The milk product at 2% had an increase for the MSM at 1.0%, a slight decrease for the control, MSM at 0.50% and 2.5%. The MSM at 5.0% and 10.0% decreased to 0.59 logs and 1.66 logs respectively. Higher fat milk products, 10.5% and 40%, show that the control continues to increase in microbial counts. Both products had the MSM at 1.0% increasing, while the product 10. 5% also had an increase of MSM to 0.5% in microbial counts, the product of 40% had a decrease in MSM to 0.5%. MSM at 2.5% decreased in microbial load for both products. 5.0% and 10% for the milk product at 10.5% had no growth. The 40% product had a microbial count of 0.35 logs for the MSM concentrations of 5.0% and 10.0%.

These studies indicate that the use of MSM as an additive to milk does not adversely affect the shelf life of milk. In particular, at day 21 there was no concentration of MSM that had a microbial count higher than the control. Furthermore, these studies indicate that in certain milk products, a concentration of MSM at 5.0% or MSM at 10.0% in fact kept the microbial load significantly lower than the control. These studies suggest that MSM at these concentrations can be used to increase the shelf life of a product, such as milk.

Example 12

Acidophilus Milk and Growth of Bacillus coagulans in Simulated Gastric Acid supplemented with MSM

This example describes acidophilus milk and growth of Bacillus coagulans in simulated gastric acid supplemented with MSM.

To analyze the effects of methylsulfonylmethane (MSM) on the growth of probiotic microorganisms reinforced with MSM in a simulated stomach fluid. Previous studies have shown that the addition of MSM to the growth medium helps in the growth rate of microorganisms. The study will measure the effect of probiotic growth reinforced with MSM in a simulated gastric acid fluid.

Microbial growth studies were performed in the presence of 0%, 0.25%, 2.0% and 5%. Time intervals for sowing plates were taken every 3 hours for 15 hours after 24 and 48 hours. The growth curves of colony forming units recovered per milliliter (cfu / mL) of the microorganisms were compared between concentrations of MSM with the concentration of MSM at 0% as a control of the sample for each microorganism. MSM raw material powder was supplied by Bergstrom Nutrition with certificate of analysis. The powder was the microgranule formula, lot # 0806809, expiration date 10/31/13. All MSM raw material media and powder were checked for sterility prior to the study. The study was carried out in two organisms over a period of two weeks.

For milk lactobacillus acidophilus, 11 mLs of milk with a count of 81,000 cfu / mL are added to 99 mLs of simulated gastric acid. For Bacillus coagulans 1 gram of powder is added to 99mL Tryptic Soy Broth (TSB = Tryptic Soy Broth) to obtain the count of 108,000 cfu per mL of Bacillus coagulans. Eleven milliliters of Bacillus coagulans TSB are added to 99 mLs of simulated gastric acid. Work MSM concentrations were prepared from a single 5.0% MSM solution and diluted according to milk or TSB to obtain the desired final concentration of MSM. All the solutions were checked for sterility before proceeding with the study. The simulated work gastric acid was incubated at 35.0 ± 0.2 ° C during the study. The pH of the simulated gastric acid was 1.2.

Lactobacillus acidophilus milk was inoculated on MRS agar at the times previously mentioned. Bacillus coagulans was inoculated in Tryptic Soy Agar (TSA) at the times previously mentioned. All preparation and coatings were carried out at room temperature. Each concentration of MSM in the simulated gastric acid was duplicated. Each dilution per organism was seeded in triplicate plates for each time interval sampled. To capture the appropriate colonies per milliliter, each organism in each time interval was coated in 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 before examination. The appropriate dilution plate was used for enumeration and average for reporting.

The MSM raw material sample and all media prepared with MSM were tested for background levels of microorganisms on MRS and TSA agar. The MSM raw material was &lt;10 cfu / g and all test media were &lt;1 cfu / mL, in all cases before inoculation (see Table below). All time intervals for plating include negative control plates during casting for quality control purposes. All the negative control plates were clean for growth of microorganisms. The results of these studies are given in Tables 16.1 to 18.2 below.

Table 16.1. Control of Raw Material Culture Numbers Before inoculation of Test Sample

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The control of numbers is derived from growth of specific organism in appropriate medium. The inoculation liquids were seeded in plates for enumeration in the appropriate media. To capture the appropriate colonies per millimeter, each liquid was coated in triplicate at four different dilutions. The appropriate dilution plate was used for enumeration and averaged for reporting. The appropriate plate for enumeration contains between 25 and 250 cfu / mL. Table 17.1. Log growth of L. acidophilus in milk in duplicate

Concentration of MSM in percent

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in oo

+ J

G

&lt;D

-I-I £ 0 0)

M

OR

Table 17.2. Log growth of L. acidophilus in average milk

<img img-format="tif" img-content="drawing" file="MX353059BD02251.tif" id="idf0065" />

Acidophilus milk placed in simulated gastric acid was aided with MSM in the recovery of Lactobacillus acidophilus. Initial recovery was less than the detection limit of the method. MSM at 5.0% had an initial log recovery of 0.41. At hour 3 it showed a recovery peak for MSM at 2.5%, while maintaining the MSM growth log at 5.0%. At the 6th hour of MSM at 0.25% it had a recovery of 0.52 logs, MSM at 5.0% showed a recovery of 0.91 logs. MSM at 2.5% had a decrease in growth to no detectable. At hour 9, there was a significant detection for all concentrations of MSM. The 0% control remains below the detectable limit. At hour 12, the control grew to 0.5 logs equaling MSM to 0.25%. The MSM samples of 2.5% and 5.0% had growth rates at 1.02 * and * TtT5 * "* I ' ogs7 'respectively. At hour 15 it showed continuous growth with the concentrations of 1.43, 1.34, and 1.43 logs. The 0% MSM control increased by 0.4 8 logs to 0.98 logs. The MSM at 0% and the MSM at 0.25% at 24 hours decreased in growth by 0.31 logs and 0.11 logs, respectively. MSM at 0.25% increased by 0.30 logs and MSM at 5.0% increased by 0.23 logs. At now 48, the MSM at 0.25% and the MSM control at 0% decreased below detectable limits. The 2.5% MSM decreased by 0.38 logs and the 5.0% MSM decreased by 0.33 logs. 25% increased by 0.30 logs and MSM by 5.0% increased by 0.23 logs. At now 48, the MSM at 0.25% and the MSM control at 0% decreased below detectable limits. The 2.5% MSM decreased by 0.38 logs and the 5.0% MSM decreased by 0.33 logs. 25% increased by 0.30 logs and MSM by 5.0% increased by 0.23 logs. At now 48, the MSM at 0.25% and the MSM control at 0% decreased below detectable limits. The 2.5% MSM decreased by 0.38 logs and the 5.0% MSM decreased by 0.33 logs.

Table 18.1. Growth Log of Bacillus coagulans in duplicate

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Table 18.2. Growth Log of Bacillus coagulans average

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The initial recovery of Bacillus coagulans indicates that there is no recovery. The MSM samples at 0.25% and 5.0% had an average of 0.26 logs, however. A low recovery is seen through the study for MSM at 0.25% and at the 15th hour for MSM at 5.0%. The recovery was too low to reach a conclusion regarding the study with gastric acid for Bacillus coagulans. It is possible that the initial exposure of 3 hours exterminated the organism.

These studies reveal that regarding milk Acidophilus there is a positive impact on bacterial growth with milk containing MSM. In the first 3 to 6 hours there is a slight increase in the log phase growth of each L. acidophilus at the MSM concentrations at 0.25%, 2.5% and 5.0%. At hour 9, there is a significant recovery db h. Acidophilus of milk at MSM concentrations against control. At hour 12 is when there was the first indication of recovery of L. acidophilus in control milk at 0.5 logs, equaling MSM at 0.25%. The MSM concentrations of 2.5% and 5.0% are at a recovery growth rate of 1 log. At the 15th hour the control reaches its maximum growth at 0.98 logs. The MSM concentration at 0.25% reaches the maximum at 1.4 3 logs. The MSM of 2.5% and 5.0% reach the maximum at hour 24 to 1.64 and 1. 66 logs, respectively. At hour 24 it shows an extinction for the control and MSM of 0.25%. At 48 hours, the control and MSM at 0.25% are below the limit detectable for the method and MSM at 2.5% and 5.0% is still about 1 log of the organism. MSM seems to help this process by accelerating adaptation and allowing microorganisms to adapt more quickly to environmental stressors.

The samples treated with MSM showed an increase in the log growth phase. This log phase increase is seen easier in the 5.0% MSM concentration. MSM at 5.0% is 0.45 logs higher than the control at hour 15, which is the maximum growth recovery for the control. The MSM at 5.0% reached a maximum of 1.66 logs or 0.68 logs higher than the control. This indicates a survival rate of daughter cells at a higher percentage than the control sample at 0%. In this way, suggesting that the environment with MSM leads to cell multiplication and survival. MSM also affects the stationary phase and the extinction phase. The stationary phase of control was shorter than the stationary phase of MSM at 2.5% and 5.0%. From hour 15 to 24, the samples not only maintain the growth rate, but they continue to increase in logs by a minimum of 0.24 logs. These results indicate that MSM as an additive allows L. acidophilus to flower longer, allowing the organism to establish itself for a better health benefit. The control was not detectable at time 48. The MSM growth of 2.5% and 5.0% was still about 1 log. This indicates that the survival of L. acidophilus in the milk was greater with the MSM additive.

The study with Bacillus coagulans does not indicate recovery. This was possible due to the exposure time in the gastric fluid. A shorter exposure time would be beneficial for the survival of Bacillus coagulans. The difference between the study of Lactobacillus acidophilus and Bacillus coagulans was the matrix. Milk provided a sufficient buffer to allow the survival of L. acidophilus in the gastric fluid.

Example 13

Feasibility Measure of Bacillus coagulans

Supplemented with MSM

This example describes the effect of 'MSM on the viability of Bacillus coagulans and colony formation.

Studies of robust microbial colonies were conducted in the presence of MSM of 0%, 1.0%, 2.0% and 5%. The microorganisms were grown 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 was also measured in an area of ​​25 mm x 25 mm of the tryptic soy agar placed between two microscope slides in a spectrophotometer.

The growth curves of the colony-forming units recovered per milliliter (cfu / mL) of the microorganisms were compared between the MSM concentrations with the 0% MSM concentration as a sample control for each microorganism. MSM raw material powder was supplied by Bergstrom Nutrition with certificate of analysis. The powder was the microgranule formula, lot # 0806809, expiration date 10/31/13. All MSM raw material media and powder were checked for sterility prior to the study. The microorganisms analyzed include Bacillus coagulans 9BB Lot # 0109E002 supplied by Ganeden.

For Bacillus coagulans, the organism was isolated and developed for 24 hours before harvesting. The microorganism collected was placed in a sterile 100 mL bottle called dilution A. Dilution A was also diluted in a working solution, with a count of 210 Bacillus coagulans per 1 mL, called dilution B. One milliliter of Dilution B was used to inoculate the 30 mL of TSB concentrations previously established. Working MSM concentrations were prepared from a single 5.0% MSM solution and diluted according to TSB to obtain the final desired concentration of MSM. All the solutions were checked for sterility before proceeding with the study.

Bacillus coagulans was inoculated in Tryptic Soy Agar (TSA) at 35 ° C ± 0.5 ° C for 72 hours for verification and population density of the colony. All preparation and sowing in plates are carried out at room temperature. Each concentration of MSM in the study was duplicated. Each dilution for the microorganism was seeded in triplicate plates for each sample. To capture the appropriate 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 appropriate plate for enumeration contains between 25 and 250 cfu / mL.

The MSM raw material sample and all media prepared with MSM were tested for background levels of MRS in agar and TSA. The MSM raw material was &lt;10 cfu / g and all test media were &lt;1 cfu / mL in all instances before inoculation. All time intervals for plating include negative control plates during casting for quality control purposes. All negative control plates were cleaned for growth of microorganisms. The results of these studies are provided below.

Table 19.1. Control of Raw Material Culture Numbers Before Inoculation of Test Sample

<img img-format="tif" img-content="drawing" file="MX353059BD02321.tif" id="idf0068" />

The control numbers are derived from growth of the specific organism in an appropriate medium. The inoculation liquids were seeded in plates for enumeration in the appropriate medium. To capture the appropriate colonies per milliliter, each liquid was plated in triplicate at four different dilutions. The appropriate dilution plate was used for enumeration and averaged for reporting. The proper plate for enumeration contains between 25 ογ &amp; 7ΤΠΙΓΓ

Table 20.1. Population table of Bacillus-Goagulana -

<img img-format="tif" img-content="drawing" file="MX353059BD02331.tif" id="idf0069" />

The population count is based on the dilution of the tryptic soy broth after 72 hours and inoculation on tryptic soy agar plates. The plates were incubated for 48 hours and enumerated.

Table 21.1. Weight of Bacillus coagulans

<img img-format="tif" img-content="drawing" file="MX353059BD02332.tif" id="idf0070" />

The broth of tryptic broth after 72 hours was centrifuged in a conical vial. The supernatant was peeled off and the precipitate was washed. The centrifugation and washing are repeated three times. At the end of the third wash, the ampules with precipitate were weighed. Each vial was weighed, emptied and recorded. The corresponding ampoule weight was then subtracted from the final weight of the precipitate and ampoule, to obtain the weight of the population of Bacillus coagulans. A 25 x 25-inch agar section was cut from each plate at the end of the 72-hour period and placed between two microscope slides. The slides were sealed to prevent the agar plug from slipping. The wavelength was adjusted to 546 nm, and two empty slides were used as target. When the plates were examined, a colony of Bacillus coagulans is observed to be larger and more robust looking with 5.0% MSM compared to the 0% MSM control. The weights indicate a higher growth or a colony that was larger in size contributing to the weight of the biomass. Table 22.1. Of transmittance percent of Bacillus coagulans

Concentration of MSM in Transmittance in ^^ ΤΓιΚΙ l> Wce - ^ percent percent

Agar 61.1% 0.0 52.3% 1.0 51.5% 2.5 49.8% 5.0 45.6%

The percent transmittance is used to indicate the colony size of Bacillus coagulans where a decrease in transmittance indicates an increase in colony size since the colony inhibits the passage of light through the agar. The addition of MSM 1 to 5% seems to be a cause of decrease in percent transmittance. It was observed that the result in percent of transmittance can be influenced by the variables sample size, sample location and agar.

From visual observation, it was noted that the colonies were larger in size after treatment with MSM compared to the blank control. The colonies also resulted in a higher total weight when the biomass is measured in the samples treated with MSM compared to the blank control. These two results combined with the measured transmittance percent indicate that MSM as an additive (at certain concentrations) influences the viability, health and size of Badilu-s-

Example 14

Effect of MSM on Recovery Growth of Lactobacillus acidophilus in a Simulated Intestinal Tract Environment

This example describes the effect of MSM on growth and recovery of Lactobacillus acidophilus in a simulated intestinal tract environment.

Microbial growth studies reinforced with MSM were performed at the following concentrations: 0%, 0.25%, 2.0% and 5%. The time intervals for coating solutions in hours were 0, 3, 8, 24, 30, 36, 48, 54, 60 and 72. The growth curves of colony forming units recovered per milliliter (cfu / mL) of. the microorganisms were compared between the MSM concentrations with the 0% MSM concentration as a control sample for each microorganism. The MSM raw material powder was supplied by Bergstrom Nutrition with certificate of analysis. The powder was the microgranule formula, lot # 0806809, expiration date 10/31/13. All media, water and powder of MSM raw material were checked for sterility before the study. The pH of the simulated gastric acid was 1.2. The pH of the simulated intestinal fluid was 6.8.

For Lactobacillus acidophilus, ..... '3a I used loQ'ho .......... bottled as the product. One milliliter of a 9-log organism solution is placed in 99 mL of bottled milk and mixed with shaking by hand. This was repeated for each MSM concentration. The suspension was listed for each concentration of MSM and is referred to as the initial inoculum. Ten milliliters of each MSM concentration and milk

Lactobacillus acidophilus are placed in 90 mL of simulated gastric acid for 20 minutes. The gastric acid was preheated to 35 ° C and kept at 35 ° C for the duration of 20 minutes. At the end of the 20-minute period, 10 mL of the simulated gastric acid, Lactobacillus acidophilus and milk mixture were placed in 90 mL of simulated intestinal fluid. The simulated intestinal fluid was previously heated to 35 ° C and kept at 35 ° C for the duration of the study. The working concentrations of MSM were prepared from a single 5.0% MSM solution and diluted according to bottled milk to obtain the final desired concentration of MSM. All the solutions were checked for sterility before proceeding with the study.

Intestinal solution of Lactobacillus acidophilus inoculum on MRS agar at the times previously mentioned. All the preparation and coating are carried out at room temperature. Each concentration of MSM is done in duplicate.

Each dilution for each organism is coated in triplicate for each time interval sampled. To capture the appropriate colonies per milliliter, each organism in each time interval is coated at four dilutions. All plates were incubated at 37 ° C ± 0.5 ° C for 72 hours in a C02 environment, before examination. The appropriate dilution plate is used for enumeration and average for reporting. The appropriate plate for enumeration contains between 25 and 250 cfu / mL.

The MSM raw material sample and all media prepared with MSM were tested for background levels of microorganisms on MRS agar and Tryptic Soy agar. The MSM raw material was &lt;10 cfu / g and all test media were negative in all instances before inoculation. All time intervals for coating include negative control plates during casting for quality control purposes. All negative control plates were absent from microorganism growth. The results of these studies are provided in the following Tables.

Table 23.1. Log growth of Lactobacillus acidophilus

Concentration of MáSf, STBIAl ^

<img img-format="tif" img-content="drawing" file="MX353059BD02391.tif" id="idf0071" />

Reviewing the data there is a benefit with the addition of MSM to the product in the growth and recovery of Lactobacillus acidophilus. Comparing the control of MSM 0% against the MSM at 5.0% there is a significant increase in the log phase of growth with Lactobacillus acidophilus. Within the first 24 hours the MSM at 5.0% was 1.81 logs higher than the control. Over the next 12 hours the control, 0.25% and 2.5% decreased. The MSM at 5.0% continues to increase over the same period of time. At the end of the MSM time window 5.0% was in 8.65 logs, 4.65 logs higher than the control. From the 36th hour to the MSM concentrations grew at a high level of rapidity of the target control. MSM 0.25% increased by 3.65 logs and MSM by 2.5% increased by 5.85 logs compared to 0.59 logs for MSM 0.0%. That trend changed the next 8 hours with the control increasing by 4.38 logs. The MSM 0.25% decreased, while MSM of 2.5% and 5.0% did not increase so significantly in the control, there was an increase of 2.07 and 3.16 logs. From the 60th hour to the 72nd hour, the target control went down as MSM concentrations increased.

Analyzing the data, another period of 24 hours of testing would have helped to better predict an extinction stage. At 72 hours, the graph indicates that Lactobacillus acidophilus reaches the stationary phase. Without indication of an extinction stage, it is difficult to predict whether the MSM concentration prolongs the life of the larger population more than the control. What we see is an increase in the speed of growth, with a higher population being achieved with MSM at 2.5% and 5.0%. MSM as an additive to Lactobacillus acidophilus products will increase the likelihood that the organism will settle in the intestinal tract. More faster organisms will increase the benefit of taking a probiotic.

With the addition of MSM there is a benefit in recovering Lactobacillus acidophilus after a decrease in population growth. At 24 hours until the end of '' 3i &amp; amp; "iFray a decrease in growth for MSM 0.2 MSM 0.0%, while MSM 0.25% and MSM at 2.5% recover in twelve hours with an increase in significant growth speed, MSM 0.0% takes another twelve hours to show a significant growth rate.For MSM at 5.0% there was no decrease in recovery for this window in time, just a slight decrease in growth velocity.MSM at 5.0% took only 6 hours to produce a increase in the rate of substantial growth after the slight decrease in growth rate at hour 24. This shows how MSM influences the recovery time for Lactobacillus acidophilus. Increasing the recovery time for Lactobacillus acidophilus will be a benefit to help establish an intestinal colony before, increasing the health benefit.

The study requires extending to 96 hours and beyond to see if MSM as an additive can further prolong the population of Lactobacillus acidophilus. A population that can be established for a longer period in the intestinal tract will be the added benefit to probiotic products and the people who take them. MSM as a supplement with Lactobacillus acidophilus helps the body to settle faster, grow at a faster rate and reach a higher population. These attributes will benefit puisuirav '^' Wff ^ 'POTOn Lactobacillus acidophilus as a probiotic.

Example 15

Effect of MSM on Grass and Value Growth

Nutritious

This example describes the effect of MSM on grass growth and nutritive value of this grass.

The effect of MSM on grass growth and nutritive value was evaluated by monitoring grass growth under the following conditions: (1) fertilizer alone; (2) MSM alone (OptiMSM® CNG - Lot # 0922904, speed 1: 500 or 0.91 kg per 92.9 m2 (2 lbs per 1,000 sq. F t.)); and (3) fertilizer and MSM (MSM in a ratio of 1: 500 or 0.91 kg per 92.9 m2 (2 lbs per 1, 000 sq. ft.) in the presence of fertilizers) applied to the same field but through an application separated. The fertilizer tested was Urea (45-0-0) and the grass type includes the following mixture of pastures (meadowsweet, ryegrass, cleft grass, Timothy, red clover, medium and middle fescue; seeds for this formulation are commercially available available on the World Wide Web at the address oregroseeds.com/allnatdairy.html). the field to be tested was measured and marked to denote different levels of MSM application and control. Using a diffusion controlled propagator, MSM and / or fertilizer were applied. The field was watered as usual (irrigated by sprinkler every four days). The pastjtf ~ qtre * * C1Sfitft'a · for seven weeks and three days before being cut for testing. The sampled grass was cut 2.54 cm (1 inch) from the ground and placed in plastic bags to dry before boarding. The results of these tests on nutrient value are shown in Table 23.2 below. a · for seven weeks and three days before cutting for testing. The sampled grass was cut 2.54 cm (1 inch) from the ground and placed in plastic bags to dry before boarding. The results of these tests on nutrient value are shown in Table 23.2 below. a · for seven weeks and three days before cutting for testing. The sampled grass was cut 2.54 cm (1 inch) from the ground and placed in plastic bags to dry before boarding. The results of these tests on nutrient value are shown in Table 23.2 below.

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Table 23.2

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Also, it was noted that while all evaluated steps grew in equal proportions, ryegrass grew 5.08 to 7.62 cm (2 to 3 ") higher in the areas treated with MSM, and it was noted that there was no visible color variation between grass treated with MSM and not treated with MSM Even more, it was noted that horses prefer grass treated with MSM against grass not treated with MSM.

These studies indicate that MSM can alter the nutritive value of 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 depend on the type of grass.

Example 16

Effect of 0.5% MSM on Fermentation Efficiency Related to Beer Production (Red Beer

Scottish)

This example describes the effect of MSM at 0.5% on the fermentation efficiency related to the production of beer, in particular Scottish red beer. ......

It has been shown here that MSM at certain concentrations has a positive effect on microorganisms, including the growth of microorganisms. This positive impact includes organisms such as fungi, yeast and bacteria. Yeast cultures are involved in the production of beer during the fermentation process to produce ethanol and carbon dioxide. This study determines whether MSM at 0.5% by weight has a positive impact, such as increasing the efficiency of the brewing process. MSM was added to the Yeast Starter (1000 mL of H20, 100 g of dried malt extract, 1 vial of Edinburgh Brewer's yeast of white labs) and the must. Mosto is liquid that is extracted from the maceration process during brewing or whiskey.

First, the Leavening Leader is prepared according to standard methods that are known to those with skill in the specialty except 0.5% MSM is added to a treatment group and without MSM added to a control group. This Procedure is detailed below. Materials include the following: 82.11 g (2.64 oz) glass jars; funnel; 2 standard air locks type brewing; 5.0 grams of MSM; 2000 mLs of Water; 200 grams of Dry Malt Extract (DME = Dried MaTt Extract); 2 ampoules of yeast extract White Labs Edinburgh Ale WLP028 and brewing disinfectant (San Star).

Lot preparation Treatment Starter includes the following stages: (1) glass jars, air locks and funnel were thoroughly cleaned and then rinsed with beer disinfectant; (2) 1000 mLs of water were boiled, then 100 grams of DME are added; (3) the sample was boiled for 10 minutes; (4) the sample is removed from the heat and 5.0 grams of MSM are added; and (5) the solution is allowed to cool to 22.2 ° C (72 ° F). The Initiator batch

Treatment was then placed in a disinfected glass jar of 1.89 liters (64 ounces) in which 1-vial of yeast White Labs Edinburgh Ale is used. The air lock was applied and the entire container was placed in a dark room at room temperature for 48 hours.

Preparation of Control Start Batch that includes the following stages: (1) glass jars, air locks and funnel were completely cleaned and then rinsed with beer disinfectant; (2) 1000 mLs of water are boiled, then 100 grams of DME are added; (3) the sample is boiled for 10 minutes; (4) the sample is removed from the heat; and (5) the solution is allowed to cool to 22.2 ° C (72 ° F). The Initial Treatment Lot is then placed in a 1.82-liter (64-ounce) glass jar disinfected using 1-vial of yeast White Labs Edinburgh Ale added. The air lock is applied and the entire container is placed in a dark room at room temperature for 48 hours.

The MSM Treatment Initiator showed signs of activity (bubbling through the trap) at approximately 2 hours after the yeast was added. The control initiator showed no signs of activity until about 10 hours after adding the yeast.

On the day of brewing (2-days after the yeast starter was made) the maceration was prepared. The materials to prepare the maceration include the following: 8.16 kg (18 Ib) of American 2-Row base grain; 1.36 kg (3 Ib) of specialty grain Crystal Malt 40L; .454 kg (1 Ib) of specialty grain Cara-Pils Malt; and water. A macerated cask (a container used in the maceration process to convert the starches into crushed grains into fermentation sugars) and the cooking kettle were cleaned with washing and rinsing of brewers in powder completely. The macerated cask was then disinfected (San Star Brew Disinfectant). The following grains were crushed and ground for maceration: 8.16 kg (18 Ib) of American 2-Row base grain; 1.36 kg (3 Ib) of specialty grain Crystal Malt 40L; and .454 kg (1 Ib) of specialty grain Cara-Pils Malt. 26.49 liters (seven gallons) of water were heated to 57.8 ° C (163 ° F) and then combined in the pre-heated macerated cask. The crushed grains were then added and the solution mixed thoroughly. The lid was connected and the solution was allowed to steep for 60 minutes. After 60 minutes, 16.09 1 (4.25 gallons) of work was drained from the macerated cask into the cooking kettle. Water for maceration was preheated to 75.6 ° C (168 ° F), added to the macerated cask and mixed thoroughly with the grain. The mixture is allowed to incubate for 10 minutes. This process is repeated twice more until a total pre-boiled volume of 48 L (12.75 gallons) is achieved in the cooking kettle. 49 liters (seven gallons) of water were heated to 57.8 ° C (163 ° F) and then combined in the pre-heated macerated cask. The crushed grains were then added and the solution mixed thoroughly. The lid was connected and the solution was allowed to steep for 60 minutes. After 60 minutes, 16.09 1 (4.25 gallons) of work was drained from the macerated cask into the cooking kettle. Water for maceration was preheated to 75.6 ° C (168 ° F), added to the macerated cask and mixed thoroughly with the grain. The mixture is allowed to incubate for 10 minutes. This process is repeated twice more until a total pre-boiled volume of 48 L (12.75 gallons) is achieved in the cooking kettle. 49 liters (seven gallons) of water were heated to 57.8 ° C (163 ° F) and then combined in the pre-heated macerated cask. The crushed grains were then added and the solution mixed thoroughly. The lid was connected and the solution was allowed to steep for 60 minutes. After 60 minutes, 16.09 1 (4.25 gallons) of work was drained from the macerated cask into the cooking kettle. Water for maceration was preheated to 75.6 ° C (168 ° F), added to the macerated cask and mixed thoroughly with the grain. The mixture is allowed to incubate for 10 minutes. This process is repeated twice more until a total pre-boiled volume of 48 L (12.75 gallons) is achieved in the cooking kettle. The crushed grains were then added and the solution mixed thoroughly. The lid was connected and the solution was allowed to steep for 60 minutes. After 60 minutes, 16.09 1 (4.25 gallons) of work was drained from the macerated cask into the cooking kettle. Water for maceration was preheated to 75.6 ° C (168 ° F), added to the macerated cask and mixed thoroughly with the grain. The mixture is allowed to incubate for 10 minutes. This process is repeated twice more until a total pre-boiled volume of 48 L (12.75 gallons) is achieved in the cooking kettle. The crushed grains were then added and the solution mixed thoroughly. The lid was connected and the solution was allowed to steep for 60 minutes. After 60 minutes, 16.09 1 (4.25 gallons) of work was drained from the macerated cask into the cooking kettle. Water for maceration was preheated to 75.6 ° C (168 ° F), added to the macerated cask and mixed thoroughly with the grain. The mixture is allowed to incubate for 10 minutes. This process is repeated twice more until a total pre-boiled volume of 48 L (12.75 gallons) is achieved in the cooking kettle. added to the macerated cask and mixed thoroughly with the grain. The mixture is allowed to incubate for 10 minutes. This process is repeated twice more until a total pre-boiled volume of 48 L (12.75 gallons) is achieved in the cooking kettle. added to the macerated cask and mixed thoroughly with the grain. The mixture is allowed to incubate for 10 minutes. This process is repeated twice more until a total pre-boiled volume of 48 L (12.75 gallons) is achieved in the cooking kettle.

After preparing the macerate, the elaboration process begins. The following materials were used for the elaboration process: .088 1 (3.0 oz) of Cascade hops; 2 tablespoons Irish moss (Irish moss); 105 grams of MSM; cooking kettle containing 48.0 L (12.75 gallons) of must; wort coolant; 2 fermentation vessels; refractometer; brewing disinfectant; rinse brewers powder (PBW = Powdered Brewers Wash); and a filtered air bubbler. The equipment was completely cleaned with PBW. The wort coolant and the filtered air bubbler is disinfected with a beer sanitizer (San Star Brewing Sanitizer). The must (48.0 L (12.75 gallons)) is boiled in a cooking kettle and a 1st aliquot of Cascade hops (..044 L (1.5 ounces)) are added to the solution. 30 minutes of boiling, a second aliquot (.014 L (0.5 oz)) of Cascade hops is added. At 40 minutes of boiling, a third aliquot (.014 L (0.5 ounce)) of Cascade hops is added as well as 2 tablespoons of Irish Moss. At 50 minutes of boiling, a fourth aliquot (.014 L (0.5 ounce)) of Cascade hops is added. The must is decanted from the processing kettle to the must refrigerant and cooled to 23.3 ° C (74 ° F). The must is then divided into two termendores (each of 21 liters in volume). 0.5% MSM (105 grams) is added to the treatment heater. The Brix reading of both fermenters is taken and the base point is recorded (Treatment fermented = 15 Brix, Control fermentor = 14.75 Brix). Each fermentor was tested in Brix every 24 hours for 21 days. Both fermenters were aerated for 25 minutes with disinfected air bubbler. MSM infused yeast was added to the treatment fermenting vessel while the unaltered yeast is added to a control fermenting vessel. Blowing tubes are connected to both fermenters and the fermentation is allowed to proceed for 21 days. The results of these studies are given in Table 23.3 below.

Table 23.3

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When an initiator is developed, the faster the activation of the yeast culture takes place, the better the efficiency and to minimize the potential environmental contamination of undesirable airborne microorganisms. The starter batch treated with MSM shows activity of 80% before the control (2 hours compared to 10 hours). The study also indicated that MSM helped in the fermentation process. As the yeast starter, the faster the activation of the yeast fermentation process the better the efficiency and the potential environmental contamination by microorganisms transported by the unwanted air will be minimized. The termendor treated with MSM showed activity 58% before the control (3.5 hours compared to 9 hours).

These results indicate that MSM is useful in brewing processing.

Example 17

Growth of Lactobacillus acidophilus in Acidophilus Milk Supplemented with MSM

This example describes the growth of

Lactobacillus acidophilus in Acidophilus milk supplemented with MSM.

Microbial growth studies performed on Acidophilus milk reinforced with MSM at 0%, U. 5%, '2 .tT%' and '5%.' Intervals of time for evaluation were at 8 and 16 hours for a total of 104 hours. Samples were then evaluated every 7 days for a total of 28 days. The growth curves of colony-forming units recovered per milliliter (cfu / mL) of the microorganisms were compared between Acidophilus milk with MSM concentrations with Acidophilus milk with the concentration of 0% MSM as a control sample. MSM raw material powder was supplied by Bergstrom Nutrition with certificate of analysis. The powder was the microgranule formula, lot # 0806809, expiration date 10/31/13. Milks were purchased at a local store. Acidophilus milk was low in fat (Darigold). The milk of Acidophilus plus Bifidus had a content of 2% milk fat (Lucerne). The milk of Acidophilus plus Bifidus was run simultaneously with a MSM concentration of 2.5% and 0% as a product containing two microorganisms. Work solutions were maintained at 4 ° C during the study. MSM milk work solutions were run in duplicate.

All preparation and coating was carried out at room temperature. All dilutions for all solutions were seeded in triplicate plates for all time intervals sampled. To capture the appropriate colonies per milliliter, all organisms at all time intervals were plated at three different dilutions. All plates were incubated at 35 degrees C ± 0.5 degrees C in C02 for 72 hours for all solutions. The appropriate dilution plate is used for enumeration and average for reporting. The appropriate plate for enumeration contains between 25 and 250 cfu / mL.

The MSM raw material sample and all media prepared with MSM were tested for background levels of microorganisms on MRS and TSA agar. The MSM raw material was &lt;10 cfu / g and all test media were &lt;1 cfu / mL in all instances before inoculation. All time intervals for plating include negative control plates during casting for quality control purposes. All control plates were cleaned for microorganism growth. At 72 hours, MSM concentrations and negative control solutions were checked for contamination. The results of these studies were provided in Table 24 below.

Table 24. Log growth of Lactobacillus acidophilus in Milk reinforced with MSM

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Cont.

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At time 0 there was at least a 1 log higher growth of Lactobacillus acidophilus in milk without MSM compared to milk boosted with MSM. The significance is, at the 8th hour, the milk reinforced with MSM showed a minimum increase in growth of 1.73 log, while the milk without MSM showed an increase of 0.26 in the growth rate. MSM within the first 8 hours of growth gave a significant increase compared to the control. The highest increase in growth is the 5% MSM with an average log increase of 2.39, while the 2.5% MSM had an average increase of 2.33 logs. At 24 hour shows a growth rate of 710 Qnt min. Control and MSM concentrations. The control had a decrease of 0.32 log in growth at time 32. MSM concentrations of 2. 5% and 5% had a decrease in growth of 0.04 and 0.03 log, while 0.5% decreased by 0.22 log at time 32. At hour 48, the control decreased from 0.31 logs, while the MSM decreased 0.54 logs to 0.5%, 0.24 logs for 2.5% and 0.51 logs for 5%. MSM concentrations maintain a higher recovery speed compared to the control. The concentration of MSM of 2.5% was on average 0.31 logs higher and 5% was 0.21 logs higher. At hour 56 it did not show significant change in growth increase or decrease. At the 72nd hour the control increased by 0.19 logs, while the MSM concentrations were stable. At hour 80 it had a significant increase in growth. The control showed an increase of 1.44 logs. The samples treated with MSM present an increase in growth at 0.5% (1.1 logs), 2.5% (1. 09 logs), and 5% (1.1 logs). At hour 96, the control stabilized. All concentrations of MSM increased on average (0.5%, 0.38 logs, 2.5%, 0.51 logs, and 5.0%, 0.41 logs) for the 96th hour. At the 96th hour, the MSM concentration at 2.5% was 0.23 logs higher than the control. At hour 104, the decrease in log growth was comparable between control and MSM concentrations (control decreased 0.57 logs, MSM decreased 0.5% and 0.60 logs, MSM decreased 2.5% 0.62 logs, and MSM decreased 5.0% 0.64). logs). Comparing the recovery of final growth between the control and MSM concentrations, the study showed that MSM at 0.5% at 0.05 logs is higher than the control, MSM at 2.5% at 0.18 logs higher than the control, and MSM 5% at 0.11 higher logs than the control. All concentrations of MSM increased on average (0.5%, 0.38 logs, 2.5%, 0.51 logs, and 5.0%, 0.41 logs) for the 96th hour. At the 96th hour, the MSM concentration at 2.5% was 0.23 logs higher than the control. At hour 104, the decrease in log growth was comparable between control and MSM concentrations (control decreased 0.57 logs, MSM decreased 0.5% and 0.60 logs, MSM decreased 2.5% 0.62 logs, and MSM decreased 5.0% 0.64). logs). Comparing the recovery of final growth between the control and MSM concentrations, the study showed that MSM at 0.5% at 0.05 logs is higher than the control, MSM at 2.5% at 0.18 logs higher than the control, and MSM 5% at 0.11 higher logs than the control. All concentrations of MSM increased on average (0.5%, 0.38 logs, 2.5%, 0.51 logs, and 5.0%, 0.41 logs) for the 96th hour. At the 96th hour, the MSM concentration at 2.5% was 0.23 logs higher than the control. At hour 104, the decrease in log growth was comparable between control and MSM concentrations (control decreased 0.57 logs, MSM decreased 0.5% and 0.60 logs, MSM decreased 2.5% 0.62 logs, and MSM decreased 5.0% 0.64). logs). Comparing the recovery of final growth between the control and MSM concentrations, the study showed that MSM at 0.5% at 0.05 logs is higher than the control, MSM at 2.5% at 0.18 logs higher than the control, and MSM 5% at 0.11 higher logs than the control. the MSM concentration at 2.5% was 0.23 logs higher than the control. At hour 104, the decrease in log growth was comparable between control and MSM concentrations (control decreased 0.57 logs, MSM decreased 0.5% and 0.60 logs, MSM decreased 2.5% 0.62 logs, and MSM decreased 5.0% 0.64). logs). Comparing the recovery of final growth between the control and MSM concentrations, the study showed that MSM at 0.5% at 0.05 logs is higher than the control, MSM at 2.5% at 0.18 logs higher than the control, and MSM 5% at 0.11 higher logs than the control. the MSM concentration at 2.5% was 0.23 logs higher than the control. At hour 104, the decrease in log growth was comparable between control and MSM concentrations (control decreased 0.57 logs, MSM decreased 0.5% and 0.60 logs, MSM decreased 2.5% 0.62 logs, and MSM decreased 5.0% 0.64). logs). Comparing the recovery of final growth between the control and MSM concentrations, the study showed that MSM at 0.5% at 0.05 logs is higher than the control, MSM at 2.5% at 0.18 logs higher than the control, and MSM 5% at 0.11 higher logs than the control. 0% decreased 0.64 logs). Comparing the recovery of final growth between the control and MSM concentrations, the study showed that MSM at 0.5% at 0.05 logs is higher than the control, MSM at 2.5% at 0.18 logs higher than the control, and MSM 5% at 0.11 higher logs than the control. 0% decreased 0.64 logs). Comparing the recovery of final growth between the control and MSM concentrations, the study showed that MSM at 0.5% at 0.05 logs is higher than the control, MSM at 2.5% at 0.18 logs higher than the control, and MSM 5% at 0.11 higher logs than the control.

On day 7 showed an increase of growth from the hour 104 for all work solutions. The control increased 0.84 logs, MSM to 0.5% increased 1.19 logs, MSM to 2.5% increased 0.87 logs and MSM to 5.0% increased 1.15 logs. MSM at 0.5% was 0.39 logs higher than the control, MSM at 2.5% was 0.21 logs higher than the control and MSM at 5.0% was 0.42 logs higher than the control. On day 14 it showed a significant decrease in growth. The largest decrease in growth was MSM at 0.5% at 4.46 logs. The control then with a decrease of 3.33 logs, MSM to 2.5% to 3.17 and MSM to 5% to 2.67 logs. MSM at 0.5% was 0.74 logs lower than the control, while MSM at 2.5% was 0.37 logs higher. The MSM sample at 5.0% was a complete superior log than the control at 1.08 logs. On day 21 the decrease in growth continued. The control was 1.66 logs smaller, MSM at 0.5% was 0.85 logs less, MSM at 2.5% was 1.87 logs less and MSM at 5.0% was 2.48 logs less. MSM at 0.5% and control were equal in growth log, with MSM at 2.5% 0.17 logs higher than the control and MSM at 5.0% 0.26 logs

ni.ΐί '· ** ιυ ^ Ί imn »rM¡A higher than the control. On day 28, decreased growth continued with 1.3 logs reduction for control, 2.37 logs for MSM at 0.5%, 2.14 logs for MSM at 2.5% and 3.04 logs for MSM at 5.0%. The growth for the negative control at Day 28 was 1.00 logs higher than MSM at 0.5%, 0.68 logs higher than MSM at 2.5% and 1.48 logs higher than MSM at 5.0%.

The milk of Acidophilus plus Bifidus over the course of the study showed similar growth rates. From hour 0 to hour 8 both showed a significant increase in growth. At hour 24, the control decreased 0.17 logs, while MSM at 2.5% increased 0.49 logs, giving MSM at 2.5% a higher count at 0.97 log than the control. At the 32nd hour, MSM at 2.5% decreased 0.29 logs and the control increased 0.54 logs, with the MSM at 2.5% which has a 0.14 log higher count than the control. From hour 48 to hour 72, there was a continuous pattern of increase and decrease in growth, with MSM at 2.5% that has an increased growth of 0.15 and 0.5 logs over the control. At the 72nd hour the growth was equal between the control and MSM at 2.5%. At the 80th hour it showed an increase in growth of 1.21 logs for control and 1.37 logs for MSM at 2.5%, with MSM at 2.5% that has a growth increase of 0.16 log. At the 96th hour, there was a significant decrease in growth for MSM at 2.5% of 1.21 logs. The control showed no significant difference from the 80th hour, resulting in a higher growth 1.07 log for comparison control with MSM at 2.5%. íT "the ..... hour Í04, the control growth decreased by 0.85 logs and the MSM samples to 2.5% increased by 0.31 logs. At hour 104 it showed that the MSM samples at 2.5% at 0.09 logs higher than the control. On Day 7, there was an increase of 1.27 logs for milk and 1.5 logs for MSM at 2.5%, with MSM at 2.5% which is 0.32 logs higher than milk. Day 14 showed a decrease in. growth, 3.27 logs for milk, 4.72 logs for MSM at 2.5%. The milk had an increase of 1.13 in growth compared to MSM at 2.5%. On Day 21 the decrease slowed, milk decreased by 1.23 logs and MSM by 2.5% in 0.33 logs, with milk that is 0.23 logs higher in growth than MSM. The milk of Day 28 decreased by 1.78 logs and MSM by 2.5% decreased by 1.43 logs, with MSM being 0.12 logs higher than milk without MSM. Table 25 shows the data by averaging the duplicates.

Table 25. Growth of Lactobacillus acidophilus in Milk reinforced with average MSM.

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Table 26. Growth of non-probiotic microorganisms in Acidophilus cfu Milk per mL.

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Cont.

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Table 26 shows the data for standard plate counts analyzed in work solutions. This was done to see how MSM would affect the normal flora found in milk. Day 0 was the day in which the samples were configured for the start of the study. The milk of Acidophilus plus Bifidus started with a higher count on Day 0 than what is typically expected. This caused the final values ​​to rise. The milk product of Acidophilus at Day 0 was at the expected values. Acidophilus milk maintained adequate growth rates throughout the study and was equivalent to typical growth rates seen in milk products. The MSM at 5.0% does not allow any significant growth for the study. MSM as an additive to this product plays a significant role in increasing the population of Lactobacillus acidophilus in a product. Within the first eight hours of reinforcing a product with MSM, there was a significant influence on probiotic in the product. There was a significant increase in the growth rate of the probiotic. Acidophilus milk without MSM had an increase of 0.26 log in growth within the first 8 hours. While the milk of Acidophilus with MSM had a minimum of 1.7 3 logs of growth. The milk sample of MSM at 2.5% had increases of 2.27 and 2.39 logs. The MSM sample at 5.0% had an increase of 2.17 and 2.61 logs. Through the study there was a continuous growth increase when the control of Acidophilus milk was compared with Acidophilus milk reinforced with MSM. The increased growth rate was in the range of 0.04 to 1.08 logs over the control. Only at two points in time were there data showing the growth of control superior to the MSM solutions, time 80 and Day 28. When analyzing the data, the reason for the higher growth at time 80 is due to the curve of peak growth Acidophilus milk without MSM reached peak before milk with MSM. Therefore, MSM was still in the growth phase, while milk with Acidophilus reached the peak of its growth. With the increase in growth due to MSM, there was a higher rate of extinction at the end of the study. Therefore, Day 28 showed lower growth for MSM solutions than control. Only at two points in time were there data showing the growth of control superior to the MSM solutions, time 80 and Day 28. When analyzing the data, the reason for the higher growth at time 80 is due to the curve of peak growth Acidophilus milk without MSM reached peak before milk with MSM. Therefore, MSM was still in the growth phase, while milk with Acidophilus reached the peak of its growth. With the increase in growth due to MSM, there was a higher rate of extinction at the end of the study. Therefore, Day 28 showed lower growth for MSM solutions than control. Only at two points in time were there data showing the growth of control superior to the MSM solutions, time 80 and Day 28. When analyzing the data, the reason for the higher growth at time 80 is due to the curve of peak growth Acidophilus milk without MSM reached peak before milk with MSM. Therefore, MSM was still in the growth phase, while milk with Acidophilus reached the peak of its growth. With the increase in growth due to MSM, there was a higher rate of extinction at the end of the study. Therefore, Day 28 showed lower growth for MSM solutions than control. the reason for the higher growth at hour 80 is due to the peak growth curve. Acidophilus milk without MSM reached peak before milk with MSM. Therefore, MSM was still in the growth phase, while milk with Acidophilus reached the peak of its growth. With the increase in growth due to MSM, there was a higher rate of extinction at the end of the study. Therefore, Day 28 showed lower growth for MSM solutions than control. the reason for the higher growth at hour 80 is due to the peak growth curve. Acidophilus milk without MSM reached peak before milk with MSM. Therefore, MSM was still in the growth phase, while milk with Acidophilus reached the peak of its growth. With the increase in growth due to MSM, there was a higher rate of extinction at the end of the study. Therefore, Day 28 showed lower growth for MSM solutions than control. there was a higher rate of extinction at the end of the study. Therefore, Day 28 showed lower growth for MSM solutions than control. there was a higher rate of extinction at the end of the study. Therefore, Day 28 showed lower growth for MSM solutions than control.

Peak growth was achieved with Acidophilus milk reinforced with MSM at 5.0%, with a log of 10.89. Acidophilus milk reached a peak growth of 10.29 logs. All concentrations of MSM exceeded the growth of Acidophilus milk. MSM at 2.5% had a peak growth of 10.59 logs and MSM at 0.5% had a peak growth rate of 10.72 logs. This further establishes the influence of MSM on a probiotic. The product once reinforced with MSM exceeded the growth of the product without MSM. With the peak growth that is higher, there was an increase in growth seen on Day 10, a whole week beyond the peak growth rate. Acidophilus milk growth was 6.96 logs; Milk reinforced with MSM was 7.82 and 8.2 6 logs, an increase of 0.86 and 1.3 logs respectively.

The probiotic effectiveness is based on three points: Survival, Colonization and Lactic Acid Production. MSM demonstrates the ability to affect the survival and colonization of probiotic bacteria. Within the first eight hours, the ability to colonize was seen with an increase in the rate of growth. On Day 14 the ability to survive was seen with the increase in log growth. The ability to increase lactic acid production was the third component of effectiveness -1 "A * ~ &lt;^ qnp will be studied In this study, there was an observed reaction of increased foam production in the MSM solutions.

The statement that MSM is an additive beneficial diet supplement is upheld by this study. Microbial flora of the gastrointestinal tract can be impacted in a positive way with the addition of MSM in the human diet. There was an increase in growth of probiotic bacteria with an increase in survival. Increasing the probability that MSM when used in a probiotic product increases the benefit to the consumer.

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 with analyzes for Recovery. The microbial growth was determined in Acidophilus milk reinforced with MSM at 0%, 0.5%, 2.5% and 5%. On days 7, 14, 21 and 28, the Acidophilus milk samples boosted DW &lt;? &lt;3ln diluted and transferred to the il-n -in ilYHl ^ for the recovery study. Intervals of time for coatings were taken every 24 hours over a period of 72 hours. The growth curves of colony forming units recovered per milliliter (cfu / mL) of the microorganisms were compared between Acidophilus milk with MSM concentrations with Acidophilus milk with MSM concentration at 0% as a sample control. All MSM raw material media and powder were checked for sterility prior to the study. The study was conducted in four organisms over a period of two weeks. The microorganisms were divided into two runs, each lasting one week, analyzing two microorganisms each week. The study was conducted in four organisms over a period of two weeks. The microorganisms were divided into two runs, each lasting one week, analyzing two microorganisms each week. The study was conducted in four organisms over a period of two weeks. The microorganisms were divided into two runs, each lasting one week, analyzing two microorganisms each week.

Acidophilus milk was low in fat (Darigold). The milk of Acidophilus plus Bifidus contains 2% milk fat (Lucerne). The milk

Acidophilus Bifidus was run simultaneously with a MSM concentration of 2.5% and 0% as a product containing two organisms. Work solutions were maintained at 4 ° C during the study. The work solutions based on milk and MSM were run in duplicate. All preparations and coatings were carried out at room temperature. All dilutions for all solutions were seeded in triplicate plates for all time intervals sampled. To capture the colonies per appropriate 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 is used for numbering and average for reporting.

The MSM raw material sample and all media prepared with MSM were tested for background levels of microorganisms on MRS and TSA agar. MSM raw material was &lt;10 cfu / g and all test media were &lt;1 cfu / mL, in all cases before inoculation. All time intervals for plating included negative control plates during casting for quality control purposes. All the control plates were clean of growth of microorganisms. At 72 hours, concentrations of MSM and negative control solutions were negative for contamination.

The study looks at the effect of MSM on the recovery of Lactobacillus acidophilus from Acidophilus milk enhanced with MSM. The recovery study was run in parallel to the study conducted on the effects of MSM on growth of Lactobacillus acidophilus in milk from

Acidophilus reinforced with MSM. The recovery growth rate was performed on Day 7, Day 14, Day 21 and Day 28. For Table 27, the growth for Day x without time is calculated for the initial growth study with the dilution factor in growth log. The growth in Table 28 is calculated from the growth log Day x without time subtracted from the log growth for the dates analyzed subsequently, for example on Day 28 it had a result of 4.00 logs, calculating the dilution on Day 28 time value 0 is 2.00 logs for Table 27. Table 28 takes the value of 2.00 as the starting value. Subsequent data for the hours analyzed, take the counts in logs and subtract the initial value of 2.00, for example, Days 28 - 24 is 11.29 logs, subtracting 2.00 logs the increase in growth speed is 9.

Table 27. Recovery Delay of Lactobacillus Acidophilus in Milk Strengthened with MSM Day 7

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Cont.

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Table 28. Recovery of Lactobacillus acidophilus in Milk reinforced with MSM Day 7 with average recovery in percent of MSM with initial data

Concentration of MSM in percent

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The recovery on Day 7 shows that all concentrations of MSM within the first 24 hours of growth had more than one increase of 1 log over the control, MSM at 0.5% 1.65, MSM at 2.5% 1.87, and MSM at 5.0% 1.42 . At time 48, the control was slightly higher in growth compared to MSM at 0.5% at 0.03 logs, and 0.36 logs higher than MSM at 2.5%, but 0.46 logs lower than MSM at 5.0%. At hour 72, the concentrations of MSM exceeded the growth of the control; MSM at 0.5% at 0.9 logs, MSM at 2.5% at 0.43 logs and MSM at 5.0% at 0.82 logs.

Control of Acidophilus plus bifidus in the first 24 was 12.88 logs for growth, while Acidophilus plus bifidus with MSM at 2.5% was a growth of 13.44 logs. The milk with MSM at 2.5% was 0.56 logs higher than the control. In the next two 24-hour periods, control of Acidophilus plus bifidus grew 13.42 logs and 13.47 logs. Acidophilus plus bifidus with MSM at 2.5% had growth at 12.50 logs and 12.77 logs over the same period of time. The control was 0.92 logs higher than MSM at 2.5% in the second 24-hour period and was 0.7 logs higher in the third 24-hour period.

Table 29. Recovery of Lactobacillus acidophilus in Milk reinforced with MSM Dia 7 in increase of recovery growth rate log from the start time of zero.

Concentration of MSM in percent

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Analyzing the recovery of Day 7 based on the speed of increase in log increments, there was a significant increase within the first 24 hours of growth. Control increases by 3 logs from the initial inoculum, while MSM concentrations increased by 4.26 logs for MSM at 0.5%, 4.66 logs for MSM at 2.5%, and 4.00 logs for MSM at 5.0%. In the second 24 hours there was a decrease in growth compared to MSM at 0.5% and MSM at 2.5%. Control growth increased by 1.42 logs and MSM growth by 5.0% increased by 0.4 7 logs. The third period of 24 hours the growth of control decreased by 0.54 logs and the growth of MSM to 5.0% decreased by 0.19 logs. The MSM at 0.5% and the MSM at 2.5% increased in log growth, 0.39 and 0.25 logs, respectively.

Milk control of Acidophilus plus bifidus showed an increase of 4.78 log in the first 24 hours, compared with an increase of 5.02 for Acidophilus plus bifidus milk reinforced with 2.5% MSM. The second 24-hour period control of Acidophilus plus bifidus increased by 0.54 logs, while 2.5% MSM milk decreased by 0.94 logs. In the third 24-hour period, control of Acidophilus plus bifidus increased by 0.05 logs, while MSM milk by 2.5% increased by 0.27 logs.

Table 30. Delay recovery in milk reinforced with Lactobacillus acidophilus MSM Day 14.

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Cont.

Concentration of MSM in percent

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Table 31. Recovery of Lactobacillus acidophilus in Milk reinforced with MSM Day 14 with average recovery in percent of MSM with start data

INDUSTRIAL

Concentration of MSM in percent

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On day 14 the control exceeded the growth of MSM concentrations in the first 24 hours. The control was 0.4 8 logs higher than MSM at 0.5%, 0.7 5 logs higher than MSM at 2.5% and 1.02 logs higher than MSM at 5.0%. At hour 48, the control solution was 0.05 logs higher than 2.5% MSM. 0.5% MSM was 0.47 logs higher than the control and 5.0% MSM was 0.65 logs higher than the control. At the 72nd hour, the MSM at 0.5% was 0.43 logs higher, MSM at 2.5% was 0.14 logs higher than the control and MSM at 5.0% was equal to the control.

Acidophilus plus bifidus reinforced with 2.5% MSM was 0.03 logs lower than the control of Acidophilus plus bifidus at hour 24. At hour 48, the control of Acidophilus plus bifidus was 0.05 logs higher than Acidophilus plus bifidus with 2.5% MSM . At 72 o'clock the Acidophilus plus bifidus with 2.5% MSM was 0.12 logs higher than the control Acidophilus plus bifidus.

Table 32. Recovery of Lactobacillus acidophilus in Milk reinforced with MSM Day 14 in growth rate increase in recovery log from the initial time of zero.

MSM concentration in percent

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Analyzing the recovery of Day 14 based on the growth rate increase in logs, the following was observed; control increased by 5.66 logs from initial inoculation, while MSM concentrations increased by 5.93 logs for 0.5% MSM, 4.54 logs for 2.5% MSM, and 3.56 logs for 5.0% MSM. In the second twenty four hours, the control growth increased by 1.75 logs while MSM by 0.5% increased by 2.69 logs, the MSM by 2.5% by 2.45 logs and MSM by 5.0% MSM increased by 3.42 logs. The third period of twenty-four hours, the growth of control increased by 0.06 logs and the growth of MSM by 5.0% decreased by 0.59 logs. The MSM at 0.5% and MSM at 2.5% increased in log growth, 0.0.03 and 0.26 logs respectively.

Milk control of Acidophilus plus bifidus showed an increase of 8.55 log in the first 24 hours, compared to an increase of 9.65 for Acidophilus plus bifidus milk reinforced with 2.5% MSM. The second 24-hour period, the control of Acidophilus plus bifidus increased by 0.14 logs, while the 2.5% MSM milk decreased by 0.12 logs. In the third 24-hour period, control of Acidophilus plus bifidus decreased by 0.21 logs, while MSM milk by 2.5% decreased by 0.04 logs.

Table 33. Log recovery of Lactobacillus acidophilus in milk reinforced with MSM Day

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Cont.

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Table 34. Recovery of Lactobacillus acidophilus in Milk reinforced with MSM Day 21 with average recovery in per cent of MSM with initial data

Concentrations of MSM in percent

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Day 21, the Acidophilus control in the first 24 hours had a log growth of 12.94. MSM at 0.5% growth was 12.91 logs, MSM at 2.5% was 10.79 logs and MSM at 5.0% was 9.69. The following 24-hour period control was equal in growth in milk with MSM at 0.5%, 0.24 logs higher than MSM at 2.5% and 0.05 logs lower than MSM at 5.0%. The final 24-hour period shows a significant increase in MSM concentrations compared to the control. MSM at 0.5% was 0.98 logs higher than the control, MSM at 2.5% was 2.78 logs higher than the controls and MSM at 5.0% was 2.83 logs higher than the control.

Acidophilus plus bifidus with 2.5% MSM was 0.65 logs higher than the control of Acidophilus plus bifidus in the first 24 hours. At hour 48, the control of Acidophilus plus bifidus was 0.08 logs higher. In the final 24 hours, the Acidophilus plus bifidus of MSM 2.5% surpassed in growth the control of Acidophilus plus bifidus by 1.47 logs.

Table 35. Recovery of Lactobacillus acidophilus in Milk reinforced with MSM Day 21 in increment in recovery growth rate log from the initial time of zero.

Concentration of MSM in percent

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Reviewing the log increase for Day 21, in the first 24 hours, Acidophilus milk control had an increase of 9.64 logs. MSM at 0.5% had an increase of 9.54 logs, MSM at 2.5% had an increase of 7.32 logs and MSM at 5.0% had an increase of 6.13 logs. In the second 24 hours, the control increased by 0.08 logs. MSM 0.5% increased by 0.11 logs, MSM by 2.5% increased by 2.00, and MSM by 5.0% increased by 3.39 logs. The final 24 hours show that the control decreases by 2.70 logs. MSM 0.5% decreased by 1.72 logs. 2.5% MSM increased by 0.32 logs and MSM by 5.0% increased by 0.08 logs.

Acidophilus plus bifidus with 2.5% MSM increased by 9.71 logs and the control of Acidophilus plus bifidus increased by 8.83 logs. In the two final 24-hour periods, control of Acidophilus plus bifidus decreased by 0.27 logs and 1.30 logs. Acidophilus plus bifidus with 2.5% MSM decreased to 1.00 logs in the second 24-hour period and increased to 0.25 logs in the final 24-hour period.

Table 36. Delay in recovery of Lactobacillus acidophilus in Milk reinforced with MSM Day 28

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Cont.

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Table 37. Recovery of Lactobacillus acidophilus in Milk reinforced with MSM Day 28 with average recovery in percent of MSM with initial data

MSM concentration in percent

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The recovery data from Day 28 show that in the first 24-hour period milk control

Acidophilus had a log growth of 13.10. The concentrations of MSM were; 6.29 logs for MSM at 0.5%, 6.37 logs for MSM at 2.5%, and 5.56 logs for MSM at 5.0%. Hour 48 the MSM at 0.5% growth log was 13.33, the MSM at 2.5% was 12.57 logs and MSM at 5.0% was 12.87 logs. The control at time 48 was 12.71 logs. The control decreased to 12.17 logs at 72 hours. MSM at 0.5% decreased to 13.00 logs and MSM at 5.0% decreased to 12.48 logs. MSM at 2.5% improved to 12.66 logs. This was an increase of 0.53 log over the control.

The control of Acidophilus plus bifidus was 13.41 logs at 24 hours, 0.74 logs higher than Acidophilus plus bifidus with 2.5% MSM. At time 48, the difference was less than the control 0.23 logs higher than Acidophilus plus bifidus with 2.5% MSM. At 72 hours, Acidophilus plus bifidus with 2.5% MSM was 1.69 logs higher than the control, which was 10.31 logs.

Table 38. Recovery of Lactobacillus acidophilus in Milk reinforced with MSM Day 28 in increase in log recovery growth rate of the initial time of zero.

MSM concentration in percent

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On Day 28, the increase in growth speed where the control of Acidophilus milk in the first 24-hour period increased 11.10 logs, MSM to 0.5% increased in 5.29 logs, MSM to 2.5% increased in 5.04 logs, and MSM to 5.0 % increased by 5.04 logs. In the second 24-hour period, there was a change to control that decreased to 2.06 logs, while milk concentrations enhanced with MSM increased 0.5% in 7.04 logs, 2.5% in 6.20 logs, and MSM to 5.0% in 7.31 logs. The final period of 24 hours showed that the control increased by 1.43, the MSM by 2.5% increased by 0.09 logs, the MSM by 0.5% decreased by 0.32 logs and MSM by 5.0% decreased by 0.39 logs.

Milk control Acidophilus plus bifidus increased by 11.59 logs in the first 24 hours and Acidophilus plus bifidus with 2.5% MSM increased by 10.73 logs. In the second 24-hour period, control of Acidophilus plus bifidus increased 0.01 logs and Acidophilus plus bifidus with 2.5% MSM increased by 0.52 logs. In the final 24-hour period, control of Acidophilus plus bifidus decreased to 3.11 logs, while Acidophilus plus bifidus with 2.5% MSM decreased by 1.19 logs.

These studies show that MSM as an additive to this product plays a significant role in the recovery of the probiotic, Lactobacillus acidophilus. In any case recovery, there was an increase in the growth rate of Lactobacillus acidophilus with product reinforced with MSM against the product without MSM.

The recovery data of Day 7 showed in the first 24 hours that MSM had an increase of 0.99 log to 1.66 log compared to the control. In the second 24-hour period for Day 7 even though the growth rate of MSM was lower than the control, the total growth numbers were higher for MSM at 5.0%, 0.46 higher logs. The third 24-hour period for Day 7, the MSM growth rate was higher than the control, 0.36 logs, 0.79 logs and 0.93 logs.

On Day 14, only 0.5% MSM grew exceeding control 0.27 logs the rate of growth in the first 24 hours. The samples of 2.5% MSM and 5.0% MSM were 1.13 and 2.10 logs, respectively, lower than the control. This is the reason why the control began to exceed growth at MSM concentrations in the first 24 hours. On Days 21 and 28, control exceeded growth at all concentrations of MSM in the first 24 hours.

The second 24 hours for each data point that is collected after Day 7, showed that MSM concentrations exceed the control performance. The second data period of Day 14 showed that the growth rates of MSM are 0.70, 0.94, and 1.67 logs higher than the control. The second data period of Day 21 showed MSM growth rates 0.03, 1.92, and 3.31 logs of superior control. Data from the second period of Day 28 show MSM growth rate at 9.10, 8.26, and 9.37 logs higher than the control. This increased growth rate does not always translate into a higher concentration of Lactobacillus acidophilus in the recovery broth. At Day 14, MSM concentrations of 0.5% and 5.0% were higher than the control, while MSM at 2.5% was lower. On Day 21 only MSM at 5. 0% was higher. At Day 28, all three concentrations of MSM were significantly higher than the control, at 2.29, 1.53, and 1.83 logs.

The third data period for Day 14 shows that only the growth rate of MSM concentration at 2.5% was higher at 0.20 logs. Even with the lowest growth rates, a higher growth log was seen for the MSM concentrations, except for 5.0% MSM that was equal to the control. On Day 21, data from the third period show that the growth rate of MSM concentrations exceeds the control by 0.98, 3.02, and 2.78 logs. This increase in growth rate translates into a higher concentration of Lactobacillus acidophilus for samples reinforced with MSM. Growth recovery counts were 0.98, 2.78, and 2.83 logs greater than control. The control exceeded the MSM concentrations for the growth rate of Day 28 in the third period.

With bacterial growth curves there is an initial delay phase where the bacteria adjust to the environment, before moving to the Exponential or log phase, where the cells are duplicated. After the log phase there is a stationary phase where the growth rate slows down. In this phase peaks and valleys are seen as growth brakes. Finally, there is a death phase where the bacteria are depleted of nutrients and die.

This study provides indicators such as MSM help in the delay phase, log phase, stationary phase and death phase. MSM in different stages shortens the delay phase, in such a way that the probiotic bacteria start the log phase in a previous time. The log phase is prolonged beyond the control in this study, so that the product with the MSM additive had a higher peak value. The stationary phase was carried out by MSM since there was an extension of higher values ​​for a longer period of time. The death rate was slowed down with MSM. At different points, there was a slower rate of decline in growth. These different observations show that MSM as an additive positively affects probiotic bacteria. The benefit of ingesting a probiotic product reinforced with MSM will be a faster response time with a longer lasting effect. The consumer will obtain a product that increases their body response to the added benefits of probiotic bacteria. MSM consistently helps in the recovery and growth of probiotic bacteria in the product studied. Within the first 24 hours of growth, there was an increase in recovery speed indicating that in a new environment, stressed microorganisms respond better with MSM as an additive.

Example 19

Growth of Bifidobacterium bifidum in Reinforced Medium with MSM

This example shows the effect of MSM on the growth of Bif idobacterium bifidum in microbial growth medium reinforced with MSM.

Microbial growth studies were performed in medium reinforced with MSM at 0%, 0.125%, 0.25%, 0.5%, 1.0%, 2.5% and 5%. Time intervals for sowing plates were taken every 8 hours for a total of 96 hours. The growth curves of colony-forming units recovered per milliliter (cfu / mL) of the microorganisms were compared between the MSM concentrations with the 0% MSM concentration as a sample control for each microorganism. MSM raw material powder was supplied by Bergstrom Nutrition with certificate of analysis. The powder was the microgranule formula, lot # 0806809, expiration date 10/31/13. All MSM raw material media and powder were checked for sterility prior to the study. The microorganism analyzed was Bifidobacterium bifidum ATCC # 29521.

Bif idobacterium bifidum (99 mL of MRS broth with the addition of 0.05% L-cysteine) is prepared with respective concentrations of MSM. Working concentrations of MSM were prepared from a single 5% MSM in MRS broth solution and diluted in accordance with MRS broth to obtain the final desired concentration of MSM. The solutions were checked for sterility before proceeding with the study.

The working solutions were inoculated at a level of 1.5 to 2 logs of microorganisms by "== MKH ~ ffiK ~ '- dé"' "caTSó'l '" ™ 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 sowing intervals on plates for the Bifidobacterium test samples.

Bifidobacteruim was inoculated on MRS agar supplemented with L-cysteine ​​at the times previously mentioned to reduce the oxidation-reduction potential of the medium. All preparation and sowing in plates are carried out at room temperature. All dilutions for all organisms were seeded in triplicate plates for all time intervals sampled. To capture the appropriate 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 appropriate plate for enumeration contains between 25 and 250 cfu / mL. The MSM raw material sample and all media prepared with MSM were tested for background levels of microorganisms on MRS and TSA agar. The MSM raw material was &lt;10 cfu / g and all the test media were &lt;1 cfu / mL in all cases before inoculation. All time intervals for plating include negative control plates during casting for quality control purposes. All negative control plates were cleaned for microorganism growth. At 72 hours, concentrations of MSM and control solutions were checked for negative contamination of strains and strains were verified with original species. All time intervals for plating include negative control plates during casting for quality control purposes. All negative control plates were cleaned for microorganism growth. At 72 hours, concentrations of MSM and control solutions were checked for negative contamination of strains and strains were verified with original species. All time intervals for plating include negative control plates during casting for quality control purposes. All negative control plates were cleaned for microorganism growth. At 72 hours, concentrations of MSM and control solutions were checked for negative contamination of strains and strains were verified with original species.

Table 39. Control of Raw Material Culture Numbers Before Inoculation of Test Sample

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The control numbers were derived from specific organism growth in the appropriate medium. After incubation, the colonies were washed from the medium and captured in a sterile vial. The vial was used as the starting solution for the numerical control (raw material). The raw material solution was then diluted to obtain an appropriate reading on the spectrophotometer using 420 wavelength with percent light transmission. Bacterial concentrations were determined according to AOAC Method 960.09, table 960.09A. The culture suspension preparation of the raw material culture was determined by spectrophotometer reading or comparison with the McFarland standard.

Table 40. Log growth of Bifidobacteruim bifidum in Reinforced Medium with MSM.

Concentration of MSM in percent

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The growth observed with Bifidobacterium bifidum shows an increase of 0.2 to 0.4 log in the growth rate for the MSM concentrations from 0.125% to 2.5% at the 8th hour. MSM concentrations from 0.125% to 2.5% at the 16th hour increased to 0.3 up to 0.7 logs. At hour 24 he showed the MSM concentrations from 0.125% to 2.5% which slow to be equal to or less than the control. MSM at a concentration of 5% showed slower growth rate compared to the control for the first 24 hours. At time 32, there was a moment in growth velocity in the range of 0.3 to 0.75 logs for all concentrations of MSM compared to the control. At hour 40, the MSM concentrations of 0.125% and 0. 25% showed a uniform decline in growth velocity to the extent that they are below the control from hour 40 to hour 96. At hour 4 0 the MSM sample showed 0.5% at a higher complete log in growth than the control. MSM at 0.5% at hour 48 until hour 96 declined in the growth rate to where it was 2 to 3 complete logs below the growth rate of control. MSM at a concentration of 1% equaled the growth growth rate from hour 40 to hour 96, except that at time 48 and time 80 where it was a complete log less. MSM at 2.5% at hour 40 was 0.7 logs higher in growth rate compared to the control. At hour 48 it showed a 0.7 log decrease in growth rate compared to the control. At hour 56 at hour 72, MSM at 2. 5% had a growth rate that was 0.7 to 2.29 logs higher than the control. At the 80th hour it showed a growth rate lower than 1 log for the MSM sample at 2.5% compared to the control and the 88 and 96 hours the growth rate was equivalent. MSM at 5% at hour 40 has a growth rate of 0.7 logs higher than the control. At hour 48, this fell to 0.7 logs lower than the control and at hour 56 the growth rate was equivalent to control. At hour 64 it shows an increased growth rate of 4.35 logs for MSM at 5% over the control. At the 72nd hour it showed a decrease in the growth rate with a return to an increase of 1.6 log in the growth rate at the 80th hour and the 88th hour. At the 96th hour it showed an MSM growth rate at 5% that It was approximately 3.

Bifidobacterium bifidum showed a significant benefit to having MSM as an additive to influence growth. All concentrations of MSM increased the growth rate to the point where Bifidobacterium bifidum reached a maximum of 16 hours before control. The control reached a maximum of 11.54 growth logs at the 48th hour. This maximum growth was reached for all MSM concentrations at the 32nd hour. The MSM concentrations of 0.125% and 0.25% showed a growth decline of the 40th hour. at hour 96, never reaching the maximum growth again. MSM at 0.5% increased growth to 0.5 logs higher than the control maximum. MSM at 0.5% slowed the growth decline from the 48th hour to the 96th hour. MSM at 0.5% delayed the extinction stage to the point where at the 96th hour there were 8.82 growth logs, which was about 2 logs higher than the control. MSM at 1% does not increase the growth of the bacteria compared to the control, but decreases the extinction stage. From hour 40 to hour 64, MSM at 1% does not show a large drop in growth there was a slight fall of 0.5 logs for hour 48, but there was no decrease for Hours 56 and 64. At hour 72 there was a fall of 2 log in growth but at hour 80 there was an increase of growth 1 log and at time 88 there was another increase of growth 1 log. At hour 96 the growth was outside the accounting range and was estimated at less than 6 logs of growth. Continuing for another 8 hours, there may have been another peak in growth that exceeds 6 logs. MSM at 2.5% at hour 40 reached 11.73 logs of growth, with a drop of 1 log at hour 48. There was a uniform increase in growth at hour 56 and hour 64, reaching a maximum of 12.26 logs, 0.72 logs higher than control. At the 72nd hour, there was a fall of 2 log, with a drop of 0.7 log at the 80th hour for MSM to 2.5%. At the 88th hour, the MSM at 2.5% increased the 1 log growth, before falling below the countable range at the 96th hour. MSM at a concentration of 5% was slower when increasing the growth rate compared to the other MSM concentrations. At the 32nd hour, the growth was 11.13 logs and the 40th hour of growth was 11.78 logs. At hour 48, the growth fell 1 log and the hour 56 there was a fall of 0.1 log. At hour 64, the growth reached the highest for all MSM concentrations of 14.32 logs for MSM at 5%. There was a drop of 6 log at the 72nd hour, but at the 80th hour the growth increased 4 logs to 12. 20. At time 88, an increase of 0.1 log was observed, before dropping to 9.60 logs of growth at hour 96. MSM at 5% slowed down the rate of extinction considerably, extending the stationary phase to 40 hours. Once the stationary phase was reached, there was a continuous increase and decrease in growth, with movement toward a lower growth pattern. These studies indicate that MSM proceeds to the fastest stationary phase for all concentrations, extending the stationary phase for concentrations above MSM to 0.5% and increasing the maximum growth for MSM concentrations to 2.5% and 5%. Once the stationary phase was reached, there was a continuous increase and decrease in growth, with movement toward a lower growth pattern. These studies indicate that MSM proceeds to the fastest stationary phase for all concentrations, extending the stationary phase for concentrations above MSM to 0.5% and increasing the maximum growth for MSM concentrations to 2.5% and 5%. Once the stationary phase was reached, there was a continuous increase and decrease in growth, with movement toward a lower growth pattern. These studies indicate that MSM proceeds to the fastest stationary phase for all concentrations, extending the stationary phase for concentrations above MSM to 0.5% and increasing the maximum growth for MSM concentrations to 2.5% and 5%.

Example 20

Effect of Purple Bromocresol in E. coli when MSM is added to the Matrix

This example shows the effect of Purple

Bromocresol in 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 in E. coli was evaluated. Purple Bromocresol is an indicator dye that turns yellow in the presence of E. coli bacteria. It is not toxic to the organism, to reduce potential ionic interference, lactose broth was selected as the preferred medium for this study because it is free of both NaCl and proteins. USP Antimicrobial Effectiveness <51> for tests was used as the template to show the lethal concentration (LC = Lethal Concentration) LCioo · Concentrations of MSM between 5% -16% in 1% increments were used. All concentrations were plated at 1CT7 dilutions to evaluate the log reduction.

Materials include the following: Lot number 0604751 from OptiMSM Flake; ATCC strain 8739 Escherichia coli lot: 57762704; glass culture tubes based on

Borosilicate 30 mL, were used for all OptiMSM material; Accumedia MacConkey Broth (MB) Lot: 100,974A; Diluent employed was Alpha Biosciences Modified Letheen Broth (MLB) Lot: 108-09; Tripto Soya Alpha Agar

Biosciences with Lecithin; and Tween 80 (TSA) Lot: F08-42.

Flake OptiMSM was weighed using a Mettler Toledo AG245 scale certified SN: 1115210833 and aliquoted for each concentration. The material was placed in 30 mL borosilicate glass culture tubes. The material was calculated in a volume of 10 mL. Material was added to each tube as follows: 5% (0.5 g), 6% (0.6 g), 7% (0.7 g), 8% (0.8 g), 9% (0.9 g), 10% (10 g) ), -11% (1.1 gj », - 12% (1.2 g), 13% (1.3 g), 14% (1.4 g), 15% (1.5 g), and 16% (1.6 g). took in aliquots in 10 mLs to each tube then sterilized for 20 minutes at 121 ° C. The tubes were cooled to room temperature which was approximately 20 ° C. All the tubes were then added the same dilution of Escherichia coli which gave a level of colony forming units at 6.0xl06 / mL (6.8).

The tubes were then incubated at 25 ° C. A daily observation for color change was made during the first seven days. The tubes were mixed periodically to ensure that OptiMSM was well balanced at all times.

A positive and negative control was evaluated.

The results of these studies are as follows (1) Day one: It showed the color change of the broth to yellow for the concentration 5-7%; 8% showed slight color clearance; and 9-16% showed no signs of change. (2) Day two: He showed the same signs as day one. (3) Day three: It showed a change in the concentration of 8% that happens to the typical yellow color. (4) Day 4 to day 6: No sign of significant change. (5) Day 7: Shows 9% change to a yellow color. There was no color change of 10% -16%. (6) Day 14: Does not show signs for the concentration range of 10% -16%.

The concentration tubes were scored on MacConkey agar to see if the organism could recover. No organisms were observed after 72 hours of incubation. Day 30 showed no signs of change for the concentration range of 10-16%. Positive control was scratched at every scratched point of time and showed signs of organism demonstrated by a classic insulation scratch.

This qualitative test indicates that OptiMSM has a certain type of carrier that affects and that reduces or exterminates the organism. This is demonstrated by the lack of yellow color in MSM concentrations lower than what was demonstrated in previous studies using growth medium or culture medium. The color showed reduction at concentrations as low as 8% against 11% in the growth medium studies.

Example 21

Antimicrobial Study of MSM and DMSO in Streptococcal Organisms

This example shows the effects of MSM and DMSO on growth of Streptococcus organisms.

It has been shown here that specific concentrations of MSM (such as 10% to 16% MSM) kill microorganisms. Dimethyl sulfoxide has also been observed to kill microorganisms at concentrations of 30-50%. This study evaluates the bactericidal properties of both compounds alone and in combination as well as their effectiveness when used with a low level of penicillin.

Streptococcus pyogenes (Lancefield group A) has a capsule of hyaluronic acid and Streptococcus pneumonia (without Lancefield Group identified to date) had a distinct polysaccharide capsule. These two organisms are responsible for many types of human streptococcal infections and present two different types of encapsulation. Both of these organisms were used in this in vitro study. In particular, this study determines the antimicrobial effects of MSM and DMSO, both individually and in combination, in Streptococcus pyogenes and Streptococcus pneumonia. This study also determined the most effective concentrations for antimicrobial properties for both compounds and in combination and if MSM and DMSO are combined it reduces the concentrations of any compound required to achieve microbial reduction. Further,

Streptococcus Pneumonia (# 10341 ™) and Streptococcus Pyogenes (Lancefield group A, # 10096 ™) were purchased from 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 was purchased from Promega (# G8230). Streptococcus Pyogenes was cultured in Heart Brain Infusion broth (BD 237500, # 44 booth) overnight. Equal amounts of broth containing bacteria were used for the studies. Streptococcus Pneumonia was also cultured in Heart Brain Infusion broth.

Bacterial viability evaluation:

The bioluminescent ATP assay kit was used to evaluate bacterial viability based on the following reaction: ATP + D-Luciferin + O2 -► Oxyluciferin + AMP + pyrophosphate + C02 + light (560 nm). Bacterial ATP can be measured by direct lysis of the bacteria with a convenient detergent; the released ATP is then free to react with the luciferin / luciferase and leads to the emission of light. The intensity of the light emitted is proportional to the concentration of ATP. 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. pneumonia were cultured under various conditions to determine the extent of 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 pneumonia and 18 hours for Streptococcus pyogenes respectively. Then, the bacterial viability was evaluated by the bioluminescent ATP assay kit. The test was performed in triplicate.

Table 41. Concentrations of MSM, DMSO and Penicillin evaluated.

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MSM and DMSO were diluted in culture medium according to Table 42 (for Streptococcus pneumonia, bottom left) and Table 43 (Streptococcus pyogenes, bottom right).

Table 42 Table 43

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To determine the effectiveness of using MSM, DMSO in conjunction with Penicillin, MSM, DMSO and Penicillin were diluted in culture medium according to Table 44-1 (S. pneumonia) and Table 44-2 {S. pyogenes).

Table 44-1

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Table 44-2

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IC50 of DMSO, MSM and Peni ni lina in Strentnr.nnnnx pneumonia were 12.86%, 15.97% and 68.54 g / L, respectively. DMSO and MSM had synergistic effect within doses of 5% to 20% (for both drugs) to inhibit Streptococcus pneumonia growth. DMSO and Penicillin also had synergistic effect within doses of 10% to 20% (for DMSO) and 25 pg / L (for Penicillin) to inhibit Streptococcus pneumonia growth. In addition, MSM and Penicillin had a synergistic effect within doses of 5% (for MSM) and 25 pg / L (for Penicillin) by inhibiting growth of Streptococcus Pneumonia. When Penicillin, DMSO and MSM were used together, the greatest synergistic effect resulted from DMSO + MSM alone instead of Penicillin + DMSO + MSM.

The IC50 of DMSO, MSM and penicillin in Streptococcus pyogenes were 9.07%, 10.26% and 15.25 pg / L, respectively. DMSO and MSM had a synergistic effect within doses of 2.5% to 5% (for both drugs) to inhibit the growth of Streptococcus Pyogenes. DMSO and Penicillin had a synergistic effect within the 5% dose (for DMSO) and 6.25 pg / L (for penicillin) to inhibit the growth of Streptococcus pyogenes. MSM and Penicillin had synergistic effect within doses of 2.5% to 5% (for MSM) and 3.125 to 6.25 pg / L (for penicillin) to inhibit the growth of Streptococcus Pyogenes. When penicillin, DMSO and MSM were used together, the resulting synergistic effect of DMSO + MSM only in place of penicillin + DMSO + MSM. _____

Table 45-1. Viability of S. pneumonia after exposure to DMSO

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Table 45-2. Viability of S. pneumonia After Exposure to MSM

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Table 45-3. Viability of S. pneumonia After Exposure to Various Concentrations of MSM in 5% DMSO

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Table 45-4. Feasibility of S. pneumonia After Exposure to Various Concentrations of MSM in 10% DMSO

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Table 45-5. Viability of S. pneumonia After Exposure to Various Concentrations of MSM in 20% DMSO

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Table 45-6. Feasibility of S. pneumonia After Exposure to Various Penicillin Concentrations

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Table 45-7. Viability of S. pneumonia After Exposure to 25 pg / L of Penicillin with Diverse Concentrations of DMSO

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Table 45-8. Feasibility of S. pneumonia After Exposure to 50 pg / L of Penicillin with Diverse Concentrations of DMSO

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Table 45-9. Feasibility of S. pneumonia after exposure to 100 pg / L of Penicillin with different concentrations of DMSO

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The combination of 5% MSM with 25 μg / L of penicillin exhibited a synergistic reduction in the viability of S. pneumonia, which leads to only 41% viability (see Table 10). Synergy compared to the expected results based on MSM alone and penicillin is only indicated in the Tables by a. In contrast, MSM at 5% only reduces viability by only about 5%, while 25 pg / L of penicillin only reduces viability by approximately 21%. In this way, the combination of 5% MSM / 25 pg / L penicillin was unexpectedly more effective than expected based on the results obtained with MSM or penicillin alone. Furthermore, as with DMSO, certain concentrations of MSM allowed lower concentrations of penicillin to reduce bacterial viability almost as effectively as higher or higher concentrations. For example, MSM at 20% with 100 pg / L of penicillin reduces the viability of S. pneumonia at 21.37%, MSM at 20% with 50 pg / L of penicillin reduces the viability of S. pneumonia at 20.75%. In this way, with the use of MSM at 20%, the required concentration of penicillin is reduced by half. Continuing this trend is the combination of 20% MSM with 25 pg / L of penicillin reduces the viability of S. nnenmom '^ a. approximately 25%. Similarly, although with a less robust reduction in bacterial viability, MSM at 5% allowed 25 pg / L of penicillin to perform almost identically at 100 pg / L penicillin (compare Tables 45-10 to 45-12 for 25 pg / L of penicillin). In this way, with the use of MSM at 20%, the required concentration of penicillin is reduced by half. Continuing this trend is the combination of 20% MSM with 25 pg / L of penicillin reduces the viability of S. nnenmom '^ a. approximately 25%. Similarly, although with a less robust reduction in bacterial viability, MSM at 5% allowed 25 pg / L of penicillin to perform almost identically at 100 pg / L penicillin (compare Tables 45-10 to 45-12 for 25 pg / L of penicillin). In this way, with the use of MSM at 20%, the required concentration of penicillin is reduced by half. Continuing this trend is the combination of 20% MSM with 25 pg / L of penicillin reduces the viability of S. nnenmom '^ a. approximately 25%. Similarly, although with a less robust reduction in bacterial viability, MSM at 5% allowed 25 pg / L of penicillin to perform almost identically at 100 pg / L penicillin (compare Tables 45-10 to 45-12 for 25 pg / L of penicillin).

Table 45-10. Viability of S. pneumonia After Exposure to 25 pg / L of Penicillin with Various Concentrations of MSM

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Table 45-11. Viability of S. pneumonia After Exposure to 50 pg / L of Penicillin with Various Concentrations of MSM

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Table 45-12. Feasibility of S. pneumonia After Exposure to 100 yg / L of Penicillin with Various Concentrations of MSM

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Based on the synergistic results seen in certain combinations of MSM or DMSO with penicillin, the present study was conducted in order to identify the various combinations of MSM, DMSO, and penicillin that result in synergistic reductions in bacterial viability compared to the effects of combining DMSO, MSM, and penicillin in bacterial viability. This study was also designed to identify combinations of the three compounds that advantageously allow one or more of the compounds to be reduced but still effectively reduce bacterial viability. DMSO at 5, 10, and 20% was individually combined with MSM to one of 5, 10, or 20% and penicillin to one of 25, 50, or 100 and g / L. Viability was estimated as described above. Feasibility data are presented in Table 45-13. comparison with the corresponding combination of DIVISO and penicillin. The symbol "ψ" represents synergistic results compared to the corresponding combination of MSM and penicillin. The values ​​for bacterial viability reduction were added together to determine the threshold reduction for synergy. For example, 5% DMSO reduces viability by approximately 25% and 25 pg / L penicillin reduces viability by approximately 21%, for an expected total combined reduction of approximately 46%. This represents viability of 64%. Thus, if the combination of 5% MSM, 5% DMSO, and 25 pg / L penicillin results in less than 64% viability, synergy between the compounds has been identified. comparison with the corresponding combination of DIVISO and penicillin. The symbol "ψ" represents synergistic results compared to the corresponding combination of MSM and penicillin. The values ​​for bacterial viability reduction were added together to determine the threshold reduction for synergy. For example, 5% DMSO reduces viability by approximately 25% and 25 pg / L penicillin reduces viability by approximately 21%, for an expected total combined reduction of approximately 46%. This represents viability of 64%. Thus, if the combination of 5% MSM, 5% DMSO, and 25 pg / L penicillin results in less than 64% viability, synergy between the compounds has been identified. represents synergistic results compared to the corresponding combination of MSM and penicillin. The values ​​for bacterial viability reduction were added together to determine the threshold reduction for synergy. For example, 5% DMSO reduces viability by approximately 25% and 25 pg / L penicillin reduces viability by approximately 21%, for an expected total combined reduction of approximately 46%. This represents viability of 64%. Thus, if the combination of 5% MSM, 5% DMSO, and 25 pg / L penicillin results in less than 64% viability, synergy between the compounds has been identified. represents synergistic results compared to the corresponding combination of MSM and penicillin. The values ​​for bacterial viability reduction were added together to determine the threshold reduction for synergy. For example, 5% DMSO reduces viability by approximately 25% and 25 pg / L penicillin reduces viability by approximately 21%, for an expected total combined reduction of approximately 46%. This represents viability of 64%. Thus, if the combination of 5% MSM, 5% DMSO, and 25 pg / L penicillin results in less than 64% viability, synergy between the compounds has been identified. The values ​​for bacterial viability reduction were added together to determine the threshold reduction for synergy. For example, 5% DMSO reduces viability by approximately 25% and 25 pg / L penicillin reduces viability by approximately 21%, for an expected total combined reduction of approximately 46%. This represents viability of 64%. Thus, if the combination of 5% MSM, 5% DMSO, and 25 pg / L penicillin results in less than 64% viability, synergy between the compounds has been identified. The values ​​for bacterial viability reduction were added together to determine the threshold reduction for synergy. For example, 5% DMSO reduces viability by approximately 25% and 25 pg / L penicillin reduces viability by approximately 21%, for an expected total combined reduction of approximately 46%. This represents viability of 64%. Thus, if the combination of 5% MSM, 5% DMSO, and 25 pg / L penicillin results in less than 64% viability, synergy between the compounds has been identified.

Several combinations of MSM, DMSO, and penicillin result in synergistic improvements in bacterial reduction. For example, the combination of 5% DMSO, 5% MSM, and 25 pg / L penicillin reduces bacterial viability to approximately 52% (see Table 45-13). 5% DMSO in combination with 25 pg / L of penicillin reduces bacterial viability to approximately 64% (eg, a reduction of approximately 46% based on the reduction of individual seen with DMSO to 5%, see Table 4 5-1, and the reduction of individual eyesight with 25 pg / L of penicillin). In this way, the combination of all 1 ^ g ^ ..... ^^ "p1 iag1; nC! Reduces bacterial viability by approximately 12% additionally, similarly, the combination of 5% MSM with 25 pg / L of penicillin resulted in bacterial viability of approximately 74%,

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 pg / L penicillin results in a synergistic improvement in antimicrobial activity compared to both reference combinations. In other combinations, synergy was detected only with respect to either DMSO plus penicillin or MSM plus penicillin. For example, the combination of 5% MSM with 10% DMSO and 25 pg / L penicillin was synergistic with respect to MSM plus penicillin, but not with respect to DMSO plus penicillin.

In addition to the synergistic effects discussed above, there are several instances where certain 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 results in very similar total bacterial viability over the range of tested penicillin concentrations (viability of -25% with 25 pg / L of penicillin to viability of -18% with 100 pg / L of penicillin). Additionally, DIVISO at 10% with MSM at 20% resulted in almost identical bacterial viabilities across the range of penicillin concentrations.

Similar results are seen with 20% DMSO in combination with 5, 10, or 20% MSM and any concentration of penicillin. These results revealed a slightly wider range of bacterial viability through different concentrations of penicillin, however since the reduction in all cases is close to approximately 90 to 95%, these combinations are still effective.

Table 45-13. Viability of S. pneumonia After Exposure to Various Combinations of DMSO, MSM, and Penicillin

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As discussed above, the structure of S. pyogenes differs from that of S. pneumonia, and therefore additional experiments were performed to evaluate the synergistic effects of various concentrations of DMSO and as combinations of DMSO, MSM, and penicillin. DMSO 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, DMSO resulted in reductions in bacterial viability in a dose-dependent manner. See Table 45-14. MSM was only 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

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Table 45-15. Viability of S. pyogenes After Exposure to MSM

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MSM and DMSO in combination were evaluated for their antibacterial effects in S. pyogenes. DMSO at 2.5%, 5%, and 8% was combined with MSM at 0% (DMSO only control), 2.5%, 5%, and 10%. As shown in Tables 16, 17, and 18 certain combinations of MSM with DMSO are synergistic in comparison to the effects of either DMSO or MSM alone. Synergistic results compared to DMSO or MSM are only indicated by a For example, addition of 2.5% MSM to DMSO at 2.5% reduces bacterial viability to approximately 65% ​​(see Table 16), while one would expect no effect from these concentrations of MSM and DMSO, since individually no compound reduces bacterial viability. The synergistic effect is also seen with DMSO at 2.5% and MSM at 5%, where bacterial viability is reduced to about 83% (compared to a 4% reduction expected based on the effects of only the compounds). The synergy is also seen with DIVISO at 5% in combination with any concentration of MSM. Thus, in some embodiments, DMSO at 5% induces synergistic reductions in bacterial viability in combination with any MSM concentration between 2.5% and 10%. In some modalities, DMSO at 2.5% and MSM at concentrations between 2.5% and 5% are synergistic in an advantageous and unexpected way to reduce bacterial viability. DMSO at 5% induces synergistic reductions in bacterial viability in combination with any MSM concentration between 2.5% and 10%. In some modalities, DMSO at 2.5% and MSM at concentrations between 2.5% and 5% are synergistic in an advantageous and unexpected way to reduce bacterial viability. DMSO at 5% induces synergistic reductions in bacterial viability in combination with any MSM concentration between 2.5% and 10%. In some modalities, DMSO at 2.5% and MSM at concentrations between 2.5% and 5% are synergistic in an advantageous and unexpected way to reduce bacterial viability.

Table -45-16. Viability of S. pyogenes After Exposure to Various Concentrations of MSM in DMSO at 2.5

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Table 45-17. Viability of S. pyogenes After Exposure to Various Concentrations of MSM in DMSO at 5

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Table 45-18. Viability of S. pyogenes After Expn. ^ - i ri ó ™ at Various Concentrations of MSM in DMSO at 8

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Various concentrations of penicillin alone were evaluated for their ability to reduce the viability of S. pyogenes. As shown in Table 45-19, penicillin decreased bacterial viability in a dose-dependent manner.

Table 45-19. Feasibility of S pyogenes After Exposure to Various Concentrations of Penicillin

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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 / L). As such, the identification of synergism between DMSO and penicillin would be less likely to be obscured mathematically.

As illustrated in Tables 45-20, 45-21, and 45-22 (identified by an "*") several combinations of DMSO and penicillin produced synergistic results. For example, 5% DMSO in combination with 3.125 pg / L penicillin, based on the efficacy of the two compounds alone, would only be expected to reduce the viability of bacteria by approximately 4%. However, when combined, the current reduction was approximately 10-fold greater (viability reduced to ~ 61%, see Table 45-20). Similar synergistic effects were seen when 5% DMSO was combined with 6.25 pg / L or 12.5 pg / L penicillin (see Table 45-21 and 45-22, respectively).

Table 45-20. Feasibility of S pyogenes After Exposure to 3.13 pg / L of Penicillin with various Concentrations of DMSO

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Table 45-21. Viability of S pyogenes After Exposure to 6.25 pg / L of Penicillin with Various Concentrations of

DMSO

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Table 45-22. Feasibility of S pyogenes After Exposure to 12.5 pg / L of Penicillin with Different Concentrations of

DMSO

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Studies similar to those using DMSO were performed by combining MSM with penicillin in the range of 3.125 to 12.5 pg / L. The results are shown iPn «i &amp; amp; amp; amp; amp; 1 45 • -111 23, 45-24, and 45-25. Synergy is indicated by Como with DMSO, previously ineffective concentrations of MSM and penicillin were effective in combination in reducing bacterial viability. When taken alone, no effect of 3.13 pg / L of penicillin with MSM at 2.5% will be expected, however a reduction in viability of 8% is detected (see Table 45-23). These effects are more pronounced with the combination of 6.25 pg / L of penicillin with MSM. For example, 5% MSM with 6.25 pg / L of penicillin would be expected to produce a viable bacterial population of 96% (see Table 45-24). However, the data indicate that the viability is reduced to approximately 17%, almost a reduction of 1 80% of the expected results. Synergy was not detected when 12.5 pg / L of penicillin was used, due to the efficacy of that concentration of penicillin alone.

Table 45-23. Feasibility of S pyogenes After Exposure to 3.13 pg / L of Penicillin with Various Concentrations of MSM

<img img-format="tif" img-content="drawing" file="MX353059BD03191.tif" id="idf0133" />

Table 45-24. Viability of S pyogenes After Exposure to 6.25 pg / L of Penicillin with Various Concentrations of MSM

<img img-format="tif" img-content="drawing" file="MX353059BD03201.tif" id="idf0134" />

Table 45-25. Feasibility of S pyogenes After Exposure to 12.5 pg / L of Penicillin with Various Concentrations of MSM

<img img-format="tif" img-content="drawing" file="MX353059BD03202.tif" id="idf0135" />

As with S. pneumonia, combinations of 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 "ψ". As can be given in Table 45-26, substantial synergy is detected through the various concentrations of compounds. Most combinations of DMSO and MSM exhibited a dose-response curve based on the concentration of penicillin used. Based on the efficacy of 12.5 g / L alone, it is not expected that combinations of this concentration of penicillin with DMSO and MSM should be more effective. It is of interest that the previously ineffective concentrations of penicillin are made effective in a dose-dependent manner by combination with DMSO and MSM. For example, DMSO at 2.5% with MSM at 5% and 3.125 and g / L penicillin would be expected to reduce bacterial viability between 100% and 96% (when compared to DMSO + penicillin and MSM + penicillin, respectively). However, the combination of all three reduces bacterial viability to approximately 19%. The expected results are similar for combinations with 6.25 g / L of penicillin, but the current combination reduces bacterial viability even more, to approximately 13%. Increased concentrations of various compounds do not result in further reductions in bacterial viability. For example, the combination of DMSO at 8% with MSM at 2.5% and 3.

Table 45-26. Viability of S. pneumonia After Exposure IWLSVJJIKSAC. - «- · to Various Combinations of DMSO, MSM, and Penicillin

<img img-format="tif" img-content="drawing" file="MX353059BD03221.tif" id="idf0136" />

These studies indicate that at certain MSM concentrations, DMSO or a combination thereof can inhibit Streptococcus pyogenes and Streptococcus pneumonia supporting a possible use of these substances to prevent or inhibit the growth of Streptococcus pyogenes and

Streptococcus Pneumonia. ______

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 medium were supplemented with MSM at 0.125, 0.25, 0.5, 1.0, 2.5, and 5%. A single MSM starting material at 5% MRS broth was prepared and used to prepare each medium composition. Medium for lactobacillus organisms is prepared by adding the appropriate amount of MSM to 99 mL of MRS broth. For Bifidobacterium bifidum, 99 mL of MRS broth is prepared with the respective MSM concentrations and 0.05% L-cysteine. For Bacillus coagulans, 99 mL of tryptic soy broth is supplemented with the appropriate amount of MSM.

These media solutions were inoculated with each probiotic organism and incubated at 35 degrees C ± 0.5 degrees C in C02 for a total of 72 hours for all solutions, except Bifidobacterium bifidum, which develops under anaerobic conditions. Samples from each medium were collected at 0, 8, 16, 24, 32, 40, 48, 56, 64, and 72 hours. Lactobacillus samples were coated on MRS agar agar, samples of Bifidobacterium bifidum were coated with MRS + L-cysteine, and samples of Bacillus · -o &amp; ag'ulaub Lti "were coated on soy agar triptych. Plates were incubated at 35 degrees C ± 0.5 degrees C in CO2 for a total of 72 hours for all solutions, except Bacillus coagulans, which was developed for 48 hours. The plates were then counted. Negative controls (raw material medium and coating controls) were free of microbial growth. The data are presented in Cfu / mL. The results of these studies are presented in the following Tables.

Table 46. Growth of Lactobacillus acidophilus in Reinforced Medium with MSM

<img img-format="tif" img-content="drawing" file="MX353059BD03241.tif" id="idf0137" />

Table 47. Growth of Lactobacillus_hulnarir.ua in MpHí n Reinforced with MSM

<img img-format="tif" img-content="drawing" file="MX353059BD03251.tif" id="idf0138" />

Table 48. Growth of Bacillus coagulans in Medium Reinforced with MSM

<img img-format="tif" img-content="drawing" file="MX353059BD03252.tif" id="idf0139" />

<img img-format="tif" img-content="drawing" file="MX353059BD03261.tif" id="idf0140" />

Table 49. Growth of Bifidobacterium bifidum in Reinforced Medium with MSM

<img img-format="tif" img-content="drawing" file="MX353059BD03262.tif" id="idf0141" />

<img img-format="tif" img-content="drawing" file="MX353059BD03271.tif" id="idf0142" />

These studies indicate that MSM can improve the growth of probiotic organisms depending on the concentration of MSM used.

Example 23

Effect of MSM on H1N1 and Herpes Simplex Virus

This example shows the ability of MSM to improve or reduce the infectivity of Porcine Influenza A H1N1 Virus strain A / California / 04/2009 (CDC ID # 2009712047), Rhinovirus type 14 virus (ATCC # VR-284), and Virus of Herpes Simplex type 1 (ATCC # VR-260). The study was carried out in a pre-treatment test of eight concentrations of MSM. The virus performance reduction / improvement test and subsequent virus titration is done in three duplicates. Inhibitory concentrations of MSM (IC50 or IC90 ~ the appropriate concentration of growth or activity is inhibited by 50% or 90%) were also determined in this study.

Cytotoxicity of MSM was determined before the test. Eight concentrations of MSM (16%, 14%, 12%, 10%, 8.0%, 6.0%, 1.0%, and 0.5%) were tested in MDCK cells (ATCC # CCL-34). Concentrations of MSM from 16% to 8% were toxic to MDCK cells and completely destroyed the cell monolayers. Concentrations of 6% to 0.5% do not produce visible cytotoxic effects. TC50 (concentration at which the compound alone kills 50% of uninfected cells) is determined to be approximately 7%. Therefore, this concentration was the first lowest non-cytotoxic dilution used in the test.

A total of eight concentrations of MSM were included in the test: 7% (~ 74,365 mM); 6% (-63,742 mM); 5% (-53.118 mM); 4% (-42,494 mM); 3% (-31,871 mM); 2% (-21.247 mM); 1% (-10.624 mM); and 0.5% (-5.312 mM). A detailed description of the material and methods are provided below. Host Cells. Madin Darby Canine Kidney Cells (MDCK [ATCC # CCL-34]), MRC-5 cells (human lung fibroblasts; [ATCC # CCL-171]), and Vero cells (African green monkey kidney [ATCC # CCL-81] ]) were kept as monolayers in cell culture laboratory equipment, discarded and used for Antiviral Pretreatment Test of the porcine type H1N1 Influenza A Virus strain / California / 04/2009, Rhinovirus type 14 (ATCC # VR-284 ), and HSV-1 (ATCC # VR-260), respectively. Before testing, host cell cultures are- " Seed 1 "to 1" appropriate cell culture plates. Cell monolayers were confluent at 80 to 90% and less than 48 hours old before inoculation with the virus. The growth medium (GM = Growth Medium) and maintenance medium (MM = Maintenance Medium) were IX EMEM and / or Advanced MEM with appropriate supplements.

Determination of cytotoxicity of test product. The highest non-cytotoxic concentration of the test product was determined before the test. Cell culture in MDCK 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 1 hour at 37 degrees ± 2 degrees C in a C02 incubator. After incubation, the treated cells were coated with MM. The plates were incubated in a C02 incubator for 3 days at 37 degrees ± 2 degrees C. The toxicity was monitored using a

Inverted Composite Microscope. A cytotoxicity test performed as established in the Study Protocol shows that product concentrations of 16% to 8% were toxic to MDCK cells and completely destroyed cell monolayers. Concentrations of product 6% to 0.5% do not produce visible cytotoxic effects. TC50 (concentration at which the compound alone kills 50% of uninfected cells) is determined to be approximately 7%. ______, ,, ----------- A. Pre-Treatment Test. Raw material solution of test product is prepared as follows: 35.0 grams of product is diluted in 100 mL of PBS and heated to 40 degrees C until dissolved. The 35% solution is maintained at 40 degrees C until higher dilutions are prepared (see Project Notes [Form No. 95-G-001] in Annex VI of this Final Report). Cultures of MDCK, MRC-5 and Vero cells were washed with PBS and incubated with the following product dilutions: 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0.5%. Incubation was 1 hour at 37 degrees + 2 degrees C in a CO2 incubator. After the incubation was completed approximately 300-1000 IU (infectious units) of each of the test viruses were added into the appropriate treated cells. The test was performed in three duplications. The plates were incubated in a CO2 incubator for 6 days at the appropriate temperature for each virus. CPE was monitored using an Inverted Compound Microscope. All data resulting from the test are included in Annex IV of this Final Report (Forms No.: 95-G-001, 91-L-002, and 07-L-002). B. Toxicity Control for Previous Treatment Test. MDCK,

Incubation was for 1 hour at 37 degrees ± 2 degrees C in a CO2 incubator. After incubation; "· '' and '' treated cells were coated with MM.The plates were incubated in a CO2 incubator for 6 days at the appropriate temperature for each virus.Toxicity was monitored using an Inverted Compound Microscope. of cytotoxicity are presented in Table 50. C. Virus control Cell cultures MDCK, MRC-5 and Vero were washed with PBS and incubated with MM.Incubation was for 1 hour at 37 degrees ± 2 degrees C in a laboratory incubator. C02 After the incubation was completed approximately 300-1000 10 (infectious units) of each of the test viruses are added to the cells.Third duplicates of Virus Control were performed. Plates were incubated in a C02 incubator for 6 days at the appropriate temperature for each virus. CPE was monitored using an Inverted Compound Microscope. D. Negative Control. Monolayers of intact cell culture served as the negative control. GM was replaced by MM in all Negative control wells. E. Determination of the reduction of and / or improvement of virus performance. After the Virus Control reached the maximum cytopathic effect (complete destruction of the monolayer), samples from the test wells and virus control wells were taken for titration. Ten-fold dilutions were performed in MM and coated on susceptible cells in four duplicates. The resnl -ile? ----- virus performance reduction / improvement tests are presented in Tables 51 to 91. CPE was monitored using an Inverted Compound Microscope. D. Negative Control. Monolayers of intact cell culture served as the negative control. GM was replaced by MM in all Negative control wells. E. Determination of the reduction of and / or improvement of virus performance. After the Virus Control reached the maximum cytopathic effect (complete destruction of the monolayer), samples from the test wells and virus control wells were taken for titration. Ten-fold dilutions were performed in MM and coated on susceptible cells in four duplicates. The resnl -ile? ----- virus performance reduction / improvement tests are presented in Tables 51 to 91. CPE was monitored using an Inverted Compound Microscope. D. Negative Control. Monolayers of intact cell culture served as the negative control. GM was replaced by MM in all Negative control wells. E. Determination of the reduction of and / or improvement of virus performance. After the Virus Control reached the maximum cytopathic effect (complete destruction of the monolayer), samples from the test wells and virus control wells were taken for titration. Ten-fold dilutions were performed in MM and coated on susceptible cells in four duplicates. The resnl -ile? ----- virus performance reduction / improvement tests are presented in Tables 51 to 91. GM was replaced by MM in all Negative control wells. E. Determination of the reduction of and / or improvement of virus performance. After the Virus Control reached the maximum cytopathic effect (complete destruction of the monolayer), samples from the test wells and virus control wells were taken for titration. Ten-fold dilutions were performed in MM and coated on susceptible cells in four duplicates. The resnl -ile? ----- virus performance reduction / improvement tests are presented in Tables 51 to 91. GM was replaced by MM in all Negative control wells. E. Determination of the reduction of and / or improvement of virus performance. After the Virus Control reached the maximum cytopathic effect (complete destruction of the monolayer), samples from the test wells and virus control wells were taken for titration. Ten-fold dilutions were performed in MM and coated on susceptible cells in four duplicates. The resnl -ile? ----- virus performance reduction / improvement tests are presented in Tables 51 to 91. samples from the test wells and virus control wells were taken for titration. Ten-fold dilutions were performed in MM and coated on susceptible cells in four duplicates. The resnl -ile? ----- virus performance reduction / improvement tests are presented in Tables 51 to 91. samples from the test wells and virus control wells were taken for titration. Ten-fold dilutions were performed in MM and coated on susceptible cells in four duplicates. The resnl -ile? ----- virus performance reduction / improvement tests are presented in Tables 51 to 91.

Analysis of data. The virus population title in cell cultures is expressed as -logio of the 50% titration endpoint for infectivity. To calculate the viral titre, - an infectious dose calculation of 50% tissue culture (TCID50) - the Quantal test (Spearman-Karber method) was applied. log TCID50 = 1 - d (s - 0.5)

Where: 1 = -log of the lowest dilution; d = difference between dilution stages; s = sum of proportions of positive wells. 1.1 The highest concentration of compound that produces a cytotoxic effect was determined as 50% of the concentration of toxic compound (TC50). 1.2 The percent reduction is calculated as follows:

<img img-format="tif" img-content="drawing" file="MX353059BD03321.tif" id="idf0143" />

1.3 TCID50 virus population recovered from the test and virus control is used para- · oleraulai · -reduction or improvement of virus infectivity. IC50 was determined using the GraphPad Prism 5, Inc. program. IC90 was determined experimentally when present.

Test Acceptance Criteria. A valid test requires that: 1) cells in the Negative control wells be viable and connected to the bottom of the well; 2) the medium is free of contamination in all wells of the plate; and 3) Virus Control shows the presence of virus-specific CPE.

Virus population reductions were observed for all test viruses. MSM at a concentration of 7% produces the following average reductions: reduction of 1.16 logio (reduction of 93.08%) of Swine Influenza A H1N1 Virus; 2.50 logio reduction (99.68% reduction) of Herpes Simplex Virus type 1 (HSV-1); reduction of 1.25 logi0 (reduction of 94.38%) of rhinovirus type 14. MSM a concentration of 6% produces the following average reductions: reduction of 1.00 logio (reduction of 90.00%) of swine-like influenza A H1N1 virus; reduction of 1.00 logio (reduction of 90.00%) of HSV-1; reduction of 0.67 logi0 (reduction of 78.62%) of rhinovirus type 14. MSM at concentration of 5% produces the following average reductions: reduction of 0.41 logio (reduction of 61.10%) of Influenza A H1N1 roduss¿ virus> & amp; of 1.34 logio (reduction of 95.43%) reduction of HSV-1; 0.09 log (reduction of 18.72%) of Rinovirus type 14. MSM at concentration of 4% produces the following average reductions: reduction of 0.16 logio (reduction of 30.82%) of Porcine Influenza H1N1A virus; reduction of 1.59 log10 (reduction of 97.43%) of HSV-1; reduction of 0.28 logio (reduction of 47.52%) of Rinovirus type 14. MSM at concentration of 3% produces the following reductions average reduction: 0.00 logio (00.00% reduction) of Influenza A H1N1 type Porcine virus; reduction of 1.00 logio (reduction of 90.00%) of HSV-1; reduction of 0.11 logio (reduction of 22.38%) of Rinovirus type 14. MSM at a concentration of 2% produces the following average reductions: reduction of 0.41 logi0 (61.10% reduction) of Porcine Influenza A H1N1 virus; reduction of 0. 84 logio (reduction of 85.55%) reduction of HSV-1; 0.42 logio (reduction of 61.98%) of Rinovirus type 14. MSM at concentration 1% produces the following average reductions: 0.25 logio reduction (43.77% reduction) of Porcine Influenza A H1N1 virus; reduction of 0.67 logio (reduction of 78.62%) of HSV-1; reduction of 0.14 logio (reduction of 27.56%) of Rinovirus type 14. MSM at concentration 0.5% produces the following average reductions: reduction of 0.66 logio (78.12% reduction) of Porcine Influenza A H1N1 virus; i »<aHn <~ pj of 0.25 logio (reduction of 43.77%) of HSV-1; reduction of 0.40 logio (reduction of 60.19%) of Rinovirus type 14. reduction of 0.25 logio (reduction of 43.77%) of Porcine Influenza A H1N1 virus; reduction of 0.67 logio (reduction of 78.62%) of HSV-1; reduction of 0.14 logio (reduction of 27.56%) of Rinovirus type 14. MSM at concentration 0.5% produces the following average reductions: reduction of 0.66 logio (78.12% reduction) of Porcine Influenza A H1N1 virus; i »<aHn <~ pj of 0.25 logio (reduction of 43.77%) of HSV-1; reduction of 0.40 logio (reduction of 60.19%) of Rinovirus type 14. reduction of 0.25 logio (reduction of 43.77%) of Porcine Influenza A H1N1 virus; reduction of 0.67 logio (reduction of 78.62%) of HSV-1; reduction of 0.14 logio (reduction of 27.56%) of Rinovirus type 14. MSM at concentration 0.5% produces the following average reductions: reduction of 0.66 logio (78.12% reduction) of Porcine Influenza A H1N1 virus; i »<aHn <~ pj of 0.25 logio (reduction of 43.77%) of HSV-1; reduction of 0.40 logio (reduction of 60.19%) of Rinovirus type 14. 12%) of Porcine Influenza A H1N1 virus; i »<aHn <~ pj of 0.25 logio (reduction of 43.77%) of HSV-1; reduction of 0.40 logio (reduction of 60.19%) of Rinovirus type 14. 12%) of Porcine Influenza A H1N1 virus; i »<aHn <~ pj of 0.25 logio (reduction of 43.77%) of HSV-1; reduction of 0.40 logio (reduction of 60.19%) of Rinovirus type 14.

Improvement / stimulus of virus infectivity is observed for Porcine Influenza A H1N1 virus treated with 3% MSM. The average improvement in virus population was 0.17 logio (32.39%). A total of three concentrations of MSM improves the infectivity of Rinovirus type 14. MSM at 5% concentration produces an average of 0.053 logio improvement (11.49%). MSM at three percent produces an average of 0.11 logio improvement (22.38%); and 1% MSM produces an average improvement of 0.11 logio (22.38%). All three virus infectivity stimuli / improvements determined in this study were within the range of normal variation for virus population and were not significant. An inhibitory concentration of MSM at which growth or activity is inhibited by 50% (IC5o), is calculated using non-linear regression dose-response (GraphPad Prism 5, software). The best fit values ​​of MSM IC50 and IC50 with 95% confidence intervals were calculated for test viruses. For Porcine Influenza A H1N1 virus, the best fit value of MSM IC5o was 5,114 mM. IC50 with a 95% confidence interval ranged from 0.008038 mM to 3253 mM. For HSV-1, the best fit value of MSM IC50 was determined to be 10.13 mM with an IC50 within the 95% confidence interval varying from 7,144 mM to 14.37 mM. For rhinovirus type 14, the value 4p mpjnr fljnstp m.qm IC50 was 38.16 mM. IC50 with a 95% confidence interval was in the range of 13.07 mM to 111.4 mM. IC90 (1.0 logi0 reduction) was experimentally determined for HSV-1 and Porcine Influenza A H1N1. However, due to intercept of multiple concentrations of MSM with the reduction axis of 90%, Experimental values ​​of IC90 can not be considered accurate. MSM tested at eight different concentrations against U-shaped dose-response curves produced by HSV-1, Porcine Influenza A H1N1 and Rhinovirus. For example: MSM at 4% (reduction of 1.00 logio) was more effective against HSV-1 than MSM at 6% (reduction of 1.59 logio); 0.5% MSM (reduction of 0.66 logio) was more or equally effective against Swine Influenza A H1N1 than MSM at 5% (reduction of 0.41 logio); 4% concentrations through 0.5% were more or equally effective against Rhinovirus than MSM at 5%. It is possible, if it is confirmed with further investigation that the effects of U-shaped MSM represent a stable event. Influenza A H1N1 type Porcine and Rhinovirus. For example: MSM at 4% (reduction of 1.00 logio) was more effective against HSV-1 than MSM at 6% (reduction of 1.59 logio); 0.5% MSM (reduction of 0.66 logio) was more or equally effective against Swine Influenza A H1N1 than MSM at 5% (reduction of 0.41 logio); 4% concentrations through 0.5% were more or equally effective against Rhinovirus than MSM at 5%. It is possible, if it is confirmed with further investigation that the effects of U-shaped MSM represent a stable event. Influenza A H1N1 type Porcine and Rhinovirus. For example: MSM at 4% (reduction of 1.00 logio) was more effective against HSV-1 than MSM at 6% (reduction of 1.59 logio); 0.5% MSM (reduction of 0.66 logio) was more or equally effective against Swine Influenza A H1N1 than MSM at 5% (reduction of 0.41 logio); 4% concentrations through 0.5% were more or equally effective against Rhinovirus than MSM at 5%. It is possible, if it is confirmed with further investigation that the effects of U-shaped MSM represent a stable event. 5% was more or equally effective against Rhinovirus than MSM at 5%. It is possible, if it is confirmed with further investigation that the effects of U-shaped MSM represent a stable event. 5% was more or equally effective against Rhinovirus than MSM at 5%. It is possible, if it is confirmed with further investigation that the effects of U-shaped MSM represent a stable event.

This study indicates that MSM can be used as an antiviral product. Non-cytotoxic concentrations of reduced populations of 7% and 6% of enveloped viruses such as HSV-1 and Swine Influenza A H1N1 in more than 1.0 logio · Tables 50 to 91 include the results for the aforementioned studies.

Table 50 presents Cytotoxicity Test for eight product concentrations performed in parallel with a pretreatment test using Vero MDCK cell cultures, MRC-5. TABLE 50

Test product: Methylsulfonylmethane, batch # 0902951

<img img-format="tif" img-content="drawing" file="MX353059BD03371.tif" id="idf0144" />

+ = CPE Present 0 = CPE not detected

Tables 2 to 9 show the infectivity of Virus Control (TCID50), the average infectivity (TCID50), and the logio reduction and in percent in the previous test of the Test Product,

Methylsulfonylmethane (Lot Number 0902951), and Swine Influenza A H1N1 virus strain A / California / 04/2009 (CDC ID # 2009712047). TABLE 51 Reduction of Infectivity

Test product: Methylsulfonylmethane, 7% (lot # 0902951)

Virus: Influenza A H1N1 Swine Type strain A / California / 04/2009 CDC ID # 2009712047

Host Cell Line: MDCK Host Cell Line ATCC # CCL-34

<img img-format="tif" img-content="drawing" file="MX353059BD03381.tif" id="idf0145" />

<img img-format="tif" img-content="drawing" file="MX353059BD03391.tif" id="idf0146" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of Virus Control TCID50 of Duplicate of Test ** - Average% of Reduction (calculated from reduction of average of logio) = 100- (1 / Reduction TCID50) * 100 TABLE 52 Reduction of Infectivity

Test product: Methylsulfonylmethane, 6% (lot # 0902951)

Virus: Influenza A H1N1 Swine type strain A / California / 04/2009 CDC ID # 2009712047 Host cell line: MDCK ATCC host cell line # CCL-34

<img img-format="tif" img-content="drawing" file="MX353059BD03401.tif" id="idf0147" />

<img img-format="tif" img-content="drawing" file="MX353059BD03411.tif" id="idf0148" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of the Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 53

Infecfivity Reduction

Test product: Methylsulfonylmethane, 5% (lot # 0902951)

Virus: Influenza A H1N1 Swine type strain A / California / 04/2009 CDC ID # 2009712047 Host cell line: MDCK ATCC host cell line # CCL-34

<img img-format="tif" img-content="drawing" file="MX353059BD03421.tif" id="idf0149" />

<img img-format="tif" img-content="drawing" file="MX353059BD03431.tif" id="idf0150" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of Test Duplicate ** - Average% Reduction (calculated from average logio reduction) = 100- (1 / Reduction TCIDso) * 100 TABLE 54

Infectivity Reduction

Test product: Methylsulfonylmethane, 4% (lot # 0902951)

Virus: Influenza A H1N1 Swine Type strain A / California / 04/2009 CDC ID # 2009712047

Host cell line: MDCK Host cell line ATCC # CCL-34

<img img-format="tif" img-content="drawing" file="MX353059BD03441.tif" id="idf0151" />

<img img-format="tif" img-content="drawing" file="MX353059BD03451.tif" id="idf0152" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of the Test Duplicate ** - Average% Reduction (Calculated from the logio reduction average) = 100- (1 / Reduction TCID50) * 100 TABLE 55

Infectivity Reduction

Test product: Methylsulfonylmethane, 3% (lot # 0902951)

Virus: Influenza A H1N1 Swine type strain A / California / 04/2009 CDC ID # 2009712047 "Host cell line: MDGK · cell phone 1 host ATCC # CCL-34

<img img-format="tif" img-content="drawing" file="MX353059BD03461.tif" id="idf0153" />

<img img-format="tif" img-content="drawing" file="MX353059BD03471.tif" id="idf0154" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE. 56

Infectivity Reduction

Test product: Methylsulfonylmethane, 2% (lot # 0902951)

Virus: Influenza A H1N1 Swine Type strain A / California / 04/2009 CDC ID # 2009712047

Host cell line: MDCK Host cell line ATCC # CCL-34

<img img-format="tif" img-content="drawing" file="MX353059BD03481.tif" id="idf0155" />

Cont.

<img img-format="tif" img-content="drawing" file="MX353059BD03491.tif" id="idf0156" />

+ = CPE Present O = CPE not detected NT = Not Tested «« "¡g *

Rep = Duplicate _ * - Reduction Log = Average TCID5o of TCID50 Virus Control of Test Duplicate ** - Average% Reduction (Calculated Average Logio Reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 57

Infectivity Reduction

Test product: Methylsulfonylmethane, 1% (lot # 0902951)

Virus: Influenza A H1N1 Swine Type strain A / California / 04/2009 CDC ID # 2009712047

Host cell line: MDCK Host cell line ATCC # CCL-34

<img img-format="tif" img-content="drawing" file="MX353059BD03501.tif" id="idf0157" />

<img img-format="tif" img-content="drawing" file="MX353059BD03511.tif" id="idf0158" />

+ = CPE Present O = CPE not detected NT = Not Tested

ι · ιι. ι · ιW

Rep = Duplicate * - Reduction Log = Average TCID50 Virus Control TCLDso of the Test Duplicate ** - Average% Reduction (calculated from average reduction of logio) = 100- (1 / Reduction TCID50) * 100 TABLE 58

Infectivity Reduction

Test product: Methylsulfonylmethane, 0.5% (lot # 0902951)

Virus: Porcine Type Influenza A H1N1 strain A / California / 04/2009 CDC ID # 2009712047

Host cell line: MDCK Host cell line ATCC # CCL-34

<img img-format="tif" img-content="drawing" file="MX353059BD03521.tif" id="idf0159" />

<img img-format="tif" img-content="drawing" file="MX353059BD03531.tif" id="idf0160" />

Cont.

<img img-format="tif" img-content="drawing" file="MX353059BD03532.tif" id="idf0161" />

<img img-format="tif" img-content="drawing" file="MX353059BD03541.tif" id="idf0162" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 Virus Control - TCID50 of Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100

Tables 59 to 67 present virus control infectivity (TCID50) / the average infectivity (TCID50), and logio and percent reductions that are observed in the pre-treatment test of the test product,

Methylsulfonylmethane (Lot number 0902951), and Herpes Simplex Virus type I (ATCC # VR-260). TABLE 59

Infectivity Reduction

Test product: Methylsulfonylmethane, 7% (lot # 0902951) _ _____

Virus: Herpes Simplex Virus strain HF ATCC # VR-260

Host cell line: Vero Host cell line ATCC # CCL-81

<img img-format="tif" img-content="drawing" file="MX353059BD03551.tif" id="idf0163" />

<img img-format="tif" img-content="drawing" file="MX353059BD03561.tif" id="idf0164" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of Test Duplicate ** - Average% Reduction (Calculated from average log10 reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 60

Infectivity Reduction

Test product: Methylsulfonylmethane, 6% (batch # 0 902 951) "unffi! MS £

Virus: Herpes Simplex Virus strain-KF ATCC # VR-2 60 Host cell line: Vero Host cell line ATCC # CCL-81

<img img-format="tif" img-content="drawing" file="MX353059BD03571.tif" id="idf0165" />

í

I i

<img img-format="tif" img-content="drawing" file="MX353059BD03581.tif" id="idf0166" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of Virus Control TCID50 strain Duplicate test ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 61

Infectivity Reduction

Test product: Methylsulfonylmethane, 5% (lot # 0902951)

Virus: Herpes Simplex Virus strain HF ATCC # VR-260

Host cell line: Vero host cell line ATCC # CCL-81 wmwnuAi. * =>: ^

<img img-format="tif" img-content="drawing" file="MX353059BD03591.tif" id="idf0167" />

<img img-format="tif" img-content="drawing" file="MX353059BD03601.tif" id="idf0168" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 62

Infectivity Reduction

Test product: Methylsulfonylmethane, 4% (lot # 0902951)

Virus: Herpes Simplex Virus strain HF ATCC # VR-260

Host cell line: Vero Host cell line ATCC # CCL-81

<img img-format="tif" img-content="drawing" file="MX353059BD03611.tif" id="idf0169" />

_ i

<img img-format="tif" img-content="drawing" file="MX353059BD03621.tif" id="idf0170" />

::: + = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 63

Infectivity Reduction

Test product: Methylsulfonylmethane, 3% (lot # 0902951)

Virus: Herpes Simplex Virus strain HF ATCC # VR-260 Host cell line: Vero Host cell line ATCC # CCL-81 ^ _________ ". . ---

<img img-format="tif" img-content="drawing" file="MX353059BD03631.tif" id="idf0171" />

<img img-format="tif" img-content="drawing" file="MX353059BD03641.tif" id="idf0172" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of the Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 64

Infectivity Reduction

Test product: Methylsulfonylmethane, 2% (lot # 0902951)

Virus: Herpes Simplex Virus strain HF ATCC # VR-260 Cell line hnc; p ^ H ^ r-ac¡_ · _Υρρλ t.í ^^ =. host cells ATCC # CCL-81

<img img-format="tif" img-content="drawing" file="MX353059BD03651.tif" id="idf0173" />

<img img-format="tif" img-content="drawing" file="MX353059BD03661.tif" id="idf0174" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 65

Reduction of Infect, vi dad

Test product: Methylsulfonylmethane, 1% (lot # 0902951)

Virus: Herpes Simplex Virus strain HF ATCC # VR-260 Host cell line: Vero Host cell line ATCC # CCL-81

<img img-format="tif" img-content="drawing" file="MX353059BD03671.tif" id="idf0175" />

<img img-format="tif" img-content="drawing" file="MX353059BD03681.tif" id="idf0176" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 66

Infection reduction

Test product: Methylsulfonylmethane, 0.5% (lot # 0902951)

Virus: Herpes Simplex Virus strain HF ATCC # VR-260 Host cell line: Vero Host cell line ATCC # CCL-81

<img img-format="tif" img-content="drawing" file="MX353059BD03691.tif" id="idf0177" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of the Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 67

Infectivity Reduction

Test product: Methylsulfonylmethane, 7% (lot # 0902951)

Viruses: Rhinovirus type 14 strain 1059 ATCC # VR-284 Host cell line: MRC-5 Host cell line ATCC # CCL-171

<img img-format="tif" img-content="drawing" file="MX353059BD03701.tif" id="idf0178" />

<img img-format="tif" img-content="drawing" file="MX353059BD03711.tif" id="idf0179" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate ......... - * - Reduction Log = Average TCID50 of Virus Control TCID50 of the Duplicate test ** - Average% Reduction (Calculated from the average logio reduction) = 100- (1 / TCID50 Reduction) * 100

Tables 68 to 74 present the virus control infectivity (TCID50), the average infectivity (TCID50), and logio Y reductions that are observed in the pretreatment test of a test product, Methylsulfonylmethane (Lot Number 0902951 ), and rhinovirus type 14 (ATCC # VR-284). TABLE 68

Infectivity Reduction

Test product: Methylsulfonylmethane, 6% (lot # 0902951)

Viruses: Rhinovirus type 14 strain 1059 ATCC # VR-284 Host cell line: MRC-5 Host cell line ATCC # CCL-171

<img img-format="tif" img-content="drawing" file="MX353059BD03721.tif" id="idf0180" />

<img img-format="tif" img-content="drawing" file="MX353059BD03731.tif" id="idf0181" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 dtí Lórit'ról 'of virus -TCID50 of the Duplicate of test ** - Average% Reduction (Calculated from the average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 69

Infectivity Reduction

Test product: Methylsulfonylmethane, 5% (lot # 0902951)

Viruses: Rhinovirus type 14 strain 1059 ATCC # VR-284 Host cell line: MRC-5 Host cell line ATCC # CCL-171

<img img-format="tif" img-content="drawing" file="MX353059BD03741.tif" id="idf0182" />

<img img-format="tif" img-content="drawing" file="MX353059BD03751.tif" id="idf0183" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of Virus Control TCTD50 of the Duplicate Test ** - Average% Reduction (Calculated from the average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 70

Infectivity Reduction

Test product: Methylsulfonylmethane, 4% (lot # 0902951)

Viruses: Rhinovirus type 14 strain 1059 ATCC # VR-284 Host cell line: MRC-5 Host cell line ATCC # CCL-171

<img img-format="tif" img-content="drawing" file="MX353059BD03761.tif" id="idf0184" />

<img img-format="tif" img-content="drawing" file="MX353059BD03771.tif" id="idf0185" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of the Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 71

Infectivity Reduction

Test product: Methylsulfonylmethane, 3% (lot # 0902951)

Viruses: Rhinovirus type 14 strain 1059 ATCC # VR-284 Host cell line: MRC-5 Host cell line ATCC # CCL-171

<img img-format="tif" img-content="drawing" file="MX353059BD03781.tif" id="idf0186" />

<img img-format="tif" img-content="drawing" file="MX353059BD03791.tif" id="idf0187" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of the Test Duplicate ** - Average% Reduction (Calculated from the average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 72

Infectivity Reduction

Test product: Methylsulfonylmethane, 2% (lot # 0902951)

Viruses: Rhinovirus type 14 strain 1059 ATCC # VR-284 Host cell line: MRC-5 Line of

host cells ATCC # CCL-171 MUimSrSuL

<img img-format="tif" img-content="drawing" file="MX353059BD03801.tif" id="idf0188" />

<img img-format="tif" img-content="drawing" file="MX353059BD03811.tif" id="idf0189" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of the Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 73

Infectivity Reduction

Test product: Methylsulfonylmethane, 1% (lot # 0902951)

Viruses: Rhinovirus type 14 strain 1059 ATCC # VR-284 Host cell line: MRC-5 Host cell line ATCC # CCL-171

<img img-format="tif" img-content="drawing" file="MX353059BD03821.tif" id="idf0190" />

<img img-format="tif" img-content="drawing" file="MX353059BD03831.tif" id="idf0191" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of the Test Duplicate ** - Average% Reduction (Calculated from average logio reduction) = 100- (1 / TCID50 Reduction) * 100 TABLE 74

Infectivity Reduction

Test product: Methylsulfonylmethane, 0.5% (lot # 0902951)

Viruses: Rhinovirus type 14 strain 1059 ATCC # VR-284

Host cell line: MRC-5_ ATCC host cell line # CCL-171

<img img-format="tif" img-content="drawing" file="MX353059BD03841.tif" id="idf0192" />

<img img-format="tif" img-content="drawing" file="MX353059BD03851.tif" id="idf0193" />

to + = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Reduction Log = Average TCID50 of TCID50 Virus Control of Test Duplicate ** - Average% Reduction (Calculated from average log10 reduction) = 100- (1 / TCID50 Reduction) * 100

Table 75 shows Virus Control infectivity (TCID50), the average infectivity (TCID50), and the logio and percent improvement that are observed in the Test Product treatment test, Methylsulfonylmethane (Lot Number 0902951), and Virus Influenza A H1N1 Swine type strain A / California / 04/2009 (CDC ID # 2009712047). TABLE 75

Improvement of Infectivity

Test product: Methylsulfonylmethane, 3% (lot # 0902951)

Virus: Porcine Type Influenza A H1N1 strain A / California / 04/2009 CDC ID # 2009712047

Host cell line: MDCK Line of

ι, ^ λ OF THE PROPERTY

host cells ATCC # CCL-34 industrial ^ ugLiP

<img img-format="tif" img-content="drawing" file="MX353059BD03861.tif" id="idf0194" />

<img img-format="tif" img-content="drawing" file="MX353059BD03871.tif" id="idf0195" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Stimulus Log = Average Test TCID50 - Virus Control Duplicate TCID50 ** - Average Stimulus% (Calculated from the mean logio stimulus) = 100- (1 / TCID50 Stimulus) * 100

Tables 76 to 78 show the infectivity of the Virus Control (TCID50), the average infectivity (TCID50) and improvement of logi0 and percent observed in Pretreatment of the Test Product, Methylsulfonylmethane (Lot Number 0902951), and Rinovirus type 14 ( ATCC # VR-284). TABLE 76 U | kit n »V! wnv

Improvement of industrial Infectivity ^ => -

Test product: Methylsulfonylideal (01) lldis "" # 0902951)

Viruses: Rhinovirus type 14 strain 1059 ATCC # VR-284 Host cell line: MRC-5 Host cell line ATCC # CCL-171

<img img-format="tif" img-content="drawing" file="MX353059BD03881.tif" id="idf0196" />

<img img-format="tif" img-content="drawing" file="MX353059BD03891.tif" id="idf0197" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Stimulus Log = Average TCID50 Test - TCID50 Duplicate Virus Control ** - Average Stimulus% (Calculated from average logio stimulus) = 100- (1 / TCID50 Stimulus) * 100 TABLE 77

Improvement of Infectivity

Test product: Methylsulfonylmethane, 3% (lot # 0902951)

Virus: Rhinovirus type 14 strain 1059 ATC ^ u ~ j ^ R Host cell line: MKC-3 'Llindd Ub! host cells ATCC # CCL-171

<img img-format="tif" img-content="drawing" file="MX353059BD03901.tif" id="idf0198" />

<img img-format="tif" img-content="drawing" file="MX353059BD03911.tif" id="idf0199" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Stimulus Log = Average Test TCID50 - Virus Control Duplicate TCID50 ** - Average Stimulus% (Calculated from average stimulus logio) = 100- (1 / TCID50 Stimulus) * 100 TABLE 78

Improvement of Infectivity

Test product: Methylsulfonylmethane, * TC # 0902951) '

Viruses: Rhinovirus type 14 strain 1059 ATCC # VR-284 Host cell line: MRC-5 Host cell line ATCC # CCL-171

<img img-format="tif" img-content="drawing" file="MX353059BD03921.tif" id="idf0200" />

<img img-format="tif" img-content="drawing" file="MX353059BD03931.tif" id="idf0201" />

+ = CPE Present O = CPE not detected NT = Not Tested

Rep = Duplicate * - Stimulus Log = Average TCID50 Test - TCID50 Duplicate Virus Control ** - Average Stimulus% (Calculated from average logio stimulus) = 100- (1 / TCID50 Stimulus) * 100 Regression No -linear, Dosage vs. Response

Dose-Response Analysis (Inhibition) were performed for concentrations of test product converted to mM (molecular weight of test product = 94.13). Non-linear regression analysis were as follows: log (inhibitor) against normalized response - slope

Variable. Concentrations are presented in Table 79. TABLE 79

<img img-format="tif" img-content="drawing" file="MX353059BD03941.tif" id="idf0202" />

Table 80 presents the data feed for Herpes Simplex Virus. TABLE 80

<img img-format="tif" img-content="drawing" file="MX353059BD03942.tif" id="idf0203" />

<img img-format="tif" img-content="drawing" file="MX353059BD03951.tif" id="idf0204" />

Table 81 presents transformed (log of dose = X = Log (X)) data for Herpes Simplex Virus. TABLE 81

<img img-format="tif" img-content="drawing" file="MX353059BD03952.tif" id="idf0205" />

Table 82 presents data normalization transform for Herpes Simplex Virus. The percent reduction was normalized as follows: 32.39% becomes 0% for all data sets; 99.79% becomes 100% for all data sets. TABLE 82

<img img-format="tif" img-content="drawing" file="MX353059BD03961.tif" id="idf0206" />

Calculation of IC5o for Virus Herpes Simplex is presented in Table 83. The best fit value for IC50 of the Herpes Simplex Virus was determined as 10.13 mM. However, due to a significant variation in virus reduction, the IC50 values ​​in the range of 7.144 mM to 14.37 mM can be considered a more plausible approach. TABLE 83

<img img-format="tif" img-content="drawing" file="MX353059BD03962.tif" id="idf0207" />

<img img-format="tif" img-content="drawing" file="MX353059BD03971.tif" id="idf0208" />

Table 84 presents the data feed for the Swine-like H1N1 Influenza A Virus. TABLE 84

<img img-format="tif" img-content="drawing" file="MX353059BD03972.tif" id="idf0209" />

<img img-format="tif" img-content="drawing" file="MX353059BD03981.tif" id="idf0210" />

Table 85 presents transformed [log dose = X = Log (X)] data for Porcine Influenza A H1N1 Virus. TABLE 85

<img img-format="tif" img-content="drawing" file="MX353059BD03982.tif" id="idf0211" />

Table 86 presents transformation of data normalization for the Swine-like H1N1 Influenza A virus. The percent reduction was normalized as follows: 0% becomes 0% for all data sets; 91.68% becomes 100% for all data sets. TABLE 86

<img img-format="tif" img-content="drawing" file="MX353059BD03991.tif" id="idf0212" />

Calculation of IC50 for Swine-like H1N1 Influenza A virus is presented in Table 87. The IC50 value of best fit for Swine-like H1N1 Influenza A virus was determined to be 5,114 mM. IC50 values ​​with 95% confidence intervals ranged from 0.008038 mM to 3253 mM. In view of the inconsistency of virus reduction (U-shaped curve) IC50s MSM were determined with a significant approximation. IC90 values ​​can not be concluded from this data set. TABLE 87

<img img-format="tif" img-content="drawing" file="MX353059BD04001.tif" id="idf0213" />

Table 88 presents the data fed for Rhinovirus type 14. TABLE 88

<img img-format="tif" img-content="drawing" file="MX353059BD04011.tif" id="idf0214" />

Table 89 presents transformed [log dose = X = Log (X)] data for Rhinovirus type 14. TABLE 89

<img img-format="tif" img-content="drawing" file="MX353059BD04012.tif" id="idf0215" />

Table 90 presents data normalization transform for type 14 Rhinovirus. The percent reduction was normalized as follows: 0% becomes 0% for all data sets; 96.20% becomes 100% for all data sets. TABLE 90

<img img-format="tif" img-content="drawing" file="MX353059BD04021.tif" id="idf0216" />

The calculation of IC50 for Rhinovirus type 14 is presented in Table 91. The IC50 value of best fit for Rhinovirus type 14 was determined to be 38.16 mM. IC50 values ​​with 95% confidence intervals were in the range of 13.07 mM to 111.4 mM. In view of the inconsistency of virus reduction (U-shaped curve) the IC50S of MSM were determined with a significant approximation. The IC90 values ​​can not be concluded from this data set. TABLE 91

<img img-format="tif" img-content="drawing" file="MX353059BD04031.tif" id="idf0217" />

Example 24

Effect of MSM on Algae

This example shows the effect of MSM on algae activity.

Two species of Chlorella were examined for growth - Chlorella sorokiniana, a species of fresh water and Chlorella minutissima a marine species. The study measured the effect of algae growth in fresh water and saltwater environment with the addition of MSM where MSM was added at the following concentrations: 0%, 0.25%, 2%, 5%, 10% and 20%. The growth was measured on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. The growth curves of percent transmittance of algae were compared with the concentration of 0% MSM as a sample control for each microorganism. MSM raw material powder was supplied by Bergstrom Nutrition with certificate of analysis. The powder was the microgranule formula, batch # 0806809. All media, water and powder of MSM raw material were checked for sterility before the study.

The algae were grown for 48 hours in the appropriate medium. The initial suspension was listed for each alga and is referred to as the initial inocula. Chlorella sorokiniana had 381 million cells per milliliter and Chlorella minutissima had 19 million cells per milliliter. One milliliter of the algae solution is placed in 9 mLs of medium and mixed by vortex. This is repeated for each concentration of MSM medium mixture. The algae tube and MSM are incubated at room temperature with exposure to sunlight. Working MSM concentrations were prepared from a 20.0% MSM solution and diluted according to medium to obtain the final desired concentration of MSM. All the solutions were checked for sterility before proceeding with the study. Each dilution of MSM for each organism was configured and analyzed in triplicate for each time interval measured. Samples were measured by percent transmittance in a UV / VIS spectrophotometer at a wavelength of 750 nm. The raw material of medium was tested by background levels of percent transmittance in each time interval that were measured. The results of these studies are provided in Tables 92 and 93 below. The lower percent of transmittance indicated a higher growth factor. These studies show that treatment with MSM can increase the growth of algae. The raw material of medium was tested by background levels of percent transmittance in each time interval that were measured. The results of these studies are provided in Tables 92 and 93 below. The lower percent of transmittance indicated a higher growth factor. These studies show that treatment with MSM can increase the growth of algae. The raw material of medium was tested by background levels of percent transmittance in each time interval that were measured. The results of these studies are provided in Tables 92 and 93 below. The lower percent of transmittance indicated a higher growth factor. These studies show that treatment with MSM can increase the growth of algae.

Table 92 - Growth of Chlorella sorokiniana

<img img-format="tif" img-content="drawing" file="MX353059BD04061.tif" id="idf0218" />

Cont.

<img img-format="tif" img-content="drawing" file="MX353059BD04062.tif" id="idf0219" />

<img img-format="tif" img-content="drawing" file="MX353059BD04071.tif" id="idf0220" />

Table 93 - Growth of Chlorella minutissima

Cont.

<img img-format="tif" img-content="drawing" file="MX353059BD04072.tif" id="idf0221" />

<img img-format="tif" img-content="drawing" file="MX353059BD04081.tif" id="idf0222" />

Example 25

Absorption of MSM in Topical Formulation is Within Recognized Safety Levels

This example shows that MSN absorption in topical formulations is within the recognized safety levels.

White New Zealand rabbits, which are an accepted animal model for dermal absorption studies, were used to estimate the absorption and blood levels resulting from MSM. The rabbits were obtained from Charles

River Cañada (Saint-Constant, Quebec). cinnn mnpins rn ^ rhn. with ages 12-13 weeks and with a weight range of 2.6 kg to 2.7 kg, they were used for thermal absorption studies. Rabbits were used because of their higher skin permeability compared to rats, pigs or humans. In this way, the test in rabbits is a more conservative approach to the safety of topical products for human use. The size of rabbits was based on the ethical restriction of collection of more than 6 mL / kg of body weight of blood within a period of two weeks. The total volume of blood to be removed during this study was 10 mL in a single day. One animal per group was used to minimize the number of animals required. The animals were housed individually in stainless steel cages with 12 hour light / dark cycles. The environment of the animal's room was monitored daily (target intervals: 18-26 ° C and relative humidity 25-50%). Fresh air was supplied to the room with sufficient velocity to provide approximately 15 to 17 changes of ambient air per hour. Clinical observations were made for all animals to ensure that the animals were in good health before the dose. Morbidity and mortality observations were also made during the study period. Clinical observations were made for all animals to ensure that the animals were in good health before the dose. Morbidity and mortality observations were also made during the study period. Clinical observations were made for all animals to ensure that the animals were in good health before the dose. Morbidity and mortality observations were also made during the study period.

Treatment groups were as shown in the

Table 94. 1 1 ___ "^ *" - ** ^

Table 94: Study Design 1

Cont.

<img img-format="tif" img-content="drawing" file="MX353059BD04101.tif" id="idf0223" />

<img img-format="tif" img-content="drawing" file="MX353059BD04102.tif" id="idf0224" />

<img img-format="tif" img-content="drawing" file="MX353059BD04111.tif" id="idf0225" />

One day before the study, the haunch of each rabbit was held tightly using hair clips. An area of ​​6 cm2 was measured and marked to ensure equivalence in the application of the various compositions. Each product was applied by 0.5 mL pipette of each composition to the center of the test area and dispersed to cover the entire test area. After 5 minutes of exposure period, the compositions were removed by flotation, rinsing and drying of the test area.

Before taking blood, the animals were sedated with acepromazine (1 mg / kg) by intramuscular injection into the muscle of the right hind paw, after which EMLA cream (lidocaine / prilocaine) was applied to both ears on the artery. of the ear. Blood was taken by inserting a 21G needle (cone or needle connector removed) into the artery of the ear. Approximately 2 mL of whole blood is taken in 4 mL vaccutainer tubes (Becton Dickinson, Mississauga, ON) containing K2EDTA. The tubes were inverted to mix with

,, INDUSTRIAL the anticoagulant and stored refrigerated until the plasma was separated by centrifugation. The plasmid is separated from whole blood by centrifugation at 3000 xg for 10 minutes. Plasma is collected, transferred and stored in a cryo-vial at -70 ° C, until further processing for MSM analysis.

Following the 5 minute exposure period to the various test products (see Table 1), the blood is collected after 10 minutes, 30 minutes, 2 hours and 8 hours. Before taking blood samples at 2 and 8 hours, EMLA cream was applied to the ears (approximately 30 minutes before each of these blood samples) since the anesthetic effect of the EMLA cream lasted approximately 1 to 2 hours. Both EMLA cream and Acepromazine were used due to ethical considerations and to provide welfare to the animals used in this study.

The concentration of MSM in plasma was quantified by mass spectrometry-gas chromatography (GC / MS = Gas Chromatography / Mass Spectrometry) based on established methods. Briefly, 450 pL of plasma sample is mixed with 50 pL of physiological saline and subjected to vortex for 30 seconds. Following this, 1 mL of Acetonitrile (Fisher, HPLC grade) is added to the mixture. The solution is vortexed vigorously for 60 seconds and centrifuged at 2000 rpm for 5 minutes. One microliter of the clear supernatant is introduced into the GC / MS system (GC / MS QP20108 El, Shimadzu, Kyoto, Japan). The analysis was performed on a Shimadzu SHR5XLB column ((Internal Diameter) 0.25 mm X length 30 m, film 0.25 um, 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 SIM profiles of MSM ion. Helium gas was used as the carrier gas, gas pressure was 0.25 kg / cm2, replenishment gas was 30 mL / min, column temperature was 80 ° C, injector temperature 120 ° C, separator temperature 200 ° C and ion source temperature 250 ° C. The ionization energy was 70 eV. An external standard chart was prepared with MSM dissolved in acetonitrile at the following concentrations: 62.5 μg / ml, 31.3 μg / ml, 15.6 μg / ml, 7.8 μg / ml, 3.9 μg / ml, 1.9 μg / ml, 0.98 μg / ml and 0.4 9 pg / ml. The concentration of MSM in plasma samples was calculated from the slope of the standard curve. The best fitting graph was linear with a R2 value of 0.998. Replenishment gas was 30 mL / min, column temperature was 80 ° C, injector temperature 120 ° C, separator temperature 200 ° C and ion source temperature 250 ° C. The ionization energy was 70 eV. An external standard chart was prepared with MSM dissolved in acetonitrile at the following concentrations: 62.5 μg / ml, 31.3 μg / ml, 15.6 μg / ml, 7.8 μg / ml, 3.9 μg / ml, 1.9 μg / ml, 0.98 μg / ml and 0.4 9 pg / ml. The concentration of MSM in plasma samples was calculated from the slope of the standard curve. The best fitting graph was linear with a R2 value of 0.998. Replenishment gas was 30 mL / min, column temperature was 80 ° C, injector temperature 120 ° C, separator temperature 200 ° C and ion source temperature 250 ° C. The ionization energy was 70 eV. An external standard chart was prepared with MSM dissolved in acetonitrile at the following concentrations: 62.5 μg / ml, 31.3 μg / ml, 15.6 μg / ml, 7.8 μg / ml, 3.9 μg / ml, 1.9 μg / ml, 0.98 μg / ml and 0.4 9 pg / ml. The concentration of MSM in plasma samples was calculated from the slope of the standard curve. The best fitting graph was linear with a R2 value of 0.998. An external standard chart was prepared with MSM dissolved in acetonitrile at the following concentrations: 62.5 μg / ml, 31.3 μg / ml, 15.6 μg / ml, 7.8 μg / ml, 3.9 μg / ml, 1.9 μg / ml, 0.98 μg / ml and 0.4 9 pg / ml. The concentration of MSM in plasma samples was calculated from the slope of the standard curve. The best fitting graph was linear with a R2 value of 0.998. An external standard chart was prepared with MSM dissolved in acetonitrile at the following concentrations: 62.5 μg / ml, 31.3 μg / ml, 15.6 μg / ml, 7.8 μg / ml, 3.9 μg / ml, 1.9 μg / ml, 0.98 μg / ml and 0.4 9 pg / ml. The concentration of MSM in plasma samples was calculated from the slope of the standard curve. The best fitting graph was linear with a R2 value of 0.998.

All the animals were observed before the start of the study and all showed good health. During the course of the study and subsequent to the study, all the animals demonstrated good health conditions. Morbidity, mortality and injury were estimated twice a day. No animal showed any morbidity, mortality or injury.

The results of the analysis study are summarized in Table 95. Plasma concentrations in MSM reference line (before exposure to the test articles) were between 4.2 pg / mL and 104.2 pg / mL. The variation in reference line is within the normal range of variation of natural MSM concentrations that have been established in previous studies. After exposure to the various test articles, the highest plasma concentrations of MSM measured were less than or equal to approximately 140 pg / mL. This peak concentration results from exposure to 10% MSM + 70% DMSO + 20% water. When it is corrected by natural variation in the

MSM concentrations of baseline, the largest change in plasma MSM is detected in the 70% DMSO + 30% water group. These data suggest that variations in MSM either due to absorption or due to the metabolism of DMSO are within the natural range of MSM concentrations. Table 95: Concentration of MSM in Plasma After Exposure to MSM and DMSO

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In view of the many possible embodiments to which the principles of the described invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be considered as limiting the scope of the invention. On the contrary, the scope of the invention is defined by the following claims. Therefore, we claim as our invention everything that falls within the scope and spirit of these claims.