US 20170 189457A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0189457 A1 Farmer et al. (43) Pub. Date: Jul. 6, 2017
(54) PROBOTC SPORTS NUTRITION Publication Classification COMPOSITIONS (51) Int. Cl. A6II 35/742 (2006.01) (71) Applicant: Ganeden Biotech, Inc., Mayfield A6II 3L/202 (2006.01) Heights, OH (US) A23K2O/It (2006.01) A23K2O/58 (2006.01) (72) Inventors: Sean Farmer, North Miami Beach, FL A23K 50/40 (2006.01) (US); David Keller, Beachwood, OH A23K 20/47 (2006.01) (US); Andrew R. Lefkowitz, Mayfield A23K2O/I 74 (2006.01) Heights, OH (US) A23K 20/20 (2006.01) A638/0 (2006.01) (21) Appl. No.: 15/466,037 A23K 50/20 (2006.01) (52) U.S. Cl. CPC ...... A61K 35/742 (2013.01); A61K 38/018 (22) Filed: Mar. 22, 2017 (2013.01); A61 K3I/202 (2013.01); A23K 20/10 (2016.05); A23K 50/20 (2016.05); A23K 50/40 (2016.05); A23K 20/147 (2016.05): Related U.S. Application Data A23K 20/174 (2016.05); A23K 20/20 (63) Continuation of application No. 13/434.401, filed on (2016.05); A23K 20/158 (2016.05) Mar. 29, 2012. (57) ABSTRACT (60) Provisional application No. 61/469.924, filed on Mar. The present application relates to nutritional compositions 31, 2011. comprising lactic acid-producing bacteria. US 2017/01894.57 A1 Jul. 6, 2017
PROBOTC SPORTS NUTRITION race horse, for which an increase in muscle development or COMPOSITIONS strength is desired. The compositions are also useful to confer clinical benefit to individuals that are suffering from RELATED APPLICATIONS or at risk of developing a muscle wasting condition, e.g., 0001. This application is a continuation application of cachexia, as a result of disease Such as cancer or infection. U.S. Ser. No. 13/434,401, filed Mar. 29, 2012, which claims 0007. The invention provides a composition comprising a the benefit of priority to U.S. Ser. No. 61/469,924, filed Mar. sports nutrition composition and an isolated Bacillus coagul 31, 2011, the contents of each of which are incorporated lans spore (e.g., in an amount of 1x10° to 1x10" colony herein by reference in their entireties. forming units (CFU) of Bacillus coagulans per unit dose. For example, the isolated Bacillus coagulans comprise FIELD OF THE INVENTION between about 0.000001% to about 50% by weight of the 0002 The present application relates to sports nutrition composition, e.g., about 1%, about 10%, about 20%, about compositions comprising lactic acid-producing bacteria. 30%, about 40%, or about 50% by weight of the composi tion. For example, the isolated Bacillus coagulans comprise BACKGROUND OF THE INVENTION between about 0.00001% and about 25% by weight of the composition, e.g., about 1%, about 2%, about 3%, about 4%, 0003. The gastrointestinal microflora plays a number of about 5%, about 6%, about 7%, about 8%, about 9%, about Vital roles in maintaining gastrointestinal tract function and 10%, about 11%, about 12%, about 13%, about 14%, about overall physiological health. The growth and metabolism of 15%, about 16%, about 17%, about 18%, about 19%, about the many individual bacterial species inhabiting the gastro 20%, about 21%, about 22%, about 23%, about 24%, or intestinal tract depend upon the Substrates available to them, about 25% by weight of the composition. In one example, most of which are derived from the diet. one unit dose, e.g., one serving, comprises one billion CFU 0004 Probiotic organisms are non-pathogenic, non-toxi Bacillus coagulans and 20-25 grams of protein (e.g., 23 genic microorganisms that are beneficial to the host organ grams of whey protein) in 30 grams of powder. The balance ism. Since probiotics do not generally permanently colonize of the composition optionally comprises nutritionally inac the host, they are typically ingested regularly for health tive ingredients such as excipients, fillers, preservatives or promoting properties to persist. nutritionally active ingredients such as vitamins or minerals. SUMMARY OF THE INVENTION 0008. The invention also provides bacterial species including Bacillus coagulans, e.g., Bacillus coagulans ham 0005. The invention is based on the discovery that lactic mer, preferably Bacillus coagulans hammer Strain Acces acid-producing bacteria, particularly Bacillus coagulans, sion No. ATCC 31284, or one or more strains derived from improve the absorption of protein compositions as well as Bacillus coagulans hammer strain Accession No. ATCC micronutrient compositions. The microorganisms, e.g., 31284 (e.g., ATCC Numbers: GBI-20, ATCC Designation spores remain viable, retain their beneficial probiotic prop Number PTA-6085; GBI-30 or BC, ATCC Designation erties, and increase metabolism and/or absorption of protein Number PTA-6086; and GBI-40, ATCC Designation Num in sports nutrition compositions. Unlike other probiotic ber PTA-6087; see U.S. Pat. No. 6,849.256 to Farmer). bacteria that die in the stomach or small intestine, the probiotic organisms described herein, e.g., Bacillus coagul 0009 Optionally, the isolated Bacillus coagulans is in the lans strain GBI-30 or BC, ATCC Designation Number form of a spore. Alternatively, the isolated Bacillus coagul PTA-6086, germinate in the stomach and/or small intestine. lans is in the form of a vegetative cell. In another aspect, the Accordingly, the invention describes probiotic sports nutri isolated Bacillus coagulans is in the form of a mixture of tion compositions. The invention provides an isolated Bacil vegetative cells and spores. The Bacillus coagulans is pre lus coagulans bacterium (e.g., a spore) in sports nutrition dominantly in spore form, e.g., about 75%, about 80%, compositions. In some cases, the composition comprises at about 85%, about 90%, about 95%, about 96%, about 97%, least 15% protein. For example, the composition comprises about 98%, about 99%, or about 100% spores. Alternatively, at least 15%, at least 20%, at least 25%, at least 30%, at least the Bacillus coagulans is predominantly in vegetative form, 35%, at least 40%, at least 45%, at least 50%, at least 55%, e.g., about 75%, about 80%, about 85%, about 90%, about at least 60%, at least 65%, at least 70%, at least 75%, at least 95%, about 96%, about 97%, about 98%, about 99%, or 80%, at least 85%, at least 90%, at least 95%, at least 98%, about 100% vegetative cells. at least 99% or more of a purified or processed protein (e.g., 0010 Provided is an isolated Bacillus coagulans and a protein extracted from a food-stuff) and a Bacillus coagul sports nutrition composition. In some cases, the composi lans spore. tions described herein comprise a large amount of calories 0006. The compositions described herein are suitable for per unit dose to assist a subject in gaining weight, e.g., consumption by a mammal, e.g., any mammal, e.g., a muscle weight or fat weight. A unit dose of the compositions human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, described herein is the amount of composition administered or a pig. In a preferred embodiment, the mammal is a human. to a consumer in a single dose, i.e., one serving. Unit-dose For example, the invention includes a method of enhancing packaging is the packaging of a single dose, e.g., in a lean muscle development or increasing lean body mass by non-reusable container. For example, a unit dose refers to a administering to a Subject a composition comprising a physically discrete unit Suitable as unitary dosages for an Bacillus coagulans spore in a composition comprising at individual, each unit containing a predetermined quantity of least 85% purified, processed, and/or isolated protein. The active material calculated to produce the desired therapeutic Subject is a human being that desires to increase muscle effect, in association with a Suitable pharmaceutical carrier, development, strength, or lean body mass, or an animal, e.g., diluent, or excipient. Suitable packaging includes single or livestock or performance animal Such as a work animal or a multiple unit dosages. US 2017/01894.57 A1 Jul. 6, 2017
0011 Compositions that provide a large amount of calo 0014. The compositions optionally further comprise ries to assist a subject in gaining weight are referred to as additional ingredients selected from the group consisting of “weight gainers.” In these cases, the composition comprises Sodium, potassium, Sugar, carbohydrates, dietary fiber, Vita between about 100 and about 10,000 food Calories (kcal) min A, vitamin C, calcium, iron, vitamin D. Vitamin E, per unit dose (i.e., serving), e.g., between 250 and 5,000 thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, kcal, between 500 and 3,000 kcal, between 750 and 2,500 biotin, pantothenic acid, phosphorus, iodine, magnesium, kcal, or between 1,000 and 2,000 kcal, e.g., about 1,000 Zinc, Selenium, copper, manganese, chromium, and molyb kcal, about 1,100 kcal, about 1,200 kcal, about 1,300 kcal, denum. about 1,400 kcal, about 1,500 kcal, about 1,600 kcal, about 0015. An exemplary composition comprises glucose 1,700 kcal, about 1,800 kcal, about 1,900 kcal, or about polymer, a protein blend (i.e., whey protein concentrate, 2,000 kcal. One exemplary amount of calories is 1.230 kcal. whey protein isolate, egg albumin, milk protein isolate, and Alternatively, the compositions do not comprise a large partially hydrolyzed whey protein), rice protein concentrate, amount of calories. For example, the compositions comprise brown rice concentrate, taurine, L-glutamine, non-dairy between about 10 and 500 kcal, e.g., between about 20 and creamer (i.e., Sunflower oil, corn Syrup Solids, sodium 250 kcal, between about 50 and 200 kcal, or between about caseinate, mono- and diglycerides, dipotassium phosphate, 100 and 150 kcal, e.g., about 100 kcal, about 110 kcal, about tricalcium phosphate, Soy lecithin, and tocopherols), natural 120 kcal, about 130 kcal, about 140 kcal, or about 150 kcal. and artificial flavors, Xantham gum, calcium citrate, potas One exemplary amount of calories is about 150 kcal. sium citrate, dipotassium phosphate, cellulose gum, trical 0012 Preferably, the composition comprises protein. For cium phosphate, magnesium aspartate, rice starch, carra example, the protein comprises about 1% to about 99% by geenan, Vitamin-mineral blend (i.e., ascorbic acid, weight of the composition, e.g., about 5%, about 10%, about niacinamide, d-Alpha tocopheryl Succinate, d-calcium pan 15%, about 20%, about 25%, about 30%, about 35%, about tothonate, Zinc citrate, pyridoxine hydrochloride, ferrous 40%, about 45%, about 50%, about 55%, about 60%, about fumarate, thiamine mononitrate, riboflavin, manganese 65%, about 70%, about 75%, about 80%, about 85%, about amino acid chelate, beta-carotene, copper gluconate, folic 90%, or about 95% by weight of the composition. For acid, biotin, potassium iodide, chromium polynicotinate, example, the composition comprises between 1 gram and molybdenum amino acid chelate, selenomethionine, cyano 500 grams of protein, e.g., about 10 grams, about 15 grams, cobalamin, and cholecalciferol), GANEDEN BC-30TM (Ba about 20 grams, about 25 grams, about 30 grams, about 35 cillus coagulans GBI-30, ATCC Designation Number PTA grams, about 40 grams, about 45 grams, about 50 grams, 6086). Sucralose, acesulfame potassium, and lactase. about 55 grams, about 60 grams, about 65 grams, about 70 0016. Another exemplary composition comprises the fol grams, about 75 grams, about 80 grams, about 85 grams, lowing ingredients: whey protein concentrate, brown rice about 90 grams, about 95 grams, about 100 grams, about 150 protein concentrate, whey protein isolate, egg albumin, milk grams, about 200grams, about 250 grams, about 300 grams, protein isolate, partially hydrolyzed whey protein, glucose about 350 grams, about 400 grams, about 450 grams, or polymer, taurine, L-glutamine, nondairy creamer (i.e., Sun about 500 grams of protein. One exemplary composition flower oil, corn syrup Solids, Sodium caseinate, mono- and comprises about 62% protein. For example, the composition diglycerides, dopotassium phosphate, tricalcium phosphate, comprises about 39 total grams, of which about 24 grams is Soy lecithin, and tocopherols), dicalcium phosphate, natural protein. Another exemplary composition comprises about and artificial flavors, Xanthan gum, cellulose gum, carra 65% protein. For example, the composition comprises about geenan, lecithin, acesulfame potassium, Sucralose, lactase, 31 total grams, of which about 20 grams is protein. Another and GANEDEN BC-30TM (Bacillus coagulans GBI-30, exemplary composition comprises about 15% protein. For ATCC Designation Number PTA-6086). example, the composition comprises about 325 total grams, 0017. The compositions contain active ingredients, e.g., of which about 50 grams is protein. A typical protein protein and GANEDEN BC-30TM (Bacillus coagulans GBI composition comprises about 50-100% protein such as whey 30, ATCC Designation Number PTA-6086), and inactive protein. For example, a unit dose comprises 1 billion CFU ingredients, e.g., excipients, binders, or fillers. Fillers fill out of Bacillus coagulans and 23 grams of whey protein in a the size of the compositions, making it practical to produce total of 30 grams of powder. and convenient for the consumer to use. By increasing the 0013. In one aspect, the sports nutrition composition bulk volume, the fillers make it possible for the final product comprises purified or processed protein, such as soy protein, to have the proper volume for consumer handling. Suitable whey protein, rice protein, hemp seed protein, casein protein fillers include Xantham gum, cellulose gum, lecithin, lactose, or milk protein. Preferably, the protein comprises whey Sucrose, glucose, mannitol, Sorbitol, calcium carbonate, and protein. The protein comprises an amino acid selected from magnesium Stearate. the group consisting of isoleucine, alanine, leucine, arginine, 0018. In some cases, the compositions do not comprise lysine, aspartate, aspartic acid, methionine, cysteine, phe certain ingredients. For example, the composition does not nylalanine, threonine, tryptophan, glycine, Valine, proline, include a Sugar (e.g., glucose, fructose, galactose, maltose or histidine, serine, tyrosine, asparagine, selenocysteine, pyr lactose), gluten, aspartame, and/or artificial coloring. rolysine, glutamate, glutamic acid, and glutamine. Option 0019 Exemplary forms for the compositions described ally, the sports nutrition composition comprises an isolated herein include a protein powder, a ready to drink protein Bacillus coagulans and protein, and further comprises cre shake, a protein bar, a protein bite, and a protein gel. atine, calcium, Sodium caseinate, whey peptides, or lacto 0020. The invention provides compositions comprising a ferrin. Alternatively, the invention provides an isolated dry mix for sports nutrition compositions comprising an Bacillus coagulans and a sports nutrition composition isolated Bacillus coagulans bacterium and a sports nutrition selected from the group consisting of creatine, calcium, composition. The dry mix is between 0.01% and 50% Sodium caseinate, whey peptides, and lactoferrin. Bacillus coagulans bacterium, e.g., about 1%, about 5%, US 2017/01894.57 A1 Jul. 6, 2017
about 10%, about 15%, about 20%, about 25%, about 35%, of Bacillus coagulans. Exemplary vitamins include vitamin about 45%, or about 50% Bacillus coagulans bacterium. In A. vitamin B, Vitamin B vitamin B vitamin Bs, vitamin Some cases, the dry mix is about 15% Bacillus coagulans B vitamin B7, Vitamin Bo, vitamin B, or vitamin C, bacterium. For example, about 100 pounds of dry mix vitamin D. Vitamin E, and vitamin K. contains about 15 pounds of Bacillus coagulans bacterium 0026. In other cases, the absorption of a chemical element and about 85 pounds of sports nutrition composition. is increased in the subject after the administration of Bacil Optionally, the isolated Bacillus coagulans is in the form of lus coagulans in combination with protein as compared to a spore. Alternatively, the isolated Bacillus coagulans is in the absorption of the chemical element after the adminis the form of a vegetative cell. In another aspect, the isolated tration of protein in the absence of Bacillus coagulans. Bacillus coagulans is in the form of a mixture of vegetative Exemplary chemical elements include Sodium, potassium, cells and spores. Preferably, the isolated Bacillus coagulans calcium, iron, and zinc. is in the form of a spore. More preferably, the bacterium is 0027 Optionally, the Bacillus coagulans aids in the present as at least 80% spores, e.g., at least 85%, at least digestion of the sports nutrition composition, e.g., protein. 90%, at least 95%, at least 98%, or at least 99% spores. For example, abdominal bloating, intestinal pain, passage of 0021 Bacterial species suitable for use in the methods gas, loose bowel movements, and excessive abdominal describe herein include Bacillus coagulans, e.g., Bacillus gurgling are reduced following consumption of protein and coagulans hammer, preferably Bacillus coagulans hammer Bacillus coagulans compared to the consumption of protein strain Accession No. ATCC 31284, or one or more strains without Bacillus coagulans. However, Subjects ingesting the derived from Bacillus coagulans hammer Strain Accession product preferably have not been diagnosed with gastroin No. ATCC 31284 (e.g., ATCC Numbers: GBI-20, ATCC testinal disease or an inflammatory bowel condition. Designation Number PTA-6085; GBI-30 or GANEDEN 0028. The compositions are also useful in the prophylac BC-30TM, ATCC Designation Number PTA-6086; and GBI tic or therapeutic treatment of conditions associated with 40, ATCC Designation Number PTA-6087; see U.S. Pat. No. gastrointestinal infection by various pathogens, thereby Sup 6,849,256 to Farmer). porting digestive health. The probiotic sports nutrition com 0022. In some cases, the dry mix also includes a protein positions of the invention are also used in the methods selected from the group consisting of soy protein, whey described herein for boosting the immune system. In some protein, rice protein, hemp seed protein, and casein protein. cases, the combination of Bacillus coagulans and protein In other cases, the sports nutrition composition also includes works Synergistically resulting in an enhanced inhibition of creatine, calcium, sodium caseinate, whey peptides, and pathogens in the gastrointestinal tract of a subject, as com lactoferrin. pared to the administration of either Bacillus coagulans or 0023 Methods of enhancing lean muscle development, protein alone. In other cases, the combination of Bacillus recovery, or repair are carried out by administering to a coagulans and protein works synergistically resulting in an Subject desiring an enhancement of the lean muscle devel enhanced boost of the immune system, as compared to the opment, recovery or repair the compositions described administration of either Bacillus coagulans or protein alone. herein, e.g., a composition comprising a sports nutrition 0029 Optionally, the compositions of the invention addi composition and an isolated Bacillus coagulans spore, tionally comprise L-alanine and/or inosine to promote the wherein the composition comprises at least 80% protein. germination of the spores in the stomach and/or Small The composition is optionally administered prior to or after intestine. an exercise period. For example, the composition is admin 0030 A probiotic lactic acid-producing bacteria suitable istered within 60 minutes of an exercise period, e.g., within for use in the methods and compositions of the invention 15 minutes, within 30 minutes, or within 45 minutes. Alter produces acid and is non-pathogenic. Purified and/or iso natively, the composition is administered within 2 hours, lated Bacillus coagulans is particularly useful as a probiotic within 5 hours, or within 8 hours of an exercise period. in the compositions described herein. By “purified’ or 0024. The absorption of the protein is increased in the “Substantially purified' is meant a Bacillus coagulans bac Subject after the administration of Bacillus coagulans in terium that is Substantially free of contaminating microor combination with protein as compared to the absorption of ganisms or other macromolecules, e.g., polysaccharides, the protein after the administration of protein in the absence nucleic acids, or proteins. of Bacillus coagulans. For example, the rate of absorption 0031 Purified defines a degree of sterility that is safe for (t) or overall absorption (from area under the curve administration to a human Subject, e.g., lacking infectious or (AUC), and C) are increased after the administration toxic agents. Specifically, as used herein, an "isolated” or of Bacillus coagulans in combination with protein as com “purified Bacillus coagulans or protein is substantially free pared to after the administration of protein in the absence of of other cellular material, culture medium, chemical precur Bacillus coagulans, as measured by changes in amino acid sors, or other chemicals when chemically synthesized. Puri levels in, e.g., blood over a one hour test period, a two hour fied compounds are at least 60% by weight (dry weight) the test period, a four hour test period, an eight hour test period, compound of interest. Preferably, the preparation is at least a twelve hour test period, or a twenty-four hour test period. 75%, more preferably at least 90%, and most preferably at For example, protein absorption is increased by at least 10%, least 99%, by weight the compound of interest. Purity is 50%, 2-fold, 5-fold, 10-fold, or more following administra measured by any appropriate standard method, for example, tion of protein and Bacillus coagulans compared to protein by column chromatography, polyacrylamide gel electropho without Bacillus coagulans. resis, or HPLC analysis. 0025. The absorption of a vitamin is increased in the 0032. The Bacillus coagulans Hammer strains of the Subject after the administration of Bacillus coagulans in invention are non-pathogenic and generally regarded as safe combination with protein as compared to the absorption of for use in human nutrition (i.e., GRAS classification) by the the vitamin after the administration of protein in the absence U.S. Federal Drug Administration (FDA) and the U.S. US 2017/01894.57 A1 Jul. 6, 2017
Department of Agriculture (USDA), and by those skilled in There are many suitable bacteria identified as described the art. Furthermore, the Bacillus coagulans Hammer strains herein, although the invention is not limited to currently of the invention germinate at or below human body tem known bacterial species insofar as the purposes and objec perature, rendering them useful as probiotics. Many Bacillus tives of the bacteria is described. The property of acid coagulans Strains outside the Hammer group have mostly production is important to the effectiveness of the probiotic industrial applications, little or no nutritional benefit, and lactic acid-producing bacteria of this invention. environmental contaminants that have not been evaluated 0037 Exemplary methods and compositions are for safety. Moreover, many other non-Hammer strains of described herein using Bacillus coagulans as a probiotic. Bacillus coagulans grow optimally at temperatures that Purified and/or isolated Bacillus coagulans is particularly exceed human body temperature and, thus, do not germinate useful as a probiotic in sports nutrition compositions. Pro efficiently in the human body. Such strains are less or not biotic B. coagulans is non-pathogenic and is generally Suitable as probiotics for human consumption. regarded as safe (i.e., GRAS classification) by the U.S. 0033. The transitional term “comprising,” which is syn Federal Drug Administration (FDA) and the U.S. Depart onymous with “including.” “containing,” or “characterized ment of Agriculture (USDA), and by those skilled in the art. by, is inclusive or open-ended and does not exclude addi 0038 Bacillus coagulans is a non-pathogenic gram posi tional, unrecited elements or method steps. By contrast, the tive spore-forming bacteria that produces L(+) lactic acid transitional phrase “consisting of excludes any element, (dextrorotatory) in fermentation conditions. It has been step, or ingredient not specified in the claim. The transitional isolated from natural Sources, such as heat-treated Soil phrase "consisting essentially of limits the scope of a claim samples inoculated into nutrient medium (Bergey’s Manual to the specified materials or steps “and those that do not off Systemic Bacteriology, Vol. 2, Sneath, P. H. A., et al., materially affect the basic and novel characteristic(s) of the eds., Williams & Wilkins, Baltimore, Md., 1986). Purified B. claimed invention. coagulans strains have served as a source of enzymes 0034. Other features and advantages of the invention will including endonucleases (e.g., U.S. Pat. No. 5,200,336), be apparent from the following description of the preferred amylase (U.S. Pat. No. 4,980,180), lactase (U.S. Pat. No. embodiments thereof, and from the claims. Unless otherwise 4.323,651), and cyclo-malto-dextrin glucano-transferase defined, all technical and scientific terms used herein have (U.S. Pat. No. 5,102,800). B. coagulans has been used to the same meaning as commonly understood by one of produce lactic acid (U.S. Pat. No. 5,079,164). A strain of B. ordinary skill in the art to which this invention belongs. coagulans (referred to as L. Sporogenes; Sakaguti & Although methods and materials similar or equivalent to Nakayama (ATCC 31284)) has been combined with other those described herein can be used in the practice or testing lactic acid producing bacteria and B. natto to produce a of the present invention, Suitable methods and materials are fermented food product from steamed soybeans (U.S. Pat. described below. All publications, patent applications, pat No. 4,110.477). ents, Genbank/NCBI accession numbers, and other refer 0039 Bacterial species include Bacillus coagulans, e.g., ences mentioned herein are incorporated by reference in Bacillus coagulans hammer, preferably Bacillus coagulans their entirety. In the case of conflict, the present specifica hammer strain Accession No. ATCC 31284, or one or more tion, including definitions, will control. In addition, the strains derived from Bacillus coagulans hammer strain materials, methods, and examples are illustrative only and Accession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20, not intended to be limiting. ATCC Designation Number PTA-6085; GBI-30 (GANEDEN BC-30TM), ATCC Designation Number PTA DETAILED DESCRIPTION OF THE 6086; and GBI-40, ATCC Designation Number PTA-6087; INVENTION see U.S. Pat. No. 6,849,256 to Farmer). 0035. The probiotic organisms of the invention are non 0040 Bacillus coagulans was previously mis-character pathogenic, non-toxigenic, retain viability during storage, ized as a Lactobacillus and labeled as Lactobacillus sporo genes. However, initial classification was incorrect because and Survive passage through the stomach and Small intes Bacillus coagulans produces spores and excretes L(+)-lactic tine. Non-pathogenic lactic acid-producing bacteria (i.e., acid through metabolism. Both of these characteristics pro “lactic acid bacteria'), such as the exemplary Bacillus vide key features to the utility of Bacillus coagulans. These coagulans, remain viable and retain their beneficial probi developmental and metabolic aspects required that the bac otic properties in sports nutrition compositions. Prior to the terium be classified as a lactic acid Bacillus. In addition, it invention described herein, probiotics were very susceptible is not generally appreciated that classic Lactobacillus spe to harsh environment of the stomach and Small intestine. cies are unsuitable for colonization of the gut due to their Unlike other probiotics that die in the stomach or small instability in the harsh (i.e., acidic) pH environment of the intestine, the probiotic organisms described herein, e.g., bile, particularly human bile. By contrast, Bacillus coagul Bacillus coagulans strain GBI-30 or GANEDEN BC-30TM, lans ATCC Designation Number PTA-6085; GBI-30 ATCC Designation Number PTA-6086, germinate in the (GANEDEN BC-30TM) is able to survive and colonize the stomach and/or small intestine. Specifically, the probiotic gastrointestinal tract in the bile environment and even grow spores described herein can Survive passage through the in this low pH range. stomach and Small intestine. Probiotic Activity of Bacillus coagulans 0041. It is well-documented clinically that many species Probiotic Lactic Acid-Producing Bacteria of bacterial, mycotic and yeast pathogens possess the ability 0036. The sports nutrition compositions include a lactic to cause a variety of gastrointestinal disorders including, but acid-producing bacterium, Such as a spore-forming Bacillus not limited to disruption of normal gastrointestinal bio species, e.g., B. coagulans. Preferably, the spore-forming chemical function, necrosis of gastrointestinal tissues, and Bacillus species of the invention is B. coagulans Hammer. disruption of the bioabsorption of nutrients, and like condi US 2017/01894.57 A1 Jul. 6, 2017
tions. The probiotic microorganism-containing composi inhibition were measured. As designated herein, “excellent tions described herein inhibit these pathogens. In the present inhibition” means the Zone was 10 mm or greater in diam case, Subjects are generally healthy, e.g., they have not been eter, and “good inhibition' means the Zone was greater than diagnosed with a gastrointestinal disease (e.g., irritable 2 mm in diameter but less than 10 mm in diameter. bowel syndrome or Crohn's disease), an inflammatory 0046. As expected, no “inhibition' was seen with the bowel condition, or an autoimmune disease. The composi negative, Saline control, and excellent “inhibition' (approxi tions and methods described herein are useful to increase mately 16.2 mm diameter; average of three tests) was seen absorption of protein and/or macro nutrients in a nutritional with the positive, glutaraldehyde control. For the enteric composition. microorganisms tested, the following inhibition by Bacillus 0042. In one aspect, a Bacillus coagulans Strain is coagulans was found: (i) Clostridium species—excellent included in the composition in the form of vegetative cells. inhibition; (ii) Escherichia coli excellent inhibition; (iii) In another aspect, the Bacillus coagulans strain is included Clostridium species—excellent inhibition, where the Zone of in the composition in the form of spores. The invention also inhibition was consistently greater than 15 mm in diameter. provides for including the Bacillus coagulans strain in the Similarly, excellent inhibition was also seen for the oppor composition in the form of a powder, a dried cell mass, a tunistic pathogens Pseudomonas aeruginosa, and Staphyllo stabilized paste, or a stabilized gel. coccus aureus. Pathogenic enteric bacteria which were 0043. Because Bacillus spores are heat and pressure inhibited by Bacillus coagulans activity include, but are not resistant and can be stored as a dry powder, they are limited to: Staphylococcus aureus, Staphylococcus epider particularly useful for formulation into and manufacture of midis, Streptococcus pyogenes, Pseudomonas aeruginosa, products such as the various sports nutrition compositions Escherichia coli (enterohemorragic species); numerous described herein. Specifically, the probiotic organisms Clostridium species (e.g., Clostridium perfingens, described herein, e.g., Bacillus coagulans strain GBI-30 or Clostridium botulinum, Clostridium tributrycum, GANEDEN BC-30TM, ATCC Designation Number PTA Clostridium sporogenes, and the like); Gardnereia vagi 6086, Survive passage through the stomach and Small intes nails, Proponbacterium aenes, Aeromonas hydrophia, tine. A Bacillus species is well suited for the present inven Aspergillus species; Proteus species; and Klebsiella species. tion, particularly species having the ability to form spores which are relatively resistant to heat and other conditions, Micro-Encapsulation making them ideal for storage (shelf-life) in product formu 0047. In one aspect, the lactic-acid producing bacteria are lations, e.g., sports nutrition compositions. Due to the shelf incorporated into a microcapsule coating prior to addition to stable properties of the Bacillus coagulans strains described the sports nutrition composition, using any micro-encapsu herein, e.g., Bacillus coagulans strain GBI-30 or lation process well-known in the art. The isolated Bacillus GANEDEN BC-30TM, ATCC Designation Number PTA coagulans are packaged, or encapsulated, within another 6086, the product formulations of the invention are not material in order to protect the bacteria from the Surrounding confined to a refrigerator and may be stored at room tem environment. The capsules of the invention range in size perature. The Bacillus coagulans of the invention Survives from one-thousandth of a millimeter to seven millimeters. storage (shelf-life) from about 12 days to about 2 years; from The internal ingredients of the microcapsule are released about 1 month to about 18 months; from about 3 months to from their shells in various ways, including mechanical about 1 year; or from about 6 months to about 9 months. rupture of the capsule wall, dissolution of the wall, melting 0044) The probiotic organisms described herein, e.g., of the wall and diffusion through the wall. Thus, micro Bacillus coagulans strain GBI-30 or GANEDEN BC-30TM, encapsulation provides additional protection to the isolated ATCC Designation Number PTA-6086, promote digestive Bacillus bacterium during manufacturing and storage of the and oral health and Support the immune system. The ability sports nutrition compositions of the invention. Physical of Bacillus coagulans to inhibit various bacterial pathogens methods of micro-encapsulation include pan coating, air was quantitatively ascertained by use of an in vitro assay. Suspension coating, centrifugal extrusion, vibrational This assay is part of a standardized bacterial pathogen screen nozzle, and spray-drying. Chemical methods of micro-en (developed by the U.S. Food and Drug Administration capsulation include interfacial polymerization, in-situ (FDA)) and is commercially available on solid support disks polymerization, and matrix polymerization. (DIFCOR) BACTROL(R) Antibiotic Disks). To perform the 0048 Alternatively, the lactic-acid producing bacteria is assay, potato-dextrose plates)(DIFCOR) were initially pre added to the sports nutrition composition without micro pared using standard procedures. The plates were then encapsulation. individually inoculated with the bacteria (approximately 1.5x10 CFU) to be tested so as to form a confluent bacterial bed. Probiotic Sports Nutrition Compositions 0045. Inhibition of microorganisms (e.g. gastrointestinal 0049. The invention includes a method of increasing pathogens) by Bacillus coagulans was Subsequently ascer protein absorption, enhancing lean muscle development, tained by placing approximately 1.8x10 CFU of Bacillus and/or increasing lean body mass by administering to a coagulans in 10 ul of broth or buffer, directly in the center Subject a composition comprising a Bacillus coagulans of the potato-dextrose plate with one test locus being spore in a composition comprising at least 50% purified or approximately 8 mm in diameter per plate. A minimum of processed protein, e.g., a composition that contains at least three test loci were used for each assay. The negative control 75% whey protein or other protein source. The sports consisted of a 10 ul volume of a sterile saline solution, nutrition compositions are suitable for human or animal whereas the positive control consisted of a 1 ul volume of consumption. A typical Subject is a healthy adult, e.g., glutaraldehyde. The plates were then incubated for approxi greater than 16 years old. Alternatively, the sports nutrition mately about 18 hr at 30° C., at which time the Zones of compositions may also be administered to children under 18 US 2017/01894.57 A1 Jul. 6, 2017 years of age, e.g., under 15 years of age, under 10 years of acid (an omega-6 fatty acid). Casein protein (or milk pro age, or under 5 years of age. Alternatively, the sports tein) has glutamine, and casomorphin. Finally, egg-white nutrition compositions are administered to children and protein is a lactose- and dairy-free protein. adults of all ages. The Subject is a human being that desires 0053 Proteins come in various forms, including protein to increase muscle development, strength or lean body mass, powder, ready to drink protein shakes, bars, bites, oats, and or an animal, e.g., livestock or performance animal Such as gels. Protein powders are available in a wide variety of a work animal or a race horse, for which an increase in flavors including pineapple, orange, fruit punch, mixed muscle development or strength is desired. The composi berry, mango, cookies and cream, Strawberry, strawberry tions are also useful to confer clinical benefit to individuals banana, French Vanilla, Vanilla, Vanilla ice cream, Vanilla that are suffering from or at risk of developing a muscle milkshake, banana, banana cream, Dutch chocolate, mocha wasting condition, e.g., cachexia, as a result of disease Such cappuccino, double rich chocolate, chocolate caramel, as cancer or infection. chocolate milkshake, extreme milk chocolate, chocolate 0050 Sports nutrition compositions are prepared by mix mint, chocolate chip, and chocolate. The protein powder is ing dry powder ingredients (e.g., protein and Bacillus mixed with water, milk or juice (e.g., grapefruit juice, grape coagulans). Sports nutrition compositions include body juice, orange juice, etc.), and often flavoring, resulting in a building or body maintenance foods, e.g., weight gainers, form known as a “protein shake' (as in milkshake) or commonly used by those involved in bodybuilding and "pudding. Protein powder is generally consumed immedi athletics. They include protein (the most widely used such ately before or after exercising, or in place of a meal. The sports nutrition composition), amino acids, glutamine, theory behind this regimen is that having a sufficient protein essential fatty acids, meal replacement products, prohor intake allows for efficient growth and repair of muscle mones, creatine, thermogenic products, testosterone boost tissue. Protein powders also aid in fat loss, muscle building, ers, and branched-chain amino acids (BCAA). The three and slow the aging process. branched-chain amino acids (BCAAS) include leucine, iso leucine, and valine. Unlike other amino acids, BCAAs are 0054 Meal replacement products (MRPs) are either pre metabolized in the muscle and have an anabolic/anti-cata packaged powdered drink mixes or edible bars designed to bolic effects. BCAAs account for 33% of muscle protein. replace prepared meals. MRPs are generally high in protein, Amino acids are the building blocks of protein, which the low in fat, have a low to moderate amount of carbohydrates, body metabolizes in the stomach and intestines. Protein and contain a wide array of vitamins and minerals. The powder contains amino acids such as isoleucine, alanine, majority of MRPs use whey protein, casein (often listed as leucine, arginine, lysine, aspartate, aspartic acid, methio calcium caseinate or micellar casein), soy protein, and/or nine, cysteine, phenylalanine, threonine, tryptophan, gly egg albumin as protein sources. Carbohydrates are typically cine, Valine, proline, histidine, serine, tyrosine, asparagine, derived from maltodextrin, oat fiber, brown rice, and/or Selenocysteine, pyrrolysine, glutamate, glutamic acid, and wheat flour. Some MRPs also contain flax seed oil powder glutamine. as a source of essential fatty acids. 0051. Glutamine is the most abundant amino acid found 0055 Sports nutrition compositions may also contain in human muscle; however, the body's natural glutamine other additional ingredients that are beneficial for body stores are depleted during anaerobic exercise. Thus, the building, including calcium, Sodium caseinate, whey pep sports nutrition compositions of the invention optionally tide, glutamine peptides, L-glutamine, calcium alpha-keto include glutamine. Serum glutamine is used by the body to glutarate, additional amino acids, lactoferrin, conjugated counteract acidosis resulting from exercise. In order to linoleic acid, medium chain triglycerides, and creatine (e.g., replenish the loss of glutamine from the bloodstream, the creatine monohydrate). Creatine is a nitrogenous organic body catabolizes glutamine from the muscle. Thus, ingestion acid that is found in the muscle tissue of vertebrates mainly of glutamine combats muscle tissue wasting. in the form of phosphocreatine replenishment of ATP, and 0052 Suitable types of protein include soy protein, whey Supplies energy for muscle contraction. Creatine improves protein, rice protein, hemp protein, casein protein, and strength, energy, muscle mass, recovery times, brain func egg-white protein (e.g., egg albumin). Soy protein isolated tion, and reduces mental fatigue. Creatine is available in a from soybeans contains isoflavones, a type of phytoestrogen. variety of forms, including creatine monohydrate and cre Whey protein contains high levels of all the essential amino atine ethyl ester. acids and branched-chain amino acids. For example, the 0056 Although it is known that athletes and bodybuild amino acids regarded as essential for humans are phenyl ers may need an increased intake of protein, the exact alanine, Valine, threonine, tryptophan, isoleucine, methio amount is highly individualized and dependent on the type nine, leucine, lysine, and histidine. Whey protein also has and duration of the exercise as well as the physiological the highest content of the amino acid cysteine, which aids in make up of the individual. Research by Tarnopolsky et al. the biosynthesis of glutathione. For this reason, whey pro (1988) showed that for bodybuilding individuals, 1.97g of tein provides amino acids to aid in muscle recovery. Whey protein per kg of body weight per day is recommended, protein is derived from the process of making cheese from whereas endurance athletes require 1.37 g/kg/d of protein. milk. There are two types of whey protein: whey concentrate Studies suggest that there are different protein requirements (29%-89% protein by weight) and whey isolate (90%+ for anaerobic and aerobic exercise. Endurance athletes in protein by weight). Another type of protein includes rice aerobic activity may have increased daily protein intake at protein, which is made from whole grain, a complete protein 1.2-1.4 g per kg body weight per day, whereas strength Source that is highly digestible and allergen free. Hemp training athletes performing anaerobic activity may have protein from hemp seed contains complete and highly increased daily protein intake needs at 1.4-1.8g per kg body digestible protein. Hemp oil is high in essential fatty acids, weight so as to enhance muscle protein synthesis or to make i.e., alpha-linolenic acid (an omega-3 fatty acid) and linoleic up for the loss of amino acid oxidation during exercise. US 2017/01894.57 A1 Jul. 6, 2017
0057 Preferably, the sports nutrition composition ingre 10062) In one aspect, the amount of bacteria is about 10 dients are blended together as dry ingredients. Exemplary to 10" colony forming units (CFU) of bacteria per gram of ingredients of the sports nutrition compositions described probiotic composition (i.e., vegetative cells and/or bacterial herein include protein and Bacillus coagulans. An exem spores), preferably 10 to 10' CFU/g of sports nutrition plary strain of Bacillus coagulans suitable in the sports composition. Alternatively, the concentrations are 10 to nutrition compositions described herein includes GBI-30 or 10' CFU/g: 10° to 10'? CFU/g; or 100 to 10 CFU/g of GANEDEN BC-30TM, ATCC Designation Number PTA sports nutrition composition. In one aspect, the amount of 6086. bacteria is about 1x10 CFU per gram of sports nutrition 0058. The dry mix for sports nutrition compositions composition. The actual amount in a sports nutrition com comprises an isolated Bacillus coagulans bacterium. The position will vary. A typical concentration is from approxi dry mix is approximately 1 billion CFU of Bacillus coagul mately 1x107 to 1x10' CFU; 1x10 to 1x10' CFU; or lans bacterium per unit dose. Optionally, the dry mix is 1x10 to 1x10' CFU of viable bacterium or spores/g of about 15% Bacillus coagulans bacterium and 85% protein. sports nutrition composition. For example, about 100 pounds of dry mix contains about 15 0063. Following drying, the sports nutrition composition pounds of Bacillus coagulans bacterium and about 85 is ready for immediate use or for storage in a sterile package, pounds of protein. The dry mix is between about 1% and e.g., a 3-ounce package (e.g., a bag or a bottle), a 6-ounce about 50% by weight of the sports nutrition composition, package, a 9-ounce package, a 12-ounce package, a e.g., about 1% to about 20%, about 5% to about 15%; about 15-ounce package, an 18-ounce package, a 24-ounce pack 6%, about 7%, about 8%, about 9%, or about 10% by weight age, a 48-ounce package, 80-ounce package, or 100-ounce of the sports nutrition composition. For example, a 3 gram package. In another example, the dried powder is packaged sports nutrition composition contains about 7% dry mix by in unit dose quantities, e.g., 5 grams, 10 grams, 20grams, 30 weight of the sports nutrition composition. A 3.8 to 4 gram grams, 40 grams, 50 grams, 60 grams, 70 grams, 80 grams, sports nutrition composition contains about 8-9% dry mix by 90 grams, or 100 gram packets. Alternatively, the dried weight of the sports nutrition composition. powder is packaged in bulk, e.g., about 500 grams, about 0059. As the recommended dietary allowances (RDA or 600 grams, about 700 grams, about 800 grams, about 900 recommended daily intake; RDI) is about 1x10 bacterium grams, about 1,000 grams, about 1,250 grams, about 1,500 (according to EU guidelines), preferably, the sports nutrition grams, about 1,750 grams, about 2,000 grams, about 2.250 composition comprises at least about 1x10 viable bacteria. grams, about 2,500 gram, or about 3,000 gram containers. In In another aspect, the sports nutrition composition com one aspect, the invention provides for storing the sports prises at least about 1x10° to 1x10"; at least about 1x10 to nutrition composition in a sterile package at room tempera 1x10; or at least about 1x10 to 1x10 viable bacteria. ture prior to consumption. Alternatively, the composition is 0060. The Bacillus and/or Bacillus coagulans in the form consumed immediately. of a dried spore composition is mixed with dry whey powder 0064. The active ingredients (i.e., Bacillus coagulans and or is applied using any of a variety of known methods protein), comprise between about 0.01% to about 10%: including, for example, applying a powder, spray-drying the 0.01% to about 1%; or about 0.05% to about 0.1% by weight probiotic onto the sports nutrition composition, or soaking of the probiotic sports nutrition composition. Optionally, the the composition in a solution containing the probiotic. Any isolated Bacillus coagulans comprise about 1 mg to about 10 of a variety of methods for placing the bacterial composition g; about 10 mg to about 1 g; or about 25 mg to about 75 mg into a sports nutrition composition can be used. In one by weight of the probiotic composition. Most preferably, the aspect, a "spray-dry method is used, in which the compo amount of Bacillus coagulans bacteria is about 5x10" colony sitions are exposed in a low humidity chamber to an atom forming units (CFU) of bacteria per gram of food matrix. ized mix containing a liquid composition, where the cham 0065. In some cases, the Bacillus coagulans spores are ber is subsequently exposed to approximately 80-110° F. to incorporated into any type of dry or lyophilized product dry the liquid, thereby impregnating the material of the which is dissolved or mixed with water, milk, or juice, so sports nutrition composition with the components. long as the temperature of the Bacillus coagulans spore containing mixture is raised to the required heat-shock 0061. In some cases, Bacillus coagulans bacteria in the temperature (i.e., 80° C. for 5 minutes) necessary for ger form of a spray-dried powder is included in or on the surface mination of the spores. The Bacillus coagulans spores may of the sports nutrition compositions described herein. Pref either be incorporated into the dry or lyophilized product by erably, the isolated Bacillus coagulans is in the form of a the manufacturer of the product or by the consumer during spore, as the Bacillus coagulans spores of the invention are preparation. protected by a hardened coating that can withstand gastric acid and bile salts for delivery to the small and large 0066. The Bacillus coagulans spores survive storage intestines. The isolated Bacillus coagulans are at least 85%, (shelf-life), i.e., retain viability or the ability to germinate at at least 90%, at least 95%, or at least 99% pure spores. physiological conditions (e.g., ingestion), from about 12 Alternatively, the isolated Bacillus coagulans is in the form days to about 2 years; from about 1 month to about 18 of a vegetative cell. In one aspect, the isolated Bacillus months; from about 3 months to about 1 year; or from about coagulans are at least 85%, at least 90%, or at least 95% pure 6 months to about 9 months. vegetative cells. In another aspect, the isolated Bacillus coagulans is in the form of a mixture of vegetative cells and Example 1: Preparation of Bacillus coagulans spores. The Bacillus coagulans mixture is 90% spores, 10% Cultures vegetative cells: 75% spores, 25% vegetative cells: 60% 0067 Bacillus coagulans Hammer bacteria (ATCC spores, 40% vegetative cells; 50% spores, 50% vegetative Accession No. 31284) was inoculated and grown to a cell cells: 60% vegetative cells, 40% spores: 75% vegetative density of about 10 to 10 cells/ml in nutrient broth con cells; 25% spores; or 90% vegetative cells, 10% spores. taining 5 g Peptone, 3 g Meat extract, 10-30 mg MnSO, and US 2017/01894.57 A1 Jul. 6, 2017
1,000 ml distilled water, adjusted to pH 7.0, using a standard buffer, serial dilutions were made until 6 tubes had been used airlift fermentation vessel at 30° C. The range of MnSO for each sample. The final dilution for the final two test tubes acceptable for sporulation is 1 mg/l to 1 g/1. The vegetative were 3x107 and 3x10, which gave a count of roughly 300 cells can actively reproduce up to 45° C., and the spores are and 30 CFU, respectively. The final 2 test tubes from each stable up to 90° C. After fermentation, the B. coagulans sample were placed into 70° C. water bath for 30 minutes. bacterial cells or spores are collected using standard meth After 30 minutes, they were cooled immediately to 45° C. ods (e.g., filtration, centrifugation) and the collected cells Three sterile petri plates per tube were set out. 1.0 ml from and spores can be lyophilized, spray-dried, air-dried or the heat-treated tube was added into each petri plate, then 15 frozen. ml of sterile molten GYE Agar medium (at 45° C.) was 0068 A typical yield from the above culture is in the poured into each of the petri plates and mixed thoroughly. range of about 10 to 10" viable spores and more typically When solidified, the plates were incubated in an inverted about 100 to 150 billion cells/spores per gram before drying. position for 48 hours at 40°C. The individual colonies were Spores maintain at least 90% viability after drying when counted. Results were expressed as CFU per gram as shown stored at room temperature for up to ten years, and thus the in Table 1 below. 1.0E+10–1x10'. effective shelf life of a composition containing B. coagulans Hammer spores at room temperature is about 10 years. TABLE 1.
20 Minutes 120 Minutes Example 2: Preparation of Bacillus coagulans Incubation Incubation Spores Spore Count, Spore Count, 0069. A culture of dried B. coagulans spores was pre Sample CFU gram CFU gram pared as follows. Ten million spores were inoculated into a Normal Saline - A 1.90E-10 188E-10 Normal Saline - B 2.12E-10 2.OOE-10 one liter culture containing 24 g potato dextrose broth, 10 g Normal Saline - C 1.64E-10 2.06E-10 of enzymic-digest of poultry and fish tissue, 5g of FOS and Average 189E-10 198E-10 10 g MnSO. The culture was maintained for 72 hours under Saline pH 1.0 - D 2O8E--09 S.98E--O7 a high oxygen environment at 37° C. to produce culture Saline pH 1.0 - E 1.47E--09 O.OOE--OO Saline pH 1.0 - F 3.59E-09 O.OOE--OO having about 150 billion cells per gram of culture. There Average 2.38E--09 1.99E-07 after, the culture was filtered to remove culture medium Saline pH 2.0 - G 3.63E--09 3.46E--09 liquid, and the bacterial pellet was resuspended in water and Saline pH 2.0 - H 4.47E-09 2.48E--09 freeze-dried. The freeze-dried powder is then ground to a Saline pH 2.0 - I 3.58E--09 2.82E--09 fine powder using standard good manufacturing practice Average 3.89E-09 2.92E--09 Saline pH 3.0 - J 1.65E-10 1.13E-10 (GMP). A dried composition that comprises at least about Saline pH 3.0 - K 1.35E-10 1.11E+10 75% (e.g., 80%, 85%, or 90%) spores is then mixed with Saline pH 3.0 - L. 180E-10 1.39E-10 protein powder. Average 16OE-10 1.21E-10 Example 3: Bacillus coagulans Spores Survive in the Gastric Environment Example 4: The Effects of a Probiotic on the 0070 This study was performed in order to determine the Absorption of Protein and Micronutrients in Survivability rate of Bacillus coagulans spores as they pass Healthy Males through the stomach. Samples of Bacillus coagulans spores were subjected to a simulated gastric environment for vary (0071. The effects of the probiotic GANEDEN BC-30TM ing lengths of time in order to attain their survivability rate. (Bacillus coagulans ATCC Designation Number PTA-6086) First, a homogeneous sample of raw material Bacillus on protein and micronutrient absorption is evaluated in coagulans of at least 12 grams was prepared. Saline Solution healthy males following a 14-day period. The protocol at pH 1 was prepared using 3N HCl (150 mls each into six outlined below determines the effect of a probiotic product 250 ml media bottles) and sterilized. Additional saline (GANEDEN BC-30TM, Bacillus coagulans ATCC Designa solutions with pH 2 and 3 were prepared similarly, resulting tion Number PTA-6086) in combination with protein as in 6 sterile 250 ml bottles, each containing 150 ml pH compared to protein in the absence of Bacillus coagulans on adjusted saline. Six sterile 250 ml media bottles each con protein absorption, both rate of absorption (t) and overall taining 150 ml normal saline solution were prepared and absorption (from area under the curve (AUC) and C), sterilized. Phosphate buffer (~400 ml) was prepared at pH as measured by changes in blood amino acid levels over a 7.2. Test tubes (24) were prepared and sterilized, each four hour test period. The protocol outlined below is also containing 9 ml of phosphate buffer pH 7.2. Test tubes (120) designed to determine the effect of a probiotic product were prepared, each containing 9 ml of normal saline. GYE (GANEDEN BC-30TM, Bacillus coagulans ATCC Designa (glucose-yeast extract) agar medium was prepared and ster tion Number PTA-6086) in combination with protein as ilized and cooled to 45° C. in a water bath. Samples (24) of compared to protein in the absence of Bacillus coagulans on raw material were weighed, each ~500 milligrams (theoreti Vitamin and mineral absorption, both rate of absorption cally equivalent to 10 billion spores). The samples were (t) and overall absorption (from AUCo., and C), as added to media bottles at 37° C. and incubated half for 20 measured by changes in blood levels of select vitamins minutes the other half for 120 minutes. After 20 and 120 (vitamins B6, B12 and C) and minerals (calcium, iron and minutes incubation, respectively, the samples were mixed to Zinc) over a four hour test period. The study also determines uniformity and pipet 1 ml into 9 ml of sterile phosphate the effect of a probiotic product (GANEDEN BC-30TM, buffer pH 7.2. After all 12 samples from each time point Bacillus coagulans ATCC Designation Number PTA-6086) were placed into test tubes containing sterile phosphate in combination with protein as compared to protein in the US 2017/01894.57 A1 Jul. 6, 2017 absence of Bacillus coagulans on gastrointestinal (GI)- naire scores (abdominal pain, abdominal bloating, gas, specific symptoms using a GI questionnaire after 14 days of bowel movements, gurgling noises and overall well-being). treatment. 0079 Efficacy endpoints generally consist of changes in 0072 The period of evaluation lasts approximately four efficacy variables from baseline to four hours post product weeks with Subjects attending a screening/randomization administration following 14 days of product use. These visit and two follow-up test visits. Eligible subjects take changes (endpoints) are then be compared between the two GANEDEN BC-30TM (Bacillus coagulans ATCC Designa products. tion Number PTA-6086) plus protein and protein alone, each for 14 days, in random order. After each 14-day period, there Statistical Methods: is a test visit at which subjects take the test product (probi otic plus protein or protein alone) and a multiple vitamin and 0080 For each efficacy endpoint (amino acid, vitamin, mineral supplement. Blood is collected at baseline (prior to and mineral blood concentration), the following non-com the test product) and at 1, 2, 3 and 4 hours following product partmental pharmacokinetic (PK) parameters are calculated: administration. Levels of various amino acids, vitamins and 0081. C. The maximum concentration observed dur minerals are measured so that the effects of the probiotic on ing the four post-dose samples; protein and micronutrient absorption are determined. The 0082 t the time at which the maximum concentra probiotic increases the absorption of the protein and micro tion was observed; nutrients by increasing the efficiency of the gut by balancing I0083 AUC, the area under the concentration-vs the intestinal flora or by increasing the acidity of the stomach time curve above the baseline (time 0) concentration, for more efficient breakdown of the nutrients. integrated from time 0 to 4 hours post-dose, by trap 0073. Although the study population is comprised of eZoidal-rule quadrature. generally healthy adults that do not have GI disorders or I0084 All safety and efficacy variables are summarized by symptoms, there may be beneficial effects in GI health with time point and by product. Numerical variables are pre the administration of GANEDEN BC-30TM (Bacillus coagul sented as mean, standard deviation, count, median, and lans ATCC Designation Number PTA-6086). A six item range (minimum to maximum value). Changes from the questionnaire is used to assess changes in GI health (e.g. appropriate baseline are Summarized and presented in the abdominal pain, bloating and gas) with lower incidence same way. Numerical variables and their changes from indicating positive effects with regard to GI health. baseline are displayed graphically, as plots of mean value VS. time, with separate lines for the two products (probiotic vs. Study Design placebo). Categorical variables are presented as tabulations 0074 The study described herein is a prospective, ran of counts and percentages of totals. domized, double blind, placebo controlled, crossover clini I0085 For each continuous variable, the mean change cal trial. A total of 10 male subjects are enrolled, with each from baseline to each Subsequent time point are tested for Subject receiving the two test products, in a randomly nominal significance by the paired Student t test, or by the assigned sequence. Each Subject serves as his own control. non-parametric Wilcoxon test if non-normally distributed. The mean differences in the variable, or in the change in that Study Population: variable from baseline, between the different products are tested for nominal significance by the paired Student t test or 0075. The study population includes 10 male subjects, by the non-parametric Wilcoxon test if non-normally dis aged 21 to 60 years, that do not have any gastrointestinal tributed. For each categorical variable, difference in the disease or inflammatory bowel condition, and who are not distribution of categories between the different product taking on a regular basis any prescription or over-the counter groups are tested for nominal significance by the Fisher medications for any gastrointestinal problems. Exact test if possible, or by the Chi-Square test if necessary. I0086 Adverse events (AEs) are listed, Medical Diction Test Products: ary for Regulatory Activities (MedDRA) encoded, grouped 0.076 Product 1 GANEDEN BC-30TM (Bacillus coagul by general type of event (gastrointestinal, neurologic, car lans strain GBI-30, ATCC Designation diac, etc.), and cross-tabulated by event type and product. Number PTA-6086) plus Protein. Differences in AE patterns between the two products are Product 2 Protein (placebo; no probiotic). tested by the Fisher Exact test. Subjective remarks are categorized to the extent possible, and analyzed for pattern Duration of Study differences between the two products in the same way as AES. 0077. Excluding the screening visit, each subject com 0087. The pharmacokinetic endpoints (C. t. and pleting the study participates for approximately four weeks, AUC) after 14 days of product use are tested for signifi tthree days. cance between the two products by a repeated-measures analysis of variance (RM-ANOVA) for each endpoint, Efficacy Assessments: where the value of the variable at each point in time is the 0078) Efficacy variables consist of protein absorption repeated dependent variable, and the product identification rate of absorption (t) and overall absorption (AUCo., (GANEDEN BC-30TM, Bacillus coagulans ATCC Designa and C) of blood amino acids, vitamin absorption rate of tion Number PTA-6086+Protein or Placebo) is the main absorption (t) and overall absorption (AUCo., and factor. If the efficacy variables are not normally distributed, C) of vitamins B6, B12 and C. mineral absorption rate and cannot be normalized by logarithmic or other transfor of absorption (t) and overall absorption (AUCo., and mations, then between-product testing are carried out using C) of minerals calcium, iron and Zinc, and GI question the Wilcoxon signed-ranks test. US 2017/01894.57 A1 Jul. 6, 2017
0088 Assuming 15% attrition during the course of the 0.111 5. Subject has had any stomach or intestinal study, there are about 8 or 9 analyzable subjects remaining Surgery (i.e. gastric bypass). in the per-protocol population. To have 80% power to obtain 0112 6. Subject takes on a regular basis (defined as significance (ps0.05) when testing a change over time (over two or more times per week) any prescription or 14 days of product use) within a product group, the mean over-the counter medications for diarrhea, constipation, change is about equal to about 15% larger than the standard heartburn or any other gastrointestinal problems. deviation of the changes (a "1.15-sigma' effect size). To 0113 7. Subject is currently taking laxatives or has show that there was a significant difference between the two taken laxatives within the 30 days prior to screening/ products, the mean difference is about 65% larger than the enrollment. within-group variability of the endpoint (a “1.65-sigma' 0114 8. Subject is currently taking antibiotics (or any effect size). drug that significantly interferes with bacterial flora) or 0089. Each efficacy endpoint is considered an indepen has taken antibiotics within the 60 days prior to screen dent question of interest, and is tested independently at the ing/enrollment. 0.05 alpha level (ps0.05 required for a conclusion of sta 0115 9. Subject is currently taking or has used in the tistical significance). Multiple testing is taken into account past 30 days probiotics/prebiotics (including yogurt when the results of the efficacy analyses are interpreted by and lacto-fermented beverages) or any digestive the statistician in the Final Statistical Report. enzymes prescription or over-the-counter (OTC). Study Population Thirty-day washout allowed. 011 6 10. Subject is on an unstable dose of medication Inclusion Criteria (defined as fewer than 90 days at the same dose). 0117 a. Anti-hypertensives and anti-hyperlipidemic (0090) 1. Male subjects, aged 21 to 60 years medications ok if stable dose. 0091 2. Subject is able to understand and sign the 0118 11. Subject is currently taking any medication informed consent to participate in the study. deemed exclusionary by PI. 0092. 3. Subject is willing and able to comply with the 0119) 12. Subject has an allergy to soy, milk, chocolate protocol including: or any of the ingredients in the test product. 0093 a. Attending three visits; 0120 13. Subject has a history of drug or alcohol abuse 0094) b. Refraining from eating any yogurt or lacto in the past 12 months. fermented beverages during the study: 0121 14. Subject has begun/stopped smoking sG 0.095 c. Refraining from using any dietary supple months ago OR has plans to begin? quit Smoking. ments or nutritionals during the study: 0.122 15. Subject has any condition or abnormality 0096 d. Not taking any new vitamin and/or mineral that, in the opinion of the investigator, would compro Supplements until after study completion and refrain mise the safety of the subject or the quality of the study ing from taking any vitamin and/or minerals Supple data. ments for 24 hours prior to the test visits. 0123 16. Subject is participating or has participated in Exclusion Criteria another research study within 30 days prior to the Screening visit. 0097. 1. Subject has any of the following medical conditions: Concomitant Medications and Other Substances 0098 a. active heart disease (0099 b. uncontrolled high blood pressure (>140/90 Substances Permitted During the Study mmHg) 0100 c. renal or hepatic impairment/disease 0.124 Subjects may take the following substances during 0101 d. Type I or II diabetes their participation in this study: 0.125 Vitamins and/or minerals subject was taking 0102 e. bipolar disorder prior to starting the study (same frequency as prior to (0103 f. Parkinson's disease study start). However, Subjects cannot take any vita 0104 g. unstable thyroid disease mins and/or minerals for the 24 hours prior to the test 0105 h. immune disorder (such as human immune visits. If the Subject was not previously taking vitamins deficiency virus (HIV)/acquired immune deficiency and/or minerals, he is asked to not start taking any syndrome (AIDS)) during the study. 0106 i. psychiatric disorders (hospitalized within the past one year) 0.126 Thyroid hormone replacement therapy, if drug 0107 j. any medical condition deemed exclusionary and dose are unchanged for at least 90 days before by the Principal Investigator (PI) Screening and throughout the study. 0108) 2. Subject has a history of cancer (except local 0127. Antihypertensive and antihyperlipidemic medi ized skin cancer without metastases) within five years cations, if drug and dose are unchanged for at least 90 prior to Screening. days before screening and throughout the study. 0109) 3. Subject has a history of or currently has any gastrointestinal disease or disorder or any inflammatory Substances not Permitted During the Study bowel condition such as Crohn's disease, short bowel, I0128 Subjects may not take the following substances for ulcerative colitis, or Irritable Bowel Syndrome (IBS). at least 60 days prior to the screening/randomization visit 0110 4. Subject is lactose intolerant (self-professed or (visit 1) or throughout the study. Any Subject taking these diagnosed). substances during the study is evaluated by the PI. US 2017/01894.57 A1 Jul. 6, 2017 11
I0129. All oral antibiotics including: Amoxil (R), Tri Other than this, subjects are allowed to continue their normal mox(R) (amoxicillin), Cipro(R) (ciprofloxacin), Flagyl(R) diet. (metronidazole), Doxy-100R) (doxycyline) and Bactrim R. Double Strength (trimethoprim-sulfame Exercise thoxazole). 0140. Subjects are asked to refrain from exercise for the 0.130. Any medications that significantly interfere with 24 hours prior to visits 2 and 3. Subjects are allowed to bacterial flora continue their usual exercise routine. Subjects may not take the following Substances for at least 30 days prior to the screening/randomization visit (visit 1) or Product Ingredients throughout the study. Any subject taking these substances during the study is evaluated by the PI. 0.141. The following lists of ingredients are provided. I0131 Laxatives: psyllium husk (Metamucil), methyl Product 1: GANEDEN BC-30TM (Bacillus coagulans ATCC cellulose (Citrucel), polycarbophil, docusate (Colace, Designation Number PTA-6086)+Protein Diocto), mineral oil, Sodium phosphate (and variants), 0142] 1 billion CFU GANEDEN BC-30TM (Bacillus magnesium citrate, magnesium hydroxide (Milk of coagulans ATCC Designation Number PTA-6086) and 23 g magnesia), magnesium Sulfate (which is Epsom salt), of whey protein (whey protein isolate (WPI) >90) in 30 g of glycerin Suppositories, Sorbitol, lactulose, and polyeth powder, chocolate flavor. ylene glycol (PEG) I0132) Digestive enzymes: CotaZym, Creon, Pancrease, Product 2: Protein (Placebo) Digex, Dygase, GastrineX, Hi-Vegi-Lip, Ku-Zyme, 0143 23 g of whey protein (WPID90) in 30 g of powder, Kutrase, Lactaid, Lapase, Lipram, Palcaps 10, Pan chocolate flavor. 2400, Pancreatin, Pancrecarb, Pangestyme, Panocaps, Panokase, Sucraid, Sure|Lac, Ultrase, Viokase, Zenpep Nutrition Facts for the Whey Protein Powder 0.133 All dietary and herbal supplements including: 0144) 0.134 Probiotic products (including fortified foods): Acidophilus, Bacid, Flora-Q, Florastor, Novaflor, RisaOuad, Superdophilus, GANEDEN BC-30TM Serving Size: 1 Packet Amount Per Serving (Bacillus coagulans ATCC Designation Number Calories 130 PTA-6086), LAFTI B94, LAFTI L10, LAFTI L26, Calories from fat 25 Bifiene, Align, Howaru Bifido, ProBactrix, Mutaflor, % Daily Value Actimel/Dan Active, Cultura, Yakult, Lactobacillus Total Fat 2.5 g. 4% fortis, GoodBelly/ProViva/TuZen, LGG, Vi?it, Saturated Fat 1 g 59% Verum, DiarSafe, Bion Flore Intime, Jarrow, Fem Trans Fat Og Dophilus, Florajen3, Bio-K, CL 1285, A Biotica; Cholesterol 65 mg 22% Sodium 50 mg 2% 0.135 Prebiotic products (including fortified foods): Potassium 170 mg 59% lactulose, lactitol oligofructose, inulin, galacto-oli Total Carbohydrate 3 g 190 gosaccharides, tagatose, isomaltooligosaccharides, Dietary Fiber 1 g 4% polydextrose, maltodextrin, fructo-oligosaccharides, Sugars 1 g arabinogalactan, polyols-lactulose; Protein 23 g Vitamin A 0.136 Those supplements purported to improve GI Vitamin C health including dandelion, devil's claw, feverfew, Calcium ginger, goldenseal, lemon balm, peppermint, roman Iron chamomile, turmeric, Valerian, yarrow, mastic gum, dill, caraway, anise, cumin. *Percent Daily Values are based on a 2,000 calorie diet, Diet Ingredients: 0.137 Subjects are required to fast (no food or beverage (0145 Whey Protein Concentrate (Whey Protein Concen other than water, no caffeine) for eight hours prior to visits trate, Soy Lecithin). Whey Protein Isolate, Cocoa Powder 2 and 3. Subjects are asked to refrain from alcohol for the 24 (Processed with Alkali), Natural & Artificial Flavors, Ace hours prior to visits 2 and 3. Subjects are not allowed to eat sulfame Potassium and Sucralose. yogurt or to drink lacto-fermented beverages throughout the 0146. At the test visits, subjects are provided a multiple study period. Examples of lacto-fermented beverages Vitamin with mineral Supplement. The Supplement that is include kefir (water and dairy), kambucha, kvass, cider and used is the One A Day(R) Men's Health Formula. ginger ale. 0.138. Subjects are not allowed to use protein powders The Ingredients in the One a Day Men's Health Formula (e.g. whey, Soy, hemp, pea, rice) while participating in the 0147 Calcium Carbonate, Magnesium Oxide, Microc study. rystalline Cellulose, Ascorbic Acid, Croscarmellose 0139 Subjects are not allowed to eat/drink foods and Sodium, Gelatin, Maltodextrin; Less than 2% of Beta beverages high in vitamin C for 24 hours prior to the test Carotene, Biotin, Cholecalciferol, Chromium Chloride, visits. Examples of foods and beverages high in Vitamin C Crospovidone, Cupric Oxide, Cyanocobalamin, D-Calcium include citrus fruits (such as oranges and grapefruit) and Pantothenate, dl-Alpha-Tocopheryl Acetate. Folic Acid, their juices, red and green pepper, kiwifruit, broccoli, Straw Hydroxypropyl Methylcellulose, Lycopene, Manganese berries, cantaloupe, baked potatoes, and tomatoes. Sulfate, Niacinamide, Phytonadione, Polyethylene Glycol, US 2017/01894.57 A1 Jul. 6, 2017
Pyridoxine Hydrochloride, Riboflavin, Silicon Dioxide, Study Procedures Sodium Selenate, Soybean Oil, Starch, Stearic Acid, Thia mine Mononitrate, Triacetin, Vitamin A Acetate and Zinc Informed Consent Oxide. 0.155. An informed consent form (ICF) is written in Product Dosing accordance with established criteria of the Institutional Review Board (IRB) and the appropriate federal regulations 0148 Product administration instructions: Subjects are (eg. 21 CFR Parts 50 and 56) to describe the study plan, instructed to take one packet per day, at approximately the procedures, and risks. The investigator and the IRB must all same time of day. approve the ICF and any ICF amendments or administrative 0149 Preparation instructions: Mix one packet with six changes before they are used. to eight ounces of cold water, milk or subjects preferred 0156 Investigators ensure that each subject is clearly and beverage for 20 to 30 seconds in a shaker or blender. fully informed of the purpose, potential risks, and require Subjects are instructed to start taking the first study product ments of study participation. Written informed consent must the day after visit 1. Subjects are instructed to not take the be obtained from each Subject before performing any screen study product prior to coming in for visits 2 and 3 (i.e. not ing or other study procedures. take study product the morning of visits 2 and 3). Subjects are required to take the study product on site for visits 2 and Medical History 3. Subjects are instructed to start the second study product 0157. A Medical History is performed at the screening the day after visit 2. visit to collect information on past and current medical Subjects cannot take anything the evening of visit 2. Sub conditions, Surgical history, allergy information, and con jects are instructed to bring the remaining product to all comitant or recently taken (in the past 90 days) medications visits. including over the counter (OTC) non-prescription products, nutritional Supplements, herbals, and investigational prod Product Administration Instructions uctS. 0150 Take one packet per day, at approximately the same time of day; mix with 6 to 8 ounces of water, milk or favorite Physical Examination beverage and shake/blend for 20 to 30 seconds. 0158 Subjects undergo a full physical examination at 0151. The product is packaged so that the subject is screening for determining eligibility. provided only the quantity needed for the time between visits (14 days) plus product to cover the window (additional Vital Signs three days of product). 0159. At each visit, subjects have blood pressure and Product Compliance heart rate measured. 0160 The following guidelines are used for the blood 0152 Compliance is measured via the packet counting pressure and heart rate: Subjects sit in a chair, resting, for at method. By documenting the number of calendar days least five minutes prior to taking the measurements. Mea between visits and the number of packets that should have Surements are done with Subjects in a seated position and the been taken, compliance is calculated. The Subjects compli subject’s left arm is used. ance is recorded (as a % of prescribed amount) for each product and this compliance % is used to establish the subjects eligibility for inclusion in the populations (Safety, Study Questionnaires Intent-to-Treat, and/or Per-Protocol) used for analysis of the 0.161 Dichotomous Questionnaire the following is study's results, in accordance with the criteria specified administered at visits 2 and 3. Each subject is asked: below. (0162 Did you: 0153. For visits 2 and 3, product administration are 0163 Not eat or drink any foods or beverages performed on site. Documentation is done in each subjects other than water or have caffeine for the eight Source confirming product administration. hours prior to the visit? 016.4 Eat or drink any foods or beverages high in Randomization vitamin C in the past 24 hours? 0154 The consulting statistician develops a randomiza 0.165 Drink any alcoholic beverages in the past tion schedule and create a randomization log for use at the 24 hours? investigative site. To minimize the effect of treatment 0166 Take any vitamin and/or mineral supple sequence (carry-over, training, fatigue, seasonality, etc.) on ments in the past 24 hours? safety and efficacy endpoints, the active product (A) and placebo (B) are administered to each subject in one of the (0167 Exercise in the past 24 hours? two possible sequences: A-B or B-A. Block-2 randomization 0168 Use any new vitamin and/or mineral is used, so that the two possible product sequences are Supplements since the screening visit? shuffled into random order for assignment for the first two 0169. Use any protein powder other than the Subjects, then shuffled again into a different random permu study product since the screening visit? tation for assignment to the next two subjects, and so on for 0170 Use any dietary supplements, any probiot the remaining Subjects to be enrolled (plus additional spare ics, or prebiotics since 30 days prior to the screen product). ing visit? US 2017/01894.57 A1 Jul. 6, 2017
0171 Eat or drink any yogurt or lacto-fermented 0.192 Subjects are allowed to do restful activities such beverages since 30 days prior to the screening as read, listen to music with earphones, watch televi visit? sion, work on the computer or rest/sleep. 0172 Did you take the study product today? 0193 Subjects are not allowed to eat or drink anything 0173 If subject answers “yes” to any of the above other than water. questions, the Subject is discontinued as per PI discretion and/or a protocol deviation is recorded. Descriptive Summarization 0.174 GI Questionnaire—a six-part questionnaire regarding general well-being and gastrointestinal 0194 For each efficacy endpoint (amino acid, vitamin, symptoms is administered at visits 1, 2 and 3. Subjects and mineral blood concentration), the following non-com are asked, “how often in the past two weeks have you' partmental pharmacokinetic (PK) parameters is calculated: experienced various GI symptoms with answer choices (0195 C. The maximum concentration observed dur of “all of the time, most of the time, sometimes, a little ing the four post-dose samples; and never'. Subjects are asked to rate how they feel 0.196 t the time at which the maximum concentra overall with answer choices of “better, same and tion was observed; worse'. See, e.g., Worthley et al., 2009 Am J Clin Nutr, 0.197 AUC, the area under the concentration-vs 90: 578-586. For example, the following questions are time curve above the baseline (time 0) concentration, asked: integrated from time 0 to 4 hours post-dose, by trap 0.175 1) How often in the past week have you had pain eZoidal-rule quadrature. in the abdomen? 0198 All safety and efficacy variables are summarized by 0176) 2) How often in the past week have you expe time point and by product. Numerical variables are pre rienced abdominal bloating? sented as mean, standard deviation, count, median, and (0177 3) How often in the past week have you been range (minimum to maximum value). Changes from the troubled by excessive passage of gas through the anus? appropriate baseline are Summarized and presented in the 0.178 4) How often in the past week have you been same way. Numerical variables and their changes from troubled by frequent of loose bowel movements? baseline are displayed graphically, as plots of mean value VS. (0179 5) How often in the past week have you been time, with separate lines for the two products (probiotic vs. troubled by excessive gurgling noises from the abdo placebo). The graphs may contain vertical error-bars around men? the mean values, indicated Standard errors of the mean, if in 0180 6) In comparison to how you felt at the start of the opinion of the statistician, this would make the graphs the study, over the past week, have you felt better, more informative. Categorical variables are presented as worse or the same? tabulations of counts and percentages of totals. 0199 For each continuous variable, the mean change Protein and Micronutrient Absorption Test from baseline to each Subsequent time point are tested for nominal significance by the paired Student t test, or by the 0181. The following test is performed at visits 2 and 3 to non-parametric Wilcoxon test if non-normally distributed. assess the effects of the products on protein, vitamin and 0200 For each continuous variable at each time point, the mineral absorption. mean differences in the variable, or in the change in that The following is performed to standardize the testing con variable from baseline, between the different products are ditions: tested for nominal significance by the paired Student t test or 0182. The test is performed after an eight hour fast: by the non-parametric Wilcoxon test if non-normally dis 0183 Subjects are not physically active during the test; tributed. 0184. No Smoking is allowed during the test. 0201 For each categorical variable, difference in the Test Procedure: distribution of categories between the different product groups are tested for nominal significance by the Fisher 0185. Collect the baseline blood sample (0 hours) for Exact test if possible, or by the Chi-Square test if necessary. the evaluation of the baseline levels of various amino 0202 All p-values appearing in these Summarizations is acids and select vitamins and minerals. considered descriptive, not inferential. No final statistical 0186 Provide subject with the multiple vitamin with conclusions are drawn from them, but they are referred to in mineral Supplement (can be taken with water or the the interpretation of the changes. study product). 0187 Provide subject with the study product which is Statistical Analysis to be consumed within 10 minutes. 0188 Timing of the test is from when the subject Safety and Efficacy Variables and Endpoints begins to drink the study product. 0203 Safety variables consist of adverse events and 0189 Collect blood samples at 1, 2, 3, and 4 hours for Subjective remarks. the evaluation of the absorption of the amino acids, 0204 Safety endpoints consist of the changes in the Vitamins and minerals. safety variables listed above with each product following 14 days of treatment. During the Time Between Blood Draws: 0205 There is no formal safety objective. 0190. Subjects are primarily sitting. 0206 Efficacy variables consist of protein absorption 0191 Subjects are allowed to walk around the site to rate of absorption (t) and overall absorption (AUCo., use the rest room and to stretch but are not allowed to and C) of blood amino acids, vitamin absorption rate of do any additional physical activity. absorption (t) and overall absorption (AUCo., and US 2017/01894.57 A1 Jul. 6, 2017
C) of vitamins B6, B12 and C. mineral absorption rate 37. The method of claim 34, wherein the lean body mass of absorption (t) and overall absorption (AUCo., and of said animal is increased. C) of minerals calcium, iron and Zinc, and GI question 38. The method of claim 34, wherein said animal is a naire scores (abdominal pain, abdominal bloating, gas, primate, a mouse, a rat, a dog, a cat, a cow, a horse, or a pig. bowel movements, gurgling noises and overall well-being). 39. The method of claim 34, wherein said animal is a 0207 Efficacy endpoints generally consist of changes in livestock animal. efficacy variables from baseline to four hours post product 40. The method of claim 34, said animal is a performance administration following 14 days of product use. These animal. changes (endpoints) are then compared between the two 41. The method of claim 34, wherein said performance products. animal is a horse or a dog. 42. The method of claim 41, wherein said performance Other Embodiments animal is a race horse. 43. The method of claim 41, wherein said performance 0208 While the invention has been described in conjunc animal is a work dog. tion with the detailed description thereof, the foregoing 44. The method of claim 34, wherein said composition description is intended to illustrate and not limit the scope of comprises at least 25% protein by weight. the invention, which is defined by the scope of the appended 45. The method of claim 34, wherein the absorption of claims. Other aspects, advantages, and modifications are said protein is increased in said animal by at least 10% as within the scope of the following claims. compared to the absorption of said protein after administra 0209. The patent and scientific literature referred to tion of protein in the absence of Bacillus coagulans. herein establishes the knowledge that is available to those 46. The method of claim 34, wherein the absorption of a with skill in the art. All United States patents and published Vitamin is increased in said Subject as compared to the or unpublished United States patent applications cited herein absorption of said vitamin after administration said vitamin are incorporated by reference. All published foreign patents in the absence of Bacillus coagulans, wherein said vitamin and patent applications cited herein are hereby incorporated comprises vitamin A, Vitamin B, Vitamin B, Vitamin B. by reference. Genbank and NCBI submissions indicated by Vitamin Bs, vitamin B vitamin B7, Vitamin Bo, Vitamin B2, accession number cited herein are hereby incorporated by or vitamin C, Vitamin D. Vitamin E, or vitamin K. reference. All other published references, documents, manu 47. The method of claim 34, wherein the absorption of a scripts and scientific literature cited herein are hereby incor chemical element is increased in said Subject as compared to porated by reference. the absorption of said chemical element after administration 0210 While this invention has been particularly shown of said chemical element in the absence of Bacillus coagul and described with references to preferred embodiments lans, wherein said chemical element comprises Sodium, thereof, it will be understood by those skilled in the art that potassium, calcium, iron, or zinc. various changes in form and details may be made therein 48. The method of claim 34, wherein said composition without departing from the scope of the invention encom further comprises a vitamin Supplement or mineral Supple passed by the appended claims. ment. 1-33. (canceled) 49. The method of claim 34, wherein said composition 34. A method for increasing the muscle development, further comprises an omega-3 fatty acid. strength, or lean body mass of an animal, comprising 50. The method of claim 34, wherein said isolated Bacil administering to said animal a composition comprising lus coagulans bacteria is present as at least 90% spores. protein and isolated Bacillus coagulans bacteria, wherein 51. The method of claim 34, wherein said protein com (a) said isolated Bacillus coagulans bacteria are present as prises whey protein, casein protein, egg albumin protein, Soy at least 75% spores; and protein, pea protein, hemp seed protein, or rice protein. (b) said composition comprises at least 20% protein by 52. The method of claim 34, wherein said protein com weight, prises whey protein. wherein said animal is not a human, 53. The method of claim 34, wherein said isolated Bacil thereby increasing the muscle development, strength, or lus coagulans bacteria are selected from the group consist lean body mass of said animal. ing of GBI-30 strain bacteria (ATCC Designation Number 35. The method of claim 34, wherein the muscle devel PTA-6086), GBI-20 strain bacteria (ATCC Designation opment of said animal is increased. Number PTA-6085), and GBI-40 strain bacteria (ATCC 36. The method of claim 34, wherein the strength of said Designation Number PTA-6087). animal is increased. k k k k k