Use of methylsulfonylmethane (msm) to modulate microbial activity.

USE OF METILSULPHONYLMETHANE (MSM) TO MODULATE ACTIVITY

MICROBIAN CROSS REFERENCE TO RELATED APPLICATIONS

This request claims priority of the Requests

<td>patent</td><td colspan="2">Provisional</td><td>of the</td><td>USA</td><td>Do not.</td><td>from</td><td colspan="2">Serie</td>

<td> 61/257,751</td><td>presented</td><td>in</td><td>November</td><td> 3, 2009,</td><td>Do not</td><td>from</td><td>I know</td><td>laugh</td>

<td> 61/259,098</td><td>presented</td><td>in</td><td>November</td><td> 6, 2009,</td><td>Do not</td><td>from</td><td>I know</td><td>laughs</td>

<td> 61/294,437</td><td>presented</td><td>in</td><td>January 12,</td><td>2010 and</td><td>Do not.</td><td>from</td><td colspan="2">Serie</td>

<td> 61/256,935,</td><td>presented</td><td>in</td><td>October 30</td><td colspan="2">, 2009 each</td><td>a</td><td>from</td><td>the</td>

which here is incorporated by reference in its entirety.

FIELD OF DESCRIPTION

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

BACKGROUND

Microorganisms (or microbes) are microscopic organisms that include bacteria, fungi, archaea, protists, plants (for example, green algae), viruses, prions, parasites and animals such as amoeba, plankton. Depending on the context, 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 can also occur in consumer products, such as food contamination. In other cases, the growth of microorganisms is beneficial and is routinely exploited in biotechnology, modern diagnostic technologies, in chemical processes (for example, fermentation), in food and beverage preparation, in environmental and industrial applications, and to maintain and / or promote human health.

COMPENDIUM

Here methods to modulate activity of microorganisms with MSM are described. MSM is a sulfur organ compound with the formula (CH3) 2SO2. In particular, the surprising ability of MSM to improve or inhibit the activity of microorganisms, such as growth or survival of microorganisms, is described herein, depending on the concentration of MSM that is provided to the microorganism (for example, in the medium in which it grows the organism). 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 in 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, concentrations of MSM between about 6 and about 17 percent by weight of the medium (or moisture content of the medium inhibit microbial activity by reducing or otherwise impairing growth, survival rate (for example, by causing or accelerate deterioration or cell death, such as programmed cell death), metabolism, reproduction (e.g. gene expression, protein expression, signal transduction, transcription, translation, protein folding, etc.), proliferation, vitality, robustness, action and / or function of the microorganism. In contrast, concentrations of MSM between about 0.04% to about 5% by weight improve microbial activity, including improving microbial fermentation efficiency, microbial growth and / or culture efficiency.

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 improve 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 providing MSM to the medium at about 0.4% to about 5% by weight of the medium or by weight of the moisture content of the medium in this way 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.

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; provide MSM to the medium at a concentration of 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 one or more microorganisms in the diagnostic test sample, in comparison with growth of one or more microorganisms in the absence of MSM.

Methods for inhibiting microbial activity are also described. In some examples, the method comprises selecting a medium that is susceptible to contamination of H1N1 influenza; and contacting the medium with MSM at a concentration of about 10% to about 16% by weight by 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, concentrations of MSM between about 6 and about 17 percent by weight of medium (or moisture content of medium inhibit microbial activity by reducing or otherwise impacting growth, survival rate (for example, by causing or accelerate the deterioration of cell death, such as programmed cell death), metabolism, reproduction (for example, gene expression, protein expression, signal transduction, transcription, translation, protein folding, etc.), proliferation, vitality, robustness, action and / or function of the microorganism. In contrast, MSM concentrations between about 0.04% to about 5% by weight improve microbial activity, including improving microbial fermentation efficiency, microbial growth and / or culture efficiency.

As such, methods for uses of 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 the MSM increases the fermentation efficiency of the microorganism compared to the fermentation efficiency in the absence of MSM. In some examples, improving fermentation efficiency comprises at least a 50% increase in alcohol, carbon dioxide or acid production in the presence of MSM by the microorganism, compared to alcohol or acid production in the absence of MSM . For example, improving fermentation efficiency comprises an increase of at least 50% in the production of ethanol, methanol or a combination thereof compared to ethanol, methanol production or a combination thereof in the absence of MSM.

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

In some examples, improving fermentation efficiency comprises an increase of at least 50% in production of lactic acid in the presence of MSM by the microorganism, as 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 to improve fermentation is for beer production. 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 concentration of sodium chloride of less than 5% of the total moisture content.

In vitro methods to improve the growth of one or more probiotic microorganisms are also described. 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 way 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.

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 acidophilus, Lactobacillus delbrueckii, Bacillus coagulans, Lactobacillus rhamnosus, Bifidobacteruim bifidum or any combination thereof.

In some examples, the medium comprises a product that contains 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; provide 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 in comparison with growth of one or more microorganisms in the absence of MSM.

Methods for inhibiting microbial activity are also described. In some examples, the method comprises selecting a medium that is susceptible to contamination of H1N1 influenza; and contacting the medium with MSM at a concentration of about 10% to about 16% by weight by 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 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 bleach free or alcohol free 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: Medium Eagle modified with Dulbecco

DMSO: Dimethyl Sulfoxide

DNA: Deoxyribonucleic acid

ELISA: Enzyme-linked immunosorbent assay

IC₅₀: 50 LAB Inhibitory Concentration: MIC Lactic Acid Bacteria: MSM Minimum Inhibitory Concentration: Methylsulfonylmethane

PACE:

Polyacrylamide gel electrophoresis

PBS: Phosphate buffered stainless salt

PDA: Potato dextrose agar SDS: Sodium dodecyl sulfate TNTC: Too many to count TSB: Tryptic soy broth

The following explanations of terms and methods are provided to better describe the present description and to guide those with ordinary skill in the art in the practice of the present description. The singular forms one, one and he / she refer to one or more than one, unless the context clearly dictates otherwise. For example, the expression comprises a bacterial cell includes single or multiple bacterial cells and is considered equivalent to the phrase comprising at least one bacterial cell. The term o 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 herein, understand means include. In this way, it comprises A or B, means including A, B or A and B, without excluding additional elements.

Unless explained otherwise, all the technical and scientific terms used here have the same meaning commonly understood by a person with ordinary skill in the technique to which this description belongs. Although methods and materials similar or equivalent to those described herein may be employed in the practice or test of the present disclosure, they describe convenient methods and materials below. The materials, methods and examples are illustrative only and are not intended to be limiting. For example, conventional methods well known in the art to which the described invention pertains, are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd 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 Protocola in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocola in Molecular Biology: A Compendium of Methods from Current Protocola in Molecular Biology, 4th ed., Wiley &amp; Sons, 1999; Harlow &amp; Lañe, Antibodiea: A Laboratory Manual, Coid Spring Harbor Laboratory Press, 1990; and Harlow &amp; Lañe, Using Antibodiea: 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 Chemiatry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemiatry,

Including Qualitative Organic Analysis, Fourth Edition, New 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 019-854287-9); 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 1-56081-5698) Additional terms commonly used in chemistry can be found in Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978.

Administration: 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 injection routes (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. (such as Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis), Burkholderia sp. (Such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (Such as Campylobacter jejuni,

Campylobacter coli, Campylobacter lari and Campylobacter fetus), Capnocytophaga sp. Cardiobacterium hominis,

Chlamydía trachomatis, Chlamydophila pneumonías,

Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, 5 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 10 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 coli)

Enterococcus sp. (such as Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, 20 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), 25 Kingella kingii, Klebsiella sp. (such as Klebsiella pneumonías, Klebsiella granulomatis and Klebsiella oxytoca), Lactobacillus sp., Lístenla monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospi ra interrogan ^, Peptostreptococcus sp., Moraxella catarrhacus, Morgacus spnelcus. , Mycobacterium sp. (such as Mycobacterium lepras, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, and

Mycobacterium marinum), Mycoplasm sp. (such as Mycoplasma pneumonías, 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 alcaáfaciens, Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi,

Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium), Serratia sp. (such as Serratia marcesans and Serratia liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus sp. (Such as Streptococcus pneumoniae (e.g. Streptococcus pneumoniae serotype chloramphenicol resistant 4, Streptococcus pneumoniae serotype resistant spectinomycin 6B, S. pneumoniae serotype streptomycin resistant 9V Streptococcus pneumoniae serotype resistant erythromycin 14, Streptococcus pneumoniae serotype resistant optochina 14, Streptococcus pneumoniae rifampicin resistant serotype 18C, Streptococcus pneumoniae tetracycline resistant serotype 19F, Streptococcus pneumoniae serotype penicillin resistant 19F, and S. pneumoniae serotype resistant trimethoprim 23F, S. pneumoniae serotype chloramphenicol resistant 4, Streptococcus pneumoniae serotype resistant spectinomycin 6B, S. pneumoniae serotype streptomycin resistant 9V Streptococcus pneumoniae serotype resistant optochina 14, Streptococcus rifampicin resistant serotype pneumoniae 18C, Streptococcus pneumoniae serotype penicillin resistant 19F, or Streptococcus pneumoniae serotype resistant trimethoprim 23F), Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A streptococci, Streptococcus pyogenes, streptococci Group B, Streptococcus agalactiae, streptococci Group C, anginal Streptococcus, Streptococcus equismilis , Group D streptococci, Streptococcus bovis, Streptococci Group F, and Streptococcus anginosus streptococci Group G), Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum, and Treponema endemicum, Tropheryma whíppelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaerionificrioususriorioususrioriousicrio Vibrio, vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Vibrio, Titanium mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela and Vibrio furnisii), Yersinia sp. (such as Yersinia enterocolitica, and Yersinia pestis) and Xanthomonas maltophilia among others.

In some embodiments, MSM is used to modify, such as 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 penicillin, cephalosporin, monobactam and carbapenem antibiotic families. Meticillin and Oxacillin are betalactam 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. 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.

Biofuel: A fuel derived from a metabolic product of a living organism. It is a renewable energy source, unlike other natural resources such as oil, coal / 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 to fuels, and some other uses, because they have a low vapor pressure, are not toxic, are 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-emitting compound, such as sodium perborate, sodium percarbonate, sodium persulfate, pyrophosphate tetrasodium, or urea peroxide together with catalysts and activators, for example, sodium tetraacetylethylenediamine and / or nonanoyloxybenzenesulfonate. 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 that, when combined, allow the microorganism to grow. Exemplary media include broths or gels. To determine if the culture conditions allow product production, the microorganism can be grown 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 develop can be tested for the presence of the desired product. When the presence of a product is tested, tests, such as those provided herein, may be used, including those presented in the Examples below.

Contact: Place in direct physical association; including in solid, liquid and gas form. Contact includes contacting between one molecule and another molecule. Contact may 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 arbitrarily arranged, keeping in mind that these values ​​may vary from laboratory to laboratory. A control group is practically 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 culture will depend on the organism that is grown and the desired result of the culture. For example, a microorganism that is grown to produce ethanol can be placed in a fermentation broth that contains a source of carbohydrates, various trace minerals and 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 at least 2-times, for example at least 3-times or at least 4 times, compared to a control (such as bacterial growth in the absence of MSM or a reference value that is known representative of bacterial growth in a subject affected with a bacterial infection). These decreases can be measured using the methods described here, as well as those known to a person with ordinary skill in the specialty. In some modalities, 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, reductions at 1 og scale 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)₂SW. 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 degrees of DMSO are commercially available (for example, product No. 472301 from Sigma-Aldrich, Corp., St. Louis, MO) and a person skilled in the art will be familiar with a source of DMSO.

Improvement or increase: To increase the quality, quantity or strength or concentration of something. In an example,

MSM increases or improves the activity of a microorganism, for example with respect to the 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 improve 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 at least 10%, at least 20%, at least 50%, or even at least 90%, including between 10% to 95%, 20% at 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 screening process for 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. Homolactic fermentation is the production of lactic acid from pyruvate; Alcoholic fermentation is the conversion of pyruvate into ethanol and carbon dioxide; and heterolytic fermentation is the production of lactic acid as well as other acids and alcohols. Fermentation should not necessarily be carried out in an anaerobic environment. For example, even in the presence of abundant oxygen, yeast cells prefer fermentation to oxidative phosphorylation, provided that sugars are readily available for consumption.

<td>Sugars</td><td>They are</td><td>a substrate</td><td colspan="2">common fermentation</td><td>and examples</td>

<td>typical</td><td>from</td><td>products of</td><td>fermentation</td><td colspan="2">they are ethanol, acid</td>

<td>lactic,</td><td>and</td><td>hydrogen.</td><td>Nevertheless,</td><td>they can</td><td>occur</td>

more exotic compounds by fermentation, such as butyric acid and acetone. Yeast carries out the fermentation in the production of ethanol in beers, wines and other alcoholic beverages, together 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, occurs relative to a control (such as in the absence of MSM) or an amount that can occur theoretically.

Fermentation medium: Any substance used to grow cells, such as mammalian cells and microorganisms. Fermentation medium includes any growth medium (for example, broth or gel) that supports the life of microorganisms (for example, 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.

Fungal pathogen: 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 orbiculare (Malassezia furfur), Candida sp. (such as Candida albicans), Aspergillus sp. (such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor and Aspergillus clavatus), Cryptococcus sp. (such as Cryptococcus neoformans, Cryptococcus gattii,

<td>Cryptococcus laurentii and</td><td>Cryptococcus albidus),</td><td>Coccidioides</td>

<td>sp., Histoplasma sp.</td><td>(such as Histoplasma</td><td>capsulatum),</td>

<td>Pneumocystis sp. (such</td><td>as Pneumocystis</td><td>jirovecii),</td>

<td>Stachybotrys sp. (such</td><td>as Stachybotrys</td><td>chartarum),</td>

<td>Paracoccidioides, Blas</td><td>Tomyce Fusarium</td><td>Sporothrix,</td>

<td>Trichosporon, Rhizopus,</td><td>Pseudallescheria,</td><td>Paecilomyces,</td>

Alternaria, Curvularia, Exophiala, Wangiella, Penicillium, and Cephalosphorium. In some embodiments, MSM is administered to inhibit or prevent an infection or disorder associated with one or more of the above-mentioned fungal pathogens.

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

Inhalant 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 size range. In some embodiments, an inhalation device is configured to generate particles of a composition that are in the size range of from about 0.1 pm to about 10 pm or from about 0.5 pm to about 5 pm.

Inhibit microbial activity or Inhibit a Disease or Infection: The phrase inhibit microbial activity refers to reducing the 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 disease or pathological condition, after it has begun to develop. As used herein, 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 lower progress of the infection. disease, a reduction in the number of relapses of the infection / disease, an improvement in the total health or well-being of the subject, or by other parameters well known in the art that are specific for the particular infection / disease, such as a particular bacterial infection.

Medium or media: An environment that contains or is suitable for supporting 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, matrix, gelatinous or gaseous environments.

Methylsulfonylmethane (MSM): A sulfur organ compound with the formula (CH₃) 2SO₂. MSM has been marketed and sold substantially as a diet supplement. MSM is also known as DMSO₂, Dimethyl sulfone and methyl sulfone.

MSM is structurally related to dimethyl sulfoxide (DMSO), but the behavior of these two is different. DMSO is a highly polar solvent and an excellent ligand, with 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:

<img file="MX367373B_D0001.tif" />

Microorganisms: A member of the prokaryotic or eukaryotic microbial species of the Archaea, Bacteria, and Eucarya domains, the latter includes yeast and filamentous fungi, protozoa, algae or upper Protist. The terms microbial cells and microbes are used interchangeably with the term microorganism. Microbes may include wild type, genetic engineering 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 used 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 within 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 required food, consume tissues and cells of the body and eliminate toxic waste, which makes people sick. Examples of fungal pathogens for use 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, Trichinelosis (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 chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject. A pharmaceutical composition may 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 vehicles: The acceptable pharmaceutical carriers (vehicles) 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 bearer will depend on the particular mode of administration employed. For example, parenteral compositions usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol or the like, as a vehicle. In a particular embodiment, the carrier is one that allows the therapeutic compound to cross the blood-brain barrier. For solid compositions (eg, powder, pill, tablet or capsule forms), conventional non-toxic solid carriers 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,

<td colspan="2">for example sodium acetate</td><td rowspan="2">or monolaurate ingredient</td><td colspan="2">of sorbitan.</td>

<td>Probiotics:</td><td>A</td><td>food</td><td>do not</td>

<td>digestible that stimulates</td><td>the</td><td>increase</td><td>and / or activity</td><td>from</td>

<td>bacteria in the tract</td><td>say</td><td>stivo that are</td><td>beneficial for</td><td>the</td>

<td>body health</td><td colspan="2">Typically, the</td><td>probiotics</td><td>They are</td>

carbohydrates (such as oligosaccharides); However, carbohydrate-free products are also sources of these ingredients. Probiotics can be short chain, long chain, and / or broad spectrum probiotics. Short chain probiotics (such as oligofructose), contains 2-8 bonds per saccharide molecule, typically fermented 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 saccharide molecule, and tend to ferment more slowly, nourishing bacteria predominantly in the colon on the left side. Spectrum probiotics with a range of molecular bond lengths of 2-64 bonds per molecule and nourish bacteria throughout the colon (such as oligofructose-enriched inulin) (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 improve mineral absorption) and the effectiveness and intrinsic strength of the immune system. A product that stimulates bifidobacteria, such as MSM, is considered a bifidogenic factor. Traditional dietary sources of probiotics include soybeans, inulin sources (such as Jerusalem artichoke, jicama and chicory root), raw oats, unrefined wheat, unrefined barley, garlic, leeks, onion, asparagus, banana and yacon Probiotic oligosaccharides are increasingly adding food for their health benefits. Some oligosaccharides used in this way are fructooligosaccharides (FOS), xylooligosaccharides (XOS), polydextrose and galactooligosaccharides (GOS). Some monosaccharides such as tagatose are also sometimes used as probiotics. As used here, MSM is a probiotic.

Probiotic: A microorganism that confers a health benefit on the host, including, but not limited to, conferring protection from 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 dairy products and foods reinforced with probiotics. However, tablets, capsules, powders and sachets containing 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 Lai Lababaillus ® 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), Lactobacillus acidophilus LA-5, Lactobacillus acidophilus NCFM (Danisco), Lactobacillus casei DN114- casei 431- Lacillus casei DN114- casei 431- Caseione1 (1) , Lactobacillus casei F19 (Arla Foods), Lactobacillus casei (Yakult), Lactobacillus paracasei Stll (or NCC2461, Nestlé), Lactobacillus johnsonii Lal (Lactobacillus LC1, Lactobacillus johnsonii NCC533, Nestlé), Lactococcus lactis LIA (Norrmejerier), Lactobacillus plantarum 299v (Probi), Lactobacillus reuteri ATTC 55730 (Lactobacillus reuteri SD2112, BioGaia Biologics), Lactobacillus rhamnosus ATCC 53013 (Valió), Lactobacillus bieberusus, Lactobacillus birbidophrifus, Lactobacillusus papus Labylbaciduserus, Bhammer , Bifidobacterium bifidum MF20 / 5, Bifidobacterium longum SP07 / 3, Streptococcus thermophilus, Lactobacillus salivarlas, Bifidobacterium longum Rosell-175, Lactococcus lactis Rosell-1058, Bifidobacterium breve Rosell70, Lactobacillus rhamnosus Rosell-11, Lactobacillus acidophilus Rosell-52, Bifidobacterium bifidum rosell-71, Bacillus subtilis var natto, Lactobacillus paracasei, Enterococcus faecium, Bifidobacterium animalis, Lavisbabrichumus caverus.

Quantify: Determination or measurement of an amount (such as a relative amount) of a molecule or the activity of a molecule, such as the analyte activity present in a sample.

Stem cell: A cell that has the ability to self-replicate indefinitely and that under the right conditions, or given the right signals, can differentiate into 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 heart 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 constitute an embryo and that support its development in utero. 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 able to differentiate 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 life of the organism. Sources of adult stem cells have been found in the 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 process that eliminates (removes) or exterminates all life forms, 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. Sterilization can be achieved by methods known to a person of ordinary skill in the art, including applying the appropriate combinations of heat, chemicals, radiation, high pressure and filtration.

Subject: Living 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 noticeable 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 to detect a disorder or disease, such as a bacterial or viral infection. In one example, reducing or inhibiting one or more symptoms or signs associated with a bacterial or viral infection, includes reducing or inhibiting bacterial growth or viral infection by a desired amount, for example by at least 20%, at least 50%, by minus 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, compared to bacterial growth or viral infectivity in the absence of MSM .

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 on a subject or 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 progress, delay progress or cause disease regression, 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%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98% or even at least 100%, compared to the sign or symptom in the absence of MSM. In a particular example, a desired response is to reduce or inhibit 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%, by minus 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). Fit

<td>alternate,</td><td>a</td><td>quantity</td><td>effective therapeutic agent</td><td>may</td>

<td>measure</td><td>how</td><td colspan="2">the amount administered to a subject by</td><td>weight</td>

<td>bodily</td><td>of the</td><td>suj eto,</td><td>for example, mg of agent / kg of</td><td>weight</td>

<td>bodily</td><td> «</td><td></td><td></td><td></td>

Untreated cells: A cell that has not contacted the desired agent, such as MSM. In one example, an untreated cell is a cell that receives the vehicle in which MSM is supplied.

Virus: A microscopic infectious organism that reproduces within living cells. A virus consists essentially of a nucleic acid nucleus surrounded by a plating on protein plates, and has the ability to replicate 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 the 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); Arenavirus (such as Guanarito virus, Lassa virus, Junin virus, Machupo and Sabia virus), Arterivirus, Ronivirus, Astrovirus, Bunyavirus (such as Crimean-Congo hemorrhagic fever virus and Hantavirus), Barnavirus, Birnavirus, Bornavirus (such as virus of Borna 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), Philovirus (such as Ebola virus and Marburg virus), Flexivirus, Hepevirus (such as Hepatitis E virus), human adenovirus (such as human adenovirus AF), human astrovirus, polyomavirus human BK, human bocavirus, human coronavirus (such as human coronavirus HKU1, NL63, and OC43), human enterovirus (such as human enteroviruses AD), human erythrovirus V9, human virus foam, human herpes virus (such as human herpes virus 1 (herpes simplex virus type 1), human herpes virus 2 (herpes simplex virus type 2), human herpes virus 3 (Varicella zoster virus), herpes virus human type 4 (Epstein-Barr type 1 virus), human herpes virus 4 type 2 (Epstein-Barr type 2 virus), human herpes virus 5 strain AD169, human herpes virus 5 Merlin strain, human herpes virus 6A , human herpes virus 6B, human herpes virus 7, human herpes virus 8 type M, human herpes virus 8 type P and human cytomegalovirus), human immunodeficiency virus HIV (HIV) (such as HIV 1 and HIV 2), human metaneumovirus, human papillomavirus (such as human papillomavirus -1, human papillomavirus-18, human papillomavirus-2, human papillomavirus-54, human papillomavirus-61, human papillomavirus-cand90, human papillomavirus RTRX7, human papillomavirus type 10, human papillomavirus type 101, type 103 human virus papilloma, type 107 human virus papilloma, type ipo 16 human virus, type 24 human virus papilloma, type 26 human virus papilloma, type 32 human virus papilloma, type 34 human virus papilloma, type 4 human papillomavirus, papilloma type 41 human virus, type 48 human virus papilloma, type 49 human virus papilloma, type 5 human virus papilloma, type 50 human virus papilloma, type 53 human virus papilloma, type 60 human virus papilloma, type 63 human papilloma virus, human papillomavirus type 6b, human papillomavirus type 7, human papillomavirus type 71, human papillomavirus type 9, human papillomavirus type 92, and human papillomavirus type 96), human influenza virus (such as influenza virus human 1-3), human parechovirus, human parvovirus (such as human parvovirus 4 and human parvovirus B19), human respiratory syncytial virus, human rhinovirus (such as human rhinovirus A and human rhinovirus B), human retrovirus foam, human T lymphotropic virus (such as human T lymphotropic virus 1 and human T lymphotropic virus 2), human polyoma virus, hypovirus, Levivirus, Luteovirus, Lymphocytic choriomeningitis virus (LCM), Marnavirus, Narnavirus, Nidoviral, Nodavirus, Orthomyxovirus (such as Influenza virus), Partitivirus, Paramyxovirus (such as

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 like 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 used to inhibit a biological activity of one or more of the aforementioned viruses.

Yeast: A eukaryotic microorganism classified in the Kingdom of Fungi, with approximately 1,500 species described. Most reproduce asexually by budding, although a few reproduce 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 that 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.

2ΈΙΙ.

MSM compositions

MSM compositions are described herein for use in modulating microbial activity, such as improving or decreasing microbial activity. In some embodiments, a composition of MSM for use in improving microbial activity includes about 0.02% to about 5% of 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 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%, approximately 0.07%, approximately 0.08%, approximately 0.09%, approximately 0.1%, approximately 3%, approximately 0.5%, approximately 1%, approximately 2%, approximately 2.5%, approximately 3%, approximately 4% or approximately 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. As an example, a composition with 5% of 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 polar solvent in the middle, excluding solids.

In some embodiments, the compositions described include a medium capable of supporting growth of a microorganism, a microorganism and MSM. In some examples, a 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 about 0.04% 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%, a approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, approximately 0.1%, approximately 0.3%, approximately 0.5%, approximately 0.7%, approximately 1%, approximately 1.5%, approximately 2.0%, approximately 2.5%, approximately 3.0%, approximately 4% or approximately 4.5% of MSM by weight of the medium or by weight of the moisture content of the medium, where the concentration of MSM is effective in improving 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 yeast packages to generate fast-activating yeast for home or commercial use.

In some modalities, improving microbial activity includes improving probiotic growth. 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 in improving the activity (for example, growth) of probiotics. In addition, a composition for improving the growth of probiotics includes approximately 0.04% to approximately 5% MSM, such as approximately 0.1% to approximately 4%, 0.5% to approximately 3%, approximately 1% to approximately 2%, including approximately 0.04% , at 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%, approximately 1%, approximately 1.5%, approximately 2.0%, approximately 2.5%, approximately 3.0%, approximately 4%, or approximately 4.5% of MSM by weight of medium or by weight of moisture content of the medium.

In some embodiments, improving microbial activity includes improving microbial biofuel production. Thus, in some examples, a composition for improving microbial biofuel production 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 approximately 4%, 0.5% to approximately 3%, approximately 1% to approximately 2%, including approximately 0.04%, to approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, approximately 0.1%, approximately 0.3%, approximately 0.5%, approximately 0.7%, approximately 1%, approximately 1.5%, approximately 2.0%, approximately 2.5%, approximately 3.0%, approximately 4 %, or about 4.5% of MSM by weight of the medium or by weight of the moisture content of the medium, where the concentration of MSM is effective in improving the production of biofuel from algae. In other examples, a composition includes algae and MSM, and optionally other ingredients to improve algae growth. In several modalities, the composition is useful for improving 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 for inhibiting microbial activity are described. 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%, about 9%, about 10%, about 11%, about 13%, about 14%, about 15%, or about 16% of MSM by weight of the medium or by weight of the moisture content of the medium, wherein the concentration of MSM is effective in inhibiting microbial activity, including, but not limited to microbial growth, rate of infection, or a combination from the same.

It is contemplated that any of the compositions described including MSM for modulating microbial activity have a sodium chloride concentration of less than 5% of total moisture content of the medium, such as about 1% to about 3% of 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, a composition of MSM consists or consists essentially of MSM and all natural non-toxic ingredients. In other examples, a described composition of MSM includes one or more additional conservatives. Conservatives include, but are not limited to one or a combination of the following: formaldehyde, potassium sorbate, methylparaben, methylchloroisothiazolinone, phthalates, cocamidopropyl betaine, parabens, decyl polyglucose, polyamine propropyl biguanide, phenoxyethanol, sodium lauret tetrasodium, EDTA glycoside, polyethylene glycol and propylene glycol.

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-times compared to products without MSM or compared to 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 instead of an unwanted conservative. In various embodiments, compositions include formulations free of preservatives or with reduced preservatives comprising MSM.

In several embodiments, 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 several 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 chemicals, heating, radiation, filtration or ultraviolet light.

In various embodiments, the compositions described include MSM in addition to one or more thickening agents, emollients and / or aromatic agents. In some embodiments, a product or other medium is supplemented with continuous or periodic additions of MSM, 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 compared to a formulation without MSM. In other embodiments, 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 conservative. 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 would not traditionally have a preservative. In still other embodiments, MSM serves as a virucidal, fungicidal and / or bacteriocidal. 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 modalities, MSM selectively inhibits aerobic bacteria. In other embodiments, MSM selectively inhibits anaerobic bacteria. In some modalities, MSM selectively inhibits gram-positive bacteria. In other modalities, 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 used to inhibit the growth of microorganisms in products packaged in single or multiple dose containers. MSM formulations according to several of the modalities 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 conditioner and lip enhancers, base, powder, blush, blush, bronzer, mask, eyeliner eyeshadow, eyeshadow, mineral powder for eyes, pencils for eye glare, eyebrow pencils, enamel, concealment, skin care products, creams, lotions, serums, moisturizer, sunscreen, skin repair products (for

<td>example,</td><td>for acne,</td><td>Burns</td><td>of the</td><td>sun, wrinkles, circuits</td>

<td>dark),</td><td colspan="2">and sunscreen. In several modalities,</td><td>MSM</td><td>is added to an array</td>

<td>of cream</td><td>cosmetic</td><td>susceptible</td><td colspan="2">to microbial contamination.</td>

<td colspan="2">In some of these</td><td>modalities</td><td>, the</td><td>cream includes jojoba,</td>

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 moisturizing needs, products for treating psoriasis or eczema, products for treating dry or irritated skin, products for treating sun and wind gums, products for before and after shaving, oils or creams for massage , personal lubricants, acne treatment products and scrubs 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, makeup remover, children's skin lotion, and cream for diaper rash In some modalities, MSM inhibits contamination while simultaneously conferring a beneficial cosmetic effect or health benefit.

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

Medicinal products include, but are not limited to treatment and prevention of cold or flu, allergy prevention and treatment, nasal irrigators, medicinal drops, eye drops, inhalants, athlete's foot treatments, herpes and canker sores or fires, creams for burns, ointments for cuts and infections, and bactericidal, fungicidal and virucidal sprays or lotions. In some modalities, MSM is used to inhibit microbial activity in inhalers, nebulisations, fans, catheters, syringes, tubes for intubation, hospital room equipment, furniture and surfaces, diagnostic equipment, fabrics, bedding and patient covers. 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 embodiments, MSM products are used nasally. In other embodiments, these products are used orally and / or as a vapor. In still other embodiments, the product is a repeated eye drop or other 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 avoid or treat athlete's foot. In some embodiments, the product is used topically. In some of these modalities, the product is a cream, ointment, dew, gel or powder. In other modalities, the product is used orally.

In several modalities, MSM inhibits the activity of mycotoxins, toxic metabolites produced by a fungal kingdom organism, including fungi, molds and yeasts. Products comprising 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 (for example, fungi, molds, and yeasts). In still other embodiments, MSM inhibits microbe toxins directly and / or indirectly by inhibiting the activity of microbes. In one embodiment, MSM inhibits the formation and / or release 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 sprays comprising MSM are provided. Products comprising MSM are also useful for decontaminating surfaces and equipment that are susceptible to viral contamination. In one embodiment, MSM is used to inhibit the influenza virus, including H1N1, either in a biological tissue or on an external surface. In some modalities, MSM is used to inhibit the human immunodeficiency virus, herpes simplex virus, papillomavirus, 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 Alexandrium and Karenia genera. In several modalities, MSM inhibits toxic metabolites (including by-products) of algae.

In some modalities, MSM is added to medicinal products used to treat a burn, cut or wound. Wounds or injuries may include, but are not limited to lacerations, split lacerations, excessive stretching, crushing compression, cut lacerations, tearing, incisions, incision injuries or wounds, abrasions, punching 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.

Skin microflora (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. Thus, in some modalities, MSM is incorporated into a topical product that inhibits growth of this microflora. Subcutaneous administration of MSM is provided in other modalities.

In other embodiments, MSM is added to optical products susceptible to microbial contamination and / or 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 storage of contact lenses. In some modalities, MSM is added to eye drops used in conjunction with contact lenses. In other embodiments, MSM is added to chemical solutions used in ocular diagnostic procedures, such as a solution for pupil dilation of multiple uses.

In several modalities, MSM is added to oral products susceptible to microbial contamination and / or oral products to improve its antimicrobial activity. In some modalities, these products are used for teeth cleaning. In some modalities, MSM is incorporated into toothpaste or tooth gel. In some embodiments, MSM is incorporated into or coated on 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 clean the tongue. In other embodiments, the product is a mouthwash, irrigator or mouthwash for professional or home dental use. Still in other embodiments, the product is a chewing gum or confectionery or sweet. In some embodiments, MSM is added for storage or cleaning solutions for dental implants, dentures and the like.

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, brine 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 ingestion of humans. 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 embodiments, MSM is added to a product that is formulated as a solid or liquid. In other modalities, MSM is added to a food during its production process, while still in other modalities, MSM is added to finished food products.

In several modalities, MSM is added to a food product that is susceptible to microbial infection. In some embodiments, MSM can be mixed, mixed, 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 on 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 storage life of food products. In several embodiments, the addition of MSM (eg, about 5% to about 25%) increases the deterioration time of ingestible 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 embodiment, if the shelf life of an edible product is 10 days, the addition of MSM will increase the shelf life 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 shelf 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 at 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 must otherwise 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 MSM compositions consists essentially of water. For example, MSM is particularly effective when combined with water or other bound components. In some examples, a described composition of MSM is bleach free, alcohol free or a combination thereof. In several 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 used in accordance with any of the modalities set forth herein may be isolated, purified or processed. MSM that is designated as Generally Recognized As Safe (GRAS = General Recognized As Safe) is used for several modalities described here.

Formulations for consumption by humans, domesticated animals and livestock, are provided according to several modalities 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 olerácea extract, strict adenofora, alaria marginata (marine plant), albumin, 25 alcohol, aldenin, alfalfa, algae extract, alkyl guanine transferase, alkyloamides, allantoin, aluminum hydroxide, almonds, aloe vera, alpha lipoic acid, aluminum benzoate, aluminum chloride, amino acids, aminopropan sulfonic acid 3, ammonium glycolate, Ammonium lauryl sulfate, anemarrhenae asphodeloides root extract, anise oil, antioxidants, apigenin, apricot or apricot, apricot kernel, arachidonic acid, arbutin, argan oil, argania spinosa leaf extract, arginine, argireline, arnica extract , artemisia dracunculus oil (tarragon), ascorbic acid, ascorbyl palmitate, ascorbyl tetraisopalmitate, aspergilus ferment, aspidosperm quebracho, astaxanthin, atelocollagen, Oat Grain Extract (Avena Sativa), Avobenzone, Azic Acid, Azuki Frilojes, Mint Balm Extract, Peru Balsam, Bamboo Cane Extract, Barley Extract (Hordeum Vulgare), Barium Sulfate, Barley, Basil , bee pollen, beeswax, bentonite, benzoyl peroxide, beta vulgaris root extract (beet), beta carotene, cranberry, biotin, bismuth oxychloride, bladder sargassum extract, borage oil, boric acid, boric oxide, liquid bovine placenta, brewer's yeast, bronopol, butyl acetate, butyl stearate, butylated hydroxyanisole, butylated hydroxytoluene, butylated glycol, butylparaben, butyrospermum parkii, C18-36 acid triglyceride, caffeine, calamine, calcium, marigold extract, carnauba wax extract of camellia oleifera leaves, extract of camellia sinensis leaves, camphor, canaga odorata flower oil (ylang ylang), candelilla wax, canela sterols, caprylic acid, caprilic / capric triglyceride, caprilil glycol, capsicum oleoresin, caramel, carmine, carotenoids, caragenin, carrot oil, carrot seed oil, carthamus tinctorius seed oil (safflower), castor oil, cellulose, Asian twig, marigold officinalis, alba wax, carnauba wax, ceramide, cerebrosides, ferric ammonium ferrocyanide, cetearet-3, cetearyl alcohol, cetearyl glucoside, cetearyl olive, cetyl alcohol, cetyl lactate, chamomile oil, chamomilla recutita (matricaria) flowers, chestnuts, chestnuts extract, chloroxylenol, chlorphenesin, cholesterol, chondrus crispus (Irish moss), green chromium hydroxide, green chromium oxide, cinnamon alcohol, citric acid, citronellol, citrus fruits , citrus nobilis oil (green tangerine), clove powder, clove flower extract, glyceryl coconate, cocamidopropyl betaine, cocoa, cocoa butter, caprylate / caprate, coconut oil, coconut wax, cod liver oil, coenzyme qlO, collagen, comfrey extract, echinacea extract, cerper copernica wax (carnauba), copper, coriander, coriander oil (coriandrum sativum), corn starch, corn flower extract, creatine , crithmum maritimum extract, cucumber, cyclomethicone, cyclopentasiloxane, dantoin 685, decyl glycoside, deionized water, diazolidinyl urea, dehydrated dicalcium phosphate, dicaprilyl carbonate, diethanolamine, dilaurate, dimethicone, dimethylaminoethanol, wild potato root extract (dioscorea villosa), dipotassium glycyrrhizinate, diestyrylbiphenyl disulfonate disodium, edta disodium, hydantoin dmdm, echinacea extract (echinacea angustifolia), edta, rose eijitsu, elaeol oleifera, oleum emifer, oleum elastin, oleum , enzymes, alpandinum extract (epilobium fleischeri), horsetail leaf extract (equisetum hiemale), erucato, essential fatty acids, essential oils, ethanol, ethoxyglycol, ethyl acetate, ethylene / acrylic acid copolymer, ethylhexyl palmitate, ethylhexylglycerin, ethylparaben, eucalyptus extract, eukarion, euterpe oleaceous fruit extract, evening primrose oil, scrubs, fatty acids, fatty alcohols, fennel oil, ferric oxide, flavanoids, flavonolignan, fish oils, flax, floralozone, fluoride, formaldehyde, fruit acids, fruit extract, fruit extracts, gaba, gamma linolenic acid, gelatin, geraniol, geranium oil, 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, glyceril oleate stearate, glycol distearate, glycolic acid, gold, gold seal extract, grape seeds, grapeseed oil, grapefruit, grapefruit oil, grapefruit seed extract, green tea, gums, hazelnut oil, Hdi / Trimethylol Hexillactone Cross Polymer, Hemp Seed Oil, Hexamidine, Hexylene Glycol, Homcsalate, Honey, Hordeum Distychum Extract, Hordihydroguayaric Acid, Hormones, Moisturizer, Humulus Lupulus Extract, Hyaluronic Acid, Gold Seal Extract (Hydrastis canadensis), hydrocortisone, hydrocotyl extract, hydrogenated castor oil laurate, hydrogenated polyisobutene, hydrogenated polyisobulene, hydrolyzed animal protein, hydrolyzed keratin, gum of hydrolyzed rhizobia, hydrolyzed soy protein, hydrolyzed wheat protein, hydroxy acids, hydroxyethylcellulose, hydroxyethyl cellulose, hydroxyisohexyl 3-cyclohexane carboxaldehyde, hydroxypropyl cellulose, hydroxypropyltrimony honey, hydroquinone, hydroxystearate, hyperinumidazole extract, urea imamide , iodine, Irish moss, iron oxides, isobutylparaben, isododecane, isohexadecane, isononyl isononanoate, isopentyldiol, isopropyl alcohol, isopropyl lanolate, isopropyl linoleate, isopropyl myristate, isostearate, isostearic acid, ivy extract, jasmine oil, jojoba butter, jojoba oil, juniper or juniper extract, juniper oil, kaolin, keratin, ketones, quinerase, kinetin, lactic acid, oil of cashew or kukui, lactic acid, lactoperoxidase, dandelion or alquenilla extract, seaweed extract (laminaria digitata), lanolin, larix sibirica wood extract, lauramide, laurate, lauryl ether, lauryl alcohol, lauryl glycoside, lavender, lavender oil, lecithin, lemon oil, licorice, lime oil, limonene, lindene extract, linoleic acid, linolenic acid, liposomes, carob, lycium barbarum fruit extract, lycium fruit extract barbarum (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, marigold, altea bonbon extract, matcha tea powder, matricaria oil, mea, queen of the meadows, melaleuca oil, melon oil, extract of organic mint (peppermint), menthol, methyl acetate, methyl ethyl ketone, methyl dihydrojasmonate, methyl paraben, mica, microdermabrasion compounds, milk protein, minerals, mineral oil, mipa, monoethanolamine, monostearate, montmorillonite (green clay), artemisa extract or artemega (artesemia vulgaris), blackberry, blackberry root extract (monis nigra), murumuru, fungi, myristate, myristate, myristic acid, myristyl myristate, green myrtle oil (myrtus comunis) , n-acetyl glucosamine, nephrite powder, neroli oil, nettle leaf, neuropeptides, niacin, nitrosamines, nonil nonoxynol-150, nucleic acids, nutmeg powder, nuts, oats, oatmeal, oat cereals, linalool ocimum basilicum oil (basil linalool), octinoxate, octsalate, oleate, oleic acid, oleyl alcohol, oligopeptides, oligosaccharides, olive fruit extract, olive oil, omega-3, orange peel oil, orthoboric acid, oxybenzone , ozoquerite, extract of pavina talina padina, palm oil, palmitate, palmitic acid, palmitoyl, panthetin, panthenol, para-aminobenzoic acid, paraben, paraffin, extract of passion fruit incarnata, passion fruit, patchouli, peach seeds, peat extract, pectin, spike, mint, peppermint oil, peptides, petrolatum, bark extract of phellodendron amurense, phenoxyethanol, phenyl trimethicone, phenylethyl resorcinol, phosphoric acid, phytochemicals, derived from pine extract, extract of pineapple, Plantago lanceolata leaf extract, banana leaf extract, pollen extract, Poligonum cuspidatum root extract, polypeptides, polysaccharides, polysilicone, polysorbate, polysorbate, polyvinylpyrrolidone, progesterone, propylene glycol, propylheptyl caprylate, propylparaben, pumpkin seed extract, pomegranate extract (Púnica granatum), púnica granatum extract / punica granatum, Picnogenol, Quaternium-15, quillaja bark extract saponaria (soap), Quillia extract, reserveratol, retinoic acid, retinoids, retinol, retinol palmate, Ribes rubrum fruit extract, rice, rice bran wax, castor oil, rose oil, rosehip, rosemary, rosemary oil, rose water, royal jelly, Rubus villosus fruit extract, Saccharum officinarum (sugarcane), salicylic acid, sage, sandalwood oil, saponins, sassafras, saw palmetto, Sarmentosa saffron extract, Sclareolide , Scutellaria baicalensis extract, seaweed, Secale cereale (rye) seed extract, aquatic moss extract (Selaginella tamariscina), selenium, sesame oil, sesguioleate, one hundred knuckle tea herb, Shea butter, silibinin, silica, silicone, sirtuins, sodium alginate, sodium ascorbate, sodium bisulfate, sodium borate, sodium carbonate, sodium chloride, sodium citrate, sodium dehydroacetate, ethylparaben, sodium glycinate, hyaluronate sodium, sodium lactobionate, sodium lauryl sulfate, sodium methylparaben, sodium polystyrene sulfonate, sodium propylparaben, sodium stearate, sodium thioglycolate, sodium acryodimethyl taurate, sorbitan isoestearate, olive, sorbitan sesguioleate, sorbitan stearate, sorbitol, sorbitol, soy, soy wax, soybean oil, spearmint oil, squalane, St. Paul's / St John's wort, stearate, stem cells, stearate sucrose, sugarcane extract, sulfate, sunflower oil, sweet almond oil, comfrey leaf extract (Symphytum officinale), Symphytum officinale leaf extract, 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, triisoestearyl citrate, trimethylolpropane triisoestearate, trimiristate, trimethylsiloxysilicate, tripeptide, turmeric, tyrosine, ubiquinone, ultramarines, undecylenyl phenylalanine, urea, uridine, fruit extract Vaccinium macrocarpon, vegetable glycerin, vetiver oil, vitamin A, vitamin B1-B12, vitamin C, vitamin C ester, vitamin D, vitamin E, vitamin K, vitamins, peel Walnut powder, water, wheat germ oil, whey protein (proteinum lactis), white birch bark extract, willow bark, gaulteria oil, magic hazelnut or witch hazel, xanthan gum, xanthan gum, yarrow extract , yeast, yerba mate, yucca, zinc oxide, zinc stearate.

In some embodiments, the compositions comprise, consist or consist essentially of MSM in combination with one, two, three, four, five or more of the ingredients identified above. In several modalities, 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 that has a preservative improves the effects of the preservative. The ingredients identified herein can be used with MSM in a cosmetic formulation (for example, oral, injectable, or topical), or in other types of formulations (for example, injectable or topical medical formulations).

In some embodiments, the composition includes MSM, but is free of one or more of the following compounds: sulfates, GMOs, synthetic fragrances, synthetic dyes, formaldehyde, potassium sorbate, methylparaben, methylchloro-thiazolinone, cocamidopropyl betaine, parabens, polyglucose decyl, polyamine propropyl Biguanide, phenoxyethanol, sodium lauret sulfate, tetrasodium EDTA, decyl glycoside, polyethylene glycol, propylene glycol, phthalates and triclosan. In some embodiments, the use of MSM allows the manufacture of the formulation that is 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 embodiments, 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 multi-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 several modalities (for example, MSM serves as an antioxidant, regenerative compound, anti-wrinkle compound, moisturizer, skin lighter , softener, circulation stimulant, neutralizing, reparative, hair / nail enhancer, healing catalyst, coating agent, etc., or combinations of two or more thereof). In several embodiments, the anti-microbial properties of MSM increase the shelf life, half-life, efficacy and / or stability of the formulation (or the specific ingredient identified herein). 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 used by makeup artists or at cosmetic counters).

Cosmetics may include, but are not limited to lipstick, lip gloss, lip liner, lip volume booster, lip balm, lip conditioner and lip enhancer, base, powder, blush, blush, bronzer, mask, eyeliner, eye shadow, mineral powder for eyes, glitter pencils for eyes, eyebrow pencils, enamel, concealment, skin care products (e.g. microdermabrasion products, softening gels) creams, lotions, serums, moisturizer, sunscreen, skin repair products (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 various modalities. Cosmetics, such as cosmetoceutic 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, hydroxy-calcium apatite, polylactic acid and botulinum toxin) are provided in several modalities Anti-wrinkle, anti-acne, anti-aging, exfoliating, moisturizing and anti-stretching formulations, fragrances, mineral makeup, and primers are provided in various modalities. Dermal gels, for cosmetic and medical use (for example, that inhibit or prevent microbial infection and / or wound healing) are provided in some modalities.

In several 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-purpose applications, and exposure to external microorganisms, such as from the 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 comprising MSM are also packed in sealed containers for single use. In one embodiment, a single-use product (such as a seasoning package, dressing package, or dressing, a cosmetic travel package, etc.) will have a longer shelf life and / or will not require more refrigeration if MSM It is used in conjunction with the product and / or packaging.

In some embodiments, MSM is incorporated directly into the 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 into containers and / or lids to improve the storage life of food, cosmetics and other products, by inhibiting unwanted microbial growth. In still other embodiments, MSM can be incorporated into plating products such as plastic wrap and plastic wrap.

In several embodiments, a composition for inhibiting microbial activity in a topical cream or ointment includes MSM, where 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 (for example, 5-10%, 10-16%, 16-20%, 2030%, 30-40%, 40-50%, 50- 75% or higher, and their overlapping intervals). In some examples, the composition is a cream free of preservatives. 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 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 modalities 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%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19% or approximately 20% MSM. Some modalities include approximately 10-16% MSM, approximately 10-14% MSM or approximately 10-12% MSM.

Exemplary additional anti-microbial agents that 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 (eg, amoxicillin, ampicillin, and epicillin);

carboxypenicillins (eg, carbenicillin, ticarcillin, and temocillin); ureidopenicillins (for example, azlocillin, piperacillin and mezlocillin); mecilinam, sulbenicillin, benzathine penicillin, penicillin G (benzylpenicillin), penicillin V (phenoxymethylpenicillin), penicillin 0 (allylmercaptomethylpenicillin), penicillin procaine, oxacillin, methicillin, naphcillin, methacillin, tacillin, methacillin, methacillin, methacillin, chlocillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacillin, methacrylamine co-amoxiclav (amoxacillin plus clavulanic acid), and piperacillion. Examples of cephalosporins include, but are not limited to, cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefamandol, cefotetan, cefoxitin, cefranide, ceftriaxone, cefotaxime, cefpodoxime proxetil, ceftazidime, cefepime, cephthymus, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex, cefopeximex. 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 [3.2.0] heptane-2-carboxylic sodium), sulbactam 4,4-acid dioxide (2S, 5R) -

3.3- dimethyl-7-oxo-4-thia-l-azabicyclo [3.2.0] heptane-2 carboxylic sodium), and clavulanic acid ((2R, 5R, Z) -3 (2-hydroxyethylidene) -7-oxo -4-oxa-l-aza-bicyclo [3.2.0] heptane2-carboxylic acid), or another Beta-lactam antibiotic.

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 pharmaceutical composition described includes an amount of Beta-lactam antibiotic equal to about 0.001 to 100 MIC for the particular bacterial pathogens described herein. In some embodiments, the pharmaceutical composition comprises about 1-5, 5-10, 10-20, 2030, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or about 90- 100 MIC of a beta-lactam antibiotic. In some embodiments, the pharmaceutical composition comprises about 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 make contact.

A person with skill in the art will know the MIC of an antibiotic for a particular bacterial pathogen, or the person with skill 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 bacterial pathogen are described herein, 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 mode of administration. For example, in addition to injectable fluids, inhalation, topical, ophthalmic, peripheral and oral formulations can be employed. Inhalation preparations may include aerosols, particles and the like. In general, the goal for the particular size for inhalation is approximately 1 pm or less so that the pharmaceutical product reaches 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 select 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 may take the form, for example, of tablets or capsules prepared by conventional means with acceptable pharmaceutical excipients such as binding agents (for example, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example, potato starch or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulfate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration may take the form, for example, of solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other convenient 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.

For administration by inhalation, the compounds for use according to the present description are conveniently supplied in the form of an aerosol spray presentation from pressurized packages or a nebulizer, with the use of a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other convenient. In the case of a pressure spray, the dose unit can be determined by providing a valve to deliver a dosed quantity. Capsules and cartridges for use in an inhaler or insufflator may be formulated that contain 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

<td>for administration</td><td>local.</td><td>The</td><td>agents</td><td>employees</td>

<td>therapeutically (such</td><td>as DMSO,</td><td>MSM</td><td>and / or others</td><td>compounds</td>

<td>therapeutic as here</td><td>It is described)</td><td>They are</td><td>easily</td><td>soluble or</td>

<td>they are suspended in water,</td><td colspan="3">and as such, this would be</td><td>useful for</td>

supply since water does not cause adverse effects on 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 an appropriate solid or liquid carrier, depending on the particular mode of administration selected. Acceptable pharmaceutical carriers and excipients useful in this description are conventional. For example, parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles, such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients that may be included for example are 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 emulsifying wetting 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 dose 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 clinical physician. Within these limits, the formulation to be administered will contain an amount of the active component (s) in effective amounts to achieve the desired effect of the subject being treated.

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

Polymers can be used for controlled release. Various degrading and non-degrading polymeric 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 supply of interleukin-2 and recombinant urease (Johnston et al., Pharm. Res. 9: 425, 1992; Pee, J. Parent. Sci. Tech. 44 (2): 58, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled protein release (Ijntema et al., Int. J. Pharm. 112: 215, 1994). Still in another aspect, liposomes are used for controlled release as well as drug target 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; US ​​Pat.

<td>the</td><td>USA</td><td>Do not.</td><td>4,501,728; Patent</td><td>of the</td><td>USA</td><td>Do not.</td>

<td> 4,837</td><td> , 028;</td><td>Patent</td><td>of the US No. 4</td><td> ,957,735;</td><td>and patent</td><td>from</td>

<td>the</td><td>USA</td><td>Do not.</td><td>5,019,369; Patent</td><td>of the</td><td>USA</td><td>Do not.</td>

<td> 5, 055</td><td> , 303;</td><td>Patent</td><td>of the US No.</td><td> 5,514,670;</td><td>Patent</td><td>from</td>

<td>the</td><td>USA</td><td>Do not.</td><td>5,413,797; Patent</td><td>of the</td><td>USA</td><td>Do not.</td>

<td> 5,268</td><td> ,164;</td><td>Patent</td><td>of the US No.</td><td> 5,004,697;</td><td>Patent</td><td>from</td>

<td>the</td><td>USA</td><td>Do not.</td><td>4,902,505; Patent</td><td>of the</td><td>USA</td><td>Do not.</td>

<td> 5, 506</td><td> , 206;</td><td>Patent</td><td>of the US No.</td><td> 5,271,961;</td><td>Patent</td><td>from</td>

<td>the</td><td>USA</td><td>No. 5</td><td>, 254,342; and patent</td><td>3 of the</td><td>USA</td><td>Do not.</td>

5,534,496).

In various embodiments, pharmaceutical compositions include DMSO and / or MSM, and a therapeutic agent to treat 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 modalities, compositions including DMSO and MSM are formulated as liquids. In some modalities, the compositions are consumed orally to treat infectious disease, while in some other modalities, the compositions are applied topically. In a particular embodiment, the compositions are supplied in an inhalant device that is configured to generate particles of the formulation in a size range from about 0.5 um to about 5 um.

In some embodiments, pharmaceutical compositions that include DMSO 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) allow antibiotics to reach deeper levels of infected tissue; (ii) allow direct contact of infected tissue; (iii) extend the exposure time of the antibiotic to 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 inhaler further comprises one or more antibiotics or other therapeutic agents.

In some embodiments, pharmaceutical compositions including DMSO and / or MSM formulations, which others include effective antiparasitic agents for treating infections caused by parasites, such as nematodes, cestodes, trematodes, protozoa or amoebas.

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 caused by pineapple or hummingbird, candidiasis and Cryptococcus (cryptococcal meningitis, for example).

In some embodiments, pharmaceutical compositions that include DMSO and / or MSM formulations also include antiviral agents that are effective in treating viral infections. In some embodiments, specific classes of antiviral agents are used, they are used to treat infections caused by a particular type of virus. In some modalities, agents that target HIV (HIV), herpes virus, hepatitis B or C virus, and influenza viruses, 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 of one: aminoglycoside, antamycin, carbacefem, carbapenem, cephalosporin (I^to, 2^to, 3^to, 4^to or 5^to generation), glycopeptides, macrolide, monobactam, penicillin, polypeptide , quinolone, sulfonamide, tetracycline and the like.

In some embodiments, specific diseases are directed 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. Similarly, penicillins, such as amoxicillin or oxacillin are incorporated into formulations used to treat a wide range of streptococcal infections.

In still other embodiments, microorganisms that cause specific disease are targeted by the specific antibiotics incorporated into a formulation comprising DMSO and / or MSM. For example, aminoglycosides, such as neomycin, are incorporated into formulations used to treat Escherichia coli infections. In several 20 modalities, antibiotics typically used to fight microbial infections are used. In certain embodiments, antibiotics that include, but are 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 include 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 additional embodiments, compositions are provided that 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 modalities, it is provided in a total daily dose range of 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, isoniazid is provided in 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

<td>day while</td><td>what</td><td>still</td><td>in others</td><td>modalities, it</td>

<td>provides in</td><td>a</td><td>interval</td><td>of dose</td><td>total daily of</td>

<td>approximately</td><td> 250</td><td colspan="2">mg to approximately</td><td>350 mg per day,</td>

<td>including 260,</td><td> 270,</td><td> 280, 290,</td><td> 300, 310,</td><td>320, 330 and 340 mg</td>

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 from about 2.0 to about 3.0 g per

<td>day while</td><td>what</td><td>still</td><td>in</td><td>other</td><td>modalities,</td><td>I know</td>

<td>provides in</td><td>a</td><td>interval</td><td>from</td><td>dose</td><td>total daily</td><td>from</td>

<td>approximately</td><td>2.0 to</td><td>2.5 g per</td><td>day,</td><td colspan="2">including 2.1, 2.2,</td><td> 2.3</td>

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 approximately 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 to approximately 1.5 g per day, including 1.1, 1.2, 1.3 and 1.4 g

In some embodiments, pharmaceutical compositions that include 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 prior treatment of patients are in the range of approximately 10% to 50% by weight by volume. In some embodiments, the pre-treatment dose of DMSO and / or MSM ranges from about 20% to about 40%, from about 25% to 35%, including 26, 27, 28, 29, 30, 31, 32, 33 and 34%. In some embodiments, about 50% to about 100% of DMSO and / or MSM are used. In several embodiments, prior treatment with DMSO and / or MSM improves the ability of an antibiotic to inhibit bacterial activity and / or sensitize a resistant strain for a drug that was previously ineffective.

In some embodiments, a pharmaceutical composition is prepared wherein antimicrobials are dissolved in DMSO and / or MSM before 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 modalities, 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 embodiments, an inhaler is used. In some embodiments, a pressure metered dose inhaler is used, and the formulation is inhaled as a liquid aerosol. In other embodiments, dry powder inhalers are employed, and the formulation is inhaled in a powdered aerosol form. In several embodiments, oral, intravenous, intramuscular or subcutaneous administration is used in addition to or instead of inhalant therapy.

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

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 inhaled DMSO and / or MSM 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 DMSO and inhaled antimicrobial compounds. In certain embodiments, the amount of inhaled MSM is in the range of about 0.01% by weight to about 70% by weight of the inhaler. In other embodiments, the inhaled formulation contains between about 0.01% and 10% of MSM by weight. Other modalities contain between approximately 10 and

20% MSM, approximately 20-30% MSM, approximately 30-40% MSM, approximately 40-50% MSM, approximately 50-60% MSM, or approximately 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% of MSM, about 15-25% MSM, about 2535% of MSM, about 35-45% of MSM, about 55-60% of 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 about 50% to about 95%, including 55, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93 and 94%.

In several embodiments, the use of MSM reduces the amount of DMSO required to achieve a comparable effect and / or improves the effectiveness 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 effectiveness 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, 3 -times, 5-times, 10 times, 50-times or 100-times. In still other embodiments, the use of DMSO and MSM reduces the amount of a therapeutic agent required to achieve a comparable effect and / or improves the effectiveness of the therapeutic agent by at least 10%, 25%, 50%, 100%, 2- times, 3-times, 5-times, 10-times, 50-times or 100-times compared to DMSO or MSM alone and / or the therapeutic agent alone.

In several embodiments, a pretreatment formulation including DMSO, alone or in combination with MSM, is administered to a subject intravenously, intramuscularly, topically or orally 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 by at least 10%, 25%, 50%, 100%, 2-times, 3-times, 5-times, 10-times, 50-times or 100-times.

In several modalities, subjects having an infectious disease treated again 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 also 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, several embodiments of the invention comprise devices to facilitate storage and administration of DMSO. In some modalities, DMSO is stored in glass bottles and is administered through non-reactive pipe. In other embodiments, inhalant devices are specially designed to be resistant to DMSO. In some embodiments, portions of the inhalant devices are disposable or replaceable. According to various modalities, formulations comprising DMSO are manufactured, stored and / or administered using materials and devices described in the Patent Application of the

US Serial No.: 12 / 066,480, which is the National Phase entry of the International Application No .: PCT / USO6 / 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 modalities because it allows a reduction in the amount of antimicrobial compounds incorporated in the formulation while maintaining or improving the effectiveness of the formulation against infectious microorganisms. In other embodiments, direct administration increases the efficacy of a given antimicrobial regimen against one or more drug resistant strains of microorganisms. Direct delivery or direct targeting, according to 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 (eg, 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 odor. In other embodiments, higher amounts of DMSO are used and tolerated.

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

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 size, thereby reducing the irritation of the mucosa of the mouth and throat. , since the droplets or particles travel deeper into the patient's lungs. In

<td>some</td><td>modalities,</td><td>the depth</td><td>travel</td><td>from</td><td>the</td>

<td>droplets</td><td>or particles</td><td>increase the</td><td>concentration</td><td>from</td><td>the</td>

<td>antibiotic</td><td>dissolved icos</td><td>In the lungs</td><td>of the patient.</td><td></td><td></td>

<td></td><td colspan="3">In various modalities, compositions of</td><td>DMSO</td><td>I</td>

MSM are combined with therapeutic agents (such as antibiotics) and are provided as an aerosol to deliver locally active drugs to the system

100 respiratory to treat respiratory illness. In one embodiment, the lower airways are contacted (or contacted exclusively) with the composition. In other embodiments, the composition is used to systemically treat diseases. For systemically active drugs, 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 local action drugs, the onset of action is immediate in some modalities. Systemically active inhaled formulations, 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 the degradation of the therapeutic agent. In another embodiment, aerosol formulations described herein reduce gastrointestinal side effects or skin irritation that may occur.

101 with oral or topical treatment.

In several embodiments, the inhaling particles are sized to minimize the deposition of these particles by inertial impact on the upper respiratory tract without reaching the site of action. In several embodiments, the particles are sized to minimize deposit in the mouth and throat, thereby minimizing swallowing / swallowing or ingestion and unwanted systemic or local side effects. In several modalities, the particles are less than 2, 5 or 10 pm. In one embodiment, the particles are approximately 3-5 pm and are transported to the smallest 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 embodiments, the use of DMSO and / or MSM allows optimizing 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 one mode,

102 homogenization, sonication, high shear or cutting fluid processing, or other mechanical methods are used to combine the therapeutic agent with DMSO and / or MSM. In other embodiments, the therapeutic agent easily dissolves in DMSO. Unlike other strong solvents, DMSO is not harmful to lung tissue. In this way, DMSO is especially advantageous in some embodiments because it can both dissolve the therapeutic agent and deliver the agent without damage to lung tissue. In some embodiments, DMSO dissolves at least 50%, 75%, 90%, 95% or 99% of the therapeutic agent, and in one embodiment, it is able to avoid unwanted precipitation of the therapeutic agent.

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

In certain embodiments, the compositions described herein are effective for treating various infectious diseases including, but not limited to, acinetobacter infection, actinomycosis, Adenovirus infection, African sleeping sickness (African trypanosomiasis),

103

AIDS (AIDS), amebiasis, anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, BV = Bacterial Vaginosis infection) , balantidiasis, Baylisascaris infection, BK virus infection, black stone, Blastocystis hominis infection, blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, botulism, Brazilian hemorrhagic fever, brucellosis, Burkholderia infection, Calicivirus infection, campylobacteriosis, candidiasis (moniliasis; canker sore), cat scratch disease, cellulite, Chagas disease, chancroid, chicken pox, chlamydia, Chlamydophila pneumoniae infection, cholera, chromoblastomycosis, clonorchiasis, clostridium difficile infection, coccidioidomycosis, Colorado tick fever, common cold, catarrhal disease Creutzfeldt-Jacob, Crimea-Congo hemorrhagic fever, cryptococcosis, cryptosporidiosis, cutaneous migrans larva (CLM = Cutaneous Larva Migrans), cyclosporiasis, cysticercosis, cytomegalovirus infection, dengue fever, dientamoebiasis, diphtheria, diphrobotriasis, dracunculiasis ebola hemorrhagic fever, echinococcosis, ehrlichiosis, enterobiasis (intestinal worm infection), Enterococcus infection, enterovirus infection, epidemic typhus, erythema, erythema

104 subitum, fasciolopsiasis, fasciolosis, fatal familial insomnia (FFI = Fatal Familial Insomnia), filariasis, food poisoning, free-living amoeba infection, Fusobacterium infection, gas gangrene (Clostridial myonecrosis), geotrichosis, Gerstmann-Stráussler syndrome Scheinker (GSS = Gerstmann-StráusslerScheinker), giardiasis, amorphous, gnatostomiasis, gonorrhea, inguinal granuloma (Donovanosis), Group A strep infection, Group B strep 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), hemorrhagic fever with renal syndrome (HFRS = Hemorrhagic Fever With Renal Syndrome), Hepatitis A, B, C, D or E, herpes simplex, histoplasmosis, tuntun infection, human bocavirus infection, Hhman ewingii ehrlichiosis, human granulocytic anaplasmosis (HGA = Human Granulocytic Anaplasmosis), human metapneumovirus infection, human monocytic erlichiosis, human papilloma infection (HPV = Human PapillomaVirus), human parainfluenza virus infection and hymenolepiasis.

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 (mono), influenza (influenza),

105

Isosporiasis, Kawasaki disease, keratitis, Kingella kingae infection, Kuru, Lassa fever, legionellosis, leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease, lymphatic filariasis, lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever (MHF), saphan fever melioidosis (Whitmore's disease), meningitis, meningococcal disease, Metagonimiasis, microsporidiosis microsporidia, 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, polio, poliovirus, primary amebic meningoencephalitis (MAP), Progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite fever, respiratory syncytial virus, Rhinosporidiosis, rhinovirus infection, Rickettsia infection, rickettsiosis, Rift Valley fever (FVR), FMR Rocky Mountain spotted fever) , rotavirus infection, rubella, salmonellosis, syndrome

Severe Acute Respiratory (SARS), scabies, schistosomiasis,

106 sepsis, shigellosis, herpes (shingles), smallpox, sporotrichosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, syphilis, teniasis, tetanus (trismus), ringworm of the beard (folliculitis), ringworm of the skin scalp), body ringworm, body ringworm), tinea cruris (inguinal ringworm), Ringworm manuum), black ringworm, tinea pedis (athlete's foot), Tinea unguium (onychomycosis), tinea versicolor (pityriasis versicolor), toxocariasis (ocular larva migrans (OLM)), toxocariasis (visceral larva migrans (VLM)), toxoplasmosis, trichinosis, trichomoniasis, trichuriasis (T. trichiura infection), tularemia, Ureaplasma urealyticum infection, Venezuelan equine hemorrhage, Venezuelan fever viral pneumonia, West Nile fever, White Stone, Yersiniosis, yellow fever, and Zygomycosis.

In several embodiments, the compositions described herein are particularly effective for treating one or more infectious diseases that are resistant to drug therapies. In addition to those infectious diseases mentioned above, which may already be or may become resistant to drugs in the future, certain modalities are effective in treating, among others, drug resistance of: measles, tetanus, malaria, upper respiratory tract infections and lower, hepatitis, typhoid fever, Staphylococcus aureus intermedia infection

107 vancomycin / glycopeptide, vancomycin resistant enterococci, methicillin-resistant Staphylococcus aureus (MRSA = Methicillin-Resistant Staphylococcus Aureus) and streptococcus pneumoniae.

In some embodiments, the treatment of an infectious disease comprises the prior treatment of a patient with DMSO, followed by the administration of a pharmaceutical composition comprising DMSO and antimicrobial agents. In other embodiments, the treatment of an infectious disease comprises the prior treatment of a patient with DMSO, followed by the administration of a formulation comprising DMSO, MSM, and antimicrobial agents. In some embodiments, prior treatment with DMSO is administered intravenously using a rapid drip IV catheter. In other embodiments, DMSO is supplied with an IV bolus injection. In yet another modality, prior treatment with DMSO is not performed. Further treatment compositions additionally include MSM, a therapeutic agent or a combination thereof in some embodiments.

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

108 In some embodiments, the formulations are administered by inhalation. In some of these embodiments, the inhalant medium comprises a nebulizer. In other embodiments, an inhaler is used.

In several embodiments, subjects are previously treated 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, prior treatment with DMSO is provided in the range of about 25 mg to about 75 mg (for example, 30 mg, 40 mg, 50 mg, 60

<td>mg, 70 mg)</td><td>in</td><td>200 mL of 5% dextrose and</td><td>Water. In a</td>

<td>modality,</td><td>I know</td><td>provide 56 mg of DMSO</td><td>in 200 mL of</td>

<td>dextrose at</td><td> 5%</td><td>and water. In one mode, it</td><td>provide the</td>

<td>following</td><td></td><td>antibiotics: rifampicin,</td><td>isoniazid,</td>

pyrazinamide, and ethambutol. In one embodiment, 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 given in a dose of 3 mL, three times a day. In one embodiment, antibiotics are combined with DMSO for inhalation delivery, with or without prior treatment with DMSO. The

109 Prior treatment with MSM is also provided in several modalities. Prior intravenous treatment of DMSO, 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 additional improvement than those treated with antibiotics only. In several modalities, the addition of MSM to the formulation improves therapeutic effects or reduces side effects. In one embodiment, MSM is used only as a pretreatment.

In various embodiments, the compositions described herein are used not only to treat undesirable symptoms and diseases, but can also act as a preventive agent. For example, a formulation can be taken on a regular basis to prevent the onset of a

110 disease. In one embodiment, risk subjects (for example, family members or subjects who are exposed to patients who have an infectious disease) are given lower doses of DMSO and / or MSM and antibiotics to prevent the onset of the disease.

IV. MSM Methods of Use

Methods for using any of the MSM compositions described (as described in Section III) to modulate microbial activity, such as to improve or inhibit the activity of microorganisms are described herein. For example, methods for improving microbial activity are described that include 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 embodiments, 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 mode, a method to improve the

111 Activity of a microorganism includes providing microorganisms, a medium capable of supporting growth of microorganisms, and MSM in an amount sufficient to improve the activity (eg, fermentation efficiency, growth, culture efficiency and / or microbial survival) of microorganisms. and bringing the MSM into contact with the medium, thus improving the growth of microorganisms in the medium. It is contemplated that MSM may be added to the medium before, concurrently with or after the medium is contacted with the microorganisms. In a particular embodiment, MSM is provided at a concentration of approximately 0.04%.

<td>until about</td><td>5% in</td><td>weight</td><td>of the middle or</td><td>in</td><td>weight of</td>

<td>moisture content</td><td>of the</td><td>medium.</td><td>As such,</td><td>in</td><td>Some</td>

<td>examples, MSM (such</td><td>how</td><td>a</td><td>composition</td><td>what</td><td>It includes</td>

approximately 0.5% to approximately 5% of MSM) is used to improve microbial growth. For example, MSM is used to improve fermentation efficiency, such as to improve fermentation efficiency associated with the production of beer, cider, wine, a biofuel,

<td>product</td><td colspan="3">dairy or any combination of</td><td>the</td><td>same.</td><td>In</td>

<td>various</td><td>examples, MSM</td><td>improve the</td><td>production</td><td>from</td><td colspan="2">a</td>

<td>process</td><td>of elaboration</td><td>of food</td><td>or drink,</td><td>what</td><td>is based</td><td>in</td>

microorganisms, such as brewing, winemaking, marinated baking, brewing

112 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 used to improve the efficiency of culture and / or survival of microorganisms.

i. Methods for Improving Efficiency of Microorganism Fermentation with MSM

In several modalities, MSM is used to facilitate energy production. Thus, methods for improving energy production, including methods for improving fermentation efficiency of microorganisms, are described herein. 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 microorganism metabolism. 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 live microbial fuel cells. In some modalities, MSM improves bacterial growth and metabolism. In some modalities, MSM improves bacterial energy production. In some modalities, MSM

113 Improves the development and metabolism of yeast. 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) ethanol fermentation or other anaerobic respiration used 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 industry; (iv) the conversion of carbohydrates into alcohols or acids under anaerobic conditions used for food preparation (for example, breads, dairy products, beans, vinegar, sour cabbage, quinchi, fish and tofu); (v) fermentation for the production of brandy, whiskey, vodka, beer, wine or cider, (vi) fermentation to produce glucosamine; and (vii) fermentation for the aerobic treatment of tea leaves for decomposition of undesirable chemicals and development of others that impact, for example, the taste and / or nutrients of tea.

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

114

0.04% to about 5% by weight of the moisture content of the medium, where the concentration of MSM increases the fermentation efficiency of the microorganism compared to the fermentation efficiency in the absence of MSM.

In one embodiment, the improved fermentation efficiency is indicated by an increase of at least 10%, such as approximately an increase of 20% to 80%, approximately an increase of 30% to 50%, including approximately an increase of 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, approximately 200%, approximately a 300% increase in alcohol, carbon dioxide or acid production in the presence of MSM by the microorganism compared to alcohol, carbon dioxide or acid production 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 fermentation efficiency includes an increase of at least 10%, such as approximately an increase of 20% to 80%, approximately an increase of 30% to 50%, including approximately 10%, approximately 20 %, approximately

115 at 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 150%, approximately 200%, approximately at 300% in ethanol, methanol or a combination thereof compared to ethanol, methanol or a combination thereof in the absence of MSM. In a particular example, the microorganism is yeast and the method for improving fermentation is for beer production. In another example, the microorganism is algae and the method to improve fermentation is for the production of a biofuel.

In some embodiments, improving fermentation efficiency includes an increase of at least 10%, such as approximately an increase of 20% to 80%, approximately an increase of 30% to 50%, including approximately an increase of approximately 10%. an increase of 20%, approximately an increase of 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately a 150%, about 200%, about 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

116 method to improve fermentation is for bread production.

In additional modalities, 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 fermentation efficiency include an increase of at least 10%, such as approximately an increase of 20% to 80%, approximately an increase of 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 modalities, the concentration of MSM

<td>effective for</td><td>to get better</td><td>the</td><td>efficiency of</td><td>fermentation</td><td>it is</td>

<td>approximately</td><td> 0.04%</td><td>to</td><td>approximately</td><td>5%, such</td><td>how</td>

<td>approximately</td><td> 0.1%</td><td>to</td><td>approximately</td><td> 4%, 0.5%</td><td>to</td>

<td>approximately</td><td colspan="4">3%, about 1% to about</td><td> 2%,</td>

which includes approximately 0.04%, to approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, approximately 0.1%,

117 approximately 0.3%, approximately 0.5%, approximately 0.7%, approximately 1%, approximately 1.5%, approximately 2.0%, approximately 2.5%, approximately 3.0%, approximately 4%, or approximately 4.5% by weight of medium or by moisture content of the medium. In some embodiments, MSM is added to yeast packages to generate a quick or immediate activation yeast for domestic or commercial use.

<td>In</td><td>Some examples,</td><td>the middle</td><td>for</td><td>the method</td><td>from</td>

<td>improve the</td><td>efficiency of a</td><td colspan="2">microorganism,</td><td>It includes</td><td>a</td>

<td colspan="2">chloride concentration of</td><td>sodium at</td><td>less</td><td>of 5%</td><td>of the</td>

<td>content</td><td colspan="2">of total humidity of</td><td>medium,</td><td colspan="2">such as</td>

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 mode, MSM is used for beer production. Yeast crops are involved in beer production during the fermentation process to produce ethanol and carbon dioxide. In some examples, MSM is used to accelerate or facilitate the activation of yeast culture, improve fermentation, reduce potential environmental contamination (such as undesirable airborne microorganisms) or a combination thereof. For example, an increase in efficiency to activate yeast (such as an increase in

118 the efficiency of the starting 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 of 20% to 80%, approximately an increase of 30% to 50%, including approximately a 10% increase, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90% , approximately 100%, approximately 150%, approximately 200%, approximately 300% compared to a control (such as efficiency of these processes in the absence of MSM).

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 embodiment, this is particularly beneficial for the cultivation of algae (algal farms), for making or making vegetable oil, biofuel, bioethanol, biogasoline, biomethane, biobutanol and / or other biofuels. In one embodiment, the addition of MSM increases the algal growth rate by approximately 25%, approximately 30%, approximately 40%, approximately 50%, approximately 100%, approximately 200%, approximately 300%, approximately 400%, approximately 500 % or higher. MSM can be particularly advantageous

119 because by improving the activity of algae (such as algae growth), biofuel production can be adjusted in scale, be economically competitive and / or commercially viable. In one embodiment, MSM improves the process by which the algae product is harvested and converted into biofuel. In other modalities, MSM improves the process by which the carbohydrate content of algae is fermented in bioethanol and biobutanol. In some modalities, MSM improves the process of algae by (i) 10 increasing the yield of algae, (ii) forming more robust algal colonies, (iii) shortening harvest time, (iv) shortening fermentation time, ( v) improve fermentation and / or otherwise support or improve growth, reproduction, proliferation, survival rate, metabolism, vitality, robustness, action and / or function of algae. Algae, including but not limited to, Botryococcus braunii, Chlorella, Dunaliella tertiolecta, Gracilaria, Pleurochrysis portfolioe, and Sargassum, are enhanced 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 (for example, 25 probiotics). In some modalities, microorganisms

120 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 compared to a comparable composition without MSM. In certain modalities, MSM significantly improves the simultaneous growth of one or more microorganisms. In some embodiments, supplemental medium with an MSM composition to improve microbial activity (such as a concentration range of about 0.4% to about 5% or any of the MSM compositions to improve microbial growth as set forth in Section III) improves The growth of microorganisms.

Some microorganisms are anaerobic organisms (anaerobes). Anaerobes do not require oxygen for growth. Anaerobes can be used for fermentation and / or cultivation. 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 anaerobic growth than other microorganisms. In other modalities, MSM has a greater positive impact on the growth of aerobic bacteria compared to others.

121 microorganisms Still in other modalities, aerobic and anaerobic are both positively impacted by the presence of MSM.

Bacteria can in general be classified as gram-positive or gram-negative, depending on the structure of their cell wall. Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas, Salmonella, Shigella, Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella, alphaproteobacteria, cyanobacteria, spirobacteria, spirobacteria, spirobacteria, spirobacteria, green bacteria Greens that are not sulfur. Enteric bacteria are Gram-negative in the form of rods; Most occur normally or pathogenicly 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 bacteria and gram positive bacteria.

122

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 at three points according to some modalities: survival, colonization and lactic acid production. To be effective in maintaining the health of the gastrointestinal tract, probiotic bacteria must be able to survive. Bacteria that are dead on arrival, in most cases, do not provide benefit. In this way, in some modalities, MSM positively affects probiotic survival. In certain modalities, MSM improves initial survival upon exposure of bacteria to a new environment. Thus, in these modalities, a product comprising a probiotic and MSM establishes a larger or healthier 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

123 growing, a larger 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. Thus, 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 supplemental with MSM, and the resulting probiotic levels achieved in the gastrointestinal tract are higher than after ingestion of food containing probiotics 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 that require 24 to 48 hours before effects are observed, become more effective because MSM increases its life span.

Bacterial growth typically has a

124 initial lateritia phase where the bacteria adjust to the environment, before entering the log phase, where the cells duplicate. After the log phase, there is a stationary phase. During the stationary phase, the growth rate slows down as a result of nutrient depletion and accumulation of metabolic by-products. This phase is reached as the microbes begin to run out of the resources that are available to them. This phase is a relatively constant value since the microbial growth rate is equal to the microbial death rate. 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 modalities, MSM shortens the latency phase, so that bacteria, such as probiotic bacteria, begin the log phase at a previous time. In several modalities, MSM prolongs the stationary phase. In certain modalities, mortality, index or mortality rate is slowed in the presence of MSM. Certain modalities of the description as described herein positively affect one or more, and in certain modalities all phases of the growth of probiotic bacteria.

In some modalities, MSM impacts the metabolism of microbes (for example, probiotics), in the phase of

125 latericia. During the latency phase, the microbes 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 media.

In some embodiments, the MSM supplement results in an increase in the log phase of microbial growth (for example, probiotics). The exponential phase (sometimes called the log phase) of growth is a period characterized by cell duplication. The number of new microbes that appear per unit of time is proportional to the present population. If growth is not limited, 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 cannot 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

126 MSM presence in the growth medium promotes microbial entry into the exponential phase more rapidly than microorganisms in MSM-free medium. The atmosphere of

<td>increase</td><td>initial with half</td><td>supplemented</td><td>with</td><td>MSM can</td>

<td colspan="4">lead to cell multiplication and survival. In several modalities, MSM affects</td><td>the phase</td>

<td>stationary</td><td>of growth</td><td>microbial</td><td>(by</td><td>example,</td>

<td>probiotic).</td><td>In an example,</td><td>the supplement</td><td>MSM</td><td>media</td>

<td>extends the</td><td>Stationary phase</td><td>by microbes</td><td colspan="2">compared</td>

with MSM free medium.

In some modalities, MSM improve probiotic growth, which in turn clutter and take nutrients from unwanted microbes. In other modalities, MSM improves probiotic activity, which in turn improves the production of lactic and acetic acids to reduce the environmental pH and inhibit the activity of undesirable bacteria. In additional modalities, MSM improves probiotic activity, which in turn stimulates the production of imunomodulatory agents (for example, cytokines), thereby improving the immune response. In certain embodiments, MSM improves probiotic activity, which in turn improves bactericidal activity with respect to undesirable microbial contamination. In one embodiment, MSM improves probiotic growth at a faster rate than undesirable microbes, thus allowing probiotics to colonize in a way

127 preferential environment (for example, edible products, intestinal tract).

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 anaplerotic 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 additional modalities, MSM increases the duration of the stationary phase of microbial growth. In some modalities, MSM decreases the population decline rate of certain microbes. In certain embodiments, MSM provides a selective or semi-selective growth environment, such that certain microbes grow faster (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.

128

As such, methods are provided to improve microbial growth. In some embodiments, methods for improving microbial growth include in vitro methods for improving the growth of one or more microorganisms. In one example, in vitro methods for improving 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% to about 5% by weight of the medium or by weight of moisture content of the medium in this way improving the growth of the 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 improve 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 of 20% to 80%, approximately an increase of 30%. at 50%, including approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, about 150%, about 200%,

129 approximately at 300% increase compared to a control (such as the weight of the microorganism or number of cells in the absence of MSM). Increases in microorganism growth 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%, such as an increase of approximately 20% to 80%, an increase of approximately 30% to 50%, including an increase of 10%, approximately 20%,

130 approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 150%, approximately 200%, approximately at 300% increase in cell growth, compared to a control (such as cell growth in the absence of MSM).

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 thereof.

It is contemplated that the method can be used to improve the growth of any probiotic microorganism, including, but not limited to Lactobacillus acidophilus, Lactobacillus delbrueckii, Bacillus coagulans, Lactobacillus rhamnosus, Bifidobacteruim 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 to improve the activity (for example, growth) of

Lactobacillus acidophilus includes contacting

131

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 approximately 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 (eg, growth) of Bifidobacterium bifidum includes contacting Bifidobacterium bifidum with a medium capable of supporting the growth of Bifidobacterium bifidum; and provide 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 thus improving the growth of Bifidobacterium bifidum compared to the growth of Bifidobacterium bifidum in the absence of MSM.

An increase in probiotic growth is indicated by an increase in weight of the probiotic microorganism or number of cells thereof, including an increase of at least 10%, such

132 as about 20% to 80% increase, approximately 30% to 50% increase, including approximately 10%, to approximately 20%, approximately 30% increase, approximately 40% increase, approximately increase of 50%, approximately a 60% increase, approximately a 70% increase, approximately an 80% increase, approximately a 90% increase, approximately a 100% increase, approximately a 150% increase, approximately a 200% increase, approximately a 300% increase compared to a control (such as weight of the probiotic microorganism or number of cells in the absence of MSM). Increases in probiotic microorganism growth can be detected by methods known to those skilled in the art including those described in the Examples.

b. Methods to Improve 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 is provided to improve the growth of a microorganism in a diagnostic test sample. In one example, the method includes contacting a diagnostic test sample (for example,

133 blood, tissue, scrapes, body 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 concentration sufficient to improve microbial growth, thereby improving the growth of 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 is provided to improve the growth of a microorganism in an industrial test sample. In one example, the method includes contacting an industrial test sample (for example, water sample, sample of domestic bacteria or mold and other similar 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 concentration sufficient to improve microbial growth, thereby improving the growth of one or more microorganisms in the industrial test sample compared to growth of the one or more microorganisms in the absence of MSM ..

In several embodiments, MSM is provided in a composition to facilitate diagnostic tests or tests of industrial test samples such as a

134 concentration of about 0.04% to about 5% by weight of the sample or by weight of the moisture content of the sample. In some embodiments, MSM is provided in a composition to facilitate diagnostic tests or tests 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 here, 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 of 20% to 80%, an increase of approximately 30% to 50%, including approximately a 10% increase, approximately a 20% increase, approximately a 30% increase, approximately a 40% increase, approximately a 50% increase, approximately a 60% increase,

135 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 with 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 screening 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 sample, thus improving the detection time of the proof. In some modalities, MSM can

136 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 scratches, vaginal or cervical samples, and the like. In one embodiment, MSM is used to provide a rapid strep 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 previously 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 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 virus growth for diagnostic testing. Viruses include, but are not limited to, human immunodeficiency virus, herpes simplex virus,

137 papilloma, parainfluenza virus, influenza, hepatitis and other viruses. Similarly, the medical diagnosis of other infections, such as those caused by bacteria, fungi, yeast and parasites is also facilitated by MSM according to several 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 contaminant of water. Many water safety test equipment evaluates the quality of drinking water through testing methods of the Environmental Protection Agency (EPA) to be tested, among other things, for the presence of bacteria. Mold that is found in home, office and school environments has been linked with lung disorders, and allergic symptoms. However, some tests used to detect bacteria or mold can be time consuming for analysis while some tests additionally detect only viable (live) organisms. In this way, in several modalities MSM is used to supplement growth medium used in commercial screening 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

138 Certain modalities, MSM restores environmentally stressed bacteria that were previously not viable. In still further embodiments, diagnostic test kits comprising medium supplemented with specific MSM of microorganisms 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 described. For example, methods to improve the survival of microorganisms or cells, such as cells in culture, include contacting one or more selected microorganisms or cells with MSM at about 0.4% to about 5% by weight of the medium or by weight of a content of humidity of the environment, thus improving the survival of one or more microorganisms or cell collection compared to the survival of one or more microorganisms or cell collection in the absence of MSM. In one example, the concentration of MSM is approximately 1%

139 at about 3% of the weight of the medium or the moisture content of the medium. An increase in survival is indicated by an increase of at least 10%, such as an increase of approximately 20% to 80%, an increase of approximately 30% to 50%, including an increase of approximately 10%, an increase of approximately 20 %, an increase of approximately 30%, an increase of approximately 40%, an increase of approximately 50%, an increase of approximately 60%, an increase of approximately 70%, an increase of approximately 80%, an increase of approximately 90 approximately 100%, an increase of approximately 150%, an increase of approximately 200%, an increase of approximately 300% in colony or number of cells compared to a control (such as colony or number of cells in the absence of MSM).

According to several modalities, MSM improves the initial survival of microorganisms (including,

<td>but not limited to,</td><td>microorganisms</td><td>probiotics)</td><td>In</td><td>a</td>

<td>modality, MSM improves</td><td>survival</td><td colspan="2">in the long term of</td><td>the</td>

<td>microorganisms In</td><td>a modality,</td><td>MSM extends</td><td>the</td><td>phase</td>

<td colspan="2">stationary of a microorganism curve.</td><td>increase</td><td>from</td><td>the</td>

<td>In several</td><td>modalities, MSM</td><td>extends the</td><td>lifetime</td><td>in</td>

storage of a product by prolonging the extension of life of beneficial bacteria compared to products without

MSM

For example, a product that contains a probiotic can

140 Having a storage 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 duration of product packaging time until 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 consumer's gastrointestinal (GI) tract), for a longer period of time after packaging.

In several embodiments, the addition of MSM increases the time to deterioration of ingestible products by supporting or improving 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% (for example, 20%, 30%, 40%, 50%, 75%, 150%, 200 % or more). For example, in one embodiment, if the shelf life of an edible product is 10 days, the addition of MSM will increase the shelf life to at least 11 days in some 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 shelf life of 14 days at room temperature and / or 30 days

141 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, irradiation, filtration , heat, chemicals, etc.).

In some embodiments, MSM is provided to improve the activity of genetic vectors, such as recombinant viral vectors in recombinant cells. This may be beneficial for diagnostic agents as well as therapeutic agents, such as gene therapy. In some embodiments, MSM is used to improve the activity (for example, 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 where one or more processes associated with gene therapy are improved or increased by treating recombinant cells or microorganisms with a concentration of MSM (such as a concentration of about 0.04% to about 5% of MSM) able to improve one or more

142 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 methods to improve cultivation efficiency with MSM are described. In one embodiment, methods for improving the efficiency of cultivation of antibiotics, spheroids, 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 modalities, 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 of waste products and / or remains of other organisms through decomposition. In this way, in some modalities, the use of MSM can

143 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 effluent, 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 contaminant microorganisms. In some modalities, MSM is added to gardening products, such as soil, fertilizers and compost hoppers, 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 for improving 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 rolled on a plant surface either before, after or simultaneously with the fertilizer. An increase in

144 Fertilizer efficiency is indicated by an increase of at least 10%, such as approximately an increase of 20% to 80%, approximately an increase of 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 a 100% increase, approximately a 150% increase, approximately a 200% increase, approximately a 300% increase in plant growth compared to a control (such as plant growth in the absence of MSM).

In another embodiment, a method for improving 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 emptying or spraying the solution) and allowing sufficient time to improve composting efficiency. An increase in compost efficiency is indicated by an increase of at least 10%, such as approximately

145 an increase of 20% to 80%, approximately an increase of 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 a 50% increase, approximately 60% increase, approximately 70% increase, approximately 80% increase, approximately 90% increase, approximately 100% increase, approximately 150% increase, approximately a 200% increase, 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 of 20% to 80%, approximately an increase of 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% in the amount of time that the decomposition of organic matter occurs compared to a control (such as speed of

146 decomposition in the absence of MSM).

B. Methods to Inhibit Microbial Activity

Methods for inhibiting microbial activity are described. In one embodiment, a method of inhibiting microbial activity includes selecting a medium that is susceptible to contamination; and contacting the medium with MSM at a concentration of about 6% to about 16% by weight by volume, thereby inhibiting microbial activity compared to 10 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 of approximately 20% to 80%, a decrease of 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%, a decrease of approximately 60%, a decrease of approximately 70%, a decrease of approximately 80%, a decrease of approximately 20%, a decrease of approximately 100%, a decrease of approximately 150%, a decrease of approximately 200%, a decrease of approximately 300% compared to a control (such as microbial activity in the absence of MSM ).

In some modalities, a method to inhibit

147 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 of inhibiting microbial activity includes selecting a medium that is susceptible to viral contamination (such as contamination by human immunodeficiency virus, H1N1, herpes simplex virus, papillomavirus, 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 by volume, thereby inhibiting viral activity.

In a particular example, a method for inhibiting microbial activity includes selecting a medium that is susceptible to contamination of H1N1 influenza; and contacting the medium with MSM at a concentration of about 10% to about 16% by weight by 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 %,

148 approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 150%, approximately 200%, approximately 300% decrease in growth or infectivity of H1N1 influenza compared to a control (such as activity or infectivity of H1N1 influenza in the absence of MSM).

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 approximately 9% and approximately 16%, between approximately 10% and approximately 16%, between approximately 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 embodiments described herein, the percentages of MSM are based on the moisture content of a product. In some modalities, MSM is particularly

149 effective when combined with water or other liquid components. In several embodiments, the percentages of MSM provided 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 growth inhibition 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 methods described include formulations of MSM that produce reduction in microbial levels (eg, bacterial) in the range of about 0.5 log to about 5 log or more within two weeks. In some embodiments, the methods described to inhibit microbial activity result in a log reduction between about 1 log reduction and about 3 log or more reductions. In other embodiments, the methods described to inhibit microbial activity lethally inhibit the growth of certain microorganisms. In one embodiment, a method that uses an MSM formulation between about 12% and about 16% lethally kill certain

150 microbes (for example, bacteria) within approximately 48 hours. In another embodiment, a formulation comprising MSM between about 8% and about 12% lethally kill certain microbes (eg, 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, lethally killing certain microbes (eg, bacteria) within about 48 hours. With higher concentrations of conservative, MSM levels can be further reduced.

In some embodiments, the methods described to inhibit microbial activity with MSM (such as with about 6% to about 16% of 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

151 cell duplication The number of new microbes that appear per unit of time is proportional to the present population. If growth is not limited, 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 cannot continue indefinitely, however, because the medium is soon depleted of nutrients and enriched with waste. In some modalities, MSM decreases the total duration of the exponential phase. In other modalities, the presence of MSM in the growth medium inhibits microbial entry into the exponential phase.

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

152

It is contemplated that a medium includes any medium or environment that contains or is suitable to withstand contamination including but not limited to cosmetics, 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, 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, contact of the medium includes spraying or rubbing the medium susceptible to microbial contamination with a composition / formulation of MSM. 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 toilets, 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 household surface may include a door handle, door knob, a boat

153 garbage, 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 may occur, for example during manufacturing, packaging or repetitive use by a consumer that includes repeated opening and closing of containers, contact with hands, skin or mucous membranes or repeated administration / withdrawal of individual doses. In the absence of antimicrobial properties, these products can allow the accidental growth of many different and potentially harmful microorganisms.

Antimicrobial preservatives can be added to products to protect them from microbial growth. Commonly used antimicrobial preservatives include calcium propionate, sodium nitrate, sodium nitrite, sulphites (sulfur dioxide, sodium bisulfite, potassium hydrogen sulphite, etc.) and EDTA disodium. Cosmetic preservatives include formaldehyde, potassium sorbate, methylparaben and methylchloroisothiazolinone.

In many cases, conservatives must be added in

154 a minimum effective concentration, as adverse reactions may occur at certain concentrations or doses. In this way, while conservatives can inhibit microbial growth, they also have the potential to cause chemical burns and / or irritate the 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 they can present unique complications with the solubility, pH limits, deactivation by some polyethylene glycol (PEG) compounds, and a change in the color, consistency or fragrance of a product. Some conservatives have only limited activity against particular classes of microorganisms.

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 (for example, 10-16%, 16-20%, 20-30%, 3040%, 40-50%, 50-75% or higher). overlapping intervals or ranges thereof).

The medium is free of

155 Conservatives in some modalities.

In some embodiments, methods for inhibiting microbial activity in a cosmetic cream at room temperature are provided. In one embodiment, the method includes selecting a medium that is susceptible to microbial contamination; and add 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 (for example, 5-10%, 10-16%, 16-20%, 2030%, 30-40%, 40-50%, 50- 75% or higher, and their intervals 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 diagnostic, 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 tissue or body fluid, such as blood. In an example, the

156 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 (e.g., 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 combinations thereof. In another embodiment, microbial contamination is caused by an infectious disease including any of the infectious diseases described herein.

In several 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.

157

<td></td><td>In</td><td>some modalities,</td><td>MSM inhibits the</td><td>exercise</td>

<td colspan="2">microbial to</td><td>reduce speed</td><td>of growth</td><td>of one or</td>

<td>plus</td><td>microbes</td><td>by more than 50%, which</td><td colspan="2">turn increases life</td>

<td>in</td><td colspan="2">media storage I know</td><td>contemplate that</td><td>MSM can</td>

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 leading to microbial activity, including 20-25 ° C, 25-30 ° C, 30-40 ° C, 40-50 ° C and above (and their overlapping ranges). In some embodiments, MSM inhibits microbial activity at favorable moisture levels for microbial activity, including 50% 60%, 60-70%, 70-80%, 80-95%, and higher (and overlapping ranges or ranges thereof). ).

<td>MSM is</td><td></td><td>particularly</td><td>advantageous in several</td>

<td colspan="2">modalities due</td><td>what can</td><td>employ superiors</td>

<td colspan="2">concentrations that</td><td colspan="2">other conservatives, that when</td>

<td>even use</td><td>in</td><td>low concent</td><td>portions may cause</td>

<td>Adverse effects.</td><td></td><td>For example,</td><td>conservatives have been</td>

<td>involved in</td><td></td><td>dermatitis</td><td>atopic, rashes,</td>

<td>redness</td><td colspan="2">abdominal pain,</td><td>nausea, asthma, rhinitis,</td>

<td colspan="2">muscle pains,</td><td>pains in</td><td>joints, fatigue,</td>

<td>numbness or</td><td colspan="2">numbness</td><td>migraines, disorder of</td>

158 hyperactivity and attention deficit, palpitations and arrhythmias. In contrast, MSM is not known to cause these effects in concentrations that are provided in accordance with the present preferred embodiments. Even more, 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 several 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, inflammation reduction, treatment of rheumatoid arthritis and osteoarthritis, cardiovascular improvements, skin lubrication, 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.

159

According to several modalities, the methods instantly inhibit microbial activity. In other embodiments, 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 several embodiments, MSM is added to cleaning agents to improve antimicrobial activity (for example, to inhibit activity of microorganisms). In some embodiments, MSM is added to a soap formulation. In some modalities, the product is a dry soap while 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 to inhibit microbial activity include methods to disinfect a surface, such as the body, equipment, floors, materials, walls, etc. In certain embodiments, the resulting disinfectant is an instant disinfectant. In other embodiments, the disinfectant acts non-instantaneously (for example, it is effective over time). In some modalities, the disinfectant is applied to the body. In still other embodiments, the product is applied to a surface. Surfaces include, but are not limited to, commercial surfaces, medical devices, medical surfaces, production equipment, production floors and production surfaces.

160 food preparation. Surfaces may include, but are not limited to domestic surfaces, vehicles, computers, clothing and toys.

In some additional embodiments, methods for inhibiting microbial activity include spraying or incorporating MSM (e.g., about 5% to about 50% in facial masks or filters. Filters may include, but are not limited to air conditioning filters, filters air, water filters Environments with recycled air, such as aircraft, can benefit especially from MSM filtration systems. Waste treatment plant and water filtration can also incorporate MSM to inhibit microbial activity. In some modalities, MSM is provided to reduce microbial contamination in flower arrangements and in garden products (such as fertilizers and soil or substrates).

In some embodiments, methods to inhibit 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 animal feed include, but are not limited to, compound feed, feed or fodder. Animal feed may consist of raw materials and / or additives. Raw food can be provided as straw or grains. Alternatively, the

161 Raw material can be manufactured and provided as food, peel or hash 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 embodiments, MSM is applied to raw food materials and thus incorporated into a particular food product. In additional embodiments, 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 for Producing Products that include MSM

Methods for producing products that include MSM are described here. In some modalities, MSM is incorporated into a stage that will reduce the crystallization of MSM. In one embodiment, MSM is incorporated into an anees product emulsification product. In another embodiment, MSM is encapsulated (for example, in a polymeric lipid or other material) before adding to a product. Micro encapsulated MSM, according to some modalities, can be designed for dose-release MSM or over time. In still other embodiments, 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.

162

In still another embodiment, MSM is added to a product at an elevated temperature (for example, 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 may be added before heating. Solutions that have a temperature greater than about 35 ° C withstand concentrations of MSM greater than 50% in some modalities. 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 greater.

Methods for manufacturing a product that has a reduced preservative concentration are also described herein. In one embodiment, the method includes providing a medium that is susceptible to microbial contamination, wherein the medium comprises a preservative and adding MSM to the medium, where MSM affects microbial contamination by inhibiting microbial growth. MSM is added in a concentration of at least 5% to about 20% according to one modality (for example, 5-8%, 8-12%, 12-15%, 15-20%, and their overlapping intervals). In one embodiment, MSM and the conservative inhibit microbial growth by at least 50% in the medium at room temperature, and MSM supplements or improves the ability of conservative to inhibit growth

163 microbial, thus reducing the concentration of the preservative 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 particular characteristics and / or 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 ¿eruginosa, Escherichia coli and Salmonella cholerasuis. All microorganisms developed in tryptic soy broth (TSB = Tryptic Soy Broth) and except for Candida and Aspergillus, all were successfully transferred to medium

164 fresh every day for 4 consecutive days before inoculation to keep organisms in an exponential growth phase. Candida and Aspergillus had 48-58 hours of growth in TSB before inoculation in half test. Aspergillus also developed on multiple potato dextrose agar plates (PDA = Potato Dextrose Agar) for 48-58 hours. The inoculum of Aspergillus was prepared by taking a surface rinse with TSB of the PDA plates with an Aspergillus lawn, and then added to the culture of 4858 hours until turbidity. For each test microorganism, 90 mL aliquots of TSB were prepared either 10% or 0% MSM. Once each set of test media was applied on plates for sterility, they were inoculated at a level of 5 inocula per 10 mLs of broth (1: 2000 dilution of inoculum) with each respective microorganism. Bacterial organisms were incubated at 30 ° C ± 2 ° C and incubated to fungal organisms at 25 ° C ± 2 ° C.

<td>Fungal organisms are</td><td>they sowed</td><td>in plates</td>

<td>daily in PDA on days 0 to</td><td>day 7 every</td><td>24 hours.</td>

<td>Preparation and sowing on plates are</td><td>made to</td><td>temperature</td>

environment. 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 express as colony forming units recovered by one milliliter (cfu / mL).

165

The effects of MSM on growth of Aspergillus niger and growth of Candida albicans are shown in Tables 1-1 (a) and 1-1 (b), respectively. Ten percent of MSM inhibited growth of Aspergillus niger on day 4 of treatment as represented by a dramatic reduction in colony formation on this date. A reduction in growth was also observed in samples treated with MSM of Candida albicans; however, the reduction was not as dramatic compared to Aspergillus niger. For example, medium supplemented with 10% MSM resulted in reduced yeast viability compared to lower concentrations of MSM as early as 2 days (plating on day 2 for Candida). Candida growth was reduced early in the study, with substantial reductions in fungal population on Day 4. After these time points, 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.

166

<td>Aspergillus niger</td><td>0% MSM</td><td>0.1% MSM</td><td>0.5% MSM</td><td>1.0% MSM</td><td>10% MSM</td>

<td>Day 0</td><td>1.0 x 10³</td><td>9.1 x 10²</td><td>7.6 x 10²</td><td>8.9 x 10²</td><td>1.2 x 10³</td>

<td>Day 1</td><td>1.3 x 10³</td><td>2.3 x 10³</td><td>2.7 x 10³</td><td>2.0 x 10³</td><td>7.0 x 10²</td>

<td>Day 2</td><td>4.3 x 10³</td><td>3.0 x 10²</td><td>4.0 x 10³</td><td>2.0 x 10³</td><td>1.6 x 10²</td>

<td>Day 3</td><td>2.0 x 10³</td><td>7.5 x 10²</td><td>1.3 x 10³</td><td>1.0 x 10³</td><td>4.6 x 10²</td>

<td>Day 4</td><td>1.4 x 10⁴</td><td>5.0 x 10³</td><td>3.7 x 10³</td><td>3.3 x 10³</td><td> 20</td>

<td>Day 5</td><td>1.3 x 10⁴</td><td>4.3 x 10³</td><td>9.7 x 10³</td><td>6.0 x 10³</td><td> 10</td>

<td>Day 6</td><td>4.4 x 10⁴</td><td>1.1 x 10⁴</td><td>7.0 x 10³</td><td>4.7 x 10³</td><td> 3</td>

<td>Day 7</td><td>4.1 x 10 '</td><td>1.3 x 10⁴</td><td>7.0 x 10³</td><td>5.7 x 10³</td><td> 3</td>

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

<td>Candida albicans</td><td>0% MSM</td><td>0.1% MSM</td><td>0.5% MSM</td><td>1.0% MSM</td><td>10% MSM</td>

<td>Day 0</td><td>2.2 x 10⁴</td><td>2.1 x 10⁴</td><td>1.8 x 10⁴</td><td>2.5 x 10⁴</td><td>2.6 x 10⁴</td>

167

<td>Day 1</td><td>1.0 x 10⁵</td><td>4.7 x 10⁶</td><td>4.9 x 10⁶</td><td>5.0 x 10⁶</td><td>&lt;1.0 x 10⁵</td>

<td>Day 2</td><td>1.0 X 10⁷</td><td>9.8 x 10^δ</td><td>1.1 x 10⁷</td><td>9.8 x 10⁶</td><td>4.0 x 10³</td>

<td>Day 3</td><td>1.3 x 10⁷</td><td>1.2 x 10⁷</td><td>1.3 x 10⁷</td><td>1.2 x 10⁷</td><td>&lt;1.0 x 10⁵</td>

<td>Day 4</td><td>1.9 x 10⁷</td><td>1.3 x 10⁷</td><td>1.4 x 10⁷</td><td>1.2 x 10⁷</td><td>7.0 x 10⁴</td>

<td>Day 5</td><td>1.6 x 10⁷</td><td>1.3 x 10⁷</td><td>1.4 x 10⁷</td><td>1.3 x 10⁷</td><td>1.7 x 10⁴</td>

<td>Day 6</td><td> 1—¹ co CO X</td><td>1.3 x 10⁷</td><td>1.4 x 10⁷</td><td>1.6 x 10⁷</td><td>2.8 x 10³</td>

<td>Day 7</td><td>1.7 x 10⁷</td><td>1.4 x 10⁷</td><td>1.6 x 10⁷</td><td>1.7 x 10⁷</td><td>3.3 x 10³</td>

The effect of MSM on growth of Staphylococcus aureus is illustrated in Table 1-2. A difference in feasibility of the presence of higher concentrations of MSM was observed. In particular, 10% MSM appeared so much that it slows the growth rate as the maximum population size of Staphylococcus aureus.

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

168

<td>Staphylococcus aureus</td><td>0% MSM</td><td>0.1% MSM</td><td>0.5% MSM</td><td>1.0% MSM</td><td>10% MSM</td>

<td>Day 0</td><td>2.4 x 10⁵</td><td>2.4 x 10⁵</td><td>2.5 x 10⁵</td><td>2.3 x 10⁵</td><td>2.5 x 10⁵</td>

<td>Day 1</td><td>3.6 x 10⁸</td><td>4.6 x 10⁸</td><td>4.1 x 10⁸</td><td>6.0 x 10⁸</td><td>5.3 x 10⁷</td>

<td>Day 2</td><td>7.5 x 10⁸</td><td>8.5 x 10⁸</td><td>7.8 x 10⁸</td><td>8.2 x 10⁸</td><td>3.0 x 10⁸</td>

<td>Day 3</td><td>9.6 x 10⁸</td><td>9.1 x 10⁸</td><td>8.4 x 10⁸</td><td>9.6 x 10⁸</td><td>5.1 x 10⁸</td>

<td>Day 4</td><td>6.4 x 10⁸</td><td>7.9 x 10⁸</td><td>4.7 x 10⁸</td><td>4.5 x 10⁸</td><td>3.7 x 10⁸</td>

<td>Day 7</td><td>2.6 x 10⁸</td><td>1.8 x 10⁸</td><td>1.7 x 10⁸</td><td>3.3 x 10⁸</td><td>9.0 x 10⁷</td>

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

169

<td colspan="2">Pseudomonas aeruginous</td><td colspan="2">0% MSM</td><td colspan="2">0.1% MSM</td><td colspan="2">0.5% MSM</td><td colspan="2">1.0% MSM</td><td colspan="2">10% MSM</td>

<td>Day</td><td> 0</td><td> 4 . 0 10⁵</td><td>X</td><td> 4.8 10⁵</td><td>X</td><td> 4.8 10⁵</td><td>X</td><td> 4.2 10⁵</td><td>X</td><td> 4.0 10⁵</td><td>X</td>

<td></td><td></td><td> 4.4</td><td>X</td><td> 5.1</td><td>X</td><td> 5.4</td><td>X</td><td> 5.8</td><td>X</td><td> 1.0</td><td>X</td>

<td>Day</td><td> 1</td><td> 10⁸</td><td></td><td> 10⁸</td><td></td><td> 10⁸</td><td></td><td> 10⁸</td><td></td><td> 10⁵</td><td></td>

<td></td><td></td><td> 1.1</td><td>X</td><td> 6.0</td><td>X</td><td> 9.2</td><td>X</td><td> 5.7</td><td>X</td><td> 5.8</td><td>X</td>

<td>Day</td><td> 2</td><td> 10⁹</td><td></td><td> 10⁸</td><td></td><td> 10⁸</td><td></td><td> 10⁸</td><td></td><td> 10³</td><td></td>

<td></td><td></td><td> 1.6</td><td>X</td><td> 1.4</td><td>X</td><td> 1.2</td><td>X</td><td> 1.0</td><td>X</td><td> 5.3</td><td>X</td>

<td>Day</td><td> 3</td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10³</td><td></td>

<td></td><td></td><td> 1.6</td><td>X</td><td> 2.0</td><td>X</td><td> 1.4</td><td>X</td><td> 1.9</td><td>X</td><td> 3.7</td><td>X</td>

<td>Day</td><td> 4</td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁶</td><td></td>

<td></td><td></td><td> 1.4</td><td>X</td><td> 2.1</td><td>X</td><td> 1.6</td><td>X</td><td> 1.2</td><td>X</td><td> 6.0</td><td>X</td>

<td>Day</td><td> 7</td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁶</td><td></td>

The effect of MSM on Pseudomonas growth

<td>aeruginous</td><td>I know</td><td>illustrates</td><td>in</td><td colspan="2">Table 1-4. Ten</td><td>percent of</td>

<td>supplement</td><td>from</td><td colspan="2">MSM medium</td><td>Resulted in</td><td colspan="2">substantially less</td>

<td>increase Table 1-4.</td><td colspan="2">from E. coli Effect of</td><td>with 10%</td><td colspan="2">time. MSM in Growth</td><td>from Escherichia</td>

cali.

<td>Escherichía coli</td><td>0% MSM</td><td>0.1% MSM</td><td>0.5% MSM</td><td>1.0% MSM</td><td>10% MSM</td>

<td>Day 0</td><td>6.9 x 10⁵</td><td>6.4 x 10⁵</td><td>7.0 x 10⁵</td><td>6.7 x 10⁵</td><td>6.8 x 10⁵</td>

170

<td></td><td></td><td> 9.2</td><td>X</td><td> 1.0</td><td>X</td><td> 1.4</td><td>X</td><td> 1.2</td><td>X</td><td> 3.0</td><td>X</td>

<td>Day</td><td> 1</td><td> 10⁸</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁴</td><td></td>

<td></td><td></td><td> 1.3</td><td>X</td><td> 1.4</td><td>X</td><td> 1.6</td><td>X</td><td> 3.2</td><td>X</td><td> 1.2</td><td>X</td>

<td>Day</td><td> 2</td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁵</td><td></td>

<td></td><td></td><td> 1.6</td><td>X</td><td> 2.0</td><td>X</td><td> 1.9</td><td>X</td><td> 1.8</td><td>X</td><td> 3.9</td><td>X</td>

<td>Day</td><td> 3</td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁷</td><td></td>

<td></td><td></td><td> 1.4</td><td>X</td><td> 1.4</td><td>X</td><td> 1.4</td><td>X</td><td> 1.4</td><td>X</td><td> 1.2</td><td>X</td>

<td>Day</td><td> 4</td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁸</td><td></td>

<td></td><td></td><td> 1.3</td><td>X</td><td> 1.2</td><td>X</td><td> 2.8</td><td>X</td><td> 1.5</td><td>X</td><td> 7.0</td><td>X</td>

<td>Day</td><td> 7</td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁹</td><td></td><td> 10⁷</td><td></td>

The effect of MSM on the 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 the time of the study.

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

<td>Salmonella cholerasuis</td><td>0% MSM</td><td>0.1% MSM</td><td>0.5% MSM</td><td>1.0% MSM</td><td>10% MSM</td>

<td>Day o</td><td>6.8 x 10⁵</td><td>8.7 x 10⁵</td><td>5.9 x 10⁵</td><td>5.5 x 10⁵</td><td>7.6 x 10⁵</td>

<td>Day 1</td><td>9.6 x 10⁸</td><td>1.2 x 10⁹</td><td>9.7 x 10⁸</td><td>1.1 x 10^s</td><td>1.7 x 10⁶</td>

<td>Day 2</td><td>1.3 x 10⁹</td><td>1.0 x 10⁹</td><td>1.2 x 10⁹</td><td>1.3 x 10^s</td><td>7.7 x 10⁷</td>

171

<td>Day 3</td><td>1.2 x 10⁹</td><td>1.2 x 10⁹</td><td>1.5 x 10⁹</td><td>1.5 x 10⁹</td><td>3.2 x 10⁸</td>

<td>Day 4</td><td>7.8 x 10⁸</td><td>6.0 x 10⁸</td><td>7.1 x 10⁸</td><td>8.0 x 10⁸</td><td>1.9 x 10⁸</td>

<td>Day 7</td><td>3.0 x 10⁸</td><td>3.4 x 10⁸</td><td>3.6 x 10⁸</td><td>9.2 x 10⁸</td><td>2.3 x 10⁸</td>

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 antimicrobial effectiveness test results 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 the

172 pollution. The product is maintained for a month, with weekly analysis of microorganism levels. Depending on the route of administration of a product, the satisfaction of the AET generally requires a reduction of 1 to 3 log in bacteria of initial levels, which should occur in one to two weeks, without further increase in bacteria after two weeks. For yeast and mold, an increase in the initial inoculum level is not allowed. Successfully meeting the AET criteria demonstrates that a product, optionally supplemented with an antimicrobial compound under evaluation, can withstand an inoculum of up to one million micrograms per gram of product without being contaminated. The AET 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 affects the storage life of a product.

The AET was performed by adding the specified microorganisms directly to test medium supplemented with MSM at concentrations that simulate microbial contamination. Active, standardized fresh cultures 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. Tryptic soy broth (TSB was used

173 = Tryptic I am Broth) 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 listed weekly to determine if they are growing, dying or remaining near 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. Test results ΆΕΤ for 1: 1 dilution of MSM.

<td>Test Body</td><td>Enter Initial</td><td>48 hrs</td><td>3 days</td><td>5 days</td><td>14 days</td><td>20 days</td><td>28 days</td><td>30 days</td>

<td>Aspexg illus Nigex</td><td>3 x 10⁵</td><td>3.5 X 10⁵</td><td>3.2 X 10⁵</td><td>3.1 X 10⁵</td><td>2.3 X 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Candida RThi na ns</td><td>2.5 x 10⁵</td><td>2.8 X 10⁵</td><td>2.6 X 10⁵</td><td>2.4 X 10⁵</td><td>1.2 X 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

174

<td>Escher ichia col i</td><td>1.3 x 10⁵</td><td>1.3 X 10⁵</td><td>1.2 X 10⁵</td><td>eleven X 10⁵</td><td>1.8 X 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Pseudo monas aerugi nosa</td><td>1.5 x 10⁵</td><td>1.8 X 10⁵</td><td>1.9 X 10⁵</td><td>1.6 X 10⁵</td><td>4.0 X 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Staphy lococc US golden</td><td>5.5 x 10⁵</td><td>5.4 X 10⁵</td><td>5.6 X 10⁵</td><td>5.2 X 10⁵</td><td>3.7 X 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Salmon Ella Typhim Urium</td><td>4.6 x 10⁵</td><td>4.9 X 10⁵</td><td>5.1 X 10⁵</td><td>4.7 X 10⁵</td><td>2.5 X 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

Table 2-2 Inorganic Log Reduction of Microorganism

Initial with 1: 1 dilution of MSM.

<td>Test Body</td><td>14 days</td><td>28 days</td>

<td>Aspergillus Niger</td><td> 3.4</td><td> 4.5</td>

<td>Candida Albicans</td><td> 3.0</td><td> 4 . 4</td>

<td>Escherichia coli</td><td> 3.3</td><td> 4.1</td>

<td>Pseudomonas aeruginosa</td><td> 3.6</td><td> 4.2</td>

175

<td>Staphylococcus aureus</td><td> 3.6</td><td> 4.7</td>

<td>Sahaonella typhimurium.</td><td> 3.4</td><td> 4.7</td>

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

MSM

<td>Test Body</td><td>Enter Initial</td><td>48 hrs</td><td>3 days</td><td>5 days</td><td>14 day s</td><td>20 days</td><td>28 days</td><td>30 days</td>

<td>Aspergí llus Niger</td><td>3 x 10⁵</td><td>4.0 x 10⁵</td><td>4.2 X 10⁵</td><td>3.8 X 10⁵</td><td>3 x 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Candida Albican s</td><td>2.5 x 10⁵</td><td>2.9 x 10⁵</td><td>3.1 X 10⁵</td><td>3.2 X 10⁵</td><td>2 x 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Escheri chia coli</td><td>1.3 x 10⁵</td><td>1.4 x 10⁵</td><td>1.7 X 10⁵</td><td>2 x 10⁵</td><td>1 x 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Pseudom onas aerugin osa</td><td>1.5 x 10⁵</td><td>1.6 x 10⁵</td><td>2.0 X 10⁵</td><td>2.2 X 10⁵</td><td>1.2 X 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

176

<td>Staphy lococc US aureus</td><td>5.5 x 10⁵</td><td>5.7 x 10⁵</td><td>6.0 X 10⁵</td><td> 5.9 10⁵</td><td>2.3 X 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Salmon Ella Typhim Urium</td><td>4.6 x 10⁵</td><td>5.0 x 10⁵</td><td>5.2 X 10⁵</td><td>5.4 X 10⁵</td><td>3.2 X 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

Table 2-4. Inorganic Log Reduction of Microorganism

Initial with 1: 5 dilution of MSM.

<td>Test Body</td><td>14 days</td><td>28 days</td>

<td>Aspergillus Niger</td><td> 3.5</td><td> 4.5</td>

<td>Candida Albicans</td><td> 3.3</td><td> 4.4</td>

<td>Escherichia coli</td><td> 3.0</td><td> 4.1</td>

<td>Pseudomonas aeruginosa</td><td> 3.2</td><td> 4.2</td>

<td>Staphylococcus aureus</td><td> 3.4</td><td> 4 . 7</td>

<td>Salmonella typhimurium</td><td> 3.5</td><td> 4.7</td>

Table 2-5. AET test results for 1:10 dilution of

MSM

<td>Organism of Test</td><td>Inoculum Initial</td><td>48 hrs</td><td>3 days</td><td>5 days</td>

<td>Aspergillus Niger</td><td>3x10⁵</td><td>5x10⁵</td><td>5.2 x 10⁵</td><td>5.4 x 10⁵</td>

<td>Candida Albicans</td><td>2.5 x 10⁵</td><td>3x10⁵</td><td>3.4 x 10⁵</td><td>4x10⁵</td>

<td>Escherichia coli</td><td>1.3 x 10⁵</td><td>2x10⁵</td><td>2.3 x 10⁵</td><td>3.2 x 10⁵</td>

<td>Pseudomonas aeruginosa</td><td>1.5 x 10⁵</td><td>1.9 x 10⁵</td><td>2.2 x 10⁵</td><td>2.9 x 10⁵</td>

<td>Staphylococcus aureus</td><td>5.5 x 10⁵</td><td>5.9 x 10⁵</td><td>6.1 x 10⁵</td><td>6.3 x 10⁵</td>

<td>Salmonella typhimuri um</td><td>4.6 x 10⁵</td><td>5.0 x 10⁵</td><td>5.2 x 10⁵</td><td>5.5x10⁵</td>

Cont.

<td>Organ i smo de Test</td><td>Inoculum Initial</td><td> 48</td><td>irs</td><td colspan="2">3 days</td><td>5 days</td>

<td>Aspergillus Niger</td><td>3x10⁵</td><td>5 x</td><td> 10⁵</td><td> 5.2 10⁵</td><td>X</td><td>5.4 x 10⁵</td>

<td>Candida Albicans</td><td>2.5 x 10⁵</td><td>3 x</td><td> 10⁵</td><td> 3.4 10⁵</td><td>X</td><td>4x10⁵</td>

<td>Escherichia coli</td><td>1.3 x 10⁵</td><td>2 x</td><td> 10⁵</td><td> 2.3 10⁵</td><td>X</td><td>3.2 x 10⁵</td>

<td>Pseudomonas aeruginosa</td><td>1.5 x 10⁵</td><td> 1.9 10⁵</td><td>X</td><td>l · -¹ M OR</td><td>X</td><td>2.9 x 10⁵</td>

178

<td>Staphylococcus aureus</td><td> 5.5 10⁵</td><td>X</td><td> 5.9 10⁵</td><td>X</td><td> 6.1 10⁵</td><td>X</td><td>6.3 x 10⁵</td>

<td>Salmonella typhimurium</td><td> 4.6 10⁵</td><td>X</td><td> 5.0 10⁵</td><td>X</td><td> 5.2 10⁵</td><td>X</td><td>5.5x10⁵</td>

Table 2-6. Log Reduction of the Inorganic Microorganism

Initial with 1:10 dilution of MSM.

<td>Test Body</td><td>14 days</td><td>28 days</td>

<td>Aspergillus Niger</td><td> 4.3</td><td> 4.5</td>

<td>Candida Albicans</td><td> 4.2</td><td> 4.4</td>

<td>Escherichia coli</td><td> 3.4</td><td> 4.1</td>

<td>Pseudomonas aeruginosa</td><td> 3.6</td><td> 4.2</td>

<td>Staphylococcus aureus</td><td> 4.5</td><td> 4.7</td>

<td>Salmonella typhimurium</td><td> 3.5</td><td> 4.7</td>

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

MSM

<td>Test Body</td><td>Inoculum Initial</td><td>48 hrs</td>

<td>Aspergillus Niger</td><td>3x10⁵</td><td>TNTC *</td>

<td>Candida Albicans</td><td>2.5x10⁵</td><td>TNTC</td>

<td>Escherichia coli</td><td>1.3 x 10⁵</td><td>TNTC</td>

<td>Pseudomonas aeruginosa</td><td>1.5 x 10⁵</td><td>TNTC</td>

<td>Staphylococcns aureus</td><td>5.5x10⁵</td><td>TNTC</td>

<td>Salmonella typhimurium</td><td>4.6x10⁵</td><td>TNTC</td>

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

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

<td>Test Body</td><td>Inoculum Initial</td><td>48 hrs</td>

<td>Aspergillus Niger</td><td>3x10⁵</td><td>TNTC *</td>

<td>Candida Albicans</td><td>2.5x10⁵</td><td>TNTC</td>

<td>Escherichia coli</td><td>1.3 x 10⁵</td><td>TNTC</td>

<td>Pseudomonas aeru ginosa</td><td>1.5 x 10⁵</td><td>TNTC</td>

<td>Staphylococcus aureus</td><td>5.5x10⁵</td><td>TNTC</td>

<td>Salmonella typhimurium</td><td>4.6x10⁵</td><td>TNTC</td>

The 1: 1, 1: 5, and 1:10 dilutions of MSM (Tables 2-1 to 2-6, above), indicate that these concentrations of MSM in microorganisms killed in the medium and do 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% of MSM. These results were confirmed by adding a 90 mL blank of TSB with 10 mLs of the dilution matrix

180 tested (for example, the medium considered does not contain more live microorganisms). None of the microorganisms can be cultured and no contamination was observed. These results demonstrated that MSM, at certain concentrations, is bactericidal for these organisms.

Example 3

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

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

The USP &lt;51&gt; AET test methodology as described in Example 2, was used as the basis for assessing the lethality to Escherichia coli (ATCC strain 8739) of various concentrations of MSM in the range of 5 to 16% in TSB or saline. The USP &lt;51&gt; AET is a compendia antimicrobial effectiveness test method! (United States Pharmacopeia) to determine if a conservative 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 scratched and developed on selective MacConkey agar for qualitative analysis of the effects of the various concentrations of MSM. This study also evaluated the effect of prior sterilization of MSM (by steam autoclave to

181

121 ° C for 15 minutes) before preparing 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 encoded as set forth in Table 3-1.

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

<td>Code of Medium</td><td>Preparation</td>

<td>NS</td><td>Sterile saline added to non-sterile MSM</td>

<td>NSA</td><td>Saline + MSM, then sterilized</td>

<td>TSB</td><td>Sterile TSB added to non-sterile MSM</td>

<td>TSBA</td><td>TSB + MSM, then sterilized</td>

<td>TSBC</td><td>TSB without MSM, added with E. coli</td>

<td>(-) TSBC</td><td>Negative control (without E. coli)</td>

<td>NSC</td><td>Saline without MSM, added with E. coli</td>

<td>(-) NSC</td><td>Negative control (without E. coli)</td>

All tubes except negative controls were added with 250 μΐ of a 1.2xl0 culture⁸, which produces an initial E. coli population density of 1.2xlO⁶/ 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 signs of growth were seen in the tubes with 10-16% of MSM in TSB / TSBA medium (see Table 3-2 below). Saline tubes do not

182 They show no sign of growth. When scratched in MacConkey medium, it resulted in heavy growth in any medium containing 5-9% of MSM, while fewer colonies were detected in the striped plates of 10% of MSM in 5 TSB / TSBA medium. Little to no growth resulted from the scratching of 11-16% of MSM in TSB / TSBA medium. Growth was detected in TSB and the positive saline control (TSB or saline without MSM, added with E. coli), while no growth was detected in the scratched plates of the medium without adding.

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

<td>MSM concentration</td><td>TSB</td><td>TSBA</td><td>NS</td><td>NSA</td>

<td> 5%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 6%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 7%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 8%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 9%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 10%</td><td>Moderate</td><td>Moderate</td><td>Heavy</td><td>Heavy</td>

<td> 11%</td><td>Little bit</td><td>Little bit</td><td>Heavy</td><td>Heavy</td>

<td> 12%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

<td> 13%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

<td> 14%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

183

<td> 15%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

<td> 16%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

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 24-hour time point, 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% 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 3-3. Growth profile of E. coli in medium containing MSM after 48 hours.

<td>Concentration from MSM</td><td>TSB</td><td>TSBA</td><td>NS</td><td>NSA</td>

<td> 5%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 6%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 7%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

184

<td> 8%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 9%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 10%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 11%</td><td>Moderate</td><td>Moderate</td><td>Heavy</td><td>Heavy</td>

<td> 12%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

<td> 13%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

<td> 14%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

<td> 15%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

<td> 16%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

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 24-hour time point, few to no observable signs of growth were observed in 12-15% MSM in TSB / TSBA culture tubes. After scratching, 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% of 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.

185

Table 3-4. Growth profile of E. coli in medium containing MSM after 72 hours.

<td>Concentration MSM</td><td>TSB</td><td>TSBA</td><td>NS</td><td>NSA</td>

<td> 5%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 6%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 7%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 8%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 9%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 10%</td><td>Heavy</td><td>Heavy</td><td>Heavy</td><td>Heavy</td>

<td> 11%</td><td>Moderate</td><td>Moderate</td><td>Heavy</td><td>Heavy</td>

<td> 12%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

<td> 13%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

<td> 14%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

<td> 15%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

<td> 16%</td><td>Little bit</td><td>Little bit</td><td>Moderate</td><td>Moderate</td>

These results show that MSM concentrations of approximately 10-16% are effective in killing bacteria at certain points in time. At 24 hours, 10% MSM reduced the viable bacterial population, while 48-72 hours were more effective higher concentrations to exterminate the majority of the bacterial population. The concentrations of 10-16% of MSM in TSB / TSBA were more effective than the same concentration in a medium based on

186 saline. The data also suggests that steam sterilization does not inherently impact the effectiveness of MSM.

Example 4

Comparison of Bactericidal Effectiveness of MSM in Saline Based Medium or Trxptica 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 USP &lt;51&gt; AET test methodology was used as the basis for assessing lethality to E. coli of various concentrations of MSM (in scales or microbeads) in the range of 5 to 16% in TSB or saline. Each medium composition was inoculated with 1.25xlO⁶/ mL of E. coli and then cultivated for seven days at 35 ° C. At the end of the incubation period, cultures were evaluated visually and then developed in tripto-soy agar at serially diluted concentrations to ensure bacterial growth (if any) at a density that was able to be quantified. Coated cultures were grown for 24 hours at 35 ° C before analysis. Media compositions were encoded as shown in Table 4-1 and the results of these studies are provided in Table 4-2.

Table 4-1. Media Compositions

187

<td>Code of Medium</td><td>Preparation</td>

<td>$</td><td>MSM microbeads added to saline</td>

<td>FS</td><td>MSM in flakes added to saline</td>

<td>PTSB</td><td>MSM microbeads added to TSB</td>

<td>FTSB</td><td>MSM on scales added to TSB</td>

<td>TSBC</td><td>TSB without MSM, added with E. coli</td>

<td>(-) TSBC</td><td>Negative control (without E. coli)</td>

<td>NSC</td><td>Saline without MSM, added with E. coli</td>

<td>(-) NSC</td><td>Negative control (without E. coli)</td>

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

<td>MSM concentration</td><td>FTSB</td><td>FS</td><td>PTSB</td><td>$</td>

<td> 16%</td><td> 1</td><td> 4.5</td><td> 1</td><td> 4.8</td>

<td> 15%</td><td> 2.6</td><td> 4.8</td><td> 2.3</td><td> 5.1</td>

<td> 14%</td><td> 2.9</td><td> 4 . 9</td><td> 2.1</td><td> 5.6</td>

<td> 13%</td><td> 2.6</td><td> 5.5</td><td> 3</td><td> 5.6</td>

<td> 12%</td><td> 2.7</td><td> 5.1</td><td> 3</td><td> 5.8</td>

<td> 11%</td><td> 3.6</td><td> 5.4</td><td> 3.3</td><td> 5.9</td>

<td> 10%</td><td> 6</td><td> 5.5</td><td> 6</td><td> 5.8</td>

<td> 9%</td><td> 6</td><td> 5.6</td><td> 6</td><td> 5.9</td>

<td> 8%</td><td> 6</td><td> 5.7</td><td> 6</td><td> 6</td>

<td> 7%</td><td> 6</td><td> 5.9</td><td> 6</td><td> 6</td>

188

<td> 6%</td><td> 6</td><td> 5.9</td><td> 6</td><td> 6.2</td>

<td> 5%</td><td> 6</td><td> 5.7</td><td> 6</td><td> 6.4</td>

Concentrations in the 11-16% range MSM has a negative growth effect of E. cali in culture for 7 days. As the concentration of MSM increases over 10% in any of 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 MSM Bactericidal Effect

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

A study was conducted using Müller-Hinton broth medium, which does not contain NaCl. Standard Müller-Hinton medium was compared with Müller-Hinton medium supplemented with NaCl at the same level as the saline-based medium of Example 4. MSM was added to each medium 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 developed in tripto-soy agar at serial diluted concentrations to ensure growth

189 bacterial (if any) at a density that was able to be quantified. The coated cultures were grown for 24 hours at 35 ° C before analysis. Media compositions were encoded as set forth in Table 5-1. Results of these studies are provided in Tables 5-2 to 5-4.

Table 5-1. Media Compositions

<td>Media Code</td><td>Preparation</td>

<td>PMHS</td><td>MSM microbeads added to medium Müller-Hinton plus NaCl</td>

<td>FMHS</td><td>MSM in flakes was added to Müller- Hinton plus NaCl</td>

<td>PMH</td><td>MSM microbeads added to medium Müller-Hinton</td>

<td>FMH</td><td>MSM in flakes or flakes added to the medium Müller-Hinton</td>

<td>MHC</td><td>Müller-Hinton medium without MSM, added with E. coli</td>

<td>(-) MHC</td><td>Negative control (without E. coli)</td>

<td>MHNSC</td><td>Müller-Hinton medium plus NaCl without MSM, added with E. coli</td>

<td>(-) MHNSC</td><td>Negative control (without E. coli)</td>

190

After 24 hours in culture, an approximate 1 log reduction of the initial inoculum was detected in all media that have 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 PMHS composition. At 11% MSM, only the FMH medium reduced the growth of E. coli.

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

<td>Concentration from MSM</td><td>PMHS</td><td>FMHS</td><td>PMH</td><td>FMH</td>

<td> 16%</td><td> 6.2</td><td> 6.1</td><td> 6.1</td><td> 6.2</td>

<td> 15%</td><td> 6.0</td><td> 6.1</td><td> 6.0</td><td> 6.2</td>

<td> 14%</td><td> 6.0</td><td> 6.0</td><td> 5.9</td><td> 6.3</td>

<td> 13%</td><td> 6.3</td><td> 6.2</td><td> 5.8</td><td> 5.9</td>

<td> 12%</td><td> 7.2</td><td> 5.7</td><td> 5.8</td><td> 5.9</td>

<td> 11%</td><td> 7.9</td><td> 7.8</td><td> 7.4</td><td> 5.9</td>

<td> 10%</td><td> 8.7</td><td> 8.2</td><td> 7.9</td><td> 7.8</td>

<td> 9%</td><td> 8.3</td><td> 8.3</td><td> 8.3</td><td> 8.2</td>

<td> 8%</td><td> 8.2</td><td> 8.4</td><td> 8.4</td><td> 8.4</td>

<td> 7%</td><td> 8.3</td><td> 8.4</td><td> 8.4</td><td> 8.5</td>

<td> 6%</td><td> 8.1</td><td> 8.4</td><td> 8.5</td><td> 8.5</td>

<td> 5%</td><td> 8.2</td><td> 8.4</td><td> 8.6</td><td> 8.6</td>

191

After 48 hours in culture, medium 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. Growth of E. coli Log in supplementary MüllerHinton medium with MSM or Müller-Hinton (plus NaCl) after 48 hours

<td>Concentration from MSM</td><td>PMHS</td><td>FMHS</td><td>PMH</td><td>FMH</td>

<td> 16%</td><td> 6.0</td><td> 5.9</td><td> 6.0</td><td> 5.9</td>

<td> 15%</td><td> 6.0</td><td> 5.9</td><td> 5.8</td><td> 4.6</td>

<td> 14%</td><td> 5.7</td><td> 5.9</td><td> 5.4</td><td> 5.3</td>

<td> 13%</td><td> 5.9</td><td> 5.9</td><td> 5.4</td><td> 5.2</td>

<td> 12%</td><td> 6.7</td><td> 6.9</td><td> 5.6</td><td> 5.3</td>

<td> 11%</td><td> 7.8</td><td> 7.8</td><td> 7.2</td><td> 7 . 1</td>

<td> 10%</td><td> 7.9</td><td> 8.0</td><td> 7.9</td><td> 8.2</td>

<td> 9%</td><td> 8.2</td><td> 8.1</td><td> 8.1</td><td> 8.2</td>

<td> 8%</td><td> 8.1</td><td> 8.1</td><td> 8.2</td><td> 8.3</td>

<td> 7%</td><td> 8.2</td><td> 8.2</td><td> 8.0</td><td> 8.4</td>

<td> 6%</td><td> 8.1</td><td> 8.2</td><td> 8.4</td><td> 8.4</td>

<td> 5%</td><td> 8.4</td><td> 8.3</td><td> 8.3</td><td> 8.4</td>

192

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

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

<td>Concentration from MSM</td><td>PMHS</td><td>FMHS</td><td>PMH</td><td>FMH</td>

<td> 16%</td><td> 4 . 4</td><td> 4.7</td><td> 3.9</td><td> 7.8</td>

<td> 15%</td><td> 4 . 7</td><td> 4.7</td><td> 3.8</td><td> 7.9</td>

<td> 14%</td><td> 4.9</td><td> 4.7</td><td> 3.3</td><td> 7.6</td>

<td> 13%</td><td> 3.9</td><td> 4.4</td><td> 3.2</td><td> 7.2</td>

<td> 12%</td><td> 6.0</td><td> 4 . 1</td><td> 6.0</td><td> 6.5</td>

<td> 11%</td><td> 6.8</td><td> 6.2</td><td> 6.6</td><td> 7.0</td>

<td> 10%</td><td> 6.8</td><td> 6.9</td><td> 7.0</td><td> 7.0</td>

<td> 9%</td><td> 6.9</td><td> 7.0</td><td> 7.0</td><td> 6.5</td>

<td> 8%</td><td> 7.2</td><td> 7.0</td><td> 7.8</td><td> 7.2</td>

<td> 7%</td><td> 7.5</td><td> 6.8</td><td> 6.7</td><td> 7.6</td>

<td> 6%</td><td> 8.0</td><td> 8.0</td><td> 7.5</td><td> 7.6</td>

<td> 5%</td><td> 8.0</td><td> 8.1</td><td> 8.0</td><td> 7.8</td>

Example 6

193

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

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

Lactose broth, free of both NaCl and protein, was used as the medium in this experiment. MSM was added to lactose broth in 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 media composition was initially inoculated with 6.75x10⁶ 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 Letheen Modified diluent) and plated on tripto-soy agar plates. The coated cultures were grown for 24 hours at 35 ° C and then analyzed. Media compositions were encoded as shown in Table 6-1. Results of these studies are shown in Tables 6-2 and 6-3.

Table 6-1 Media Compositions

<td>Media Code</td><td>Preparation</td>

<td>LBM</td><td>MSM added to lactose broth</td>

<td>LBMD</td><td>MSM added to lactose broth supplemented with 1% DMSO</td>

194

<td>LB</td><td>Lactose broth without MSM, added with E. coli.</td>

<td>(-) LB</td><td>Negative control (without E. coli.)</td>

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. Bacterial growth inhibition is reduced by 1 log or more from 9-16% MSM. Table 6-2. 24-hour E. coli growth log in MSM-lactose broth with or without DMSO

<td>MSM concentration</td><td>LBM</td><td>LBMD</td>

<td> 16%</td><td> 5.8</td><td> 5.3</td>

<td> 15%</td><td> 5.6</td><td> 5.5</td>

<td> 14%</td><td> 5.5</td><td> 5.3</td>

<td> 13%</td><td> 5.1</td><td> 5</td>

<td> 12%</td><td> 5.5</td><td> 4.9</td>

<td> 11%</td><td> 4.6</td><td> 5.6</td>

<td> 10%</td><td> 7</td><td> 4.8</td>

<td> 9%</td><td> 7.1</td><td> 5.7</td>

<td> 8%</td><td> 8</td><td> 7.8</td>

<td> 7%</td><td> 8</td><td> 9.2</td>

<td> 6%</td><td> 8.1</td><td> 8.4</td>

<td> 5%</td><td> 8.4</td><td> 8.8</td>

195

After 1 day of culture, a more defined pattern of bacterial growth inhibition was evident (Table 6-3). 10% MSM in lactose broth kept the E. coli population approximately equivalent to the initial inoculum.

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

<td>MSM concentration</td><td>LBM</td><td>LBMD</td>

<td> 16%</td><td> 4</td><td> 4</td>

<td> 15%</td><td> 3.9</td><td> 3.6</td>

<td> 14%</td><td> 4</td><td> 3.6</td>

<td> 13%</td><td> 3.3</td><td> 3.2</td>

<td> 12%</td><td> 3.4</td><td> 2.9</td>

<td> 11%</td><td> 2.5</td><td> 3.1</td>

<td> 10%</td><td> 6.9</td><td> 4.2</td>

<td> 9%</td><td> 7.9</td><td> 7.9</td>

<td> 8%</td><td> 8.2</td><td> 8.4</td>

<td> 7%</td><td> 8 . 4</td><td> 8.6</td>

<td> 6%</td><td> 8.4</td><td> 8.6</td>

<td> 5%</td><td> 8.5</td><td> 8.6</td>

Example 7

Evaluation of Bactericidal Effect of MSM in Cosmetics

This example shows the bactericidal effect of MSM in cosmetics.

196

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 4.6xl0⁵ cfu / mL and incubated at 25 ° C for 48 hours. After 48 hours, aliquots of each culture were diluted and plated on tryptic soy agar, which was then incubated at 35 ° C for 24 hours before counting. The results of these studies are shown in Table 7-1.

Table 7-1 Growth log for 48 hours of E. coli in cosmetic matrix containing MSM

<td>MSM concentration</td><td>Cream</td>

<td> 16%</td><td> 1</td>

<td> 15%</td><td> 1</td>

<td> 14%</td><td> 1</td>

<td> 13%</td><td> 1</td>

<td> 12%</td><td> 1</td>

<td> 11%</td><td> 0.78</td>

<td> 10%</td><td> 1.5</td>

<td> 9%</td><td> 1.9</td>

<td> 8%</td><td> 1.8</td>

<td> 7%</td><td> 2.5</td>

197

<td> 6%</td><td> 2</td>

<td> 5%</td><td> 3.16</td>

These data indicate that bacteria that grow in a cosmetic cream are particularly sensitive to MSM. Surprisingly, lower concentrations of MSM (for example, the concentration range of 5-9%) substantially inhibit bacterial growth in this study. Thus, 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 Conservative 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 assess the ability of MSM to function as a long-term antimicrobial on a cosmetic basis, 10% MSM is incorporated into a matrix of cosmetic cream added with E. coli, which is evaluated for a period of 28 days using the protocol USP &lt;51&gt; 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.

198

Table 8-1 Effect of 10% MSM on Microbial Growth in a Cosmetic Cream Without Conservative

<td>Test Body</td><td>Inoculum Initial</td><td>48 hrs</td><td>7 days</td><td>14 days</td><td>28 days</td>

<td>Aspergillus Niger</td><td>1.1 x 10⁵</td><td>8 x 10³</td><td>8 x 10³</td><td>6 x 10³</td><td>5 x 10²</td>

<td>Candida Albicans</td><td>2.1 x 10⁵</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Escherichia coli</td><td>4.8 x 10⁶</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Pseudomonas aeruginosa</td><td>1.89 x 10⁶</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Staphylococcus aureus</td><td>4.0 x 10⁶</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

Table 8-2 Initial Inoculum Log Reduction of

Microorganism with 10% MSM in a Cream MSM Dilution

Conservative Free Jojoba Cosmetics

<td>Test Body</td><td>14 days</td><td>28 days</td>

<td>Aspergillus Niger</td><td> 1.2</td><td> 2.3</td>

<td>Candida Albicans</td><td> 4.3</td><td> 4.3</td>

<td>Escherichia coli</td><td> 5.7</td><td> 5.7</td>

<td>Pseudomonas aeruginosa</td><td> 5.3</td><td> 5.3</td>

<td>Staphylococcus aureus</td><td> 5.6</td><td> 5.6</td>

199

Table 8-3 Microbial Growth in a Cosmetic Cream that

It contains a Conservative

<td>Organism of Test</td><td>Inoculum Initial</td><td>48 hrs</td><td>7 days</td><td>14 days</td><td>28 days</td>

<td>Aspergillus Niger</td><td>1.1 x 10⁵</td><td>2 x 10⁶</td><td>1.6 x 10²</td><td>18 x 10¹</td><td>3 x 10¹</td>

<td>Candi da Albicans</td><td>2.1 x 10⁵</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Escherichia coli</td><td>4.8 x 10⁶</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Pseudomonas aeruginosa</td><td>1.89 x 10⁶</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Staphylococcus aureus</td><td>4.0 x 10⁶</td><td>3 x 10⁴</td><td>1.6 x 10⁴</td><td> &lt;10</td><td> &lt;10</td>

<td>Table 8-4</td><td>Log reduction</td><td>from</td><td>Inoculum</td><td>from</td><td>Microorganism</td>

<td>Initial in Conservative</td><td>Cosmetic Cream</td><td>from</td><td>Joj Oba</td><td>what</td><td>It contains a</td>

<td>Test Body</td><td>14 days</td><td>28 days</td>

<td>Aspergillus Niger</td><td> 2.7</td><td> 3.5</td>

<td>Candida Albicans</td><td> 4.3</td><td> 4.3</td>

<td>Escherichia coli</td><td> 5.7</td><td> 5.7</td>

<td>Pseudomonas aeruginosa</td><td> 5.3</td><td> 5.3</td>

<td>Staphylococcus aureus</td><td> 5.6</td><td> 5.6</td>

200

These studies show that a cosmetic cream base containing MSM is effective in substantially inhibiting 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. Even more, S. aureus is reduced to almost undetectable levels to 48 hours in the cream containing MSM. In contrast, the cream containing conservative showed a modest bacterial population of 3x10⁴ bacteria after 48 hours. Despite a less robust initial phase, the bacterial load of the preservative-containing cream is reduced in the same proportion at the end of the study compared to the preservative-containing cream.

Example 9 Evaluation of MSM Antimicrobial Activity in Two Conservative Free Cosmetic Compositions

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

As described in Example 8, above, 10% MSM is incorporated into cosmetic matrices, which were added with various initial inoculum of microbes. According to the USP &lt;51&gt; AET test, these cultures of aggregate microbes

201 they were incubated for 28 days, with samples removed at 48 hours, 7 days, 14 days and 28 days for plating and subsequent colony counting. The results of these studies are shown in Tables 9-1 to 9-4 below. Table 9-1 Effect of 10% MSM on Microbial Growth in Conservative Free Cosmetic Composition # 1

<td>Organism of Test</td><td>Inoculum Initial</td><td>48 hrs</td><td>7 days</td><td>14 days</td><td>28 days</td>

<td>Aspergillus Niger</td><td>8.0 x 10⁵</td><td>9.0 x 10³</td><td>4.0 x 10³</td><td>5.0 x 10¹</td><td> &lt;10</td>

<td>Candida Albicans</td><td>2.0 x 10⁶</td><td>2.1 x 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Escherichia coli</td><td>5.8 x 10⁶</td><td>5.4 x 10⁴</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Pseudomonas aeruginosa</td><td>5.7 x 10⁶</td><td>7.3 x 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Staphylococcus aureus</td><td>5.3 x 10⁶</td><td>1.9 x 10⁴</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

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

<td>Test Body</td><td>14 days</td><td>28 days</td>

<td>Aspergillus Niger</td><td> 4.2</td><td> 4.9</td>

202

<td>Candida Albicans</td><td> 5.3</td><td> 5.3</td>

<td>Escherichia coli</td><td> 5.8</td><td> 5.8</td>

<td>Pseudomonas aeruginosa</td><td> 5.8</td><td> 5.8</td>

<td>Staphylococcus aureus</td><td> 5.7</td><td> 5.7</td>

Table 9-3 Effect of 10% MSM on Microbial Growth in

Conservative Free Cosmetic Composition # 2

<td>Organism of Test</td><td>Inoculum Initial</td><td>48 hrs</td><td>7 days</td><td>14 days</td><td>28 days</td>

<td>Aspergillus Niger</td><td>8.0 x 10⁵</td><td>9.0 x 10³</td><td>3.0 x 10³</td><td>1.3 x 10³</td><td>6.0 x 10¹</td>

<td>Candida Albicans</td><td>2.0 x 10⁶</td><td>9.0 x 10²</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Escherichia coli</td><td>5.8 x 10⁶</td><td>1.7 x 10⁵</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Pseudomonas aeruginous</td><td>5.7 x 10®</td><td>2.1 x 10³</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

<td>Staphylococcus aureus</td><td>5.3 x 10⁶</td><td>1.5 x 10⁵</td><td> &lt;10</td><td> &lt;10</td><td> &lt;10</td>

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

<td>Test Body</td><td>14 days</td><td>28 days</td>

<td>Aspergillus Niger</td><td> 2.8</td><td> 5.1</td>

203

<td>Candida Albicans</td><td> 5.3</td><td> 5.3</td>

<td>Escherichia coli</td><td> 5.8</td><td> 5.8</td>

<td>Pseudomonas aeruginosa</td><td> 5.8</td><td> 5.8</td>

<td>Staphylococcus aureus</td><td> 5.7</td><td> 5.7</td>

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

Example 10

Select MSM Concentrations Support Microbial Activity

This example shows that selected 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, are

<td>compared with</td><td>the</td><td>curve of</td><td>growth of</td><td>miscellaneous</td>

<td>microorganisms</td><td>to one</td><td>concentrate</td><td>0% of MSM</td><td>in sample</td>

<td>of control.</td><td>Each</td><td>organism</td><td>(Lactobacillus</td><td>rhamnosus,</td>

Lactobacillus acidophilus, and Bifidobacterium bifidum) was grown in MRS bacterial growth medium (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, 100 mL aliquots of MRS broth were prepared with the respective concentration of MSM as established. Initially, a test solution

204 of 1%, 0.4%, and 0.2% MSM (+/- 0.01%) was prepared by adding 1 g or 0.45 g of MSM in 110 g of MRS broth and 0.20 g of MSM in 100 g, respectively. The 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 was coated by sterility, they were inoculated at a level of 100 pl of inoculum per 100 g or mL 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 plates at times 0, 12, 36, 48, 60 and 72 hours (+/- 45 minutes). All preparations and plating were performed at room temperature. All plating events were incubated at 35 ° C +/- 2 ° C for at least 2 days or 3 days for Bifidobacterium. Test samples were plated in triplicate plates on each test date and the averages are reported. The results of these studies are provided in Tables 10-1 to 10-3.

For samples of Lactobacillus rhamnosus (Table ΙΟΙ), within the first 12 hours all 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 test values ​​were online 24 hours before

205 leveling, the crops were highly murky suggesting that the organism was going to the stationary phase. However, after leveling slightly at 36 and 48 hours, the counts in the MSM samples continued to increase while the 0% control began to fall.

Table 10-1 Lactobacillus rhamnosus growth

<td>Time (hours)</td><td>0% MSM</td><td>0.04% MSM</td><td>0.1% MSM</td><td>0.2% MSM</td><td>0.4% MSM</td><td>one% MSM</td>

<td> 0</td><td>1.4 x 10⁶</td><td>1.7 x 10⁶</td><td>1.7 x 10⁶</td><td>1.9 x 10⁶</td><td>2.2 x 10⁶</td><td>1.5 x 10⁶</td>

<td> 12</td><td>1.7 x 10⁷</td><td>2.1 x 10⁷</td><td>1.9 x 10⁷</td><td>2.4 x 10⁷</td><td>Η * M OR</td><td>2.5 x 10⁷</td>

<td> 24</td><td>1.4 x 10⁹</td><td>1.5 x 10⁹</td><td>1.3 x 10⁹</td><td>1.4 x 10⁹</td><td>1.4 x 10⁹</td><td>1.4 x 10⁹</td>

<td> 36</td><td>2.0 x 10⁹</td><td>1.8 x 10⁹</td><td>1.8 x 10⁹</td><td>2.0 x 10⁹</td><td>2.1 x 10⁹</td><td>2.7 x 10⁹</td>

<td> 48</td><td>2.0 x 10⁹</td><td>2.1 x 10⁹</td><td>2.3 x 10⁹</td><td>2.3 x 10⁹</td><td>2.1 x 10⁹</td><td>2.4 x 10⁹</td>

<td> 60</td><td>3.0 x 10⁹</td><td>2.6 x 10⁹</td><td>3.0 x 10⁹</td><td>2.3 x 10⁹</td><td>2.8 x 10⁹</td><td>2.6 x 10⁹</td>

<td> 72</td><td>2.7 x 10⁹</td><td>2.6 x 10⁹</td><td>3.0 x 10⁹</td><td>3.2 x 10⁹</td><td>3.0 x 10⁹</td><td>3.8 x 10⁹</td>

These studies suggest that MSM concentrations

206 from about 0.1% to about 1% improve the growth / function of Lactobacillus rhamnosus, with microbial levels in the range of 11% to 41% higher at the end of 72 hours.

For samples of Lactobacillus acidophilus (Table 10-2), concentrations of 0.04% and 0.1% MSM were the first in production growth, followed by 0.2% and 0.4% MSM at 36 hours and the sample of 1% MSM for 48 hours . No 0% control growth was 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 Lactobacillus acidophilus growth

<td>Weather (hr)</td><td>0% MSM</td><td>0.04% MSM</td><td>0.1% MSM</td><td>0.2% MSM</td><td>0.4% MSM</td><td>one% MSM</td>

<td> 0</td><td>1.0 x 10³</td><td>1.0 x 10³</td><td>1.0 x 10³</td><td>1.0 x 10³</td><td>1.0 x 10³</td><td>1.0 x 10³</td>

<td> 12</td><td>1.0 x 10³</td><td>1.0 x 10³</td><td>1.0 x 10³</td><td>1.0 x 10³</td><td>1.0 x 10³</td><td>1.0 x 10³</td>

207

<td> 24</td><td>1.0 x 10³</td><td>1.5 x 10⁴</td><td>1.2 x 10⁴</td><td>1.0 x 10⁵</td><td>1.0 x 10⁵</td><td>1.0 x 10⁵</td>

<td> 36</td><td>1.0 x 10³</td><td>8.7 x 10⁷</td><td>3.4 x 10⁷</td><td>7.3 x 10⁵</td><td>1.6 x 10⁸</td><td>1.0 x 10³</td>

<td> 48</td><td>1.0 x 10³</td><td>2.2 x 10⁸</td><td>2.1 x 10⁸</td><td>7.4 x 10⁶</td><td>2.2 x 10⁸</td><td>4.7 x 10⁴</td>

<td> 60</td><td>1.0 x 10⁵</td><td>3.4 x 10⁸</td><td>1.7 x 10⁸</td><td>1.7 x 10⁷</td><td>3.1 x 10⁸</td><td>2.8 x 10⁷</td>

<td> 72</td><td>1.0 x 10⁵</td><td>4.7 x 10⁸</td><td>2.7 x 10⁸</td><td>8.9 x 10⁶</td><td>3.6 x 10⁸</td><td>1.6 x 10⁸</td>

Bifidobacterium bifidum, a common microbe used in probiotics, was also tested. All samples of Bifidobacterium were incubated under anaerobic conditions. Oxygen indicators were used to verify anaerobic conditions between plating intervals for Bifidobacterium test samples and plating events.

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

As observed with Lactobacillus rhamnosus (Table 10-2), samples from 0.1% to 1% MSM continued to grow while control was directed to a growth phase

208 stationary descending (Table 10-3). Increase of 1 and 2 log of Bifidobacterium were observed with concentrations of 0.04% and 0.2% MSM, respectively (Table 10-3).

Table 10-3 Bifidobacterium bifidum growth

<td>Time (hours)</td><td>0% MSM</td><td>0.04% MSM</td><td>0.1% MSM</td><td>0.2% MSM</td><td>0.4% MSM</td><td>one% MSM</td>

<td> 0</td><td>8.7 x 10⁴</td><td>6.6 x 10⁴</td><td>7.0 x 10⁴</td><td>7.0 x 10⁴</td><td>1.1 x 10⁵</td><td>6.8 x 10⁴</td>

<td> 12</td><td>2.0 x 10³</td><td>3.5 x 10³</td><td>4.4 x 10⁴</td><td>3.1 x 10⁴</td><td>2.0 x 10³</td><td>2.3 x 10⁴</td>

<td> 24</td><td>1.4 x 10⁵</td><td>1.2 x 10⁵</td><td>1.3 x 10⁵</td><td>1.3 x 10⁵</td><td>1.0 x 10⁵</td><td>2.0 x 10⁵</td>

<td> 36</td><td>2.4 x 10⁵</td><td>2.3 x 10⁵</td><td>2.0 x 10⁵</td><td>2.3 x 10⁵</td><td>2.3 x 10⁵</td><td>4.0 x 10⁵</td>

<td> 48</td><td>2.6 x 10⁵</td><td>5.2 x 10⁶</td><td>2.3 x 10⁵</td><td>5.8 x 10⁶</td><td>4.0 x 10⁵</td><td>3.1 x 10⁵</td>

<td> 60</td><td>3.3 x 10⁵</td><td>3.4 x 10⁷</td><td>4.7 x 10⁵</td><td>4.8 x 10⁷</td><td>8.7 x 10⁵</td><td>3.7 x 10⁵</td>

<td> 72</td><td>1.6 x 10⁶</td><td>8.0 x 10⁷</td><td>5.3 x 10⁵</td><td>1.2 x 10⁸</td><td>1.2 x 10⁶</td><td>5.7 x 10⁵</td>

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 medium

209

0% and a half containing 5% MSM are compared (see Example 13 for detailed description).

Example 11

MSM Influence Evaluation in 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 stability of storage life of a product based on microbial count. Milk that has a relatively short shelf 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 shelf life for milk is 18 to 21 days, the study is carried out at 28 days. Storage life study was conducted on milk products reinforced with MSM at 0.0%, 0.5%, 1.0%, 2.5%, 5.0% and 10%. The time intervals for planting on solution plates in days were on days 0, 7, 14, 21, 24 and 28. The influences of percent solids in concentrations of MSM to the following percent were evaluated: 0.0%, 1.0%, 2%, 10.5% and 40%. The growth curves of colony forming units recovered

210 per milliliter (cfu / mL) of microorganisms were compared between concentrations of 0% MSM as a control sample. The MSM raw material powder was supplied by Bergstrom Nutrition with certificate of analysis. The powder was the formula of microgranules, lot # 0806809, expiration date 10/31/13. All media, water and MSM raw material powder were verified by sterility before the study. The MSM concentrations of the work were prepared from a single 10.0% MSM solution and diluted in accordance with bottled milk to obtain the desired final concentration of MSM. The product was supplied by a local milk processing plant. Samples were collected and the study began on processing day. The product included two bottles of each type of product for each concentration of MSM for each day of analysis. The total 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. Product samples were kept at 4 ° C for the duration of the study. All preparation and sowing on plates was done at room temperature. Each concentration of MSM was performed in duplicate. Each dilution was plated in duplicate for each sampled time interval.

211

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 exam. 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 MSM prepared media were tested for background levels of microorganisms in 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 emptying for quality control purposes. All negative control plates were absent for microorganism growth. The results of these studies are shown in Tables 11.1 15.1 below.

Table 11.1. Log Growth of Fat Free Milk

Conc. Time (days)

<td>from MSM</td><td> 0</td><td> 7</td><td> 14</td><td> 21</td><td> 24</td><td> 28</td>

<td> 0%</td><td> 1.50</td><td> 2.05</td><td> 0.94</td><td> 4.40</td><td> 1.44</td><td> 2.20</td>

<td> 0.5%</td><td> 0.00</td><td> 1.00</td><td> 0.70</td><td> 4.41</td><td> 4.68</td><td> 3.79</td>

<td> 1%</td><td> 0.74</td><td> 0.70</td><td> 0.00</td><td> 4.56</td><td> 1.28</td><td> 2.02</td>

212

<td> 2.5%</td><td> 0.74</td><td> 0.70</td><td> 1.65</td><td> 4.37</td><td> 3.69</td><td> 0.00</td>

<td> 5%</td><td> 0.00</td><td> 0.70</td><td> 0.00</td><td> 1.15</td><td> 3.20</td><td> 0.00</td>

<td> 10%</td><td> 0.92</td><td> 0.70</td><td> 0.59</td><td> 0.00</td><td> 0.00</td><td> 0.00</td>

Table 12.1. Log Growth of 1% Milk Fat

Conc. Time (days)

<td>from MSM</td><td> 0</td><td> 7</td><td> 14</td><td> 21</td><td> 24</td><td> 28</td>

<td> 0.0%</td><td> 0.35</td><td> 1.36</td><td> 0.00</td><td> 4.44</td><td> 4.44</td><td> 5.74</td>

<td> 0.5%</td><td> 1.05</td><td> 0.50</td><td> 0.70</td><td> 4.37</td><td> 3.27</td><td> 6.09</td>

<td> 1.0%</td><td> 0.35</td><td> 1.23</td><td> 0.70</td><td> 4.37</td><td> 5.27</td><td> 5.36</td>

<td> 2.5%</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 4.28</td><td> 4.28</td><td> 2.53</td>

<td> 5.0%</td><td> 0.00</td><td> 2.55</td><td> 0.59</td><td> 0.59</td><td> 1.28</td><td> 0.00</td>

<td> 10.0%</td><td> 0.70</td><td> 0.00</td><td> 0.50</td><td> 0.85</td><td> 0.50</td><td> 0.00</td>

Table 13.1. Log Growth of 2% Milk Fat

Conc. Time (days)

<td>from MSM</td><td> 0</td><td> 7</td><td> 14</td><td> 21</td><td> 24</td><td> 28</td>

<td> 0.0%</td><td> 1.90</td><td> 1.60</td><td> 2.44</td><td> 4.45</td><td> 4.44</td><td> 4.93</td>

<td> 0.5%</td><td> 1.26</td><td> 1.59</td><td> 2.09</td><td> 4.43</td><td> 3.27</td><td> 5.48</td>

<td> 1.0%</td><td> 1.04</td><td> 3.21</td><td> 0.00</td><td> 4.07</td><td> 5.27</td><td> 6.56</td>

<td> 2.5%</td><td> 1.06</td><td> 0.00</td><td> 0.70</td><td> 2.96</td><td> 4.28</td><td> 5.81</td>

<td> 5.0%</td><td> 1.39</td><td> 0.35</td><td> 0.00</td><td> 3.97</td><td> 1.28</td><td> 0.59</td>

<td> 10.0%</td><td> 0.35</td><td> 0.50</td><td> 0.70</td><td> 1.97</td><td> 0.50</td><td> 1.66</td>

213

Table 14.1. 10.5% Milk Fat Log Growth

Conc. Time (days)

<td>from MSM</td><td> 0</td><td> 7</td><td> 14</td><td> 21</td><td> 24</td><td> 28</td>

<td> 0.0%</td><td> 0.35</td><td> 0.42</td><td> 1.98</td><td> 4.43</td><td> 2.94</td><td> 4.7</td>

<td> 0.5%</td><td> 0.00</td><td> 1.09</td><td> 0.00</td><td> 4.36</td><td> 2.73</td><td> 3.84</td>

<td> 1.0%</td><td> 0.50</td><td> 0.00</td><td> 0.94</td><td> 3.30</td><td> 2.24</td><td> 3.91</td>

<td> 2.5%</td><td> 0.35</td><td> 0.35</td><td> 0.00</td><td> 2.34</td><td> 3.14</td><td> 1.84</td>

<td> 5.0%</td><td> 0.00</td><td> 0.85</td><td> 0.00</td><td> 0.35</td><td> 1.99</td><td> 0.00</td>

<td> 10.0%</td><td> 1.00</td><td> 0.85</td><td> 0.35</td><td> 2.14</td><td> 0.35</td><td> 0.00</td>

Table 15.1. Log Growth of 40% Milk Fat

Conc. Time (days)

<td>from MSM</td><td> 0</td><td> 7</td><td> 14</td><td> 21</td><td> 24</td><td> 28</td>

<td> 0.0%</td><td> 0.00</td><td> 1.89</td><td> 2.08</td><td> 4.47</td><td> 4.23</td><td> 4.75</td>

<td> 0.5%</td><td> 0.00</td><td> 0.35</td><td> 2.32</td><td> 4.46</td><td> 4.02</td><td> 1.95</td>

<td> 1.0%</td><td> 0.74</td><td> 0.00</td><td> 2.37</td><td> 4.35</td><td> 3.33</td><td> 3.86</td>

<td> 2.5%</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 2.16</td><td> 3.79</td><td> 0.95</td>

<td> 5.0%</td><td> 0.00</td><td> 0.80</td><td> 0.50</td><td> 1.25</td><td> 1.03</td><td> 0.35</td>

<td> 10.0%</td><td> 0.00</td><td> 0.00</td><td> 1.95</td><td> 4.45</td><td> 0.85</td><td> 0.35</td>

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

214 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 batch production day. Microbial counts for a single batch of milk products may vary by 0.5 to 1.5 logs. Seeing the 0 day of growth, the intervals for each product are within 1.5 logs of each other.

<td></td><td>Day 7 shows</td><td>what</td><td>for him</td><td>control</td><td>Y</td><td>the</td>

<td colspan="3">MSM concentrations there was a</td><td>light</td><td>increase</td><td>in</td><td>the</td>

<td>counts</td><td>microbial There was</td><td colspan="3">a microbial count</td><td>what</td><td>It was</td>

<td>higher</td><td>than the others for</td><td>each</td><td>shows,</td><td>except</td><td>for</td><td>the</td>

10.5% milk fat product. The microbial counts of the product at 10.5% were all within 0.75 logs of each other, (control and concentrations of MSM). The fat-free and 40% milk products had an increase in the control sample. While 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. No abnormal growth is seen in the products. Products with lower milk fat are within the expected microbial load variability, when white is compared to MSM concentrations. 40% milk product demonstrates

215 a lower microbial count for the concentrations of MSM at 5.00% and 2.50% while the blank and the other concentrations of MSM are all within 0.40 logs in microbial counts. Milk product at 10.5% shows that the target is 1 log higher than the concentrations of MSM with the MSM at 1.00% and 10.0% that are the only two with a microbial count.

On Day 21, the microbial counts of products are separated from the target and the MSM concentrations. 1% and fat-free milk products indicated that MSM in higher concentrations (5.0% and 10.0%) slowed the growth of normal flora. While microbial control counts and lower MSM concentrations increased to 4 logs. In the 2% milk product, the concentration of 10.0% MSM 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 at 4 logs. The 10.5% milk product showed 5.00% MSM at 0.35 logs, slowing the growth rate compared to the control, which went to 4.43 logs. The concentration of MSM at 0.50% was 4.36 logs. 1.00% MSM went to 3.30 logs, 2.50% MSM went to 2.34 logs and 10.0% MSM went to 2.14 logs. With the increase in concentrations of MSM, microbial counts decreased, except for MSM to 5.00%. The microbial load of

216 Milk product at 40% was relatively equal in count for control, MSM at 0.5%, 1.0% and 10%. The concentration of 2.5% MSM was two logs lower than the control at 2.16 logs, while 5.0% MSM was lower by 3.22 logs.

On Day 24, the control and the 1.0% MSM for the fat-free milk product dropped around 1.3 logs, while the 0.50% MSM kept microbial counts. Microbial counts of MSM at 2.50% fell, while MSM increased to 5.0%. There was no growth observed in the MSM at 10.0%. The 1% milk product had a decreased microbial count in the MSM at 0.50%, increased in the MSM at 1.00%, and without control alteration and MSM at 2.5%. The 5.0% MSM increased and in 10.0% MSM it decreased. These two concentrations of higher 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 the microbial count. The milk product at 10.5% showed a decrease in the control and concentrations of MSM: 0.5%, 1.0%, and 10.0%. The 2.5% MSM and the 5.0% MSM continued to grow. The 40% milk product showed a slight decrease in growth for the control, 5.0% MSM and the two lowest concentrations of MSM. Microbial counts of 2.5% MSM increased to 24 hours, while 10.0% MSM had a decrease

217 significant in microbial growth. 5.0% MSM and 10.0% MSM were counts close to the microbial loads of the initial day 0.

On Day 28, fat-free milk products were greater than the 2-log microbial load. The concentration of 0.5% MSM was 3.79 logs. The higher concentrations of MSM, 2.50%, 5.0% and 10.0% had no growth by day 28. The 1% milk product showed an increase for the control, 0.5% and 1.0% MSM. The 2.5% MSM sample had a decrease in microbial load, while the 5.0% and 10.0% MSM had no growth. The 2% milk product had an increase for the MSM to 1.0%, a slight decrease for the control, MSM to 0.50% and 2.5%. The MSM at 5.0% and 10.0% decreased to 0.59 logs and 1.66 logs respectively. Products of higher fat in milk, 10.5% and 40% show that control continues to increase in microbial counts. Timbos products had the MSM at 1.0% increasing, while the 10.5% product also had an increase of MSM to 0.5% in microbial counts, the product of 40% had a decrease in MSM to 0.5%. The MSM at 2.5% decreased in microbial load for both products. 5.0% and 10% for the 10.5% milk product 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 a

218 Milk additive does not adversely affect the storage life of milk. In particular, on day 21 there was no concentration of MSM that had a microbial count higher than the control. Moreover, these studies indicate that in certain milk products, a concentration of MSM at 5.0% or MSM at 10.0% actually maintained 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 Bacillus coagulans Growth in Simulated Gastric Acid supplemented with MSM

This example describes acidophilus milk and Bacillus coagulans growth in simulated gastric acid supplemented with MSM. To analyze the effects of methylsulfonylmethane (MSM) on the growth of MSM-reinforced probiotic microorganisms 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 MSM-reinforced probiotic growth on a simulated gastric acid fluid.

Microbial growth studies were performed in

219 the presence of 0%, 0.25%, 2.0% and 5%. Time intervals for plating 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 sample control for each microorganism. The MSM raw material powder was supplied by Bergstrom Nutrition with certificate of analysis. The powder was the formula of microgranules, lot # 0806809, expiration date 10/31/13. All MSM media and powder were checked for sterility before the study. The study was carried out in two organizations over a period of two weeks. The microorganisms analyzed include Lactobacillus acidophilus and Badilas coagulaos milk (15BB Lot # 9OBCOO4A1MZ supplied by Ganeden).

For lactobacillus acidophilus milk, 11 mLs of milk with a count of 81,000 cfu / mL are added to 99 mLs of simulated gastric acid. For Bacillus coagulaos 1 gram of powder is added to 99mL of Tryptic Soy Broth (TSB = Tryptic Soy Broth) to obtain the count of 108,000 cfu per mL of Bacillus coagulans. Eleven milliliters of Bacillus coagulaos TSB are added to 99 mLs of simulated gastric acid. Working MSM concentrations were prepared from a single 5.0% MSM solution and diluted from

220 agreement with milk or TSB to obtain the desired final concentration of MSM. All solutions were verified by sterility before proceeding with the study. Simulated working gastric acid was incubated at 35.0 + 0.2 ° C during the study. The pH of the simulated gastric acid was 1.2.

Milk Lactobacillus acidophilus was inoculated in MRS agar at the times previously mentioned. Bacillus coagulans was inoculated in Tryptic Soy Agar (TSA) in the times previously mentioned. All preparation and coatings were performed at room temperature. Each concentration of MSM in the simulated gastric acid was performed in duplicate. Each dilution for each organism was plated in triplicate for each time interval mastered. 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 appropriate plate for enumeration contains between 25 and 250 cfu / mL.

The MSM raw material sample and all MSM prepared media were tested for background levels of microorganisms in 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

221 next). All time intervals for plating include negative control plates during emptying for quality control purposes. All negative control plates were clean for microorganism growth. The results of these studies are provided in Tables 16.1 to 18.2 below.

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

<td></td><td>Milk Lactobaci- llus acidophilus</td><td>Bacillus coagulans</td>

<td>Inoculum cfu / mL</td><td>8.1 x 10⁴</td><td>1.08x10⁵</td>

<td>Cf u added to 99 mL</td><td>8.1 x 10⁵</td><td>1.08x10⁶</td>

<td>Cfu / mL in half to time 0</td><td>8.1 x 10³</td><td>1.08x10⁴</td>

The control of numbers derives from growth of specific organism in appropriate medium. The inoculation liquids were plated for enumeration in the appropriate media. To capture the appropriate colonies per millimeter, each liquid was coated in triplicate to four

222 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 duplicate milk

MSM concentration in percent

<td>Time in (Hours)</td><td> 0</td><td> 0</td><td> 0.25</td><td> 0.25</td><td> 2.5</td><td> 2.5</td><td> 5</td><td> 5</td>

<td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0.82</td>

<td> 3</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 1.3</td><td> 1</td><td> 0</td><td> 0.82</td>

<td> 6</td><td> 0</td><td> 0</td><td> 0.52</td><td> 0.52</td><td> 0</td><td> 0</td><td> 0.82</td><td> 1</td>

<td> 9</td><td> 0</td><td> 0</td><td> 0.82</td><td> 0</td><td> 0.82</td><td> 0</td><td> 0.52</td><td> 0.52</td>

<td> 12</td><td> 0</td><td> 1.00</td><td> 1.00</td><td> 0</td><td> 1.22</td><td> 0.82</td><td> 0.82</td><td> 1.37</td>

<td> 15</td><td> 0.52</td><td> 1.43</td><td> 1.56</td><td> 1.3</td><td> 1.3</td><td> 1.37</td><td> 1.43</td><td> 1.43</td>

<td> 24</td><td> 0.52</td><td> 0.82</td><td> 1.37</td><td> 1.27</td><td> 1.67</td><td> 1.6</td><td> 1.67</td><td> 1.64</td>

<td> 48</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 1.3</td><td> 1.22</td><td> 1.22</td><td> 1.43</td>

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

223

<td></td><td></td><td colspan="4">MSM concentration in percent</td>

<td></td><td>Time in (Hrs)</td><td> 0</td><td> 0.25</td><td> 2.5</td><td> 5</td>

<td rowspan="6">Log Growth</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0.41</td>

<td> 3</td><td> 0</td><td> 0</td><td> 1.15</td><td> 0.41</td>

<td> 6</td><td> 0</td><td> 0.52</td><td> 0</td><td> 0.91</td>

<td> 9</td><td> 0</td><td> 0.41</td><td> 0.41</td><td> 0.52</td>

<td> 12</td><td> 0.5</td><td> 0.5</td><td> 1.02</td><td> 1. 10</td>

<td> 15</td><td> 0.98</td><td> 1.43</td><td> 1.34</td><td> 1.43</td>

<td></td><td> 24</td><td> 0.67</td><td> 1.32</td><td> 1.64</td><td> 1.66</td>

<td></td><td> 48</td><td> 0</td><td> 0</td><td> 1.26</td><td> 1.33</td>

Acidophilus milk placed in simulated gastric acid was 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 peak in recovery for MSM at 2.5%, while maintaining the growth log of MSM at 5.0%. At hour 6 of MSM at 0.25% 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 not detect it. At hour 9, there was a significant detection for all concentrations of MSM. The 0% control remains below the detection limit. At hour 12, the control grew to 0.5 logs

224 matching MSM at 0.25%. The 2.5% and 5.0% MSM samples had growth rates at 1.02 and 1.10 logs, 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 0.48 logs to 0.98 logs. The

0% MSM and 0.25% MSM at 24 hours decreased in growth by 0.31 logs and 0.11 logs, respectively. The 0.25% MSM increased by 0.30 logs and the 5.0% MSM increased by 0.23 logs. At now 48, the 0.25% MSM and the 0% 0% MSM control 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. Log growth of Bacillus coagulans in duplicate

<td></td><td></td><td colspan="8">MSM concentration in percent</td>

<td></td><td>Time (Hrs)</td><td> 0</td><td> 0</td><td> 0.25</td><td> 0.25</td><td> 2.5</td><td> 2.5</td><td> 5</td><td> 5</td>

<td rowspan="8">Log Growth</td><td> 0</td><td> 0</td><td> 0.52</td><td> 0.52</td><td> 0</td><td> 0</td><td> 0</td><td> 0.52</td><td> 0</td>

<td> 3</td><td> 0</td><td> 0</td><td> 0.52</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 6</td><td> 0</td><td> 0</td><td> 0.52</td><td> 0.52</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 9</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 12</td><td> 0</td><td> 0</td><td> 1.12</td><td> 0.52</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 15</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0.52</td><td> 0</td>

<td> 24</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 48</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

225

Table 18.2. Log growth of average Bacillus coagulans

<td></td><td></td><td colspan="4">MSM concentration in percent</td>

<td></td><td>Time (Hours)</td><td> 0</td><td> 0.25</td><td> 2.5</td><td> 5</td>

<td rowspan="8">Log Growth</td><td> 0</td><td> 0</td><td> 0.26</td><td> 0</td><td> 0.26</td>

<td> 3</td><td> 0</td><td> 0.26</td><td> 0</td><td> 0</td>

<td> 6</td><td> 0</td><td> 0.52</td><td> 0</td><td> 0</td>

<td> 9</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 12</td><td> 0</td><td> 0.82</td><td> 0</td><td> 0</td>

<td> 15</td><td> 0</td><td> 0</td><td> 0</td><td> 0.26</td>

<td> 24</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td> 48</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

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

These studies reveal that with respect to Acidophilus milk 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

226

L. acidophilus at the concentrations of MSM at 0.25%, 2.5% and 5.0%. At hour 9, there is a significant recovery of L. acidophilus from milk at the MSM concentrations against the control. At hour 12 is when there was the first indication of recovery of L. acidophilus in the control milk at 0.5 logs, matching MSM at 0.25%. The MSM concentrations of 2.5% and 5.0% are at a 1 log recovery growth rate. At hour 15 the control reaches its maximum growth at 0.98 logs. The MSM concentration at 0.25% peaks at 1.43 logs. The MSM of 2.5% and 5.0% peak at 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% go below the detectable limit for the method and MSM of 2.5% and 5.0% is still over 1 log of the organism. MSM seems to help this process by accelerating adaptation and allows microorganisms to adapt more quickly to environmental stressors.

The samples treated with MSM showed an increase in the log growth phase. This increase in the log phase is easier in the MSM concentration at 5.0%. 5.0% MSM is 0.45 logs higher than the control at hour 15, which is the maximum growth recovery for the control. The 5.0% MSM reached a maximum of 1.66 logs or 0.68 logs higher than the control. This indicates a survival rate of

227 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 control phase was shorter than the stationary phase of MSM at 2.5% and 5.0%. From 15 to 24 hours, the samples not only maintain the growth rate, but continue to increase in logs by a minimum of 0.24 logs. These results indicate that MSM as an additive allows L. acidophilus to flourish for longer, allowing the body to establish itself for a better health benefit. The control was not detectable at 48 hours. The MSM growth of 2.5% and 5.0% was still over 1 log. This indicates that the survival of L. acidophilus in 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

Viability Measurement of Bacillus coagulans

228

Supplemental with MSM

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

Robust microbial colony formation studies were performed in the presence of MSM of 0%, 1.0%, 2.0% and 5%. The microorganisms developed 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 in tryptic soy agar. The percent transmittance was also measured in a 25 mm x 25 mm area of ​​the tryptic soy agar placed between two microscope slides on a spectrophotometer.

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

Win.

229

For Bacillus coagulans, the organism was isolated and developed for 24 hours before collecting. The collected microorganism 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 previously established TSB concentrations. The concentrations of working MSM were prepared from a single solution of the MSM at 5.0% and diluted according to TSB to obtain the desired final concentration of MSM. All solutions were verified by 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 colony population verification and density. All preparation and sowing on plates are done at room temperature. Each concentration of MSM in the study was performed in duplicate. Each dilution for the microorganism was plated in triplicate 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

230 It contains between 25 and 250 cfu / mL.

The MSM raw material sample and all MSM prepared media were tested for background levels of MRS on 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 emptying 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 Test Sample Inoculation

<td></td><td>Bacillus coagulaos</td>

<td>Inoculum cfu / mL</td><td>2.1 x 10⁴</td>

<td>Cfu added to 99 mL</td><td>2.1 x 10⁴</td>

<td>Cfu / mL in medium at time 0</td><td>2.1 x 10²</td>

Control numbers are derived from growth of the specific organism in appropriate medium. The inoculation liquids were seeded on plates for enumeration in the appropriate medium. To capture the appropriate colonies per milliliter, each liquid was plated in triplicate plates at four different dilutions. The appropriate dilution plate was used for enumeration and averaged for reporting. The plate

231 Appropriate for enumeration contains between 25 and 250 cfu / mL.

Table 20.1. Population table of Bacillus coagulans

<td>MSM concentration in percent</td><td>Population Count Average</td>

<td> 0</td><td>4.3 x 10¹⁰</td>

<td> 1.0</td><td>1.01x10¹¹</td>

<td> 2.5</td><td>3.3 x 10ⁿ</td>

<td> 5.0</td><td>2.8x10¹¹</td>

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

Table 21.1. Bacillus coagulans weight

<td>MSM concentration in percent</td><td>Population Count Average</td>

<td> 0</td><td> 0.0586</td>

<td> 1.0</td><td> 0.1363</td>

<td> 2.5</td><td> 0.9828</td>

<td> 5.0</td><td> 0.1260</td>

232

The tryptic soy broth after 72 hours was centrifuged in a conical vial. The supernatant detached and the precipitate was washed. The spin and wash are repeated three times. At the end of the third wash, the 5 vials with precipitate were weighed. Each vial was weighed, emptied and registered. The corresponding ampule weight was then subtracted from the final weight of the precipitate and ampule, to obtain the population weight of Bacillus coagulans. A 25 x 25 mm section of agar 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 blank. When the plates were examined, a colony of Bacillus 15 coagulans is observed to be larger and more robust in appearance with 5.0% MSM compared to the 0% MSM control. The weights indicate a larger growth or a colony that was larger in size contributing to the weight of the biomass. Table 22.1. Transmittance percent of Bacillus coagulans

233

<td>Concentration</td><td>from</td><td>MSM</td><td>in Transmittance</td><td>in</td>

<td>percent</td><td></td><td></td><td>percent</td><td></td>

<td>Agar</td><td></td><td></td><td> 61.1%</td><td></td>

<td> 0.0</td><td></td><td></td><td> 52.3%</td><td></td>

<td> 1.0</td><td></td><td></td><td> 51.5%</td><td></td>

<td> 2.5</td><td></td><td></td><td> 49.8%</td><td></td>

<td> 5.0</td><td></td><td></td><td> 45.6%</td><td></td>

The percent transmittance is used to indicate colony size of Bacillus coagulans where a decrease in transmittance indicates an increase in colony size since the colony inhibits light passing 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 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 white control. Colonies also resulted in a higher total weight when measuring biomass in samples treated with MSM compared to the blank control. These two results combined with the percent transmittance measured indicate that MSM as a

2. 3. 4 Additive (at certain concentrations) positively influences the viability, health and size of Bacillus coagulans.

Example 14

Effect of MSM on Growth of Recovery for 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 microorganisms they were compared between the concentrations of MSM with the concentration of MSM at 0% 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 formula of microgranules, lot # 0806809, expiration date 10/31/13. All media, water and MSM raw material powder were verified by sterility before the study. The pH of the simulated gastric acid was 1.2. The pH of the simulated intestinal fluid was 6.8. The microorganisms analyzed include Lactobacillus acidophilus

235

ATCC # 4356:

For Lactobacillus acidophilus, bottled milk was used as the product. One milliliter of a 9 log organism solution is placed in 99 mL of bottled milk and mixed with hand shaking. This was repeated for each concentration of MSM. 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 maintained at 35 ° C for the duration of 20 minutes. At the end of the 20 minute period, 10 mL of the simulated gastric acid, the mixture of Lactobacillus acidophilus and milk was placed in 90 mL of simulated intestinal fluid. The simulated intestinal fluid was previously heated to 35 ° C and maintained 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 in accordance with bottled milk to obtain the desired final concentration of MSM. All solutions were verified by sterility before proceeding with the study.

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

236

Each dilution for each organism is in triplicate for each time interval sampled. To capture the appropriate colonies per milliliter, each organism in each time interval is coated in four dilutions. All plates were incubated at 37 ° C ± 0.5 ° C for 72 hours in a CO environment₂, before exam. 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 MSM prepared media were tested for background levels of microorganisms in 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 emptying 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

237

MSM concentration

<td>Weather</td><td>0% MSM</td><td>MSM 0.25%</td><td>MSM at 2.5%</td><td>MSM at 5.0%</td>

<td>Initial inoculum</td><td> 6.83</td><td> 6.86</td><td> 6.86</td><td> 7.02</td>

<td>20 min Gastric</td><td> &lt;1.00</td><td> &lt;1.00</td><td> &lt;1.00</td><td> &lt;1.00</td>

<td>Hour 3</td><td> 1.33</td><td> 1.30</td><td> 1.46</td><td> 1.62</td>

<td>Hour 8</td><td> 2.22</td><td> 2.18</td><td> 2.30</td><td> 2.33</td>

<td>24 hour</td><td> 4.47</td><td> 4.40</td><td> 5.94</td><td> 6.39</td>

<td>30 hour</td><td> 4.10</td><td> 4.66</td><td> 5.39</td><td> 6.63</td>

<td>Hour 36</td><td> 4.00</td><td> 4.38</td><td> 5.32</td><td> 8.65</td>

<td>48 hour</td><td> 4.59</td><td> 8.03</td><td> 11.17</td><td> 10.17</td>

<td>54 hour</td><td> 8.97</td><td> 8.02</td><td> 13.23</td><td> 13.33</td>

<td>60 hour</td><td> 9.23</td><td> 9.14</td><td> 11.27</td><td> 12.76</td>

<td>72 hour</td><td> 9.21</td><td> 9.34</td><td> 12.87</td><td> 12.78</td>

Reviewing the data there is a benefit with the addition of MSM to the product in the growth and recovery of Lactobacillus acidophilus. Comparing the control of 0% MSM against the 5.0% MSM there is a significant increase in the log growth phase with Lactobacillus acidophilus. Within the first 24 hours the 5.0% MSM was 1.81 logs higher than the control. Over the next 12 hours the control, 0.25% and 2.5% decreased. The 5.0% MSM continues to increase over the same period of time. At the end of the time window MSM 5.0% was at 8.65 logs, 4.65 logs higher

238 That control. From hour 36 to hour 48, MSM concentrations grew at a faster rate than the target control. 0.25% MSM increased by 3.65 logs and 2.5% MSM increased by 5.85 logs compared to 0.59 logs for 0.0% MSM. That trend changed the next 8 hours with control increasing by 4.38 logs. The 0.25% MSM decreased, while the 2.5% and 5.0% MSM did not increase so significantly in the control, there was an increase of 2.07 and 3.16 logs. From hour 60 to hour 72, the target control dropped while MSM concentrations increased.

Analyzing the data, another 24-hour trial period 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 population greater than the control. What we see is an increase in the speed of growth, with a higher population achieved with MSM at 2.5% and 5.0%. MSM as an additive to Lactobacillus acidophilus products will increase the likelihood of the organism establishing itself in the intestinal tract. More organisms faster will increase the benefit of taking a probiotic.

With the addition of MSM there is a recovery benefit of Lactobacillus acidophilus after a decrease in

239 population growth At hour 24 until hour 36 there is a decrease in growth for MSM 0.25%, MSM at 2.5% and MSM 0.0%. While 0.25% MSM and 2.5% MSM recover within twelve hours with a significant increase in growth rate, it takes 0.0% MSM another twelve hours to show a significant growth rate. For 5.0% MSM there was no decrease in recovery for this window in time, just a slight decrease in growth speed. 5.0% MSM took only 6 hours to produce an increase in substantial growth rate after the slight decrease in growth rate at 24 hours. 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 earlier, 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 organism to settle faster, grow faster and reach a higher rate

240 population. These attributes will benefit people who take Lactobacillus acidophilus as a probiotic.

Example 15

Effect of MSM on Pasture Growth and Nutritional Value

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

The effect of MSM on grass growth and nutritional value was evaluated by monitoring grass growth under the following conditions: (1) fertilizer alone; (2) MSM only (OptiMSM® GNC - Lot # 0922904, speed of 1: 500 or 0.91 kg per 92.9 m² (2 Ibs 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 Ibs per 1,000 sq. Ft.) In the presence of fertilizers) applied to the same field but through a separate application. The fertilizer tested was Urea (45-00) and the type of grass includes the following mixture of grasses (stucco of the meadows, ballico, clew grass, Timotea, red clover, medium and intermediate fescue; seeds for this formulation are commercially available in the Global Network 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 propagation controlled diffuser, MSM and / or fertilizer were applied. The field was watered as usual (irrigated by

241 sprinkler every four days). The grass was allowed to grow 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 shipment. The results of these tests in nutrient value are shown in Table 23.2 below.

<td></td><td></td><td> 1:500</td><td></td>

<td></td><td colspan="3">Results reported in a dry base</td>

Table 23.2

<td colspan="2"></td><td>MSM / Fert percent</td><td>MSM percent</td><td>Fertilizer percent</td><td>Method AOAC</td>

<td>Subject</td><td>Dry</td><td> 43.26</td><td> 39.57</td><td> 44.62</td><td> 934.02</td>

<td>Humidity</td><td></td><td> 56.74</td><td> 60.43</td><td> 55.33</td><td> 934.02</td>

<td>Protein</td><td>Raw</td><td> 20.03</td><td> 24.36</td><td> 22.26</td><td> 2001.11</td>

<td>Fiber Acid</td><td>Detergent</td><td> 30.95</td><td> 25.77</td><td> 29.44</td><td> 973.18</td>

<td>Fiber Neutral</td><td>Detergent</td><td> 52.84</td><td> 35.23</td><td> 48.44</td><td> 2002.04</td>

<td>Soluble</td><td>of cells</td><td> 47.16</td><td> 64.71</td><td> 51.56</td><td>Cale</td>

242

<td>Lignin</td><td> 4.21</td><td> 4 . 62</td><td> 3.98</td><td> 973.18</td>

<td>Ashes</td><td> 10.89</td><td> 11.25</td><td> 11.26</td><td> 942.05</td>

<td>Dear TDN / DDM</td><td> 64.47</td><td> 68.39</td><td> 65.61</td><td>Calculation</td>

<td>Energy Lact</td><td> 0.687</td><td> 0.639</td><td> 0.709</td><td>NFTA</td>

<td>net (Mcal / kg (Ib))</td><td> (0.66)</td><td> (0.71)</td><td> (0.67)</td><td>cale</td>

<td>Net Energy</td><td></td><td></td><td></td><td></td>

<td>Estimated (Mcal / kg</td><td> 0.859</td><td> 0.756</td><td> 0.796</td><td>NFTA</td>

<td>(Ib))</td><td> (0.55)</td><td> (0.6)</td><td> (0.57)</td><td>cale</td>

<td>Calcium</td><td> 0.47</td><td> 0.96</td><td> 0.55</td><td> 968.08</td>

<td>Match</td><td> 0.46</td><td> 0.43</td><td> 0.42</td><td> 964.06</td>

<td>Magnesium</td><td> 0.16</td><td> 0.28</td><td> 0.17</td><td> 968.08</td>

<td>Potassium</td><td> 3.97</td><td> 3.48</td><td> 4.09</td><td> 968.08</td>

<td>Sodium</td><td> 0.03</td><td> 0.3</td><td> 0.05</td><td> 983.04</td>

<td>Copper</td><td> 14.57</td><td> 8.82</td><td> 8.18</td><td> 968.08</td>

<td>Iron</td><td> 94.51</td><td> 157.77</td><td> 128.15</td><td> 968.08</td>

<td>Zinc</td><td> 25.75</td><td> 24.79</td><td> 25.76</td><td> 968.08</td>

<td>Manganese</td><td> 40.23</td><td> 33.08</td><td> 33.73</td><td> 968.08</td>

<td>Selenium</td><td> 2.61</td><td> 3.23</td><td> 2.87</td><td> 996.17</td>

<td>Nitrate</td><td></td><td></td><td></td><td></td>

<td>Quantitative</td><td> 0.3</td><td> 0.05</td><td> 0.64</td><td> 968.07</td>

<td>Food Value</td><td></td><td></td><td></td><td>NFTA</td>

<td>Relative</td><td> 114.06</td><td> 181.73</td><td> 126.68</td><td>cale</td>

<td>Dear RFV-Ash</td><td></td><td></td><td></td><td></td>

<td>corrected</td><td></td><td> 191.08</td><td> 132</td><td>Calculation</td>

243

<td>Chloride</td><td> 0.94</td><td> 0.62</td><td> 0.34</td><td> 915.01</td>

<td>Sulfide</td><td> 0.32</td><td> 0.25</td><td> 0.36</td><td> 923.01</td>

<td>Solubility of Protein</td><td> 46.48</td><td> 59.52</td><td> 52.56</td><td> 923.04</td>

<td>Carbo No Structural</td><td> 12.74</td><td> 25.66</td><td> 14.54</td><td>Calculation</td>

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

These studies indicate that MSM can alter the nutritional value of grass (for example, it can increase the relative nutritional value compared to fertilizer alone), possibly the taste 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 (Scottish Red Beer)

This example describes the effect of 0.5% MSM on fermentation efficiency related to the production of

244 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 crops are involved in beer production 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 Initiator (1000 mL of H₂Or, 100 g of dry malt extract, 1 ampoule of Edinburgh White Yeast Edinburgh Yeast) and must. Wort is a liquid that is extracted from the maceration process during brewing or whiskey. The musts contain the sugars that are fermented by the processed yeast to produce alcohol.

First, the Yeast Initiator is prepared according to standard methods that are known to those with specialty skills 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 brewing type; 5.0 grams of MSM; 2000 mLs of water;

245

<td>200 grams of Extract</td><td>from Dry Malta</td><td>(DME =</td><td>Dried</td><td>Malt</td>

<td>Extract); 2 vials</td><td>of extract of</td><td>yeast</td><td>White</td><td>Labs</td>

<td>Edinburgh Ale WLPO28</td><td>and disinfectant</td><td colspan="2">of elaboration</td><td>from</td>

beer (San Star).

Batch Preparation Treatment Initiator includes the following stages: (1) glass jars, exclusive of air 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 heat and 5.0 grams of MSM are added; and (5) the solution is allowed to cool to 22.2 ° C (72 ° F). The Treatment Initiator batch was then placed in a 1.89 liter (64 ounce) disinfected glass jar in which White Labs Edinburgh Ale 1-ampoule is used. The air exclusa was applied and the entire container was placed in a dark room at room temperature for 48 hours.

Preparation of Control Start Lot that includes the following stages: (1) glass jars, exclusive of air and funnel were thoroughly 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

246 a glass jar of 1.82 liters (64 ounces) disinfected using 1-ampoule White Labs Edinburgh Ale added. The exclusive air 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 (bubbled through the trap) about 2 hours after the yeast was added. The control initiator showed no signs of activity until approximately 10 hours after the yeast was added.

On the day of brewing (2-days after the yeast initiator was made) the maceration was prepared. 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 Crystal Malt 40L specialty grain; .454 kg (1 Ib) of Cara-Pils Malt specialty grain; and water. A macerate barrel (a container used in the maceration process to convert the starches into crushed grains into sugars for fermentation) and the processing pot, were cleaned with washing and rinsing of fully brewed powders. The macerate barrel was then disinfected (San Star Beer Sanitizer). 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 Crystal Malt 40L specialty grain; and .454 kg (1 Ib)

247 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 preheated macerate barrel. The crushed grains were then added and the solution was mixed thoroughly. The lid was connected and the solution was allowed to macerate for 60 minutes. After 60 minutes, 16.09 1 (4.25 gallons) of work was drained from the macerate barrel to the processing pot. Water for maceration was preheated to 75.6 ° C (168 ° F), added to the macerate barrel and mixed thoroughly with the grain. The mixture is allowed to incubate for 10 minutes. This process is repeated two more times until a total pre-boiling volume of 48 L (12.75 gallons) is achieved in the processing pot.

After preparing the maceration, the elaboration process begins. The following materials were used for the manufacturing process: .088 1 (3.0 oz) Cascade hops; 2 tablespoons Irish moss (Irish moss); 105 grams of MSM; processing pot containing 48.0 L (12.75 gallons) of must; must refrigerant; 2 fermentation vessels; refractometer; beer disinfectant; rinse powder brewers (PBW = Powdered Brewers Wash); and a filtered air bubbler. The equipment was completely cleaned with PBW. The must coolant and filtered air bubbler are disinfected with beer disinfectant (San Star Brewing Sanitizer). The

248 must (48.0 L (12.75 gallons)) is boiled in a processing pot and a 1^er aliquot of Cascade hops (.044 L (1.5 oz)) are added to the solution. After 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 oz)) of Cascade hops are added as well as 2 tablespoons of Irish Moss. At 50 minutes of boiling, a fourth aliquot (.014 L (0.5 oz)) 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 fermenters (each 21 liters in volume). 0.5% MSM (105 grams) is added to the treatment treater. The Brix reading of both fermenters is taken and the base point is recorded (Treatment fermented = 15 Brix; Control fermenter = 14.75 Brix). Each fermenter was tested at Brix every 24 hours for 21 days. Both fermenters were aerated for 25 minutes with a disinfected air bubbler. MSM infused yeast was added to the treatment fermenting vessel while the unaltered yeast is added to a control fermenting vessel. Blow tubes are connected to both fermenters and the fermentation is allowed to proceed for 21 days. The results of these studies are provided in Table 23.3 below.

Table 23.3

249

<td>Day</td><td>MSM FG: (Adjusted for Ale and Temp)</td><td>FG Control (Adjusted for Ale and Temp)</td>

<td> 1</td><td> 1.026</td><td> 1.028</td>

<td> 2</td><td> 1.018</td><td> 1.020</td>

<td> 3</td><td> 1.016</td><td> 1.018</td>

<td> 4</td><td> 1.016</td><td> 1.017</td>

<td> 5</td><td> 1.015</td><td> 1.017</td>

<td> 6</td><td> 1.015</td><td> 1.015</td>

<td> 7</td><td> 1.015</td><td> 1.015</td>

<td> 8</td><td> 1.015</td><td> 1.015</td>

<td> 9</td><td> 1.015</td><td> 1.015</td>

<td> 10</td><td> 1.015</td><td> 1.015</td>

<td> 11</td><td> 1.015</td><td> 1.015</td>

<td> 12</td><td> 1.015</td><td> 1.015</td>

<td> 13</td><td> 1.015</td><td> 1.015</td>

<td> 14</td><td> 1.015</td><td> 1.015</td>

<td> 15</td><td> 1.015</td><td> 1.015</td>

<td> 16</td><td> 1.015</td><td> 1.015</td>

<td> 17</td><td> 1.015</td><td> 1.015</td>

<td> 18</td><td> 1.015</td><td> 1.015</td>

<td> 19</td><td> 1.015</td><td> 1.015</td>

<td> 20</td><td> 1.015</td><td> 1.015</td>

<td> 21</td><td> 1.015</td><td> 1.015</td>

250

When a yeast initiator is made, the faster the activation of the yeast culture is carried out, the better the efficiency and to minimize the potential environmental contamination of undesirable airborne microorganisms. The MSM treated starter batch shows 80% activity before the control (2 hours compared to 10 hours). The study also indicated that MSM helped in the fermentation process. As the yeast initiator, the faster the activation of the yeast fermentation process, the better the efficiency and the potential environmental contamination by microorganisms transported by unwanted air will be minimized. The MSM treated thermostat showed 58% activity before the control (3.5 hours compared to 9 hours). The MSM treatment batch also reached maximum fermentation in 5 days while the control batch took 6 days (a time to complete from 17-25% earlier).

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.

251

Microbial growth studies performed on Acidophilus milk reinforced with MSM at 0%, 0.5%, 2.5% and 5%. Time intervals for evaluation were at 8 and 16 hours for a total of 104 hours. Then samples were evaluated every 7 days for a total of 28 days. The growth curves of colony forming units recovered per milliliters (cfu / mL) of the microorganisms were compared between Acidophilus milk with MSM concentrations with Acidophilus milk with 0% MSM concentration as a control sample. The MSM raw material powder was supplied by Bergstrom Nutrition with certificate of analysis. The powder was the formula of microgranules, lot # 0806809, expiration date 10/31/13. The milks were purchased at a local store. Acidophilus milk was low in fat (Darigold). Acidophilus plus Bifidus milk had a 2% milk fat content (Lucerne). Acidophilus plus Bifidus milk was run simultaneously with an 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 working solutions were run in duplicate.

All preparation and coating was carried out at room temperature. All dilutions for all solutions were plated in triplicate plates for all

252 Sampled time intervals. 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 CO₂ 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 MSM prepared media were tested for background levels of microorganisms in 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 emptying for quality control purposes. All control plates were cleaned for microorganism growth. At 72 hours, MSM concentrations and negative control solutions were negative for contamination. The results of these studies were provided in Table 24 below.

Table 24. Log Growth of Lactobacillus acidophilus in MSM-reinforced Milk

253

Cont.

<td></td><td colspan="4">Concentration of MSM in percent of acidophilus milk</td>

<td>Weather</td><td>Milk Acid</td><td>A / B 0</td><td>A / B 2.5%</td><td> 0.50%</td>

<td> 0</td><td> 8.82</td><td> 5.33</td><td> 6.41</td><td> 7.33</td>

<td> 8</td><td> 9.08</td><td> 8.36</td><td> 8.67</td><td> 9.06</td>

<td> 24</td><td> 9.17</td><td> 8.19</td><td> 9.16</td><td> 9.41</td>

<td> 32</td><td> 8.85</td><td> 8.73</td><td> 8.87</td><td> 9.2</td>

<td> 48</td><td> 8.54</td><td> 8.15</td><td> 8.3</td><td> 8.55</td>

<td> 56</td><td> 8.46</td><td> 8.51</td><td> 8.56</td><td> 8.77</td>

<td> 72</td><td> 8.65</td><td> 8.45</td><td> 8.45</td><td> 8.56</td>

<td> 80</td><td> 10.09</td><td> 9.66</td><td> 9.82</td><td> 9.92</td>

<td> 96</td><td> 10.02</td><td> 9.68</td><td> 8.61</td><td> 9.93</td>

<td> 104</td><td> 9.45</td><td> 8.83</td><td> 8.92</td><td> 9.47</td>

<td>Day 7</td><td> 10.29</td><td> 10.1</td><td> 10.42</td><td> 10.64</td>

<td>Day 14</td><td> 6.96</td><td> 6.83</td><td> 5.70</td><td> 6.81</td>

<td>Day 21</td><td> 5.30</td><td> 5.60</td><td> 5.37</td><td> 5.37</td>

<td>Day 28</td><td> 4.00</td><td> 3.82</td><td> 3.94</td><td> 2.52</td>

<td></td><td colspan="5">Concentration of MSM in percent of acidophilus milk</td>

<td>Weather</td><td> 0.50%</td><td> 2.50%</td><td> 2.50%</td><td> 5%</td><td> 5%</td>

<td> 0</td><td> 6.62</td><td> 6.67</td><td> 6.7</td><td> 6.56</td><td> 6.47</td>

254

<td> 8</td><td> 9.00</td><td> 9.06</td><td> 8.97</td><td> 8.73</td><td> 9.08</td>

<td> 24</td><td> 9.35</td><td> 9.24</td><td> 9.01</td><td> 9.32</td><td> 9.26</td>

<td> 32</td><td> 9.12</td><td> 9.06</td><td> 9.11</td><td> 9.33</td><td> 9.19</td>

<td> 48</td><td> 8.69</td><td> 8.93</td><td> 8.77</td><td> 8.78</td><td> 8.72</td>

<td> 56</td><td> 8.69</td><td> 8.7</td><td> 8.81</td><td> 8.88</td><td> 8.7</td>

<td> 72</td><td> 8.67</td><td> 8.71</td><td> 8.6</td><td> 8.75</td><td> 8.63</td>

<td> 80</td><td> 9.51</td><td> 9.52</td><td> 9.96</td><td> 9.87</td><td> 9.71</td>

<td> 96</td><td> 10.25</td><td> 10.18</td><td> 10.31</td><td> 10.14</td><td> 10.26</td>

<td> 104</td><td> 9.52</td><td> 9.8</td><td> 9.45</td><td> 9.74</td><td> 9.38</td>

<td>Day 7</td><td> 10.72</td><td> 10.4</td><td> 10.59</td><td> 10.89</td><td> 10.52</td>

<td>Day 14</td><td> 5.63</td><td> 8.00</td><td> 6.66</td><td> 7.82</td><td> 8.26</td>

<td>Day 21</td><td> 5.37</td><td> 5.56</td><td> 5.37</td><td> 5.64</td><td> 5.48</td>

<td>Day 28</td><td> 3.48</td><td> 3.43</td><td> 3.22</td><td> 2.52</td><td> 2.52</td>

At time 0 there was at least 1 log growth of Lactobacillus acidophilus in MSM-free milk compared to MSM-reinforced milk. The significance is, at hour 8, the milk reinforced with MSM showed a minimum increase in growth of 1.73 log, while 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

255 shows a growth rate that is leveled between control concentrations and MSM. The control had a 0.32 log decrease in growth at hour 32. The 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 hour 32. At hour 48, the control decreased from 0.31 logs, while the MSM decreased 0.54 logs for 0.5%, 0.24 logs for 2.5% and 0.51 logs for 5%. MSM concentrations maintain a higher recovery rate compared to the control. The MSM concentration of 2.5% was on average 0.31 logs higher and 5% was 0.21 logs higher. At hour 56 it showed no significant change in increase or decrease in growth. At 72 hours the control increased by 0.19 logs, while the MSM concentrations were stable. At the 80 hour it had a significant increase in growth. The control showed an increase of 1.44 logs. The samples treated with MSM show an increase in growth to 0.5% (1.1 logs), 2.5% (1.09 logs), and 5% (1.1 logs). At time 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 hour 96. At time 96, the concentration of MSM at 2.5% was 0.23 logs higher than the control. At hour 104, the log growth decrease was comparable between the control and the MSM concentrations (the

256 control decreased 0.57 logs; 0.5% MSM decreased and 0.60 logs; 2.5% MSM decreased 0.62 logs; and 5.0% MSM decreased 0.64 logs). Comparing the final growth recovery between the control and concentrations of MSM, the study showed that 0.5% MSM at 0.05 logs is higher than the control, the 2.5% MSM at 0.18 logs higher than the control, and 5% MSM at 0.11 logs higher than the control.

Day 7 showed an increase in growth from the 104th hour for all work solutions. 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. Day 14 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 sample of MSM at 5.0% was a complete log superior than the control at 1.08 logs. Day 21 continued the decline in growth. The control was 1.66 logs lower, MSM at 0.5% was 0.85 logs lower, MSM at 2.5% was 1.87 logs lower and MSM at 5.0% was 2.48 logs lower. MSM at 0.5% and control were equal in log growth, with MSM at 2.5%

257

0.17 logs higher than the control and MSM at 5.0% 0.26 logs higher than the control. On day 28, the decreased growth continued with a decrease of 1.3 logs for the 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 on 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%.

Acidophilus plus Bifidus milk 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 to 2.5% increased 0.49 logs, giving MSM to 2.5% a higher count at 0.97 log than the control. At hour 32, MSM at 2.5% decreased 0.29 logs and control increased 0.54 logs, with the MSM at 2.5% having a 0.14 log count higher than the control. From hour 48 to hour 72, there was a continuous pattern of increase and decrease in growth, with 2.5% MSM having an increased growth of 0.15 and 0.5 logs over the control. At 72 hours the growth was equal between the control and MSM at 2.5%. At hour 80 it showed an increase in growth of 1.21 logs for the control and 1.37 logs for MSM at 2.5%, with MSM at 2.5% having an increase in growth of 0.16 log. At hour 96, there was a significant decrease in growth for MSM to 2.5% of 1.21 logs. The control showed no significant difference from the 80 hour,

258 resulting in a 1.07 log higher growth for comparison control with MSM at 2.5%. At hour 104, control growth decreased by 0.85 logs and MSM samples at 2.5% increased by 0.31 logs. At hour 104 he showed that the MSM samples at 2.5% at 0.09 logs higher than the control. On Day 7 it had 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%. Milk had an increase of 1.13 in growth compared to MSM at 2.5%. On Day 21 the decline slowed, milk decreased by 1.23 logs and MSM to 2.5% by 0.33 logs, with milk that is 0.23 logs higher in growth than MSM. Milk on Day 28 decreased by 1.78 logs and MSM to 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.

<td colspan="7">MSM concentration in percent in Acidophilus milk</td>

<td>Weather</td><td>Milk Acid</td><td>A / B</td><td>A / B 2.5%</td><td> 0.50%</td><td> 2.50%</td><td> 5.00%</td>

<td> 0</td><td> 8.82</td><td> 5.33</td><td> 6.41</td><td> 6.98</td><td> 6.69</td><td> 6.52</td>

259

<td> 8</td><td> 9.08</td><td> 8.36</td><td> 8.67</td><td> 9.03</td><td> 9.02</td><td> 8.91</td>

<td> 24</td><td> 9.17</td><td> 8.19</td><td> 9.16</td><td> 9.38</td><td> 9.13</td><td> 9.29</td>

<td> 32</td><td> 8.85</td><td> 8.73</td><td> 8.87</td><td> 9.16</td><td> 9.09</td><td> 9.26</td>

<td> 48</td><td> 8.54</td><td> 8.15</td><td> 8.3</td><td> 8.62</td><td> 8.85</td><td> 8.75</td>

<td> 56</td><td> 8.46</td><td> 8.51</td><td> 8.56</td><td> 8.73</td><td> 8.76</td><td> 8.79</td>

<td> 72</td><td> 8.65</td><td> 8.45</td><td> 8.45</td><td> 8.62</td><td> 8.66</td><td> 8.69</td>

<td> 80</td><td> 10.09</td><td> 9.66</td><td> 9.82</td><td> 9.72</td><td> 9.74</td><td> 9.79</td>

<td> 96</td><td> 10.02</td><td> 9.68</td><td> 8.61</td><td> 10.09</td><td> 10.25</td><td> 10.20</td>

<td> 104</td><td> 9.45</td><td> 8.83</td><td> 8 . 92</td><td> 9.50</td><td> 9.63</td><td> 9.56</td>

<td>Day 7</td><td> 10.29</td><td> 10.1</td><td> 10.42</td><td> 10.68</td><td> 10.50</td><td> 10.71</td>

<td>Day 14</td><td> 6.96</td><td> 6.83</td><td> 5.7</td><td> 6.22</td><td> 7.33</td><td> 8.04</td>

<td>Day 21</td><td> 5.3</td><td> 5.6</td><td> 5.37</td><td> 5.37</td><td> 5.465</td><td> 5.56</td>

<td>Day 28</td><td> 4.00</td><td> 3.82</td><td> 3.94</td><td> 3.00</td><td> 3.325</td><td> 2.52</td>

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

<td colspan="5">MSM concentration in percent</td>

<td>Day</td><td>Acid Milk</td><td>A / B 0</td><td>A / B 2.5%</td><td> 0.50%</td>

<td> 0</td><td> 10</td><td> 300</td><td> 370</td><td> 20</td>

<td> 3</td><td> 20</td><td> 830</td><td> 480</td><td> 10</td>

<td> 7</td><td> 10</td><td> 1250</td><td> 570</td><td> 10</td>

<td> 14</td><td> 20</td><td> 2500</td><td> 1460</td><td> 20</td>

<td> 21</td><td> 20</td><td> 8000</td><td> 3500</td><td> 20</td>

<td> 28</td><td> 650</td><td> 120000</td><td> 110000</td><td> 30</td>

260

Cont.

<td colspan="6">MSM concentration in percent</td>

<td>Day</td><td> 0.50%</td><td> 2.50%</td><td> 2.50%</td><td> 5%</td><td> 5%</td>

<td> 0</td><td> 30</td><td> 10</td><td> 10</td><td> 10</td><td> 20</td>

<td> 3</td><td> 10</td><td> 30</td><td> 10</td><td> 10</td><td> 10</td>

<td> 7</td><td> 10</td><td> 20</td><td> 10</td><td> 10</td><td> 20</td>

<td> 14</td><td> 20</td><td> &lt;10</td><td> &lt;10</td><td> 20</td><td> 10</td>

<td> 21</td><td> 40</td><td> 60</td><td> 170</td><td> 20</td><td> 10</td>

<td> 28</td><td> 2250</td><td> 2460</td><td> 2700</td><td> 10</td><td> 10</td>

Table 26 shows the data for standard plate counts analyzed in work solutions. This was done to see how MSM would affect the normal flora found in milks. Day 0 was the day the samples were set up for the start of the study. Acidophilus plus Bifidus milk started with a higher count on Day 0 than is typically expected. This caused the final values ​​to rise. Acidophilus milk product on 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 5.0% MSM does not allow any significant growth by the study.

MSM as an additive to this product plays a role

261 significant to increase the population of the probiotic, Lactobacillus acidophilus in a product. Within the first eight hours of strengthening a product with MSM, there was a significant probiotic influence on the product. There was a significant increase in the growth rate of the probiotic. Acidophilus milk without MSM had a 0.26 log increase in growth within the first 8 hours. While Acidophilus milk with MSM had a minimum of 1.73 growth logs. The MSM milk sample 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 an increase in continuous growth when the control of Acidophilus milk is 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 control growth superior to the MSM solutions, the 80 hour and the 28th day. When the data is analyzed, the reason for the higher growth at 80 hours 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 Acidophilus milk 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. By

62

<td>so much</td><td>the</td><td>Day</td><td> 28</td><td>showed a</td><td>less</td><td>increase</td><td>for</td><td>the</td>

<td>solutions</td><td>from</td><td>MSM</td><td>what</td><td>the control.</td><td></td><td></td><td></td><td></td>

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. Every concentration 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 additionally establishes the influence of MSM on a probiotic. The product once reinforced with MSM exceeded product growth 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; MSM-reinforced milk was 7.82 and 8.26 logs, an increase of 0.86 and 1.3 logs respectively.

Probiotic effectiveness is based on three points: Survival, Colonization and Production of Lactic Acid. 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 increased growth speed. On Day 14 the ability to survive was seen with the increase in log growth. The ability to increase lactic acid production was

263 The third component of the effectiveness of a probiotic to be studied. In this study, there was an observed reaction of increased foam production in MSM solutions.

The statement that MSM is a beneficial dietary supplement additive is supported 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 likelihood that MSM when used in a probiotic product increases the consumer benefit.

Example 18

Recovery of Lactobacillus acidophilus in Milk

<td rowspan="2">from</td><td rowspan="2">Acidophilus East</td><td rowspan="2">Supplemented example</td><td colspan="3">with MSM</td>

<td>shows</td><td>the</td><td>recovery of</td>

<td colspan="3">Lactobacillus acidophilus supplemented with MSM.</td><td>in</td><td>milk</td><td>of Acidophilus</td>

<td></td><td>For</td><td>analyze the</td><td>effects</td><td>from MSM</td><td>in recovery</td>

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 rates. 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

264 Acidophilus milk samples reinforced with MSM were diluted and transferred to the appropriate broths without MSM for the recovery study. Time intervals 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 0% MSM concentration as a sample control. All media and MSM raw material powder were verified by sterility before the study. The study was conducted in four organisms over a period of two weeks. The microorganisms were divided into two runs, each one week long, analyzing two microorganisms each week.

Acidophilus milk was low in fat (Darigold). Acidophilus plus Bifidus milk contains 2% milk fat (Lúceme). Acidophilus Bifidus milk was run simultaneously with an MSM concentration of 2.5% and 0% as a product containing two organisms. Work solutions were maintained at 4 ° C during the study. Working solutions based on milk and MSM were run in duplicate. All preparations and coatings were made at room temperature. All dilutions for all

265 solutions were plated in triplicate plates for all sampled time intervals. 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 ° 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 appropriate plate for enumeration contains between 25 and 250 cfu / mL.

The MSM raw material sample and all MSM prepared media were tested for background levels of microorganisms in 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. All time slots for plating included negative control plates during emptying for quality control purposes. All control plates were clean of microorganism growth. At 72 hours, concentrations of MSM and negative control solutions were verified negative for contamination.

The study looks at the effect of MSM on the recovery of Lactobacillus acidophilus from Acidophilus milk reinforced 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

266

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 growth dilution factor log. The growth in Table 28 is calculated from the log growth Day x without time subtracted from the log growth for the dates subsequently analyzed, for example Day 28 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 the initial value of 2.00 is subtracted, for example, Days 28 - 24 is 11.29 logs, subtracting 2.00 logs the increase in growth speed is 9.29 logs.

Table 27. Lactobacillus acidophilus Recovery Delay in Milk reinforced with MSM Day 7

<td></td><td colspan="4">MSM concentration in percent</td>

<td>Day hour</td><td> 0%</td><td>A / B 0%</td><td>A / B 2.5%</td><td> 0.50%</td>

<td>Day 7 -24</td><td> 11.29</td><td> 12.88</td><td> 13.44</td><td> 13.1</td>

<td>Day 7-48</td><td> 12.71</td><td> 13.42</td><td> 12.5</td><td> 12.7</td>

<td>Day 7-72</td><td> 12.17</td><td> 13.47</td><td> 12.77</td><td> 12.4</td>

Cont.

267

<td></td><td colspan="5">MSM concentration in percent</td>

<td>Day hour</td><td> 0.5%</td><td> 2.50%</td><td> 2.5%</td><td> 5%</td><td> 5%</td>

<td>Day 7 -24</td><td> 12.78</td><td> 13.16</td><td> 13.15</td><td> 12.12</td><td> 13.29</td>

<td>Day 7-48</td><td> 12.67</td><td> 12.04</td><td> 12.67</td><td> 13.09</td><td> 13.25</td>

<td>Day 7-72</td><td> 13.74</td><td> 11.92</td><td> 13.28</td><td> 13.1</td><td> 12.87</td>

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

MSM concentration in percent

<td>Day - Time</td><td>Milk Acid- phílus</td><td>Milk Ac / Bf</td><td>Ac / Bf 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>

<td>Day 7</td><td> 8.29</td><td> 8.1</td><td> 8.42</td><td> 8.68</td><td> 8.50</td><td> 8.71</td>

<td>Day 7-24</td><td> 11.29</td><td> 12.88</td><td> 13.44</td><td> 12.94</td><td> 13.16</td><td> 12.71</td>

<td>Day 7-48</td><td> 12.71</td><td> 13.42</td><td> 12.50</td><td> 12.69</td><td> 12.36</td><td> 13.17</td>

<td>Day 7-72</td><td> 12.17</td><td> 13.47</td><td> 12.77</td><td> 13.07</td><td> 12.60</td><td> 12.99</td>

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

268 logs higher than MSM at 2.5%, but 0.46 logs lower than MSM at 5.0%. At 72 hours, MSM concentrations exceeded control growth; 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 2.5% MSM was a growth of 13.44 logs. Milk with 2.5% MSM was 0.56 logs higher than the control. In the next two 24-hour periods, the control of Acidophilus plus bifidus grew 13.42 logs and 13.47 logs. Acidophilus plus bifidus with 2.5% MSM had growth at 12.50 logs and 12.77 logs over the same period of time. The control was 0.92 logs higher than 2.5% MSM 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 Day 7 in increasing the log recovery growth rate from the start time of zero.

MSM concentration in percent

<td>Hours</td><td>Milk Acid- philus</td><td>Milk Ac / Bf</td><td>Ac / Bf 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>

<td> 0</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td>

269

<td> 24</td><td> 3.00</td><td> 4.78</td><td> 5.02</td><td> 4.26</td><td> 4.66</td><td> 4.00</td>

<td> 48</td><td> 1.42</td><td> 0.54</td><td> -0.94</td><td> -0.26</td><td> -0.8</td><td> 0.47</td>

<td> 72</td><td> -0.54</td><td> 0.05</td><td> 0.27</td><td> 0.39</td><td> 0.25</td><td> -0.19</td>

Analyzing the recovery of Day 7 based on the rate of increase increase in logs, there was a significant increase within the first 24 hours of growth. Control increases by 3 logs from the initial inoculum, while the concentrations of MSM increased by 4.26 logs for MSM to 0.5%, 4.66 logs for MSM to 2.5%, and 4.00 logs for MSM to 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 to 5.0% increased by 0.47 logs. The third 24-hour period control growth decreased by 0.54 logs and the MSM growth 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.

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

270 hours, the control of Acidophilus plus bifidus increased by 0.05 logs, while 2.5% MSM milk increased by 0.27 logs.

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

<td></td><td colspan="5">MSM concentration in percent</td>

<td>Day hour</td><td> 0%</td><td>A / B 0%</td><td>A / B 2.5</td><td> 0.5%</td><td> 0.5%</td>

<td>Day 14-24</td><td> 10.62</td><td> 13.38</td><td> 13.35</td><td> 10.19</td><td> 10.10</td>

<td>Day 14-48</td><td> 12.37</td><td> 13.52</td><td> 13.47</td><td> 12.95</td><td> 12.72</td>

<td>Day 14-72</td><td> 12.43</td><td> 13.31</td><td> 13.43</td><td> 13.02</td><td> 12.70</td>

Cont.

MSM concentration in percent

<td>Day hour</td><td> 2.50%</td><td> 2.50%</td><td> 5.0%</td><td> 5.0%</td>

<td>Day 14 - 24</td><td> 10.00</td><td> 9.73</td><td> 9.6</td><td> 9.6</td>

<td>Day 14 - 48</td><td> 12.43</td><td> 12.2</td><td> 12.99</td><td> 13.04</td>

<td>Day 14 - 72</td><td> 12.51</td><td> 12.63</td><td> 12.65</td><td> 12.21</td>

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

271

MSM concentration in percent

<td>Day Time</td><td>Acidophilus milk</td><td>Ac / Bf milk</td><td>Ac / Bf MSM at 2.5%</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>

<td>Day 14-0</td><td> 4.96</td><td> 4.83</td><td> 3.70</td><td> 4.22</td><td> 5.33</td><td> 6.04</td>

<td>Day 14-24</td><td> 10.62</td><td> 13.38</td><td> 13.35</td><td> 10.15</td><td> 9.87</td><td> 9.60</td>

<td>Day 14-48</td><td> 12.37</td><td> 13.52</td><td> 13.47</td><td> 12.84</td><td> 12.32</td><td> 13.02</td>

<td>Day 14-72</td><td> 12.43</td><td> 13.31</td><td> 13.43</td><td> 12.86</td><td> 12.57</td><td> 12.43</td>

On day 14 the control exceeded the growth of MSM concentrations in the first 24 hours. The control was 0.48 logs higher than 0.5% MSM, 0.75 logs higher than 2.5% MSM and 1.02 logs higher than 5.0% MSM. 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 72 hours, the 0.5% MSM was 0.43 logs higher, the 2.5% MSM was 0.14 logs higher than the control, and the 5.0% MSM was equal to the control.

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

272 2.5% MSM bifidus was 0.12 logs higher than the Acidophilus plus bifidus control.

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

MSM concentration in percent

<td></td><td>Milk</td><td></td><td>Ac / Bf</td><td>MSM</td><td>MSM</td><td>MSM</td>

<td></td><td>Acid-</td><td>Milk</td><td>MSM at</td><td>to the</td><td>to the</td><td>to the</td>

<td>Time</td><td>philus</td><td>Ac / Bf</td><td> 2.5%</td><td> 0.5%</td><td> 2.5%</td><td> 5.0%</td>

<td> 0</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td>

<td> 24</td><td> 5.66</td><td> 8.55</td><td> 9.65</td><td> 5.93</td><td> 4.54</td><td> 3.56</td>

<td> 48</td><td> 1.75</td><td> 0.14</td><td> 0.12</td><td> 2.69</td><td> 2.45</td><td> 3.42</td>

<td> 72</td><td> 0.06</td><td> -0.21</td><td> -0.04</td><td> 0.03</td><td> 0.26</td><td> -0.59</td>

Analyzing the recovery of Day 14 based on the increase in growth rate 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, control growth increased by 1.75 logs while 0.5% MSM increased by 2.69 logs, the 2.5% MSM by 2.45 logs and the 5.0% MSM increased by 3.42 logs. The third twenty-four hour period, control growth increased by 0.06 logs and MSM growth to 5.0%

273 decreased by 0.59 logs. The 0.5% MSM and 2.5% MSM increased in log growth, 0.0.03 and 0.26 logs respectively.

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

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

<td></td><td colspan="4">MSM concentration in percent</td>

<td>Day hour</td><td> 0%</td><td>A / B 0%</td><td>A / B 2.5%</td><td> 0.50%</td>

<td>Day 21-24</td><td> 12.94</td><td> 12.43</td><td> 13.08</td><td> 12.89</td>

<td>Day 21 - 48</td><td> 13.02</td><td> 12.16</td><td> 12.08</td><td> 12.94</td>

<td>Day 21 - 72</td><td> 10.32</td><td> 10.86</td><td> 12.33</td><td> 12.29</td>

Cont.

<td></td><td colspan="5">MSM concentration in percent</td>

<td>Day hour</td><td> 0.50%</td><td> 2.50%</td><td> 2.50%</td><td> 5%</td><td> 5.00%</td>

<td>Day 21-24</td><td> 12.93</td><td> 10.62</td><td> 10.95</td><td> 9.52</td><td> 9.85</td>

<td>Day 21 - 48</td><td> 13.1</td><td> 12.44</td><td> 13.12</td><td> 13.08</td><td> 13.07</td>

<td>Day 21 - 72</td><td> 10.31</td><td> 13.14</td><td> 13.05</td><td> 13.26</td><td> 13.04</td>

274

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

MSM concentrations in percent

<td>Day - Time</td><td>Milk Acid- philus</td><td>Milk Ac / Bf</td><td>Ac / Bf 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>

<td>Day twenty-one</td><td> 3.30</td><td> 3.60</td><td> 3.37</td><td> 3.37</td><td> 3.47</td><td> 3.56</td>

<td>Day 21-24</td><td> 12.94</td><td> 12.43</td><td> 13.08</td><td> 12.91</td><td> 10.79</td><td> 9.69</td>

<td>Day 21-48</td><td> 13.02</td><td> 12.16</td><td> 12.08</td><td> 13.02</td><td> 12.78</td><td> 13.08</td>

<td>Day 21-72</td><td> 10.32</td><td> 10.86</td><td> 12.33</td><td> 11.30</td><td> 13.10</td><td> 13.15</td>

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

275 control, 2.5% MSM was 2.78 logs higher than controls and 5.0% MSM was 2.83 logs higher than control.

Acidophilus plus bifidus with 2.5% MSM was 0.65 logs higher than the Acidophilus plus bifidus control 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 2.5% MSM Acidophilus plus bifidus outperformed the control of Acidophilus plus bifidus by 1.47 logs.

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

MSM concentration in percent

<td>Time</td><td>Milk Acid- philus</td><td>Milk Ac / Bf</td><td>Ac / Bf 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>

<td> 0</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td>

<td> 24</td><td> 9.64</td><td> 8.83</td><td> 9.71</td><td> 9.54</td><td> 7.32</td><td> 6.13</td>

<td> 48</td><td> 0.08</td><td> -0.27</td><td> -1.00</td><td> 0.11</td><td> 2.00</td><td> 3.39</td>

<td> 72</td><td> -2.70</td><td> -1.30</td><td> 0.25</td><td> -1.72</td><td> 0.32</td><td> 0.08</td>

Reviewing the log increase for Day 21, in the

276 First 24 hours, the 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, control increased by 0.08 logs. 0.5% MSM increased by 0.11 logs, 2.5% MSM increased by 2.00, and 5.0% MSM increased by 3.39 logs. The final 24 hours show that the control decreases by 2.70 logs. 0.5% MSM decreased by 1.72 logs. 2.5% MSM increased by 0.32 logs and 5.0% MSM 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 final two 24-hour periods, the 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. Lactobacillus acidophilus recovery delay in milk reinforced with MSM Day 28

<td></td><td colspan="4">MSM concentration in percent</td>

<td>Day hour</td><td> 0%</td><td>A / B 0</td><td>A / B 2.5%</td><td> 0.50%</td>

<td>Day 28 - 24</td><td> 13.1</td><td> 13.41</td><td> 12.67</td><td> 6.11</td>

<td>Day 28 - 48</td><td> 11.04</td><td> 13.42</td><td> 13.19</td><td> 13.66</td>

<td>Day 28 - 72</td><td> 12.47</td><td> 10.31</td><td> 12.00</td><td> 12.99</td>

277

Cont.

<td></td><td colspan="5">MSM concentration in percent</td>

<td>Day hour</td><td> 0.50%</td><td> 2.50%</td><td> 2.50%</td><td> 5.0%</td><td> 5.0%</td>

<td>Day 28 - 24</td><td> 6.47</td><td> 6.37</td><td> 6.37</td><td> 5.64</td><td> 5.48</td>

<td>Day 28 - 48</td><td> 12.99</td><td> 12.52</td><td> 12.62</td><td> 12.58</td><td> 13.16</td>

<td>Day 28 - 72</td><td> 13.01</td><td> 12.98</td><td> 12.34</td><td> 12.49</td><td> 12.47</td>

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

MSM concentration in percent

<td>Day - Time</td><td>Milk Acid- philus</td><td>Milk Ac / Bf</td><td>Ac / Bf 2.5% MSM</td><td>0.5% MSM</td><td>2.5% MSM</td><td>5.0% MSM</td>

<td>Day 28</td><td> 2.00</td><td> 1.82</td><td> 1.94</td><td> 1.00</td><td> 1.33</td><td> 0.52</td>

<td>Day 28 - 24</td><td> 13.10</td><td> 13.41</td><td> 12.67</td><td> 6.29</td><td> 6.37</td><td> 5.56</td>

<td>Day 28 - 48</td><td> 11.04</td><td> 13.42</td><td> 13.19</td><td> 13.33</td><td> 12.57</td><td> 12.87</td>

<td>Day 28 - 72</td><td> 12.47</td><td> 10.31</td><td> 12.00</td><td> 13.00</td><td> 12.66</td><td> 12.48</td>

Recovery data from Day 28 shows in the first 24-hour period that the milk control

278

<td>Acidophilus had</td><td>a</td><td>increase</td><td>log</td><td>from</td><td> 13.10.</td><td>The</td>

<td>concentrations of</td><td>MSM</td><td>were; 6.29</td><td>logs</td><td>for</td><td>MSM at</td><td> 0.5%,</td>

<td>6.37 logs for MSM</td><td>to the</td><td>2.5%, and 5.56</td><td>logs</td><td>for</td><td>MSM at</td><td> 5.0%.</td>

<td>Hour 48 the MSM at</td><td> 0.5%</td><td>increase .</td><td>log fu</td><td>e 13.</td><td>33, the</td><td>MSM at</td>

2.5% was 12.57 logs and 5.0% MSM was 12.87 logs. The control at hour 48 was 12.71 logs. Control decreased to 12.17 logs at 72 hours. 0.5% MSM decreased to 13.00 logs and 5.0% MSM decreased to 12.48 logs. 2.5% MSM 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 hour 24, 0.74 logs higher than Acidophilus plus

<td></td><td>bifidus</td><td>with</td><td>MSM</td><td>at 2.5%</td><td>At the time</td><td colspan="2">48, the difference</td><td>It was</td>

<td></td><td colspan="2">less than</td><td colspan="2">control 0.23</td><td>super logs</td><td>why</td><td>Acidophilus</td><td>plus</td>

<td></td><td>bifidus</td><td>with</td><td>MSM</td><td>at 2.5%</td><td>At the time</td><td>72, the</td><td>Acidophilus</td><td>plus</td>

<td> 15</td><td>bifidus</td><td>with</td><td>MSM</td><td>at 2.5%</td><td>It was 1.69</td><td>logs</td><td colspan="2">superior than the</td>

<td></td><td>control,</td><td>what</td><td>It was</td><td>10.31 logs</td><td> •</td><td></td><td></td><td></td>

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

279

MSM concentration in percent

<td></td><td>Milk</td><td></td><td>Ac / Bf</td><td>MSM</td><td>MSM</td><td>MSM</td>

<td></td><td>Acid-</td><td>Milk</td><td>MSM at</td><td>to the</td><td>to the</td><td>to the</td>

<td>Weather</td><td>philus</td><td>Ac / Bf</td><td> 2.5%</td><td> 0.5%</td><td> 2.5%</td><td> 5.0%</td>

<td> 0</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td><td> 0.00</td>

<td> 24</td><td> 11.10</td><td> 11.59</td><td> 10.73</td><td> 5.29</td><td> 5.04</td><td> 5.04</td>

<td> 48</td><td> -2.06</td><td> 0.01</td><td> 0.52</td><td> 7.04</td><td> 6.20</td><td> 7.31</td>

<td> 72</td><td> 1.43</td><td> -3.11</td><td> -1.19</td><td> -0.32</td><td> 0.09</td><td> -0.39</td>

On Day 28, the increase in growth rate where the control of Acidophilus milk in the first 24-hour period increased 11.10 logs, 0.5% MSM increased by 5.29 logs, 2.5% MSM increased by 5.04 logs, and MSM 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 concentrations of MSM-reinforced milk increased 0.5% in 7.04 logs, 2.5% in 6.20 logs, and 5.0% MSM in 7.31 logs. The final 24-hour period showed that the control increased by 1.43, the 2.5% MSM increased by 0.09 logs, the 0.5% MSM decreased by 0.32 logs and the 5.0% MSM decreased by 0.39 logs.

Control of milk 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

280 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 MSM reinforced product against the product without MSM.

Recovery data from 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 loqs higher. 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 0.27 logs the growth rate in the first 24 hours. The samples of 2.5% MSM and 5.0% MSM were 1.13 and 2.10 logs, respectively, less than the control. This is why the control began to exceed in growth at MSM concentrations in the first 24

281 hours. On Days 21 and 28, the control exceeded in 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 on Day 14 showed that MSM growth rates are 0.70, 0.94, and 1.67 logs higher than the control. The second data period on Day 21 showed MSM growth rates 0.03, 1.92, and 3.31 logs of higher 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. On Day 14, the concentrations of MSM of 0.5% and 5.0% were higher than the control, while MSM at 2.5% was lower. On Day 21, only 5.0% MSM was higher. On 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 demonstrates that only the 2.5% MSM concentration growth rate was greater by 0.20 logs. Even with the lowest growth rates, a higher growth log was seen for MSM concentrations, except for 5.0% MSM

282 That was equal to control. On Day 21, data from the third period show that the growth rate of MSM concentrations exceeds control by 0.98, 3.02, and 2.78 logs. This increase in growth speed 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 higher than the control. The control exceeded the concentrations of MSM for the growth rate of Day 28 in the third period. Although the growth recovery counts for the MSM concentrations of 0.5, 2.5 and 5% were 0.53, 0.19 and 0.01 logs, respectively, higher than the control.

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

This study provides indicators such as MSM help in the delay phase, log phase, stationary phase and death phase. MSM at different stages shortens the delay phase, so that probiotic bacteria begin the phase

283 log in previous time. The log phase extends 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 5 higher values ​​for a longer period of time.

The death rate slowed 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 get 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 the speed of recovery indicating that in a new environment, stressed microorganisms respond better with MSM as an additive.

Example 19

Growth of Bifidobacterium bifidum in Medium Reinforced with MSM

This example shows the effect of MSM on the growth of Bifidobacterium bifidum in the middle of

284 microbial growth 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 plating were taken every 8 hours for a total of 96 hours. The growth curves of colony forming units recovered per milliliters (cfu / mL) of the microorganisms were compared between the concentrations of MSM with the concentration of MSM at 0% as a sample control for each microorganism. The MSM raw material powder was supplied by Bergstrom Nutrition with certificate of analysis. The powder was the formula of microgranules, lot # 0806809, expiration date 10/31/13. All MSM media and powder were checked for sterility before the study. The microorganism analyzed was Bifidobacterium bifidum ATCC # 29521.

Bifidobacterium bifidum (99 mL of MRS broth with the addition of 0.05% L-cysteine) is prepared with respective concentrations of MSM. The 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 desired final concentration of MSM. The solutions were verified by sterility before proceeding with the study.

Work solutions were inoculated at a level

285 1.5 to 2 logs of microorganisms per 100 mL of broth. Bifidobacterium bifidum was incubated under anaerobic conditions at 35 ° C ± 0.5 ° C for 72 hours. Oxygen indicators were used to verify anaerobic conditions between plating intervals for Bifidobacterium test samples.

Bifidobacteruim was inoculated in MRS agar supplemental with L-cysteine ​​at the times previously mentioned to reduce the oxidation-reduction potential of the medium. All preparation and sowing on plates are done at room temperature. All dilutions for all organisms were plated in triplicate for all sampled time intervals. 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 MSM prepared media were tested for background levels of microorganisms in 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. All time intervals for plating include negative control plates during emptying

286 For quality control purposes. All negative control plates were cleaned for microorganism growth. At 72 hours, concentrations of MSM and control solutions were verified for negative by strain contamination and the strains were verified with original species.

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

<td></td><td>Bifidobacterium bifidum</td>

<td>cfu / mL inoculum</td><td>1.48x10⁴</td>

<td>Cfu gregated to 100 mL</td><td>1.48x10⁴</td>

<td>Cfu / mL in medium at time 0</td><td>1.48x10²</td>

Control numbers were derived from growth of specific organism 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 numerical control (raw material). The raw material solution was then diluted to obtain an appropriate reading in the spectrophotometer using wavelength 420 with percent light transmission. Bacterial concentrations were determined according to Method AOAC 960.09, table 960.09A. The crop suspension preparation of the raw material crop is

287 determined by spectrophotometer reading or comparison with the McFarland standard.

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

MSM concentration in percent

<td>Hr</td><td> 0</td><td> 0.125</td><td> 0.</td><td> 25</td><td> 0.</td><td> 5</td><td> 1</td><td> 2 .</td><td> 5</td><td> 5</td>

<td> 0</td><td> 1.22</td><td> 1.12</td><td> 1.</td><td> 00</td><td> 1.</td><td> 00</td><td> 1.00</td><td> 1.</td><td> 30</td><td> 1.12</td>

<td> 8</td><td> 2.55</td><td> 2.89</td><td> 2 .</td><td> 93</td><td> 2.</td><td> 84</td><td> 2.73</td><td> 2 .</td><td> 71</td><td> 2.04</td>

<td> 16</td><td> 7.16</td><td> 7.85</td><td> 8.</td><td> 07</td><td> 7.</td><td> 45</td><td> 7.73</td><td> 7.</td><td> 56</td><td> 6.10</td>

<td> 24</td><td> 8.94</td><td> 9.06</td><td> 8 .</td><td> 49</td><td> 8.</td><td> 81</td><td> 8.81</td><td> 8 .</td><td> 64</td><td> 8.43</td>

<td> 32</td><td> 10.81</td><td> 11.45</td><td> 11</td><td> .41</td><td> 11</td><td> .22</td><td> 11.49</td><td> 11</td><td> .56</td><td> 11.13</td>

<td> 40</td><td> 11.03</td><td> 10.80</td><td> 10</td><td> . 31</td><td> 12</td><td> .09</td><td> 11.19</td><td> 11</td><td> .73</td><td> 11.78</td>

<td> 48</td><td> 11.54</td><td> 8.22</td><td> 8 .</td><td> 10</td><td> 9.</td><td> 39</td><td> 10.43</td><td> 10</td><td> .72</td><td> 10.70</td>

<td> 56</td><td> 10</td><td> . 64</td><td> 7.25</td><td> 7.22</td><td> 9.51</td><td> 10.57</td><td> 11.34</td><td> 10.59</td>

<td> 64</td><td> 9.</td><td> 97</td><td> 8.86</td><td> 6.77</td><td> 10.34</td><td> 10.66</td><td> 12.26</td><td> 14.32</td>

<td> 72</td><td> 8.</td><td> 56</td><td> 6.59</td><td> 6.39</td><td> 8.35</td><td> 8.34</td><td> 10.26</td><td> 8.65</td>

<td> 80</td><td> 10</td><td> . 56</td><td> 9.12</td><td> 8.52</td><td> 10.38</td><td> 9.43</td><td> 9.52</td><td> 12.20</td>

<td> 88</td><td> 10</td><td> . 64</td><td> 9.12</td><td> 8.52</td><td> 9.70</td><td> 10.41</td><td> 10.64</td><td> 12.31</td>

<td> 96</td><td> &lt;6</td><td> . 00</td><td> &lt;6.00</td><td> &lt;6.00</td><td> 8.82</td><td> &lt;6.00</td><td> &lt;6.00</td><td> 9.60</td>

The growth observed with Bifidobacterium bifidum shows a 0.2 to 0.4 log increase in growth rate for MSM concentrations from 0.125% to 2.5% at hour 8. MSM concentrations from 0.125% to 2.5% in

288 the 16th hour increased to 0.3 to 0.7 logs. At hour 24 it showed the concentrations of MSM from 0.125% to 2.5% that 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 hour 32, there was a moment in growth velocity in the range of 0.3 to 0.75 logs for all MSM concentrations compared to the control. At hour 40, the MSM concentrations of 0.125% and 0.25% showed a uniform decline in growth rate as they are below the control of hour 40 at time 96. At hour 40, the sample was shown. 0.5% MSM at a complete growth log higher than the control. MSM at 0.5% at hour 48 until hour 96 declined in growth rate where it was 2 to 3 full logs below the control growth rate. MSM at a concentration of 1% equaled the growth growth rate from hour 40 to hour 96, except because at hour 48 and time 80 where it was a complete less log. 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 speed 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 time 80 showed a

289 growth rate less than 1 log for the MSM sample at 2.5% compared to the control and the 88 and 96 hours growth rate was equivalent. MSM at 5% at hour 40 has a growth rate of 0.7 logs higher than the control. At 48 hours, it fell to 0.7 logs lower than the control and at 56 hours the growth rate was equivalent to the control. At hour 64 it shows an increased growth rate of 4.35 logs for MSM at 5% over the control. At hour 72 it showed a decrease in the growth rate with a return to a 1.6 log increase in the growth rate at hour 80 and at hour 88. At hour 96 it showed a growth rate of MSM at 5% which It was approximately 3.6 logs higher than the control.

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 logs of growth at hour 48. This maximum growth was reached for all concentrations of MSM at hour 32. MSM concentrations of 0.125% and 0.25% showed a decline in growth from hour 40 to hour 96, never again reaching maximum growth. MSM at 0.5% increased the growth to 0.5 logs higher than the maximum of the control. MSM at 0.5% stopped the

290 Growth decrease from hour 48 to hour 96. MSM to 0.5% delayed the extinction stage to the point where at hour 96 there were 8.82 logs of growth, which was approximately 2 logs higher than the control. MSM to 1% does not increase the growth of bacteria compared to the control, but decreases the extinction stage. From hour 40 to hour 64, the MSM at 1% does not show a large decline in growth

<td>there was</td><td>a</td><td>light</td><td>0.5 drop</td><td>logs</td><td>for</td><td>the</td><td>time</td><td> 48,</td><td>but</td><td>do not</td>

<td>there was</td><td colspan="2">decrement</td><td>for the Hours</td><td>56 and</td><td> 64 .</td><td>To</td><td>time</td><td> 72</td><td>there was</td><td>a</td>

<td>drop</td><td>from</td><td>2 log</td><td colspan="3">growing but at</td><td>the</td><td>time</td><td> 80</td><td>there was</td><td>a</td>

1 log growth increase and at 88 hours there was another 1 log growth increase. At hour 96 the growth was out of 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 fall 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 greater than control. At 72 hours, there was a 2 log drop, with a 0.7 log drop at 80 hour for MSM at 2.5%. At hour 88, the MSM to 2.5% increased growth by 1 log, before falling below the accounting range at hour 96. MSM at a concentration of 5% was slower as growth rate increased compared to the others. MSM concentrations. At the time

291

32, the growth was 11.13 logs and the 40 hour of growth was 11.78 logs. At hour 48, growth fell 1 log and 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 5% MSM. There was a 6 log drop at 72 hours, but at 80 hours the growth increased 4 logs to 12.20. At hour 88, an increase of 0.1 log was observed, before falling to 9.60 logs of growth at hour 96. MSM at 5% slowed the extinction rate 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 towards a lower growth pattern. These studies indicate that MSM proceeds to the fastest stationary phase for all concentrations, extending the stationary phase for concentrations over MSM to 0.5% and increasing the maximum growth for concentrations at MSM to 2.5% and 5%.

Example 20 Effect of Purple Bromocresol on 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 a

292 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. The USP &lt;51&gt; Antimicrobial Effectiveness for tests was used as the template to show lethal concentration (LC = Lethal Concentration) LCioo · MSM concentrations between 5% -16% in 1% increments were employed. All concentrations were plated on ICC⁷ dilutions to evaluate log reduction.

Materials include the following: Lot number 0604751 from OptiMSM Flake; ATCC strain 8739 Escherichia coli lot: 57762704; 30 mL Borosilicate-based glass culture tubes were used for all OptiMSM material; Accumedia MacConkey Broth (MB) Lot: 100,974A; Diluent used was Alpha Biosciences Modified Letheen Broth (MLB) Lot: 108-09; Tripto Soya Alpha Biosciences agar with Lecithin; and Tween 80 (TSA) Lot: F08-42.

Flake OptiMSM was weighed using a Mettler Toledo AG245 SN certified scale: 1115210833 and took aliquots for each concentration. The material was placed in 30 mL borosilicate glass culture tubes. The material was calculated in a volume of 10 mL. The material was added to each tube as follows: 5% (0.5 g), 6% (0.6 g), 7%

293 (0.7 g), 8% (0.8 g), 9% (0.9 g), 10% (1.0 g), 11% (1.1 g), 12% (1.2 g), 13% (1.3 g), 14% ( 1.4 g), 15% (1.5 g), and 16% (1.6 g). MacConkey Broth was taken in aliquots in 10 mLs to each tube then sterilized for 20 minutes at 121 ° C. The tubes were cooled to room temperature that was approximately 20 ° C. All the tubes were then added the same dilution of Escherichia coli that 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 all the time. 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: Showed the same signs as day one.

(3) Day three: It showed a change in concentration of 8% that passes to the typical yellow color.

(4) Day 4 to day 6: Does not show significant signs of change.

(5) Day 7: Shows the 9% change to a yellow color. There was no color change of 10% -16%.

294 (6) Day 14: It shows no signs for the concentration range of 10% -16%.

The concentration tubes were scratched 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 by each point of time scratched and showed signs of organism demonstrated by a scratch of classical isolation.

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

<td>low as 8% against</td><td>11% in</td><td>the</td><td>studies</td><td>of the</td><td>source of</td>

<td colspan="2">increase. Example Antimicrobial Study</td><td>twenty-one from</td><td>MSM and DMSO</td><td>in</td><td>organisms</td>

<td>of streptococci This example</td><td colspan="2">show the</td><td>effects of</td><td>MSM</td><td>and DMSO in</td>

Streptococcus organisms growth.

It has been shown here that specific concentrations of MSM (such as 10% to 16% MSM) exterminate microorganisms. Dimethyl sulfoxide has also been observed that

295 exterminates 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 A group) has a hyaluronic acid capsule and Streptococcus Pneumonia (no Lancefield Group identified to date) had a different polysaccharide capsule. These two organisms are responsible for many types of human streptococcal infections and have two different types of encapsulation. Timbos 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 combined MSM and DMSO reduces the concentrations of any compound required to achieve microbial reduction. In addition, the effectiveness of using MSM, DMSO, and the combination of the two in conjunction with an antibiotic agent was evaluated.

Streptococcus Pneumonia (# 10341 ™) and Streptococcus Pyogenes (Lancefield group A, # 10096 ™) were purchased from ATCC. MSM (# 41631) and DMSO (# D8418) were purchased from Sigma

296

Aldrich Penicillin was purchased from Henry Schein. Bacterial culture medium was purchased from Becton-Dickinson and company (# 297963). The bioluminescent ATP test kit was purchased from Promega (# G8230). Streptococcus Pyogenes was grown 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 grown in Heart Brain Infusion Broth.

Evaluation of bacterial viability:

The bioluminescent ATP test kit was used to evaluate bacterial viability based on the following reaction:

ATP + D-Luciferin + O₂ -► Oxyluciferin + AMP + pyrophosphate + CO₂ + 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 luciferin / luciferase and leads to light emission. The intensity of the emitted light 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 live microorganisms.

Both S. pyogenes and S. pneumonia were grown

297 under various conditions to determine optimal conditions to evaluate 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 ΆΤΡ bioluminescent test set. The test was performed in triplicate.

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

<td>MSM (%)</td><td>DMSO (%)</td><td>Penicillin</td>

<td> 20</td><td> 20</td><td> 100</td>

<td> 10</td><td> 10</td><td> 50</td>

<td> 5</td><td> 5</td><td> 25</td>

<td> 2.5</td><td> 2.5</td><td> 12.5</td>

<td> 1.25</td><td> 1.25</td><td> 6.25</td>

<td> 0.625</td><td> 0.625</td><td> 3.125</td>

<td> 0.3125</td><td> 0.3125</td><td> 1.5625</td>

<td> 0</td><td> 0</td><td> 0</td>

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

298

Table 42

<td>DMSO (%)</td><td>MSM (%)</td>

<td rowspan="4"> 0</td><td> 0</td>

<td> 5</td>

<td> 10</td>

<td> 20</td>

<td rowspan="4"> 5</td><td> 0</td>

<td> 5</td>

<td> 10</td>

<td> 20</td>

<td rowspan="4"> 10</td><td> 0</td>

<td> 5</td>

<td> 10</td>

<td> 20</td>

<td rowspan="5"> 20</td><td> 0</td>

<td> 5</td>

<td> 10</td>

<td> 20</td>

<td></td>

Table 43

<td>DMSO (%)</td><td>MSM (%)</td>

<td rowspan="4"> 0</td><td> 0</td>

<td> 2.5</td>

<td> 5</td>

<td> 10</td>

<td rowspan="4"> 2.5</td><td> 0</td>

<td> 2.5</td>

<td> 5</td>

<td> 10</td>

<td rowspan="4"> 5</td><td> 0</td>

<td> 2.5</td>

<td> 5</td>

<td> 10</td>

<td rowspan="5"> 8</td><td> 0</td>

<td> 2.5</td>

<td> 5</td>

<td> 10</td>

<td></td>

To determine the 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).

299

Table 44-1

<td>DMSO</td><td>MSM (%)</td><td>Penicillin (pg / L)</td>

<td rowspan="3"> 5</td><td> 5</td><td rowspan="9"> 25</td>

<td> 10</td>

<td> 20</td>

<td rowspan="3"> 10</td><td> 5</td>

<td> 10</td>

<td> 20</td>

<td rowspan="3"> 20</td><td> 5</td>

<td> 10</td>

<td> 20</td>

Table 44-2

<td>DMSO</td><td>MSM</td><td>Penicillin (pg / L)</td>

<td rowspan="6"> 2.5</td><td> 2.5</td><td rowspan="3"> 3.125</td>

<td> 5</td>

<td> 10</td>

<td> 2.5</td><td rowspan="3"> 6.25</td>

<td> 5</td>

<td> 10</td>

<td rowspan="2"> 5</td><td rowspan="4"> 2.5</td><td> 3.125</td>

<td> 6.25</td>

<td rowspan="2"> 8</td><td> 3.125</td>

<td> 6.25</td>

300

IC50 of DMSO, MSM and Penicillin in Streptococcus 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 growth of Streptococcus pneumonia. DMSO and Penicillin also had synergistic effect within doses of 10% to 20% (for DMSO) and 25 pg / L (for Penicillin) to inhibit growth of Streptococcus pneumonia. 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 only 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 synergistic effect within doses of 5% (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 synergistic effect resulting from DMSO +

301

MSM only instead of penicillin + DMSO + MSM.

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

<td>DMSO concentration</td><td>Viability of S. pneumonia (%)</td>

<td> 5</td><td> 75.45</td>

<td> 10</td><td> 40.18</td>

<td> 20</td><td> 27.19</td>

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

<td>Concentration from MSM</td><td>Viability of S. pneumonia (%)</td>

<td> 5</td><td> 95.34</td>

<td> 10</td><td> 48.57</td>

<td> 20</td><td> 39.08</td>

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

<td>DMSO (%)</td><td>MSM (%)</td><td>Viability of S. pneumonia (%)</td>

<td> 5</td><td> 0</td><td> 75.45</td>

<td> 5</td><td> 5</td><td> 50.94 *</td>

<td> 5</td><td> 10</td><td> 45.40</td>

<td> 5</td><td> 20</td><td> 27.22</td>

302

Table 45-4 Viability of S. pneumonia after exposure to various concentrations of MSM in 10% DMSO

<td>DMSO (%)</td><td>MSM (%)</td><td>Viability of S. pneumonia (%)</td>

<td> 10</td><td> 0</td><td> 40.18</td>

<td> 10</td><td> 5</td><td> 47.81</td>

<td> 10</td><td> 10</td><td> 37.42</td>

<td> 10</td><td> 20</td><td> 11.95</td>

Table 45-5 Viability of S. pneumonia after exposure to various concentrations of MSM in 20% DMSO

<td>DMSO (%)</td><td>MSM (%)</td><td>Viability of S. pneumonia (%)</td>

<td> 20</td><td> 0</td><td> 27.19</td>

<td> 20</td><td> 5</td><td> 17.60 *</td>

<td> 20</td><td> 10</td><td> 7.76</td>

<td> 20</td><td> 20</td><td> 5.15</td>

Table 45-6. Viability of S. pneumonia After

Exposure to Various Concentrations of Penicillin

<td>Penicillin (pg / L)</td><td>Viability of S. pneumonia (%)</td>

<td> 25</td><td> 79.82</td>

<td> 50</td><td> 42.70</td>

<td> 100</td><td> 40.93</td>

303

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

<td>Penicillin (pg / L)</td><td>DMSO (%)</td><td>Viability of pneumonia (%)</td><td>S.</td>

<td> 25</td><td> 0</td><td colspan="2"> 79.82</td>

<td> 25</td><td> 5</td><td colspan="2"> 46.13</td>

<td> 25</td><td> 10</td><td colspan="2"> 39.78</td>

<td> 25</td><td> 20</td><td colspan="2"> 22.08</td>

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

<td>Penicillin (pg / L)</td><td>DMSO (%)</td><td>Viability of S. pneumonia (%)</td>

<td> 50</td><td> 0</td><td> 42.70</td>

<td> 50</td><td> 5</td><td> 46.27</td>

<td> 50</td><td> 10</td><td> 37.09</td>

<td> 50</td><td> 20</td><td> 19.14</td>

Table 45-9. Viability of S. pneumonia After Exposure to 100 pg / L of Penicillin with Various DMSO Concentrations

<td>Penicillin (pg / L)</td><td>DMSO (%)</td><td>S. variability pneumonia (%)</td>

<td> 100</td><td> 0</td><td> 40.93</td>

<td> 100</td><td> 5</td><td> 45.09</td>

304

<td> 100</td><td> 10</td><td> 35.80</td>

<td> 100</td><td> 20</td><td> 21.76</td>

The combination of 5% MSM with 25 pg / 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 expected results based on MSM alone and penicillin is only indicated in the Tables by a Contrast, 5% MSM only reduces viability by only about 5%, while 25 pg / L of penicillin only reduces viability in approximately 21% Thus, the combination of MSM at 5% / 25 pg / L of 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 to 21.37%, MSM at 20% with 50 pg / L of penicillin reduces the viability of S. pneumonia to 20.75%. Thus, with the use of MSM at 20%, the required concentration of penicillin is reduced by half. Continuing with this trend is the combination of MSM at 20% with 25 pg / L of

305 Penicillin reduces the viability of S. pneumonia to 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 to 100 pg / L of penicillin (compare Tables 45-10 to 45-12 for 25 pg / L of penicillin).

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

<td>Penicillin (pg / L)</td><td>MSM (%)</td><td>Viability of S. pneumonia (%)</td>

<td> 25</td><td> 0</td><td> 79.82</td>

<td> 25</td><td> 5</td><td> 41.23 *</td>

<td> 25</td><td> 10</td><td> 41.83</td>

<td> 25</td><td> 20</td><td> 25.36</td>

Viability of S. pneumonia After

Table 45-11.

50 pg / L Exposure of Penicillin with Various

MSM concentrations

<td>Penicillin (pg / L)</td><td>MSM (%)</td><td>Viability of S. pneumonia (%)</td>

<td> 50</td><td> 0</td><td> 42.70</td>

<td> 50</td><td> 5</td><td> 41.23</td>

<td> 50</td><td> 10</td><td> 47.47</td>

<td> 50</td><td> 20</td><td> 20.75</td>

306

Table 45-12. Viability of S. pneumonia After

100 pg / L Exposure of Penicillin with Various

MSM concentrations

<td>Penicillin (pg / L)</td><td>MSM (%)</td><td>Viability of S. pneumonia</td>

<td> 100</td><td> 0</td><td> 40.93</td>

<td> 100</td><td> 5</td><td> 41.75</td>

<td> 100</td><td> 10</td><td> 36.67</td>

<td> 100</td><td> 20</td><td> 21.37</td>

Based on the 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 at one of 5, 10, or 20% and penicillin at one of 25, 50, or 100 pg / L. Viability was estimated as described above. Feasibility data are presented in Table 45-13. The

307 Symbol represents synergistic results compared to the corresponding combination of DMSO and penicillin. The symbol ψ represents synergistic results compared to the corresponding combination of MSM and penicillin. The values ​​for reduction of bacterial viability were added together to determine the threshold reduction for synergy. For example, DMSO at 5% reduces viability by approximately 25% and 25 pg / L of 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% (for example, a reduction of approximately 46% based on the individual reduction seen with 5% DMSO, see Table 45 -1, and the reduction of individual sight with 25 pg / L of penicillin). From

308 In this way, the combination of all three compounds reduces bacterial viability by an additional approximately 12%. Similarly, the combination of 5% MSM with 25 pg / L of penicillin resulted in bacterial viability of approximately 74%, while the combination of all three compounds reduces viability by an additional 22%.

In some combinations, synergistic results were detected with respect to both DMSO and penicillin as well as MSM plus penicillin. For example, 10% DMSO in combination with 20% MSM and 25 pg / L of 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 of 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

309 Proven penicillin concentrations (from -25% viability with 25 pg / L of penicillin to -18% viability with 100 pg / L of penicillin). Additionally, 10% DMSO with 20% MSM resulted in almost identical bacterial viability across the penicillin concentration range. Similar results are seen with 20% DMSO in combination with 5, 10, or 20% MSM and any concentration of penicillin. These results revealed a slightly wider range of bacterial viability across the different concentrations of penicillin, however, since the reduction in all cases is close to approximately 90 to 95%, these combinations are still effective.

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

<td>DMSO (%)</td><td>MSM (%)</td><td>Penicillin (pg / L)</td><td>Viability of S. pneumonia (%)</td>

<td> 5</td><td> 5</td><td> 25</td><td> 52.11 *, ^ψ</td>

<td> 5</td><td> 5</td><td> 50</td><td> 43.36</td>

<td> 5</td><td> 5</td><td> 100</td><td> 53.03</td>

<td> 5</td><td> 10</td><td> 25</td><td> 51.82 *</td>

<td> 5</td><td> 10</td><td> 50</td><td> 44.52</td>

<td> 5</td><td> 10</td><td> 100</td><td> 31.33</td>

310

<td> 5</td><td> 20</td><td> 25</td><td> 24.91 *</td>

<td> 5</td><td> 20</td><td> 50</td><td> 19.20</td>

<td> 5</td><td> 20</td><td> 100</td><td> 18.12</td>

<td> 10</td><td> 5</td><td> 25</td><td> 44.41 ^ψ</td>

<td> 10</td><td> 5</td><td> 50</td><td> 38.24</td>

<td> 10</td><td> 5</td><td> 100</td><td> 36.19</td>

<td> 10</td><td> 10</td><td> 25</td><td> 39.38</td>

<td> 10</td><td> 10</td><td> 50</td><td> 33.73</td>

<td> 10</td><td> 10</td><td> 100</td><td> 25.98</td>

<td> 10</td><td> 20</td><td> 25</td><td> 11.87 *, ^ψ</td>

<td> 10</td><td> 20</td><td> 50</td><td> 11.03</td>

<td> 10</td><td> 20</td><td> 100</td><td> 10.96</td>

<td> 20</td><td> 5</td><td> 25</td><td> 12.74 *, ^ψ</td>

<td> 20</td><td> 5</td><td> 50</td><td> 13.39 ^ψ</td>

<td> 20</td><td> 5</td><td> 100</td><td> 9.2 8 ^ψ</td>

<td> 20</td><td> 10</td><td> 25</td><td> 7.69 *, ^ψ</td>

<td> 20</td><td> 10</td><td> 50</td><td> 7.74</td>

<td> 20</td><td> 10</td><td> 100</td><td> 5.58</td>

<td> 20</td><td> 20</td><td> 25</td><td> 4.93 *, ^ψ</td>

<td> 20</td><td> 20</td><td> 50</td><td> 5.60</td>

<td> 20</td><td> 20</td><td> 100</td><td> 1.80</td>

As discussed above, the structure of S. pyogenes differs from that of S. pneumonia, and therefore additional experiments were performed to assess the effects

311 synergists of various concentrations of DMSO and MSM, as well as combinations of DMSO, MSM, and penicillin.

DMSO was added to cultures of S. pyogenes 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 cultures of S. pyogenes 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 DMSO Exposure

<td>DMSO concentration</td><td>Viability of S. pyogenes (%)</td>

<td> 0.31</td><td> 100</td>

<td> 0.63</td><td> 100</td>

<td> 1.25</td><td> 100</td>

<td> 2.50</td><td> 100</td>

<td> 5.00</td><td> 96.66</td>

<td> 10.0</td><td> 14.50</td>

<td> 20.0</td><td> 5.14</td>

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

312

<td>MSM concentration</td><td>Viability of S. pyogenes (%)</td>

<td> 0.31</td><td> 100</td>

<td> 0.63</td><td> 100</td>

<td> 1.25</td><td> 100</td>

<td> 2.50</td><td> 100</td>

<td> 5.00</td><td> 95.88</td>

<td> 10.0</td><td> 23.94</td>

<td> 20.0</td><td> 15.18</td>

MSM and DMSO in combination were evaluated for their antibacterial effects in S. pyogenes. 2.5%, 5%, and 8% DMSO was combined with 0% MSM (DMSO control only), 2.5%, 5%, and 10%. As shown in Tables 16, 17, and 18 certain combinations of MSM with DMSO are synergistic compared to the effects of either DMSO or MSM alone. Synergistic results compared to DMSO or MSM are only indicated by an For example, adding MSM at 2.5% to DMSO at 2.5% reduces bacterial viability to approximately 65% ​​(see Table 16), while no effect of these concentrations would be expected of MSM and DMSO, since individually no compound reduces bacterial viability. The synergistic effect is also seen with 2.5% DMSO and 5% MSM, where bacterial viability is reduced to about 83% (compared to an expected 4% reduction based on the effects of the compounds only). The

313 Synergy is also seen with 5% DMSO in combination with any concentration of MSM. Thus, in some modalities, 5% DMSO induces synergistic reductions in bacterial viability in combination with any concentration of MSM between 2.5% and 10%. In some modalities, 2.5% DMSO and MSM in concentrations between 2.5% and 5% are advantageously and unexpectedly synergistic to reduce bacterial viability.

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

<td>DMSO (%)</td><td>MSM (%)</td><td>Viability of S. pyogenes (%)</td>

<td> 2.5</td><td> 0</td><td> 100</td>

<td> 2.5</td><td> 2.5</td><td> 65.06 *</td>

<td> 2.5</td><td> 5.0</td><td> 17.71 *</td>

<td> 2.5</td><td> 10.0</td><td> 16.37</td>

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

<td>DMSO (%)</td><td>MSM (%)</td><td>Viability of S. pyogenes (%)</td>

<td> 5.0</td><td> 0</td><td> 96.66</td>

<td> 5.0</td><td> 2.5</td><td> 36.21 *</td>

<td> 5.0</td><td> 5.0</td><td> 7.87 *</td>

<td> 5.0</td><td> 10.0</td><td> 7.64 *</td>

314

Table 45-18. Viability of S. pyogenes After Exposure to Various Concentrations of MSM in DMSO at 8

<td>DMSO (%)</td><td>MSM (%)</td><td>Viability of S. pyogenes (%)</td>

<td> 8.0</td><td> 0</td><td> 9.96</td>

<td> 8.0</td><td> 2.5</td><td> 14.37</td>

<td> 8.0</td><td> 5.0</td><td> 5.97</td>

<td> 8.0</td><td> 10.0</td><td> 5.60</td>

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

Table 45-19. Viability of S pyogenes after Exposure to Various Concentrations of Penicillin

<td>Penicillin (pg / L)</td><td>Viability of S. pyogenes (%)</td>

<td> 1.56</td><td> 100</td>

<td> 3.13</td><td> 100</td>

<td> 6.25</td><td> 100</td>

<td> 12.5</td><td> 13.16</td>

<td> 25.0</td><td> 9.07</td>

<td> 50</td><td> 9.57</td>

<td> 100</td><td> 9.40</td>

315

Due to the highly effective nature of penicillin concentrations at or above 25 pg / L, DMSO was combined with penicillin concentrations that were less effective (in the range of 3,125 to 12.5 pg / 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 a *) several combinations of DMSO and penicillin produced synergistic results. For example,

<td>5% DMSO</td><td>in combination</td><td>with</td><td>3,125 pg / L</td><td>from</td><td>penicillin,</td><td>with</td>

<td>base on the</td><td>effectiveness of</td><td>the</td><td colspan="2">two compounds</td><td>alone</td><td>I know</td>

<td>would wait</td><td>to reduce</td><td>the</td><td>viability</td><td>from</td><td>bacteria</td><td>in</td>

approximately 4% However, when combined, the current reduction was approximately 10-fold greater (viability was 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 of penicillin (see Table 45-21 and 45-22, respectively).

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

<td>Penicillin (pg / L)</td><td>DMSO (%)</td><td>Viability of S. pyogenes (%)</td>

<td> 3.13</td><td> 0</td><td> 100</td>

<td> 3.13</td><td> 2.5</td><td> 100</td>

316

<td> 3.13</td><td> 5.0</td><td> 60.85 *</td>

<td> 3.13</td><td> 8.0</td><td> 12.90</td>

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

DMSO

<td>Penicillin (pg / L)</td><td>DMSO (%)</td><td>Viability of S. pyogenes (%)</td>

<td> 6.25</td><td> 0</td><td> 100</td>

<td> 6.25</td><td> 2.5</td><td> 100</td>

<td> 6.25</td><td> 5.0</td><td> 60.23 *</td>

<td> 6.25</td><td> 8.0</td><td> 6.91 *</td>

Table 45-22. Viability of S pyogenes After Exposure to 12.5 pg / L of Penicillin with Various DMSO Concentrations

<td>Penicillin (pg / L)</td><td>DMSO (%)</td><td>Viability of S. pyogenes (%)</td>

<td> 12.5</td><td> 0</td><td> 13.16</td>

<td> 12.5</td><td> 2.5</td><td> 19.63</td>

<td> 12.5</td><td> 5.0</td><td> 14.77 *</td>

<td> 12.5</td><td> 8.0</td><td> 6.43</td>

Similar studies to those using DMSO were performed by combining MSM with penicillin in the range of

317

3,125 to 12.5 pg / L. Results are shown in Tables 4523, 45-24, and 45-25. Synergy is indicated by As with DMSO, previously ineffective concentrations of MSM and penicillin were effective in combination by reducing bacterial viability. When taken alone, no effect of 3.13 pg / L of penicillin with 2.5% MSM will be expected, however a viability reduction 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 indicates 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. Viability of S pyogenes After Exposure to 3.13 pg / L of Penicillin with Various Concentrations of MSM

<td>Penicillin (pg / L)</td><td>MSM (%)</td><td>Viability of S. pyogenes (%)</td>

<td> 3.13</td><td> 0</td><td> 100</td>

<td> 3.13</td><td> 2.5</td><td> 92.89 *</td>

<td> 3.13</td><td> 5.0</td><td> 78.31 *</td>

<td> 3.13</td><td> 8.0</td><td> 9.91*</td>

318

Table 45-24. Viability of S pyogenes After Exposure to

6.25 pg / L of Penicillin with Various Concentrations of MSM

<td>Penicillin (pg / L)</td><td>MSM (%)</td><td>Viability pyogenes (%)</td><td>from</td><td>s.</td>

<td> 6.25</td><td> 0</td><td colspan="3"> 100</td>

<td> 6.25</td><td> 2.5</td><td> 90.11</td><td> *</td><td></td>

<td> 6.25</td><td> 5.0</td><td> 17.42</td><td> *</td><td></td>

<td> 6.25</td><td> 8.0</td><td> 10.77</td><td> *</td><td></td>

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

<td>Penicillin (pg / L)</td><td>MSM (%)</td><td>Viability of S. pyogenes (%)</td>

<td> 12.5</td><td> 0</td><td> 13.16</td>

<td> 12.5</td><td> 2.5</td><td> 16.33</td>

<td> 12.5</td><td> 5.0</td><td> 12.85</td>

<td> 12.5</td><td> 8.0</td><td> 16.02</td>

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

319 Penicillin is indicated by ψ. As can be seen from the data in Table 45-26, substantial synergy is detected across 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 pg / 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 become effective in a dose-dependent manner by combination with DMSO and MSM. For example, 2.5% DMSO with 5% MSM and 3,125 pg / L of 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 pg / 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 8% DMSO with 2.5% MSM and 3,125 pg / L of penicillin appears to be more effective than 8% DMSO with 2.5% MSM and 12.5 pg / L of penicillin.

Table 45-26. Viability of S. pneumonia After Exposure

320 to Various Combinations of DMSO, MSM, and Penicillin

<td>DMSO (%)</td><td>MSM (%)</td><td>Penicillin (pg / L)</td><td>Viability pyogenes (%)</td><td>from</td><td>S.</td>

<td> 2.5</td><td> 2.5</td><td> 3.125</td><td> 91.74 *,</td><td>ψ</td><td></td>

<td> 2.5</td><td> 2.5</td><td> 6.25</td><td> 60.55 *,</td><td>ψ</td><td></td>

<td> 2.5</td><td> 2.5</td><td> 12.5</td><td> 8.08 *,</td><td>ψ</td><td></td>

<td> 2.5</td><td> 5</td><td> 3.125</td><td> 18.72 *,</td><td>ψ</td><td></td>

<td> 2.5</td><td> 5</td><td> 6.25</td><td> 13.38 *,</td><td>ψ</td><td></td>

<td> 2.5</td><td> 5</td><td> 12.5</td><td colspan="3"> 9.41 *</td>

<td> 2.5</td><td> 10</td><td> 3.125</td><td> 16.05 *,</td><td>ψ</td><td></td>

<td> 2.5</td><td> 10</td><td> 6.25</td><td> 11.78 *,</td><td>ψ</td><td></td>

<td> 2.5</td><td> 10</td><td> 12.5</td><td colspan="3"> 11.77 *</td>

<td> 5</td><td> 2.5</td><td> 3.125</td><td> 14.60 *,</td><td>ψ</td><td></td>

<td> 5</td><td> 2.5</td><td> 6.25</td><td> 10.44 *,</td><td>ψ</td><td></td>

<td> 5</td><td> 2.5</td><td> 12.5</td><td> 9.55 *,</td><td>ψ</td><td></td>

<td> 8</td><td> 2.5</td><td> 3.125</td><td> 9.55 *,</td><td>ψ</td><td></td>

<td> 8</td><td> 2.5</td><td> 6.25</td><td colspan="3"> 10.28 ^ψ</td>

<td> 8</td><td> 2.5</td><td> 12.5</td><td colspan="3"> 15.55 ^ψ</td>

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

321

Streptococcus Pneumonia.

Example 22

Probiotic Growth in Supplemented Medium 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, 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 Bifidobacteri um 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 CO2 for a total of 72 hours for all solutions, except Bifidobacterium bifidum, which develops under anaerobic conditions. Samples of each medium were collected at 0, 8, 16, 24, 32, 40, 48, 56, 64, and 72 hours. Samples of Lactobacillus were coated on MRS agar,

322 Bifidobacterium bifidum samples were coated on MRS + L-cysteine ​​agar, and Bacillus coagulans samples were coated on tryptic soy agar. 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. Data are presented in Cfu / mL. The results of these studies are presented in the following Tables.

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

<td>Weather</td><td>0% MSM</td><td>0.125 % MSM</td><td>0.2% MSM</td><td>0.50% MSM</td><td>1 2-L or MSM</td><td>2.5% MSM</td><td>5 % MSM</td>

<td> 0</td><td> 1.48</td><td> 1.37</td><td> 1.37</td><td> 1.30</td><td> 1.48</td><td> 1.48</td><td> 1.52</td>

<td> 8</td><td> 1.48</td><td> 2.19</td><td> 1.43</td><td> 2.01</td><td> 2.25</td><td> 2.20</td><td> 1.37</td>

<td> 16</td><td> 4 . 87</td><td> 4.83</td><td> 5.74</td><td> 3.82</td><td> 3.79</td><td> 4.24</td><td> 2.69</td>

<td> 24</td><td> 6.97</td><td> 7.14</td><td> 8.19</td><td> 6.47</td><td> 6.77</td><td> 5.78</td><td> 5.36</td>

<td> 32</td><td> 9.47</td><td> 9.15</td><td> 9.85</td><td> 9.15</td><td> 9.05</td><td> 9.16</td><td> 8.91</td>

<td> 40</td><td> 7.08</td><td> 9.53</td><td> 9.50</td><td> 9.58</td><td> 9.49</td><td> 9.35</td><td> 9.25</td>

<td> 48</td><td> 7.27</td><td> 9.59</td><td> 9.32</td><td> 10.45</td><td> 10.98</td><td> 10.16</td><td> 10.88</td>

<td> 56</td><td> 7.20</td><td> 10.16</td><td> 9.26</td><td> 9.33</td><td> 10.90</td><td> 11.10</td><td> 10.01</td>

<td> 64</td><td> 7.29</td><td> 9.40</td><td> 9.37</td><td> 9.53</td><td> 10.34</td><td> 11.58</td><td> 8.95</td>

<td> 72</td><td> 7.19</td><td> 8.56</td><td> 8.36</td><td> 8.57</td><td> 8.68</td><td> 8.44</td><td> 6.66</td>

323

Table 47. Growth of Lactobacillus bulgaricus in Medium

Reinforced with MSM

<td>Weather</td><td>0% MSM</td><td>0.125% MSM</td><td>0.2% MSM</td><td>0.50% MSM</td><td>one% MSM</td><td>2.5% MSM</td><td>5 % MSM</td>

<td> 0</td><td> 2.22</td><td> 2.29</td><td> 2.25</td><td> 2.23</td><td> 2.29</td><td> 2.26</td><td> 2.19</td>

<td> 8</td><td> 3.56</td><td> 4.15</td><td> 4.08</td><td> 4 . 42</td><td> 4.83</td><td> 4.57</td><td> 3.28</td>

<td> 16</td><td> 8.09</td><td> 8.22</td><td> 8.34</td><td> 8.28</td><td> 8.36</td><td> 8.09</td><td> 7.39</td>

<td> 24</td><td> 8.71</td><td> 9.04</td><td> 9.10</td><td> 9.05</td><td> 9.03</td><td> 9.01</td><td> 8.50</td>

<td> 32</td><td> 9.29</td><td> 8.55</td><td> 9.60</td><td> 9.54</td><td> 9.31</td><td> 9.15</td><td> 9.29</td>

<td> 40</td><td> 9.32</td><td> 9.29</td><td> 9.11</td><td> 9.40</td><td> 9.34</td><td> 9.27</td><td> 9.37</td>

<td> 48</td><td> 10.8 1</td><td> 10.94</td><td> 11.07</td><td> 10.82</td><td> 11.2 3</td><td> 11.3 7</td><td> 10.92</td>

<td> 56</td><td> 7.69</td><td> 8.00</td><td> 8.79</td><td> 9.14</td><td> 8.11</td><td> 8.23</td><td> 10.07</td>

<td> 64</td><td> 8.78</td><td> 8.59</td><td> 8.79</td><td> 8.80</td><td> 6.50</td><td> 8.75</td><td> 10.96</td>

<td> 72</td><td> 6.56</td><td> 6.74</td><td> 6.72</td><td> 6.72</td><td> 6.45</td><td> 6.51</td><td> 8.62</td>

Table 48. Growth of Bacillus coagulans in Medium

Reinforced with MSM

<td>Weather</td><td>0% MSM</td><td>0.125 % MSM</td><td>0.2% MSM</td><td>0.50% MSM</td><td>one% MSM</td><td>2.5% MSM</td><td>5 % MSM</td>

<td> 0</td><td> 1.43</td><td> 1.52</td><td> 1.67</td><td> 1.48</td><td> 1.30</td><td> 1.43</td><td> 1.56</td>

<td> 8</td><td> 5.05</td><td> 4.81</td><td> 4.94</td><td> 4.42</td><td> 4.98</td><td> 5.13</td><td> 4.61</td>

<td> 16</td><td> 6.75</td><td> 6.95</td><td> 7.19</td><td> 6.94</td><td> 7.29</td><td> 7.05</td><td> 7.56</td>

324

<td> 24</td><td> 10.34</td><td> 9.87</td><td> 10.34</td><td> 10.29</td><td> 10.3 0</td><td> 10.2 2</td><td> 10.4 8</td>

<td> 32</td><td> 10.70</td><td> 11.06</td><td> 11.25</td><td> 11.05</td><td> 11.4 2</td><td> 11.7 0</td><td> 11.5 5</td>

<td> 40</td><td> 10.70</td><td> 11.72</td><td> 11.34</td><td> 10.25</td><td> 11.0 2</td><td> 10.5 5</td><td> 10.8 5</td>

<td> 48</td><td> 11.07</td><td> 11.56</td><td> 9.94</td><td> 10.40</td><td> 10.3 8</td><td> 10.8 8</td><td> 10.2 2</td>

<td> 56</td><td> 11.35</td><td> 9.60</td><td> 11.45</td><td> 10.76</td><td> 10.8 6</td><td> 10.8 6</td><td> 11.1</td>

<td> 64</td><td> 11.01</td><td> 12.13</td><td> 11.37</td><td> 10.45</td><td> 10.4 0</td><td> 10.9 7</td><td> 10.7 5</td>

<td> 72</td><td> 10.92</td><td> 10.14</td><td> 10.94</td><td> 10.86</td><td> 10.7 ⁰</td><td> 11.0 5</td><td> 11.8 1</td>

Table 49. Growth of Bifidobacteruim bifidum in Medium

Reinforced with MSM

<td>Weather</td><td>0% MSM</td><td>0.125 % MSM</td><td>0.2% MSM</td><td>0.50% MSM</td><td>one% MSM</td><td>2.5% MSM</td><td>5 % MSM</td>

<td> 0</td><td> 1.67</td><td> 1.64</td><td> 1.82</td><td> 1.85</td><td> 1.48</td><td> 1.64</td><td> 1.00</td>

<td> 8</td><td> 2.30</td><td> 1.73</td><td> 2.08</td><td> 1.99</td><td> 1.70</td><td> 1.60</td><td> 2.29</td>

<td> 16</td><td> 5.33</td><td> 6.55</td><td> 5.22</td><td> 6.53</td><td> 6.81</td><td> 6.21</td><td> 6.71</td>

<td> 24</td><td> 5.86</td><td> 2.70</td><td> 6.15</td><td> 3.14</td><td> 2.75</td><td> 2.52</td><td> 5.72</td>

<td> 32</td><td> 8.80</td><td> 3.37</td><td> 5.03</td><td> 3.52</td><td> 3.37</td><td> 3.37</td><td> 10.32</td>

325

<td> 40</td><td> 9.71</td><td> 4.19</td><td> 8.14</td><td> 4.62</td><td> 3.48</td><td> 3.52</td><td> 12.02</td>

<td> 48</td><td> 10.60</td><td> 6.41</td><td> 8.55</td><td> 4.51</td><td> 3.90</td><td> 3.95</td><td> 10.54</td>

<td> 56</td><td> 10.42</td><td> 9.97</td><td> 9.00</td><td> 6.05</td><td> 8.32</td><td> 8.35</td><td> 10.92</td>

<td> 64</td><td> 10.65</td><td> 11.34</td><td> 10.19</td><td> 9.55</td><td> 8.02</td><td> 8.30</td><td> 12.04</td>

<td> 72</td><td> 11.21</td><td> 10.00</td><td> 9.10</td><td> 7.52</td><td> 9.52</td><td> 10.1 2</td><td> 12.43</td>

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 H1N1 Influenza Virus A swine type 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 conducted in a pretreatment test of eight concentrations of MSM. The virus performance reduction / improvement test and subsequent virus titration is performed in three duplicates. MSM inhibitory concentrations (IC₅₀ or IC₉₀ - the appropriate concentration of growth of or activity is inhibited by 50% or 90%) were also determined in this study.

Cytotoxicity of MSM was determined before

326 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). MSM concentrations of 16% to 8% were toxic to MDCK cells and completely destroyed cell monolayers. Concentrations of 6% to 0.5% do not produce visible cytotoxic effects. TC₅₀ (concentration at which the compound only 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.

Hosting cells. Madin Darby Canine Kidney Cells (MDCK [ATCC # CCL-34]), MRC-5 cells (Human Pulmonary Fibroblasts; [ATCC # CCL-171]), and Vero Cells (African Green Monkey Kidney [ATCC # CCL-81 ]) were maintained as monolayers in disposable cell culture laboratory equipment and were used for Antiviral Test of Pretreatment of the swine Influenza A H1N1 virus strain / California / 04/2009, Rhinovirus type 14 (ATCC # VR-284 ), and HSV-1 (ATCC # VR-260), respectively. Prior to

327 test, host cell cultures were seeded in the appropriate cell culture plates. Cell monolayers were confluent at 80 to 90% and less than 48 hours of age 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 = Phosphate Buffered Saline) and incubated with the following dilutions of a product: 16%, 14%, 12%, 10%, 8.0%, 6.0%, 1.0%, and 0.5% The incubation was 1 hour at 37 degrees ± 2 degrees C in a CO2 incubator. After incubation, the treated cells were coated with MM. Plates were incubated in a CO2 incubator for 3 days at 37 degrees ± 2 degrees C. Toxicity was monitored using an Inverted Compound Microscope. A cytotoxicity test performed as set out in the Study Protocol, shows that product concentrations 16% to 8% were toxic to MDCK cells and completely destroyed cell monolayers. Product concentrations 6% to 0.5% produce no visible cytotoxic effects. TC50 (concentration at which the compound, alone, kills 50% of cells not

328 infected) is determined to be approximately 7%.

A. Pre-Treatment Test. Test product raw material solution 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 were prepared (see Project Notes [Form No. 95-G-001] in Annex VI of this Final Report). MDCK, MRC-5 and Vero cell cultures were washed with PBS and incubated with the following product dilutions: 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0.5%. The incubation was 1 hour at 37 degrees ± 2 degrees C in a CO incubator₂ - After the incubation was completed approximately 300-1000 IU (infectious units) of each of the test viruses were added in the appropriate treated cells. The test was performed in three duplications. The plates were incubated in a CO incubator₂ 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 Prior Treatment Test. MDCK, MRC-5 and Vero cell cultures were washed with PBS and incubated with product dilution of 7% to 0.5%. Incubation was for 1 hour at 37 degrees ± 2 degrees C in a

329 CO incubator₂. After incubation, the treated cells were coated with MM. The plates were incubated in a CO incubator₂ for 6 days at the appropriate temperature for each virus. Toxicity was monitored using an Inverted Compound Microscope. The results of the cytotoxicity tests are presented in Table 50.

C. Virus Control. MDCK, MRC5 and Vero cell cultures were washed with PBS and incubated with MM. The incubation was for 1 hour at 37 degrees ± 2 degrees C in a CO incubator₂. After the incubation is completed approximately 300-1000 IU (infectious units) of each of the test viruses are added to the cells. Three duplicates of Virus Control were performed. The plates were incubated in a CO incubator₂ 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 made in MM and coated on cells

330 susceptible in four duplicates. The results of the virus performance reduction / improvement tests are presented in Tables 51 to 91.

Analysis of data. The virus population titer in cell cultures is expressed as -logio of the 50% titration endpoint for infectivity. To calculate the viral titer, - a 50% tissue culture infectious dose calculation (TCID50) was applied - the Quantal test (Spearman-Karber Method).

log TCID50 = 1 - d (s - 0.5)

Where:

= -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:

% reduction

TCID₅₀Test

TCID₅₀virus control

X100

1.3

TCID50 of virus population recovered from the test

331 and virus control is used to calculate 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 plate wells; 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: 1.16 log reduction (93.08% reduction) of Swine Influenza A H1N1 Virus; 2.50 log reduction (99.68% reduction) of Herpes Simplex Virus type 1 (HSV-1); 1.25 log reduction (94.38% reduction) of type 14 rhinovirus. MSM a concentration of 6% produces the following average reductions: 1.00 log reduction (90.00% reduction) of swine influenza A H1N1 virus; 1.00 log reduction (90.00% reduction) of HSV-1; 0.67 log reduction (78.62% reduction) of Rhinovirus type 14.

MSM at 5% concentration produces the following average reductions: 0.41 log reduction₁₀ (reduction of

332

61.10%) of Swine Influenza A H1N1 virus; 1.34 log reduction (95.43% reduction) HSV-1 reduction; 0.09 log (18.72% reduction) of Rhinovirus type 14. MSM at 4% concentration produces the following average reductions: 0.16 log reduction (30.82% reduction) of H1N1A Influenza virus of Porcine type; 1.59 log reduction (97.43% reduction) of HSV-1; 0.28 log reduction (47.52% reduction) of Rhinovirus type 14. MSM at a concentration of 3% produces the following average reduction reductions: 0.00 log (00.00% reduction) of Swine Influenza A H1N1 virus; 1.00 log reduction (90.00% reduction) of HSV-1; 0.11 log reduction (22.38% reduction) of Rhinovirus type 14. MSM at a concentration of 2% produces the following average reductions: 0.41 log reduction (61.10% reduction) of Swine Influenza A H1N1 virus; 0.84 log reduction (85.55% reduction) HSV-1 reduction; 0.42 log (61.98% reduction) of Rhinovirus type 14. MSM at 1% concentration produces the following average reductions: 0.25 log reduction (43.77% reduction) of Swine Influenza A H1N1 virus; 0.67 log reduction (78.62% reduction) of HSV-1; 0.14 log reduction (27.56% reduction) of Rhinovirus type 14. MSM at 0.5% concentration produces the following average reductions: 0.66 log reduction (78.12% reduction) of

333 Swine Influenza A H1N1 virus; 0.25 log reduction (43.77% reduction) of HSV-1; 0.40 log reduction (60.19% reduction) of Rhinovirus type 14.

Improvement / stimulation of virus infectivity is observed for Swine Influenza A H1N1 virus treated with 3% MSM. The average improvement in virus population was 0.17 log (32.39%). A total of three concentrations of MSM improves the infectivity of Rhinovirus type 14. MSM at 5% concentration produces an average of 0.053 log improvement (11.49%). Three percent MSM produces an average of 0.11 log₁₀ improvement (22.38%); and 1% MSM produces an average improvement of 0.11 log (22.38%). All three virus infectivity stimuli / improvements determined in this study were within the normal variation range for virus population and were not significant. An inhibitory concentration of MSM at which growth or activity is inhibited by 50% (IC50), is calculated using Nonlinear 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 Swine Influenza A H1N1 virus, the best fit value of MSM IC₅₀ It was 5,114 mM. IC₅₀ with 95% confidence interval varied from 0.008038 mM to 3253 mM. For HSV-1, the best fit value of MSM IC₅₀ was determined as 10.13 mM with an IC₅₀ Within the 95% confidence interval it varies from 7,144 mM to 14.37

334 mM. For Rhinovirus type 14, the best fit value of MSM IC50 was 38.16 mM. IC50 with 95% confidence interval was in the range of 13.07 mM to 111.4 mM. IC90 (1.0 log reduction) was determined experimentally for HSV-1 and Influenza A H1N1 Porcine type. However, due to interception of multiple concentrations of MSM with the 90% reduction axis, experimental IC90 values ​​cannot be considered accurate.

MSM tested at eight different concentrations against U-dose dose response curves produced by HSV-1, Influenza A H1N1 Porcine type and Rhinovirus. For example: MSM at 4% (1.00 log reduction₁₀) was more effective against HSV-1 than MSM at 6% (1.59 log reduction); 0.5% MSM (0.66 log reduction) was more or equally effective against Swine Influenza A H1N1 than 5% MSM (0.41 log reduction); 4% concentrations through 0.5% were more or equally effective against rhinovirus than MSM at 5%. It is possible, if more research is confirmed 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 logon. Tables 50 to 91 include the results for the aforementioned studies.

335

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

TABLE 50

Test product: Methylsulfonylmethane, lot # 0902951

Designation of Cytotoxicity of Test Product

<td>culture cell phone</td><td> 7%</td><td> 6%</td><td> 5%</td><td> 4%</td><td> 3%</td><td> 2%</td><td> 1%</td><td> 0.5%</td>

<td>Vero</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td>

<td>MDCK</td><td> ++</td><td> ++</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td>

<td>MRC-5</td><td> ++</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td><td> 00</td>

+ = CPE Present = CPE not detected

Tables 2 to 9 present the infectivity of Virus Control (TCID₅₀), the average infectivity (TCID50), and the reduction of logium and in percent in test of 20 previous treatment of the Test Product, Methylsulfonylmethane (Lot Number 0902951), and Influenza A H1N1 Swine type virus strain A / California / 04/2009 (CDC ID # 2009712047).

TABLE 51 Infectivity Reduction

Test product: Methylsulfonylmethane, 7% (lot #

336

0902951)

Virus: Influenza A H1N1 Swine Type strain

A / California / 04/2009 CDC ID # 2009712047

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

<td rowspan="2">Dilution is (- log)</td><td colspan="3">Virus control</td><td colspan="4">Test product</td><td rowspan="2">Cell control</td>

<td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td><td colspan="2">Rep. one</td><td>Rep. two</td><td>Rep. 3</td>

<td colspan="8"></td><td> 0000</td>

<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td colspan="2"> + +++</td><td> ++++</td><td> + + + +</td><td></td>

<td> -3</td><td> + 4- + +</td><td> ++ + +</td><td> ++ + +</td><td colspan="2"> ++++</td><td> ++++</td><td> + + + +</td><td></td>

<td> -4</td><td> ++++</td><td> ++++</td><td> + + + +</td><td colspan="2"> 00 + 0</td><td> 0000</td><td> 0 + 00</td><td></td>

<td> -5</td><td> 00 + 0</td><td> + 000</td><td> + 0 + 0</td><td colspan="2"> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td colspan="2"> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td colspan="2"> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>tcid₅₀</td><td>4,751 ogio</td><td>4.75 log</td><td>5.00 log</td><td colspan="2">3.75 log</td><td>3,501 ogio</td><td>3.75 log</td><td></td>

<td>Average TCID₅₀</td><td colspan="3">4.83 log</td><td colspan="4">3.67 log</td><td></td>

<td>Reduced n Log *</td><td colspan="3"></td><td>1.08 logw</td><td colspan="2">1.33 logw</td><td>1.08 log</td><td></td>

337

<td>Reduction Log Average</td><td></td><td colspan="3">1.16 logw</td><td></td>

<td>Percent from Reduction</td><td></td><td> 91 . 6 8%</td><td> 95.32 0, 0</td><td> 91.68%</td><td></td>

<td>Average Reduction Percent * *</td><td></td><td colspan="3"> 93.08%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log Reduction = Average TCID50 Virus Control

TCID50 Test Duplicate ** - Average% Reduction (calculated from log average reduction) = 100- (1 / TCID Reduction₅o) * lOO

TABLE 52 Infectivity Reduction

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

Virus: Influenza Ά H1N1 Swine Type strain

A / California / 04/2009 CDC ID # 2009712047

338

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

<td rowspan="2">Dilutions (- log)</td><td colspan="3">Virus control</td><td colspan="5">Test product</td><td rowspan="2">Control mobile</td>

<td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td><td>Rep. one</td><td colspan="2">Rep. two</td><td colspan="2">Rep. 3</td>

<td colspan="9"></td><td> 0000</td>

<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> + + + +</td><td colspan="2"> ++++</td><td colspan="2"> +++ +</td><td></td>

<td> -3</td><td> + + + +</td><td> + +++</td><td> +++ +</td><td> + + + +</td><td colspan="2"> ++++</td><td colspan="2"> + + + +</td><td></td>

<td> -4</td><td> ++++</td><td> ++++</td><td> ++ + +</td><td> 0 + 00</td><td colspan="2"> 000 +</td><td colspan="2"> + 00 +</td><td></td>

<td> -5</td><td> 00 + 0</td><td> + 000</td><td> + 0 + 0</td><td> 0000</td><td colspan="2"> 0000</td><td colspan="2"> 0000</td><td></td>

<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td colspan="2"> 0000</td><td colspan="2"> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td colspan="2"> 0000</td><td colspan="2"> 0000</td><td></td>

<td>TCID₅or</td><td>4.75 log</td><td>4.75 log</td><td>5.00 log</td><td>3.75 log</td><td colspan="2">3.75 log</td><td colspan="2">Four . 00 log</td><td></td>

<td>Average TCID50</td><td colspan="3">4.83 log</td><td colspan="5">3.67 log</td><td></td>

<td>Reduction Log*</td><td colspan="3"></td><td colspan="2">1.08 log</td><td colspan="2">1.08 logw</td><td>0.83 log</td><td></td>

<td>Reduction Log Average</td><td colspan="3"></td><td colspan="5">1.00 log</td><td></td>

339

<td>Percent reduction</td><td></td><td> 91.68%</td><td> 91.68%</td><td> 85.21%</td><td></td>

<td>Average Reduction Percent</td><td></td><td colspan="3"> 90.00%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log reduction = TCID average₅₀ Virus Control

TCID50 of the Test Duplicate ** - Average% Reduction (Calculated from the average log reduction) = 100- (1 / TCID₅₀ Reduction) * 100

TABLE 53

Infectivity 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

340

<td rowspan="14"> 5 10 15 20</td><td rowspan="2">Dilutions (- log)</td><td colspan="3">Virus control</td><td colspan="3">Test product</td><td rowspan="2">Cell control</td>

<td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td><td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td>

<td colspan="7"></td><td> 0000</td>

<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> +++ +</td><td> + + + +</td><td rowspan="8"></td>

<td> -3</td><td> + + + +</td><td> ++ + +</td><td> +++ +</td><td> ++++</td><td> + + + +</td><td> ++++</td>

<td> -4</td><td> + + + +</td><td> +++ +</td><td> ++++</td><td> +++ +</td><td> 0+++</td><td> + + + 0</td>

<td> -5</td><td> 00 + 0</td><td> +000</td><td> + 0 + 0</td><td> 0000</td><td> + 000</td><td> 0000</td>

<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td>

<td>tcid₅₀</td><td>4.75 log</td><td>4.75 log₁₀</td><td>5.00 log</td><td>4.50 logi₀</td><td>4,501 ogio</td><td>4,251 ogre</td>

<td>I averaged or TCID₅₀</td><td colspan="3">4.83 log</td><td colspan="3">4.42 log</td>

<td>Reduction ón Log *</td><td colspan="3"></td><td>0.33 log</td><td>0.33 log</td><td>0.58 log</td><td></td>

<td>I averaged 0 of Reduction ón Log</td><td colspan="3"></td><td colspan="3">0.41 log</td><td></td>

<td>Percent of</td><td colspan="3"></td><td> 53.2 3%</td><td>53.23 or. 0</td><td> 73.70 0. 0</td><td></td>

341

<td>Reduction</td><td></td><td></td><td></td>

<td>Average Rate Reduction 0 **</td><td></td><td> 61.10%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log Reduction = Average TCID50 Virus Control

TCTD₅₀ of the Test Duplicate ** - Average% Reduction (calculated from the average log reduction) = 100- (1 / TCID Reduction₅o) * lOO

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 Cell line

342 ATCC Hosts # CCL-34

<td rowspan="2">Dilutions | 'log)</td><td colspan="3">Virus control</td><td colspan="2">Product test</td><td>from</td><td rowspan="2">With- troll cell lar</td>

<td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td><td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td>

<td colspan="7"></td><td> 0000</td>

<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> +++ +</td><td> ++++</td><td> ++++</td><td></td>

<td> -3</td><td> +++ +</td><td> ++++</td><td> +++ +</td><td> +++ +</td><td> ++++</td><td> + +++</td><td></td>

<td> -4</td><td> ++++</td><td> ++++</td><td> +++ +</td><td> + +++</td><td> 0 +++</td><td> + +++</td><td></td>

<td> -5</td><td> 00 + 0</td><td> + 000</td><td> + 0+0</td><td> 00 + 0</td><td> 000 +</td><td> 000 +</td><td></td>

<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>TCID50</td><td>4,751 ogio</td><td>4.75 logi 0</td><td>5.00 logi 0</td><td>4.75 logi 0</td><td>4.50 logi 0</td><td>4,751 ogio</td><td></td>

<td>Average io TCID₅₀</td><td colspan="3">4.83 log</td><td> 4.67</td><td>log</td><td></td><td></td>

<td>Reduce ion Log*</td><td colspan="3"></td><td>0.08 logi 0</td><td>0.33 logw</td><td>0.08 log</td><td></td>

343

<td>Average Reduction Log</td><td></td><td colspan="3">0.16 log</td><td></td>

<td>By hundred of Reduction</td><td></td><td> 16.8 2%</td><td> 53.23 0 0</td><td> 16.8 2%</td><td></td>

<td>Average Percent of Reduction * *</td><td></td><td colspan="3"> 30.82%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log reduction = TCID average₅₀ Virus Control

TCID50 of Test Duplicate ** - Average% Reduction (Calculated from the average log reduction) = 100- (1 / TCID Reduction₅₀)*100

TABLE 55

Ineffectiveness Reduction

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

Virus: Influenza A H1N1 Swine Type strain

344

A / California / 04/2009 CDC ID # 2009712047

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

<td rowspan="2">Dilute nes (-logio)</td><td colspan="3">Virus control</td><td colspan="3">Product of test</td><td rowspan="2">Celu control -lar</td>

<td>Rep. 1</td><td>Rep. two</td><td>Rep. 3</td><td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td>

<td colspan="7"></td><td> 0000</td>

<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> +++ +</td><td> ++++</td><td></td>

<td> -3</td><td> +++ +</td><td> + + + +</td><td> + + + +</td><td> + + + +</td><td> + +++</td><td> + + + +</td><td></td>

<td> -4</td><td> ++ + +</td><td> + +++</td><td> + + + +</td><td> ++++</td><td> ++++</td><td> + + + +</td><td></td>

<td> -5</td><td> 00 + 0</td><td> + 000</td><td> + 0 + 0</td><td> 00 + +</td><td> + 00 +</td><td> 0 + + 0</td><td></td>

<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>TCID50</td><td>4,751o gio</td><td>4.75 log</td><td>5.00 log</td><td>5.00 log</td><td>5.00 log</td><td>5.00 log</td><td></td>

<td>I averaged OR TCID50</td><td colspan="3">4.83 log</td><td colspan="3">5.00 log</td><td></td>

<td>Reduction ón Log</td><td colspan="3"></td><td>0.00 log</td><td>0.00 log</td><td>0.00 log</td><td></td>

<td>Average 0 Reduction ón Log</td><td colspan="3"></td><td colspan="3">0.00 log</td><td></td>

345

<td>Percent from Reduction</td><td></td><td> 00.00%</td><td> 00.0 0%</td><td> 00.0 0%</td><td></td>

<td>Average Percent from Reduction</td><td></td><td colspan="3"> 00.00%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log Reduction - Average TCID50 Virus Control

TCID50 of the Test Duplicate ** - Average% Reduction (Calculated from the average log 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 ATCC host cell line # CCL-34

346

<td rowspan="2">Dilutions (-log₁₀)</td><td colspan="3">Virus control</td>

<td></td><td></td><td></td>

<td> -2</td><td>NT</td><td>NT</td><td>NT</td>

<td> -3</td><td> +++ +</td><td> ++++</td><td> ++ + +</td>

<td> -4</td><td> + +++</td><td> + +++</td><td> ++ + +</td>

<td> -5</td><td> 00 + 0</td><td> + 000</td><td> + 0 + 0</td>

<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td>

<td>tcid₅₀</td><td>4,751og 10</td><td>4.75 logw</td><td>5.00 log</td>

<td>TCID average₅that</td><td colspan="3">4.83 achievement</td>

<td>Log reduction *</td><td></td><td></td><td></td>

<td>Average Log reduction</td><td></td><td></td><td></td>

<td>Percent of Reduction</td><td></td><td></td><td></td>

<td>Average By hundred of Reduction**</td><td></td><td></td><td></td>

Cent.

347

<td rowspan="2">Dilutions (-logi₀)</td><td colspan="3">Test product</td><td>Cell control</td>

<td></td><td></td><td></td><td> 0000</td>

<td> -2</td><td> ++ + +</td><td> ++ + +</td><td> + +++</td><td></td>

<td> -3</td><td> ++++</td><td> +++ +</td><td> +++ +</td><td></td>

<td> -4</td><td> + + + +</td><td> ++++</td><td> + 000</td><td></td>

<td> -5</td><td> 0000</td><td> 000 +</td><td> 0 + 00</td><td></td>

<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>tcid₅₀</td><td>4,501ogio</td><td>4,751og₁₀</td><td>4.001og₁₀</td><td></td>

<td>TCID average₅₀</td><td>4.42 log</td><td></td><td></td><td></td>

<td>Log reduction *</td><td>0.33 log</td><td>0.08 log</td><td>0.83 log</td><td></td>

<td>Average Log reduction</td><td>0.41 log</td><td></td><td></td><td></td>

<td>Percent of Reduction</td><td> 53.23%</td><td> 16.82%</td><td> 85.21%</td><td></td>

<td>Average By hundred of Reduction**</td><td colspan="3"> 61.10%</td><td></td>

+ = CPE Present

CPE not detected

348

NT = Not Tested

Rep = Duplicate * - Log reduction = TCID average₅₀ Virus Control

TCID50 of Test Duplicate ** - Average% Reduction (Calculated from average log 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 ATCC host cell line # CCL-34

<td>Dilu-</td><td colspan="3">Virus control</td><td colspan="2">Product of</td><td>test</td><td>I-</td>

<td>clones</td><td>Rep.</td><td>Rep.</td><td>Rep.</td><td>Rep.</td><td>Rep.</td><td>Rep.</td><td>troll</td>

<td> (-</td><td> 1</td><td> 2</td><td> 3</td><td> 1</td><td> 2</td><td> 3</td><td>celu</td>

<td>log)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>-lar</td>

<td colspan="7"></td><td> 0000</td>

<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> +++ +</td><td></td>

<td> -3</td><td> ++++</td><td> ++++</td><td> +++ +</td><td> ++++</td><td> ++++</td><td> + +++</td><td></td>

<td> -4</td><td> ++++</td><td> +++ +</td><td> ++++</td><td> ++0 +</td><td> ++++</td><td> ++++</td><td></td>

<td> -5</td><td> 00 + 0</td><td> + 000</td><td> + 0 + 0</td><td> 00 + 0</td><td> 000 +</td><td> 0000</td><td></td>

<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

349

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>TCID50</td><td>4,751 ogio</td><td>4.75 log</td><td>5.00 logw</td><td>4,501 ogio</td><td>4.75 log</td><td>4,501 ogio</td><td></td>

<td>TCID average₅₀</td><td colspan="3">4.83 log</td><td colspan="3">4,581ogio</td><td></td>

<td>Reduction Log*</td><td colspan="3"></td><td>0.33 log₁₀</td><td>0.08 log</td><td>0.33 log</td><td></td>

<td>Log Reduction Average</td><td colspan="3"></td><td colspan="3">0.25 log</td><td></td>

<td>Percent Reduction</td><td colspan="3"></td><td>53.23 Q. OR</td><td> 16.8 2%</td><td> 53.23 0 0</td><td></td>

<td>Average Percent Reduc tion</td><td colspan="3"></td><td colspan="3"> 43.77%</td><td></td>

+ = CPE Present

CPE not detected

350

NT = Not Tested

Rep = Duplicate * - Log reduction = TCID average₅₀ of TCID50 Virus Control of Test Duplicate ** - Average% Reduction (calculated from average log reduction₁₀) = 100- (1 / TCID reduction₅₀) *100

TABLE 58

Infectivity Reduction

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

Virus: Swine Type Influenza A H1N1 strain

A / California / 04/2009 CDC ID # 2009712047

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

<td>Dilutions</td><td>Control of</td><td>virus</td><td></td>

<td>(-logio)</td><td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td>

<td> -2</td><td>NT</td><td>NT</td><td>NT</td>

<td> -3</td><td> ++++</td><td> ++ + +</td><td> +++ +</td>

<td> -4</td><td> ++++</td><td> ++ + +</td><td> + + + +</td>

<td> -5</td><td> 00 + 0</td><td> + 000</td><td> + 0 + 0</td>

<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td>

<td>tcid₅₀</td><td>4,751ogi₀</td><td>4.75 log₁₀</td><td>5.00 log</td>

<td>TCID average₅₀</td><td colspan="3">4.83 log</td>

351

<td>Log reduction *</td><td></td><td></td><td></td>

<td>Average Log reduction</td><td></td><td></td><td></td>

<td>Percent of Reduction</td><td></td><td></td><td></td>

<td>Average By hundred of Reduction**</td><td></td><td></td><td></td>

Cont.

<td rowspan="2">Dilutions (-logio)</td><td colspan="3">Test product</td><td>With- troll cell lar</td>

<td>Rep. 1</td><td>Rep. 2</td><td>Rep. 3</td><td> 0000</td>

<td> -2</td><td> + + + +</td><td> ++++</td><td> ++ + +</td><td></td>

<td> -3</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>

<td> -4</td><td> + 000</td><td> ++++</td><td> + + + 0</td><td></td>

<td> -5</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -6</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>TCID₅₀</td><td>3.7 51og</td><td>Four . SOlogio</td><td>4,251gio</td><td></td>

<td>TCID average₅₀</td><td>4.17 log</td><td></td><td></td><td></td>

<td>Log reduction *</td><td>1.08 log₁₀</td><td>0.33 log</td><td>0.58 log</td><td></td>

352

<td>Average Log reduction</td><td colspan="3">0.66 logi₀</td><td></td>

<td>Percent of Reduction</td><td> 91.68%</td><td> 53.23%</td><td> 73.70%</td><td></td>

<td>Average By hundred of Reduction**</td><td colspan="3"> 78.12%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log reduction = TCID average₅₀ Virus Control

TCID50 of the Test Duplicate ** - Average% Reduction (Calculated from the average log reduction) = 100- (1 / TCID₅₀ Reduction) * 100

Tables 59 to 67 show infectivity of virus control (TCID50), the average infectivity (TCID₅₀), and log and reductions in percent that are observed in the pretreatment 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 #

353

0902951)

Virus: Herpes Simplex Virus strain HF ATCC # VR260

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

<td rowspan="2">I Dilutions (-logio)</td><td colspan="3">Virus control</td><td colspan="2">Product test</td><td>from</td><td rowspan="2">Celu control -lar</td>

<td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td><td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td>

<td colspan="7"></td><td> 0000</td>

<td> -1</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> + + + +</td><td> ++++</td><td></td>

<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> +++ +</td><td> ++++</td><td> + +++</td><td></td>

<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> + +++</td><td> + 000</td><td> 0 + 0 +</td><td></td>

<td> -4</td><td> + + + +</td><td> + + + +</td><td> ++++</td><td> 0000</td><td> 00 + 0</td><td> 0000</td><td></td>

<td> -5</td><td> + + + +</td><td> ++ + 0</td><td> + + + +</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -6</td><td> 0000</td><td> 00 + 0</td><td> + 0 + 0</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td>NT</td><td>NT</td><td>NT</td><td></td>

<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td>NT</td><td>NT</td><td>NT</td><td></td>

<td>TCID₅₀</td><td>5,501 ogio</td><td>5.50 log</td><td>6.00 log</td><td>3.50 log</td><td>3.00 log</td><td>3.00 log</td><td></td>

<td>Average tcid₅₀</td><td colspan="3">5,671ogio</td><td> 3.17</td><td>log</td><td></td><td></td>

354

<td>Reduction Log*</td><td></td><td>2.17 log</td><td>2.67 log</td><td colspan="2">2.67 log</td><td></td>

<td>Average Reduction Log</td><td></td><td colspan="4">2.50 log</td><td></td>

<td>Percent from Reduction</td><td></td><td> 99.3 2%</td><td colspan="2"> 99.79%</td><td> 99 . 7 9%</td><td></td>

<td>Average Percent from Reduction* *</td><td></td><td colspan="4"> 99.68%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log reduction = TCID average₅₀ Virus Control

TCID50 of the Test Duplicate ** - Average% Reduction (Calculated from the average log reduction) = 100- (1 / TCID₅₀ Reduction) * 100

TABLE 60

Infectivity Reduction

Test product: Methylsulfonylmethane, 6% (lot

355 # 0902951)

Virus: Herpes Simplex Virus strain HF ATCC # VR-2 60

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

<td>I Diluci ones</td><td colspan="2">Control of</td><td>virus</td><td colspan="2">Product test</td><td>from</td><td>With- troll</td>

<td> (-</td><td>Rep</td><td>Rep.</td><td>Rep.</td><td>Rep.</td><td>Re</td><td>Rep.</td><td>cell</td>

<td>log)</td><td> . 1</td><td> 2</td><td> 3</td><td> 1</td><td>P- two</td><td> 3</td><td>lar</td>

0000

<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> ++ + +</td><td> ++++</td><td> ++++</td><td></td>

<td> -4</td><td> + + + +</td><td> ++++</td><td> + + + +</td><td> + + + +</td><td> + +++</td><td> + + + 0</td><td></td>

<td> -5</td><td> + + + +</td><td> +++0</td><td> + + + +</td><td> 000 +</td><td> 0 + 00</td><td> + 000</td><td></td>

<td> -6</td><td> 0000</td><td> 00 + 0</td><td> + 0 + 0</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>TCID fifty</td><td>5.50 log</td><td>5.50 log</td><td>6.00 log</td><td>4,751 ogio</td><td>4.75 logw</td><td>4.50 logw</td><td></td>

<td>Average TCID fifty</td><td colspan="3">5.67 log</td><td colspan="3">4.67 log</td><td></td>

356

<td>Reduction Log*</td><td></td><td>0.92 achievement</td><td>0.92 achievement</td><td>one . 17 achievement</td><td></td>

<td>Average Log reduction</td><td></td><td colspan="3">1.00 achievement</td><td></td>

<td>Percent of Reduction</td><td></td><td> 87.9 8%</td><td> 87.9 8%</td><td> 93.2 4%</td><td></td>

<td>Average By hundred of Reduction**</td><td></td><td colspan="3"> 90.00%</td><td></td>

+ = CPE Present = CPE not detected

NT - Not Tested

Rep = Duplicate * - Log reduction = TCID average₅or Virus Control

TCID₅₀ strain Duplicate test ** - Average% Reduction (Calculated from average achievement reduction) = 100- (1 / TCID50 Reduction) * 100

TABLE 61

Infectivity Reduction

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

Virus: Herpes Simplex Virus strain HF ATCC # VR260

Host cell line: Vero Line

357 host cells ATCC # CCL-81

<td rowspan="2">Dilution is (- log)</td><td colspan="3">Virus control</td><td colspan="3">Product of test</td><td rowspan="2">Contr ol cellul ar</td>

<td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td><td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td>

<td colspan="7"></td><td> 0000</td>

<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>

<td> -4</td><td> ++++</td><td> +++ +</td><td> ++++</td><td> ++0 +</td><td> ++++</td><td> 000 +</td><td></td>

<td> -5</td><td> +++ +</td><td> +++0</td><td> +++ +</td><td> 0 + 00</td><td> 0000</td><td> 0000</td><td></td>

<td> -6</td><td> 0000</td><td> 00+0</td><td> + 0 + 0</td><td> 000 +</td><td> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>TCID50</td><td>5.50 log</td><td>5.50 logi 0</td><td>6.00 logi 0</td><td>4.75 logi 0</td><td>4.50 logi 0</td><td>3,751 ogio</td><td></td>

<td>Average TCID₅₀</td><td colspan="3">5,671ogio</td><td colspan="3">4.33 log</td><td></td>

<td>Reduced n Log *</td><td colspan="3"></td><td>0.92 logi 0</td><td>1.17 logi 0</td><td>1.92 log</td><td></td>

<td>Average Log reduction</td><td colspan="3"></td><td colspan="3">1.34 log</td><td></td>

358

<td>Percent of Reduction</td><td></td><td> 87.9 8%</td><td>C \] in _or\ o Oh</td><td> 98.80</td><td></td>

<td>Average By hundred of * * Reduction</td><td></td><td colspan="3"> 95.43%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log Reduction = Average TCID50 Virus Control

TCID50 of the Test Duplicate ** - Average% Reduction (Calculated from the average log reduction) = 100- (1 / TCID₅₀ Reduction) * 100

TABLE 62

Infectivity Reduction

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

Virus: Herpes Simplex Virus strain HF ATCC # VR260

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

359

<td rowspan="2">Dilutions logw)</td><td colspan="3">Control of virus</td><td colspan="3">Test product</td><td rowspan="2">Control mobile</td>

<td>Rep. one</td><td>Rep . two</td><td>Rep. 3</td><td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td>

<td colspan="7"></td><td> 0000</td>

<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> + +++</td><td> ++++</td><td> +++ +</td><td></td>

<td> -4</td><td> + + + +</td><td> + + + +</td><td> + + + +</td><td> 00 + 0</td><td> + 0 + 0</td><td> + 0 + 0</td><td></td>

<td> -5</td><td> + + + +</td><td> + + + 0</td><td> + + + +</td><td> 0000</td><td> + 00 +</td><td> 0000</td><td></td>

<td> -6</td><td> 0000</td><td> 00 + 0</td><td> + 0 + 0</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 000 0</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -8</td><td> 0000</td><td> 000 0</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>TCID₅or</td><td>5.50 logi 0</td><td>5.5 01o 9io</td><td>6.00 logi 0</td><td>3.75 log</td><td>4,501 ogio</td><td>4,001 ogio</td><td></td>

<td>I averaged 0 tcid₅₀</td><td colspan="3">5,671ogio</td><td colspan="3">4.08 log</td><td></td>

<td>Reduction ón Log *</td><td colspan="3"></td><td>1.92 log</td><td>1.17 log</td><td>1.67 log</td><td></td>

360

<td>Average Reduction Log</td><td></td><td colspan="3">1.59 log</td><td></td>

<td>Percent from Reduction</td><td></td><td> 98.8 0%</td><td>93.24 Q, OR</td><td>97.86 Or 0</td><td></td>

<td>Average Percent from Reduction**</td><td></td><td colspan="3"> 97.43%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log Reduction = Average TCID50 Virus Control

TCID50 of the Test Duplicate ** - Average% Reduction (Calculated from the average log 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 Line

361 host cells ATCC # CCL-81

<td rowspan="2">Dilution is (- logm)</td><td colspan="2">Control virus</td><td>from</td><td colspan="2">Product test</td><td>from</td><td rowspan="2">Control mobile</td>

<td>Rep. one</td><td>Rep . two</td><td>Rep . 3</td><td>Rep . one</td><td>Rep . two</td><td>Rep. 3</td>

<td colspan="7"></td><td> 0000</td>

<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> +++</td><td> +++</td><td> +++ +</td><td></td>

<td></td><td></td><td></td><td></td><td> +</td><td> +</td><td></td><td></td>

<td> -4</td><td> +++ +</td><td> + + +</td><td> +++</td><td> 00 +</td><td> +++</td><td> ++++</td><td></td>

<td></td><td></td><td> +</td><td> +</td><td> +</td><td> +</td><td></td><td></td>

<td> -5</td><td> +++ +</td><td> + + +</td><td> +++</td><td> 00 +</td><td> 000</td><td> 00++</td><td></td>

<td></td><td></td><td> 0</td><td> +</td><td> 0</td><td> 0</td><td></td><td></td>

<td> - 6</td><td> 0000</td><td> 00 +</td><td> + 0 +</td><td> 000</td><td> 000</td><td> 0000</td><td></td>

<td></td><td></td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td></td><td></td>

<td> -7</td><td> 0000</td><td> 000</td><td> 000</td><td> 000</td><td> 000</td><td> 000 +</td><td></td>

<td></td><td></td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td></td><td></td>

<td> -8</td><td> 0000</td><td> 000</td><td> 000</td><td> 000</td><td> 000</td><td> 0000</td><td></td>

<td></td><td></td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td></td><td></td>

<td>tcid₅₀</td><td> 5.50</td><td> 5.5</td><td> 6.0</td><td> 4.2</td><td> 4.5</td><td> 5.251</td><td></td>

<td></td><td>logi</td><td>01o</td><td>01o</td><td>51st</td><td>01o</td><td>ogio</td><td></td>

<td></td><td> 0</td><td>gio</td><td>gio</td><td>gio</td><td>gio</td><td></td><td></td>

<td>Average</td><td colspan="2">5,671ogio</td><td></td><td> 4.67</td><td>logic</td><td></td><td></td>

<td>tcid₅₀</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

362

<td>Reduction Log*</td><td rowspan="4"></td><td>1.4 2 log 10</td><td>1.17 log</td><td>0.42 log</td><td></td>

<td>Average Log reduction</td><td colspan="3">1.00 log</td><td></td>

<td>Percent of Reduction</td><td> 96. 20%</td><td> 93.2 4%</td><td>61.98 O. 0</td><td></td>

<td>Average By hundred of Reduction**</td><td colspan="3"> 90.00%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

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

TABLE 64

Infectivity Reduction

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

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

363

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

<td rowspan="2">Dilutions (-logio)</td><td colspan="4">Virus control</td><td colspan="4">Product of test</td><td rowspan="2">With- troll celu -lar</td>

<td>Rep. one</td><td>Rep. two</td><td colspan="2">Rep. 3</td><td>Rep . one</td><td colspan="2">Rep. two</td><td>Rep. 3</td>

<td colspan="9"></td><td> 0000</td>

<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td colspan="2"> ++++</td><td colspan="2"> + +++</td><td> + +++</td><td></td>

<td> -4</td><td> + + + +</td><td> ++++</td><td> ++ + +</td><td colspan="2"> ++++</td><td colspan="2"> ++ + +</td><td> ++++</td><td></td>

<td> -5</td><td> + + + +</td><td> +++0</td><td> + + + +</td><td colspan="2"> 0+++</td><td colspan="2"> 0000</td><td> 00 + 0</td><td></td>

<td> -6</td><td> 0000</td><td> 00 + 0</td><td> + 0 + 0</td><td colspan="2"> 0000</td><td colspan="2"> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td colspan="2"> 0000</td><td colspan="2"> 0000</td><td> 0000</td><td></td>

<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td colspan="2"> 0000</td><td colspan="2"> 0000</td><td> 0000</td><td></td>

<td>TCIDso</td><td>5.50 log</td><td>5.50 logi 0</td><td>6.00 icgio</td><td colspan="2">5.25 log</td><td colspan="2">4.50 log</td><td>4,751 ogio</td><td></td>

<td>Average TCID₅or</td><td colspan="3">5,671ogio</td><td colspan="5">4.83 log</td><td></td>

<td>Reduction Log</td><td colspan="3"></td><td colspan="3">0.42 log</td><td>1.1 7 log 10</td><td>0.92 log</td><td></td>

364

<td>Average Reduction Log</td><td colspan="2"></td><td colspan="3">0.84 log</td><td></td>

<td>Percent from Reduction</td><td colspan="2"></td><td> 61.98 0 0</td><td> 93. 24%</td><td>87. 98 Q. 0</td><td></td>

<td>Average Percent from Reduction</td><td></td><td></td><td colspan="3"> 85.55%</td><td></td>

+ - CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log reduction = TCID average₅₀ Virus Control

TCID50 of the Test Duplicate ** - Average% Reduction (Calculated from the average log reduction) = 10 0- (1 / TCID₅q Reduction) * 100

TABLE 65

Infectivity Reduction

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

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

365 host cells ATCC # CCL-81

<td rowspan="2">Dilutions (-log ™)</td><td colspan="4">Virus control</td><td colspan="4">Product of test</td><td rowspan="2">With- troll cell lar</td>

<td>Rep. one</td><td>Rep. two</td><td colspan="2">Rep. 3</td><td colspan="2">Rep. one</td><td>Rep. two</td><td>Rep. 3</td>

<td colspan="9"></td><td> 0000</td>

<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td colspan="3"> ++++</td><td> +++ +</td><td> + +++</td><td></td>

<td> -4</td><td> ++++</td><td> ++++</td><td> ++++</td><td colspan="3"> ++++</td><td> ++++</td><td> +++ +</td><td></td>

<td> -5</td><td> ++++</td><td> +++0</td><td> ++++</td><td colspan="3"> 00++</td><td> 00++</td><td> 00++</td><td></td>

<td> -6</td><td> 0000</td><td> 00 + 0</td><td> + 0+0</td><td colspan="3"> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td colspan="3"> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td colspan="3"> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>TCID₅₀</td><td>5.50 log</td><td>5.50 log</td><td>6.00 log</td><td colspan="3">5,001 ogio</td><td>5.00 logw</td><td>5.00 logi 0</td><td></td>

<td>Average tcid₅₀</td><td colspan="3">5,671ogio</td><td colspan="5">5.00 log</td><td></td>

<td>Reduction Log</td><td colspan="3"></td><td colspan="2">0.67 log</td><td colspan="2">0.67 log_w</td><td>0.67 logi 0</td><td></td>

<td>Average Reduction Log</td><td colspan="3"></td><td colspan="5">0.67 log</td><td></td>

366

<td>Percent reduction</td><td></td><td> 78.6 2%</td><td> 78.62%</td><td> 78. 62%</td><td></td>

<td>Average By hundred of Reduction</td><td></td><td colspan="3"> 78.62%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log reduction = TCID average₅₀ Virus Control

TCID50 of the Test Duplicate ** - Average% Reduction (Calculated from the average log reduction) = 100- (1 / TCIDso Reduction) * 100

TABLE 66

Infectivity Reduction

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

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

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

367

<td>Dilutions</td><td colspan="2">Control of</td><td>virus</td><td colspan="2">Product test</td><td>EC</td><td>With- troll</td>

<td>log)</td><td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td><td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td><td>celu -lar</td>

<td colspan="7"></td><td> 0000</td>

<td> -3</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> + + + +</td><td> ++++</td><td></td>

<td> -4</td><td> +++ +</td><td> + + + +</td><td> + + + +</td><td> ++++</td><td> + + + +</td><td> ++++</td><td></td>

<td> -5</td><td> ++++</td><td> + + + 0</td><td> + + + +</td><td> +++ +</td><td> ++0 +</td><td> + + 0 +</td><td></td>

<td> -6</td><td> 0000</td><td> 00 + 0</td><td> + 0 + 0</td><td> 0000</td><td> 0000</td><td> 000 +</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -8</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>TCID₅₀</td><td>5.50 logw</td><td>5.50 log</td><td>6.00 log</td><td>5.50 logw</td><td>5.25 log</td><td>5.50 logw</td><td></td>

<td>Average TCID50</td><td colspan="3">5,671ogio</td><td> 5.42</td><td>log</td><td></td><td></td>

<td>Reduction Log</td><td></td><td></td><td></td><td>0.17 log</td><td>0.42 log</td><td>0.17 log</td><td></td>

<td>Average Reduction Log</td><td></td><td></td><td></td><td> 0.25</td><td>log</td><td></td><td></td>

<td>Percent of reduction</td><td></td><td></td><td></td><td> 32.3 9%</td><td> 61.9 8%</td><td> 32.3 9%</td><td></td>

<td>Average By hundred of Reduction</td><td></td><td></td><td></td><td> 43.77</td><td> 2. 0</td><td></td><td></td>

368 + = CPE Present = CPE not detected

NT = Not Tested

Rep = Duplicate * - Log Reduction = Average TCID50 Virus Control

TCID₅₀ of Test Duplicate ** - Average% Reduction (Calculated from the average log reduction) = 100- (1 / TCID50 Reduction) * 100

TABLE 67

Infectivity Reduction

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

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

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

<td rowspan="2">Dilutions (-logio)</td><td colspan="2">Control of</td><td>virus</td><td colspan="2">Product test</td><td>from</td><td rowspan="2">Contr ol cellul ar</td>

<td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td><td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td>

<td colspan="7"></td><td> 0000</td>

<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> +++ +</td><td> ++ + +</td><td></td>

<td> -3</td><td> + +++</td><td> ++++</td><td> + +++</td><td> ++++</td><td> ++++</td><td> + +++</td><td></td>

<td> -4</td><td> + +++</td><td> ++++</td><td> ++++</td><td> + + 0 +</td><td> +++ +</td><td> ++ + +</td><td></td>

<td> -5</td><td> ++++</td><td> +++ +</td><td> +++ +</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

369

<td> - 6</td><td> 0000</td><td> 0000</td><td> 0+0 +</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td> -7</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td></td>

<td>tcid₅₀</td><td>5.50 logw</td><td>5.50 logw</td><td>6.00 log</td><td>4.25 log</td><td>4.50 lodge</td><td>4.50 logi 0</td><td></td>

<td>Average TCID₅₀</td><td colspan="3">5.67 log</td><td colspan="3">4.42 log</td><td></td>

<td>Reduction Log</td><td colspan="3" rowspan="4"></td><td>1.42 log</td><td>1.17 lodge</td><td>1.17 logi 0</td><td></td>

<td>Average Reduction Log</td><td colspan="3">1.25 log</td><td></td>

<td>Percent from Reduction</td><td> 96.2 0%</td><td> 93.2 4%</td><td> 93.2 4%</td><td></td>

<td>Average Percent from Reduction</td><td colspan="3"> 94.38%</td><td></td>

+ = CPE Present = CPE not detected

NT = Not Tested

370

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

Tables 68 to 74 present the infectivity of virus control (TCID₅₀), the average infectivity (TCID50), and log and percent reductions 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)

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

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

<td rowspan="2">1 dilutions (- log)</td><td colspan="2">Control of</td><td>virus</td><td colspan="2">Product test</td><td>from</td><td rowspan="2">With- troll cell lar</td>

<td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td><td>Rep. one</td><td>Rep. two</td><td>Rep. 3</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0000</td>

<td> -2</td><td>NT</td><td>NT</td><td>NT</td><td> ++++</td><td> ++++</td><td> ++++</td><td></td>

371

<td> -3</td><td> + +++</td><td> + + + +</td><td> ++++</td><td> ++++</td><td colspan="2"> + + + +</td><td> ++ + +</td><td></td>

<td> -4</td><td> ++++</td><td> + + + +</td><td> ++++</td><td> ++++</td><td colspan="2"> ++ + +</td><td> + 4- + 4-</td><td></td>

<td> -5</td><td> + + + +</td><td> +++ +</td><td> ++++</td><td> + 000</td><td colspan="2"> + 0 + 0</td><td> + 0 + +</td><td></td>

<td> -6</td><td> 0000</td><td> 0000</td><td> 0 + 0 +</td><td> 0000</td><td colspan="2"> 0000</td><td> 0000</td><td></td>

<td> -1</td><td> 0000</td><td> 0000</td><td> 0000</td><td> 0000</td><td colspan="2"> 0000</td><td> 0000</td><td></td>

<td>TCID₅₀</td><td>5.50 log</td><td>5.50 log</td><td>6.00 log</td><td>4.75 log</td><td colspan="2">5.00 log</td><td>5,251 ogio</td><td></td>

<td>Average TCID₅₀</td><td colspan="3">5.67 log</td><td colspan="4">5.00 log</td><td></td>

<td>Reduction Log</td><td colspan="3" rowspan="2"></td><td>0.92 logi₀</td><td colspan="2">0.67 log</td><td>0.42 log</td><td></td>

<td>Average Reduction Log</td><td colspan="4">0.67 log</td><td></td>