US 2010O21 0000A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0210000 A1 Farmer et al. (43) Pub. Date: Aug. 19, 2010

(54) PROBIOTIC GRAIN-BASED COMPOSITIONS (21) Appl. No.: 12/580,622 (75) Inventors: Sean Farmer, Miami Beach, FL (22) Filed: Oct. 16, 2009 (US); Andrew R. Lefkowitz, University Heights, OH (US); Related U.S. Application Data Michael Bush, Brecksville, OH (60) Provisional application No. 61/106,116, filed on Oct. (US); David Maske, Chagrin Falls, 16, 2008. OH (US) Publication Classification Correspondence Address: (51) Int. Cl. MINTZ, LEVIN, COHN, FERRIS, GLOVSKY CI2N L/20 (2006.01) AND POPEO, PC ONE FINANCIAL CENTER (52) U.S. Cl...... 435/252.5 BOSTON, MA 02111 (US) (57) ABSTRACT (73) Assignee: Ganeden Biotech, Inc., Mayfield The present application relates to probiotic grain-based com Heights, OH (US) positions comprising lactic acid-producing bacteria.

3.1 x 10" cfu/g BC30 survival in potato pasta " 10 cfu/g

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E-4 Giocchi. BC30 TO Grocchi. BC3OT Grocchi +BC3OT2 Gnocchi +BC3T3 Gnocchi +BC3 T4 (no treat) (preboiling 100°C (pasteurization in (horne Cooking (half shelflife 4°C) 1'30") pack 500g, 95°C 100°C 130" h2O) Preparation steps Patent Application Publication Aug. 19, 2010 US 2010/0210000 A1

Figure 1.

3.1 x 10" cfu/g BC30 survival in potato pasta cfu/g

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OOE-04 Gnocchi- BC3OO Gnocchi. BC3OT1 Gnocchi +BC3O2 Gnocchi +BC303 Gnocci +BC30 A. (no treat) (preboiling 100°C (pasteurization in (home cooking (half shelflife 4°C) 1'30") pack 500g, 95°C 100°C '30" 1 h2O) Preparation steps

Figure 2.

BC30 Survival in the fresh egg pasta filling

1.OO+8 1.OOE+7 1.OOE+06 100+5 100E+04 Total bacteria 1.OOE+(3 100+C2 1.OOE-- 100+OO Cheese based Cheese based Cheese based Fitting TO (no treat Filing + BC30 TO (no Filing + BC301 treat) (pasteurization 100°C 5) Preparation steps US 2010/021 0000 A1 Aug. 19, 2010

PROBOTC GRAIN-BASED COMPOSITIONS PTA-6086; and GBI-40, ATCC Designation Number PTA 6087; see U.S. Pat. No. 6,849,256 to Farmer). RELATED APPLICATIONS 0008 Optionally, the isolated Bacillus coagulans is in the 0001. This application claims the benefit of U.S. Ser. No. form of a spore. In one aspect, the Bacillus coagulans spores 61/106,116, filed Oct. 16, 2008, the contents of which are activate upon contacting hot liquid. Preferably, the hot liquid incorporated by reference herein in their entirety. is water or milk. Alternatively, the isolated Bacillus coagul lans is in the form of a vegetative cell. In another aspect, the FIELD OF THE INVENTION isolated Bacillus coagulans is in the form of a mixture of Vegetative cells and spores. Preferably, the Bacillus coagul 0002 The present application relates to probiotic grain lans is predominantly in spore form, e.g., about 75%, about based compositions comprising lactic acid-producing bacte 80%, about 85%, about 90%, about 95%, about 99%, or about ria. 100% spores. 0009. The invention also provides compositions compris BACKGROUND OF THE INVENTION ing a dry mix for grain-based compositions comprising a 0003. The gastrointestinal microflora plays a number of grain and an isolated Bacillus coagulans bacterium. Also Vital roles in maintaining gastrointestinal tract function and provided are compositions comprising a dry mix for soup overall physiological health. The growth and metabolism of comprising a dehydrated matter and an isolated Bacillus the many individual bacterial species inhabiting the gas coagulans bacterium. trointestinal tract depend primarily upon the Substrates avail 0010 Also provided are methods of making a grain-based able to them, most of which are derived from the diet. Since composition comprising providing a grain-containing base probiotics do not generally permanently colonize the host, mix and a liquid portion; mixing the grain-containing base they need to be ingested regularly for any health promoting mix and the liquid portion to form a batter or dough; combin properties to persist. ing an isolated Bacillus coagulans bacterium with the batter or dough; and heat processing the batter or dough to cook the SUMMARY OF THE INVENTION grain-based composition. Suitable liquid portions include 0004. The invention is based on the discovery that lactic water and milk. In one aspect, the isolated Bacillus coagulans acid-producing bacteria, particularly Bacillus species, is in the form of a spore. In another aspect, the isolated remain viable and retain their beneficial probiotic properties Bacillus coagulans is in the form of a vegetative cell. In one in food compositions, such as those prepared at high tempera aspect, the isolated Bacillus coagulans comprise between tures (e.g., 80,90, 100, 120, or 150° C.) in boiling water. The about 0.1% to about 50% by weight of the grain-based com invention describes probiotic grain-based compositions. Spe position. Preferably, the isolated Bacillus coagulans com cifically, the invention provides an isolated Bacillus coagul prise between about 1% and about 10% by weight of the lans bacterium in a grain-based composition. grain-based composition. Most preferably, the amount of 0005. The invention provides compositions comprising a Bacillus coagulans bacteria is about 5x10" colony forming cooked or uncooked composition of a grain and an isolated units (CFU) of bacteria per gram of food matrix. Bacillus coagulans bacterium or spore. The grain is pro 0011 Bacillus coagulans bacteria are included in the cessed, e.g., altered from its naturally-occurring state. For grain-based or Soup compositions of this invention. Bacterial example, the grain is husked, crushed, cracked, or ground. species include Bacillus coagulans, e.g., Bacillus coagulans The grain is in the form of or a composition made from hammer, preferably Bacillus coagulans hammer Strain further manipulation of a grain-based flour. Exemplary grains Accession No. ATCC 31284, or one or more strains derived include wheat, rice, buckwheat, barley, Kamut, corn and oats. from Bacillus coagulans hammer strainAccession No. ATCC Exemplary cooked compositions include pasta, oatmeal, and 31284 (e.g., ATCC Numbers: GBI-20, ATCC Designation grits. Suitable pastas include egg pasta, spaghetti (solid, thin Number PTA-6085; GBI-30 or BC, ATCC Designation cylinders), macaroni (tubes or hollow cylinders), fusilli (spi Number PTA-6086; and GBI-40, ATCC Designation Number ral-shaped), lasagna (sheets), tagliatelle (flat ribbons), Vermi PTA-6087; see U.S. Pat. No. 6,849.256 to Farmer). celli (thin spaghetti), ravioli (filled pasta), spätzle and gnoc 0012. In one aspect, the isolated Bacillus coagulans is in chi. Other suitable pastas include penne rigate (furrowed the form of a spore. The invention provides for the activation cylinder-shaped pasta), penne lisce (Smooth cylinder-shaped of Bacillus coagulans spores upon heating. Optionally, the pasta), rotini (corkscrew-shaped pasta), and rigatoni (tube isolated Bacillus coagulans is in the form of a vegetative cell. shaped pasta). In another aspect, the isolated Bacillus coagulans is in the 0006. In one aspect, the isolated Bacillus coagulans com form of a mixture of vegetative cells and spores. prise between about 0.01% to about 50% by weight of the 0013 The Bacillus coagulans Hammer strains of the grain-based composition. Optionally, the isolated Bacillus invention are non-pathogenic and generally regarded as safe coagulans comprise between about 0.01% and about 10% by for use in human nutrition (i.e., GRAS classification) by the weight of the grain-based composition. Preferably, the iso U.S. Federal Drug Administration (FDA) and the U.S. lated Bacillus coagulans comprise between about 0.01% and Department of Agriculture (USDA), and by those skilled in about 0.1% by weight of the grain-based composition. the art. Furthermore, the Bacillus coagulans Hammer strains 0007. The invention also provides bacterial species of the invention germinate at or below human body tempera including Bacillus coagulans, e.g., Bacillus coagulans ham ture, rendering them useful as probiotics. Many Bacillus mer, preferably Bacillus coagulans hammer Strain Accession coagulans Strains outside the Hammer group have mostly No. ATCC 31284, or one or more strains derived from Bacil industrial applications, little or no nutritional benefit, and lus coagulans hammer strain Accession No. ATCC 31284 environmental contaminants that have not been evaluated for (e.g., ATCC Numbers: GBI-20, ATCC Designation Number safety. Moreover, many other non-Hammer strains of Bacil PTA-6085; GBI-30 or BC, ATCC Designation Number lus coagulans grow optimally at temperatures that exceed US 2010/021 0000 A1 Aug. 19, 2010

human body temperature and, thus, do not germinate effi endonucleases (e.g., U.S. Pat. No. 5,200,336); amylase (U.S. ciently in the human body. Such strains are less or not suitable Pat. No. 4,980,180); lactase (U.S. Pat. No. 4.323,651); and as probiotics for human consumption. cyclo-malto-dextrin glucano-transferase (U.S. Pat. No. 0014 Cited publications are incorporated herein by refer 5,102,800). B. coagulans has been used to produce lactic acid ence. Both the foregoing general description and the follow (U.S. Pat. No. 5,079,164). A strain of B. coagulans (referred ing detailed description and examples are exemplary and to as L. Sporogenes; Sakaguti & Nakayama (ATCC 31284)) explanatory only and are not restrictive of the invention as has been combined with other lactic acid producing bacteria claimed. and B. natto to produce a fermented food product from BRIEF DESCRIPTION OF THE DRAWINGS steamed soybeans (U.S. Pat. No. 4,110.477). 0022. Bacterial species include Bacillus coagulans, e.g., 0015 FIG. 1 is a bar graph demonstrating the survival of Bacillus coagulans hammer, preferably Bacillus coagulans BC" in gnocchi (potato pasta). hammer strain Accession No. ATCC 31284, or one or more 0016 FIG. 2 is a bar chart illustrating the survival of BC strains derived from Bacillus coagulans hammer strain in fresh egg pasta filling. Left bar: untreated pasta with Accession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20, cheese-based filling (containing bacterial cheese cultures); ATCC Designation Number PTA-6085; GBI-30, ATCC Des middlebar: pasta with cheese-based filling treated with BC': ignation Number PTA-6086; and GBI-40, ATCC Designation right bar pasta with cheese-based filling treated with BC Number PTA-6087; see U.S. Pat. No. 6,849.256 to Farmer). and pasteurized at 100° C. for 5 minutes. 0023 Bacillus coagulans was previously mis-character ized as a Lactobacillus and labeled as Lactobacillus sporo DETAILED DESCRIPTION OF THE INVENTION genes (See Nakamura et al. 1988. Int. J. Syst. Bacteriol. 38: 0017 Probiotic organisms are non-pathogenic, non-toxi 63-73). However, initial classification was incorrect because genic, retain viability during storage, and Survive passage Bacillus coagulans produces spores and excretes L(+)-lactic through the stomach and Small intestine. Non-pathogenic acid through metabolism. Both of these characteristics pro lactic acid-producing bacteria (i.e., "lactic acid bacteria'), vide key features to the utility of Bacillus coagulans. These Such as the exemplary Bacillus coagulans, remain viable and developmental and metabolic aspects required that the bac retain their beneficial probiotic properties in grain-based and terium be classified as a lactic acid Bacillus. In addition, it is Soup compositions, such as those prepared in boiling water. not generally appreciated that classic Lactobacillus species Specifically, the probiotic organisms described herein, e.g., are unsuitable for colonization of the gut due to their insta Bacillus coagulans strain GBI-30 or BC, ATCC Designa bility in the harsh (i.e., acidic) pH environment of the bile, tion Number PTA-6086, survive the harsh manufacturing and particularly human bile. By contrast, Bacillus coagulans is cooking processes of the grain-based and Soup compositions able to Survive and colonize the gastrointestinal tract in the described below. bile environment and even grown in this low pH range. Probiotic Activity of Bacillus coagulans Probiotic Lactic Acid-Producing Bacteria 0024. It is well-documented clinically that many species 0018. A probiotic lactic acid-producing bacteria suitable of bacterial, mycotic and yeast pathogens possess the ability for use in the methods and compositions of the invention to cause a variety of gastrointestinal disorders including, but produces acid and is non-pathogenic. There are many Suitable not limited to: disruption of normal gastrointestinal bio bacteria identified as described herein, although the invention chemical function, necrosis of gastrointestinal tissues, and is not limited to currently known bacterial species insofar as disruption of the bioabsorption of nutrients, and like condi the purposes and objectives of the bacteria is described. The tions. The probiotic microorganism-containing compositions property of acid production is important to the effectiveness described herein inhibit these pathogens. Thus, the composi of the probiotic lactic acid-producing bacteria of this inven tions are useful in the prophylactic ortherapeutic treatment of tion. conditions associated with infection by these aforementioned 0019. The invention provides using a lactic acid-produc pathogens. ing bacteria, such as a spore-forming Bacillus species, such as 0025. In one aspect, a Bacillus coagulans Strain is B. coagulans. Preferably, the spore-forming Bacillus species included in the composition in the form of vegetative cells. In of the invention is B. coagulans Hammer. another aspect, the Bacillus coagulans strain is included in 0020 Exemplary methods and compositions are the composition in the form of spores. The invention also described herein using Bacillus coagulans as a probiotic. provides for including the Bacillus coagulans strain in the Purified and/or isolated Bacillus coagulans is particularly composition in the form of a powder, a dried cell mass, a useful as a probiotic in grain-based or soup compositions. stabilized paste, or a stabilized gel. Probiotic B. coagulans is non-pathogenic and is generally 0026. Because Bacillus spores are heat and pressure-resis regarded as safe (i.e., GRAS classification) by the U.S. Fed tant and can be stored as a dry powder, they are particularly eral Drug Administration (FDA) and the U.S. Department of useful for formulation into and manufacture of products Such Agriculture (USDA), and by those skilled in the art. as the various grain-based and Soup compositions described 0021 Bacillus coagulans is a non-pathogenic gram posi herein. A Bacillus species is well suited for the present inven tive spore-forming bacteria that produces L(+) lactic acid tion, particularly species having the ability to form spores (dextrorotatory) in fermentation conditions. It has been iso which are relatively resistant to heat and other conditions, lated from natural sources, such as heat-treated soil samples making them ideal for storage (shelf-life) in product formu inoculated into nutrient medium (Bergey’s Manual off Sys lations, e.g., grain-based and Soup compositions. Due to the temic Bacteriology, Vol. 2, Sneath, P. H. A., et al., eds., Wil shelf-stable properties of the Bacillus coagulans strains liams & Wilkins, Baltimore, Md., 1986). Purified B. coagul described herein, e.g., Bacillus coagulans strain GBI-30 or lans strains have served as a source of enzymes including BC, ATCC Designation Number PTA-6086, the product US 2010/021 0000 A1 Aug. 19, 2010

formulations of the invention are not confined to a refrigerator process well-known in the art. The isolated Bacillus coagul and may be stored at room temperature. lans are packaged, or encapsulated, within another material in 0027. The Bacillus coagulans of the invention survives order to protect the bacteria from the surrounding environ storage (shelf-life) from about 12 days to about 2 years; from ment. The capsules of the invention range in size from one about 1 month to about 18 months; from about 3 months to thousandth of a millimeter to seven millimeters. The internal about 1 year; or from about 6 months to about 9 months. ingredients of the microcapsule are released from their shells in various ways, including mechanical rupture of the capsule Anti-Microbial Probiotic Activity wall, dissolution of the wall, melting of the wall and diffusion 0028. The probiotic organisms described herein, e.g., through the wall. Thus, micro-encapsulation provides addi Bacillus coagulans strain GBI-30 or BC, ATCC Designa tional protection to the isolated Bacillus bacterium during tion Number PTA-6086, promote digestive health and Sup heat processing of the grain-based compositions of the inven port the immune system. The ability of Bacillus coagulans to tion. Physical methods of micro-encapsulation include pan inhibit various bacterial pathogens was quantitatively ascer coating, air-Suspension coating, centrifugal extrusion, vibra tained by use of an in vitro assay. This assay is part of a tional nozzle, and spray-drying. Chemical methods of micro standardized bacterial pathogen screen (developed by the encapsulation include interfacial polymerization, in-situ U.S. Food and Drug Administration (FDA)) and is commer polymerization, and matrix polymerization. cially available on solid support disks (DIFCOR) BAC 0032. Alternatively, the lactic-acid producing bacteria is TROL(R) Antibiotic Disks). To perform the assay, potato-dex added to the grain-based composition without micro-encap trose plates (DIFCOR) were initially prepared using standard Sulation. procedures. The plates were then individually inoculated with the bacteria (approximately 1.5x10 CFU) to be tested so as to Probiotic Grain-Based and Soup Compositions form a confluent bacterial bed. 0033. The invention is directed to the surprising discovery 0029. Inhibition of microorganisms (e.g. gastrointestinal that lactic acid-producing bacteria, particularly Bacillus spe pathogens) by Bacillus coagulans was Subsequently ascer cies, remain viable and retain their beneficial probiotic prop tained by placing approximately 1.8x10 CFU of Bacillus erties in grain-based and Soup compositions, such as those coagulans in 10 ul of broth or buffer, directly in the center of prepared in boiling water. The compositions are prepared by the potato-dextrose plate with one test locus being approxi combining dry matter and a liquid, e.g., water or milk. In one mately 8 mm in diameter per plate. A minimum of three test aspect, the composition is prepared by combining dry matter loci were used for each assay. The negative control consisted and a liquid, and heating the resulting combination. Option of a 10 ul volume of a sterile saline solution, whereas the ally, the combination is heated (heat-processed) using applied positive control consisted of a lul Volume of glutaraldehyde. heat, a flame, or a microwave. The grain-based or soup com The plates were then incubated for approximately about 18 hr position is boiled in hot water, e.g., Stovetop boiling, addition at 30° C., at which time the Zones of inhibition were mea of boiling water to a container, or microwaving the grain sured. As designated herein, “excellent inhibition' means the based or soup composition along with water. Preferably, boil Zone was 10 mm or greater in diameter; and “good inhibition' means the Zone was greater than 2 mm in diameter but less ing water (about 100° C.) is added to a combination of grain than 10 mm in diameter. based composition and Bacillus coagulans bacteria. 0034. In one aspect, at least about 5%-25% of the bacteria 0030. As expected, no “inhibition' was seen with the are viable after heating, e.g., at least about 25%-50%; at least negative, saline control, and excellent “inhibition' (approxi about 50% to 75%; or at least about 75%-99% of the bacteria mately 16.2 mm diameter, average of three tests) was seen are viable after heating. As the recommended dietary allow with the positive, glutaraldehyde control. For the enteric ances (RDA or recommended daily intake; RDI) is about microorganisms tested, the following inhibition by Bacillus 1x10 bacterium (according to EU guidelines), preferably, coagulans was found: (i) Clostridium species—excellent the grain-based or soup composition comprises at least about inhibition; (ii) Escherichia coli excellent inhibition; (iii) Clostridium species—excellent inhibition, where the Zone of 1x10 viable bacteria after heating. In another aspect, the inhibition was consistently greater than 15 mm in diameter. grain-based or soup composition comprises at least about Similarly, excellent inhibition was also seen for the opportu 1x10° to 1x107; at least about 1x107 to 1x10: orat least about nistic pathogens Pseudormonas aeruginosa, and Staphyllo 1x10 to 1x10 viable bacteria after heating. coccus aureus. Pathogenic enteric bacteria which were inhib 0035. The compositions are formulated in many configu ited by Bacillus coagulans activity include, but are not limited rations, because the bacterium is present as a vegetative cellor to: Staphylococcus aureus, Staphylococcus epidermidis, as a spore, or both, depending on the species and form of the Streptococcus pyogenes, Pseudomonas aeruginosa, Escheri probiotic organism. The cells/spores are formulated in a vari chia coli (enterohemorragic species); numerous Clostridium ety of compositions Suited for use in a grain-based or soup species (e.g., Clostridium perfingens, Clostridium botulinum, composition. In one aspect, the bacterium is present as a Clostridium tributrycum, Clostridium sporogenes, and the mixture of spores and vegetative cells. In another aspect, the like); Gardnereia vaginails, Proponbacterium aenes, Aero bacterium is present as at least 90% spores, e.g., 95%, 98%, or monas hydrophia, Aspergillus species; Proteus species; and 99% spores. Optionally, prior to addition to the grain-based or Klebsiella species. Soup compositions of the invention, the Bacillus coagulans cells are cultured in liquid in the absence of or with limited Micro-Encapsulation quantities of a food source to induce sporulation. In another aspect, heat gun spray drying kills about 50%, about 75%, 0031. In one aspect, the lactic-acid producing bacteria are about 90%, about 95%, or about 99% of vegetative cells prior incorporated into a microcapsule coating prior to addition to to addition to the grain-based or soup compositions of the the grain-based composition, using any micro-encapsulation invention. US 2010/021 0000 A1 Aug. 19, 2010

0036 Grain-based compositions, such as those described or cut into Small pieces to make Steel-cut oats, or steamed and herein, are made from a variety of grains known to those rolled to make rolled oats. In the case of rolled oats (old skilled in the art. Suitable grains include rice, wheat, , fashioned oats), oat groats are steamed, pressed with a roller, barley, rye, oats, buckwheat, Sorghum, millets, triticale, and dried. Rolled oats take about 15 minutes to cook. The fonio, and quinoa. Other types of grains used to make the quick-cooking rolled oats (“quick oats”) are cut into Small grain-based compositions of the invention include teff, wild pieces before being steamed and rolled. “Instant' oatmeal is rice, and durum. pre-cooked and dried. Optionally, the oatmeal includes: 0037 Exemplary grain-based compositions include pasta, sweetener and flavor additives. Suitable Sweeteners and fla oatmeal, grits, cereal, etc. The invention provides probiotic Vor additives include salt, white , brown Sugar, Stevia, enhanced pasta, e.g., isolated Bacillus coagulans and pasta. cinnamon, honey, jam, molasses, maple syrup, , choco Pasta (Italian for “dough') is a generic term for Italian vari late, soy sauce, soy milk, milk, Vinegar, condensed or evapo ants of noodles, food made from a dough of flour, water rated milk, and cream. Various types of fruit and nuts are also and/or eggs. Pasta is cooked in hot water/boiling water prior often added, including: Strawberries, blueberries, apples, to consumption. The probiotic organisms described herein, peaches, mangos, bananas, , dried cherries, dried cran e.g., Bacillus coagulans strain GBI-30 or BC, ATCC Des berries, pecans, walnuts, and peanut butter. Oatmeal is used to ignation Number PTA-6086, uniquely survive the harsh make , as an ingredient as in oatmeal cookies and oat manufacturing and cooking processes of the grain-based and cakes, or as an accent as in the topping on many oat bran Soup compositions. In one aspect, the pasta is the primary breads and the coating on Caboc cheese. Oatmeal is used as a ingredient, served with sauce or seasonings. Common vari thickener in some foods such as canned chilicon came. Oat eties of pasta include tubular pasta, Straight round rod pasta, meal is also used in Some alcoholic drinks, cosmetics, Soaps, ribbon pasta, micro pasta, stuffed pasta, and irregular-shaped external medical treatments, and is sometimes added to ani pasta. Exemplary pastas include spaghetti (Solid, thin cylin mal feed products. ders), macaroni (tubes or hollow cylinders), fusilli (spiral 0040. In another aspect, the probiotic composition of the shaped), lasagna (sheets), tagliatelle (flat ribbons), Vermicelli invention is grits with isolated Bacillus coagulans. Grits is an (thin spaghetti), and ravioli (filled pasta). Other suitable pas American Indian corn-based food common in the Southern tas include penne (cylinder-shaped pasta), rotini (corkscrew United States, consisting of coarsely ground corn. Tradition shaped pasta), and rigatoni (tube-shaped pasta). In Italy, ally the corn for grits is ground by a stone mill. The results are penne are produced in two variants: "penne lisce” (Smooth) passed through screens, with the finer part being corn meal, and "penne rigate' (furrowed), the latter having ridges on and the coarser part being grits. each noodle. Two other noodles, gnocchi and spätzle, are 0041. Also provided are probiotic instant soups including Sometimes counted as pasta because they are traditional in isolated Bacillus coagulans in Soups that require hot water. Italy; however, their “native' distributions (and perhaps their Soup is a food that is made by combining ingredients such as origins) are outside Italy, and these noodles have more in meat and vegetables in Stock or hot/boiling water, until the common with dumplings than with typical pasta. The two flavor is extracted, forming a broth. Traditionally, Soups are basic styles of pasta are dried and fresh. Dried pasta has a classified into two broad groups: clear soups and thick Soups. firmer, denser texture when cooked and is Suited to chunky, Thick Soups are classified depending upon the type of thick meaty or oily sauces. Fresh pasta has a softer, more absorbent ening agent used: purées are vegetable soups thickened with texture and is Suited to buttery or creamy sauces or sauces ; bisques are made from puréed shellfish thickened with with delicate flavors. There are also variations in the ingredi cream; cream soups are thickened with bechamel sauce; and ents used in pasta. The time for which pasta can be stored veloutés are thickened with eggs, butter and cream. Other varies from days to years depending upon whether the pasta is ingredients commonly used to thicken Soups and broths made with egg or not, and whether it is dried or fresh. include rice, flour, and grain. Oriental-style soup mixes con 0038. Many ingredients are used to make pasta dough, taining ramen noodles are marketed as an inexpensive instant ranging from a simple flour and water mixture, to those that lunch, requiring only hot water for preparation. Various types call for the addition of eggs, spices and cheeses, or even Squid of Soups include tomato Soup, cream of mushroom soup, ink to the dough. Optionally, the pasta contains a filling, e.g., chicken noodle Soup, vegetable beef Soup, minestrone soup, cheese, vegetables, fruit, and/or meat. In one aspect, dry pasta leek and potato Soup, lentil soup, fish Soup, miso Soup, is made from durum wheat or semolina flour. Durum flour has Soup, fruit Soup, clam chowder, gumbo, and bisque. Many a yellow tinge in color. Alternatively, dry pasta is made from Soups, such as Vegetable, chicken base, potato, pasta and other types of flour (such as farina), which yields a softer cheese soups, are also available in dry mix form, ready to be product. Particular varieties of pasta may also use other grains served by adding hot water. Dry mix Soup includes dehy and/or milling methods to make the flour. Some pasta variet drated matter, e.g., dehydrated meat, such as poultry and beef, ies, such as Pizzoccheri, are made from buckwheat flour. dehydrated vegetables, dehydrated herbs, dehydrated spices, Various types of fresh pasta include eggs (egg pasta). Gnocchi dehydrated noodles, etc. A packet of dry Soup stock (e.g., are often considered pasta dishes, although they are quite ramen) typically does not contain water. The instant Soup is different in ingredients (mainly milled potatoes). prepared by adding water first, and then heating the product 0039. Also provided are probiotic grain-based composi for a short time (usually 3-5 minutes) or by adding hot water tions in the form of oatmeal with isolated Bacillus coagulans. directly to the dry soup mix. Instant Soup can also be pre Oatmeal is a product of ground oat groats (i.e., oat-meal, served into a dry powder which can be stored in, e.g., a packet cornmeal, peasemeal, etc.) or a porridge made from this prod or a cup. Bacillus coagulans bacteria in the form of spray uct (also called oatmeal cereal). In regions such as the United dried powder is added prior to or subsequent to addition of the States and Canada, “oatmeal can refer to other products dry mix Soup powder to hot water. made from oat groats, such as cut oats, crushed oats, and 0042. In one aspect, Bacillus coagulans bacteria in the rolled oats. The groats are coarsely ground to make oatmeal, form of a spray-dried powder is included in or on the surface US 2010/021 0000 A1 Aug. 19, 2010 of the probiotic grain-based composition described herein. CFU/g; or 10' to 10' CFU/g of food matrix. In one aspect, Preferably, the isolated Bacillus coagulans is in the form of a the amount of bacteria is about 1x10 CFU per gram of food spore. The isolated Bacillus coagulans are at least 85%, at matrix. The actual amount in a grain-based or soup compo least 90%, at least 95%, or at least 99% pure spores. Alterna sition will vary depending upon the amounts of composition tively, the isolated Bacillus coagulans is in the form of a to be dispersed into the food composition and upon routes of Vegetative cell. In one aspect, the isolated Bacillus coagulans dispersal. are at least 85%, at least 90%, or at least 95% pure vegetative 0048. In one aspect, the invention provides for storing the cells. In another aspect, the isolated Bacillus coagulans is in grain-based or dry mix Soup composition in a sterile package the form of a mixture of vegetative cells and spores. The at room temperature prior to consumption. Alternatively, the Bacillus coagulans mixture is 90% spores, 10% vegetative composition is consumed immediately. cells: 75% spores, 25% vegetative cells; 60% spores, 40% 0049. In another aspect, the composition comprises at vegetative cells; 50% spores, 50% vegetative cells; 60% veg least 85%, at least 90%, at least 95% or 100% isolated Bacil etative cells, 40% spores; 75% vegetative cells; 25% spores: lus coagulans spores. or 90% vegetative cells, 10% spores. 0050. By way of example, and not of limitation, Bacillus 0043. The Bacillus and/or Bacillus coagulans isolated coagulans spores may be incorporated into any type of dry or active agent is applied using any of a variety of known meth lyophilized product which is dissolved or mixed with hot ods including, for example, applying a powder, spray-drying water, so long as the temperature of the Bacillus coagulans the probiotic onto the grain-based or dry mix Soup composi spore-containing mixture is raised to the required heat-shock tion, or soaking the composition in a solution containing the temperature (i.e., 80°C. for 5 minutes) necessary for germi probiotic. Optionally, the Bacillus bacterium is added to the nation of the spores. The Bacillus coagulans spores may dough and dried into the product (e.g., pasta). Alternatively, either be incorporated into the dry or lyophilized product by the Bacillus bacterium is mixed with the dry mix product the manufacturer of the product or by the consumer during (e.g., oatmeal or soup) prior to boiling in water. In another preparation. These dry or lyophilized products include, but aspect, Bacillus coagulans bacteria in the form of spray-dried are not limited to: dry mix soups, pasta, oatmeal, grits, etc. powder is added directly to the grain-based or Soup compo The grain-based or Soup composition is Subsequently boiled sition itself. In yet another aspect, maltodextrin along with in hot water, e.g., Stovetop boiling, addition of boiling water Bacillus coagulans bacteria in the form of spray-dried pow to a container, or microwaving the grain-based or Soup com der is added directly to the grain-based or soup composition position along with water. itself. Optionally, about 5x107 CFU Bacillus coagulans bac 0051. In one aspect, the Bacillus coagulans spores survive teria (per gram of food matrix) in the form of spray-dried storage (shelf-life), i.e., retain viability or the ability to ger powder along with maltodextrin is added directly to the food minate at physiological conditions (e.g., ingestion), from composition itself. about 12 days to about 2 years; from about 1 month to about 0044 Any of a variety of methods for placing the bacterial 18 months; from about 3 months to about 1 year; or from composition into a grain-based or Soup composition can be about 6 months to about 9 months. used. However, preferred methods include a “spray-dry” Example 1 method in which the compositions are exposed in a low humidity chamber to an atomized mix containing a liquid Preparation of Bacillus coagulans Cultures composition, where the chamber is Subsequently exposed to 0.052 Bacillus coagulans Hammer bacteria (ATCC approximately 80-110° F. to dry the liquid, thereby impreg Accession No. 31284) was inoculated and grown to a cell nating the material of grain-based or dry mix Soup composi density of about 10 to 10 cells/ml in nutrient broth contain tion with the components. ing 5 g Peptone, 3 g Meat extract, 10-30 mg MnSO, and 0045. A typical concentration is from approximately 1,000 ml distilled water, adjusted to pH 7.0, using a standard 1x107 to 1x10'? CFU; 1x10 to 1x10 CFU; or 1x10 to airlift fermentation vessel at 30° C. The range of MnSO 1x10" CFU of viable bacterium or spores/g of food matrix. acceptable for sporulation is 1 mg/l to 1 g/l. The vegetative Following drying, the food is ready for immediate use or for cells can actively reproduce up to 45° C., and the spores are storage in a sterile package, e.g., a 3-ounce package, a stable up to 90° C. After fermentation, the B. coagulans 6-ounce package, a 9-ounce package, a 12-ounce package, a bacterial cells or spores are collected using standard methods 15-ounce package, an 18-ounce package, or a 24-ounce pack (e.g., filtration, centrifugation) and the collected cells and age. spores can be lyophilized, spray-dried, air-dried or frozen. As 0046. The active ingredients (i.e., live bacteria or extracel described herein, the supernatant from the cell culture is lular components), comprise between about 0.01% to about collected and used as an extracellular agent Secreted by B. 10%; 0.01% to about 1%; or about 0.05% to about 0.1% by Coagulans. weight of the probiotic grain-based or soup composition. 0053 A typical yield from the above culture is in the range Optionally, the isolated Bacillus coagulans comprise about 1 of about 10 to 10" viable spores and more typically about mg to about 10 g; about 10 mg to about 1 g, or about 25 mg to 100 to 150 billion cells/spores per gram before drying. Spores about 75 mg by weight of the probiotic composition. Most maintain at least 90% viability after drying when stored at preferably, the amount of Bacillus coagulans bacteria is about room temperature for up to ten years, and thus the effective 5x107 colony forming units (CFU) of bacteria per gram of shelf life of a composition containing B. coagulans Hammer food matrix. spores at room temperature is about 10 years. 0047. In one aspect, the amount of bacteria is about 10 to 10' colony forming units (CFU) of bacteria per gram of Example 2 probiotic composition (i.e., vegetative cells and/or bacterial Preparation of Bacillus coagulans Spores spores), preferably 10 to 10' CFU/g of food matrix. Alter 0054. A culture of dried B. coagulans spores was prepared natively, the concentrations are 10 to 10' CFU/g: 10 to 10' as follows. Ten million spores were inoculated into a one liter US 2010/021 0000 A1 Aug. 19, 2010 culture containing 24 g potato dextrose broth, 10 g of enzy mic-digest of poultry and fish tissue, 5 g of FOS and 10 g TABLE 1-continued MnSO. The culture was maintained for 72 hours under a high 20 Minutes 120 Minutes oxygen environment at 37° C. to produce culture having Incubation Incubation about 150 billion cells per gram of culture. Thereafter, the Spore Count, Spore Count, culture was filtered to remove culture medium liquid, and the Sample CFU gram CFU gram bacterial pellet was resuspended in water and freeze-dried. Average 189E-10 198E-10 The freeze-dried powder is then ground to a fine powder using Saline pH 1.0 - D 2O8E--09 S.98E--O7 standard good manufacturing practice (GMP). Saline pH 1.0 - E 1.47E--09 O.OOE--OO Saline pH 1.0 - F 3.59E-09 O.OOE--OO Example 3 Average 2.38E--09 1.99E-07 Saline pH 2.0 - G 3.63E--09 3.46E--09 Saline pH 2.0 - H 4.47E--09 2.48E--09 Bacillus coagulans Spores Survive in the Gastric Saline pH 2.0 - I 3.58E--09 2.82E--09 Environment Average 3.89E-09 2.92E--09 Saline pH 3.0 - J 1.65E-10 1.13E-10 0055. This study was performed in order to determine the Saline pH 3.0 - K 1.35E-10 1.11E+10 Survivability rate of Bacillus coagulans spores as they pass Saline pH 3.0 - L. 180E-10 1.39E-10 through the stomach. Samples of Bacillus coagulans spores Average 16OE-10 1.21E-10 were subjected to a simulated gastric environment for varying lengths of time in order to attain their survivability rate. First, a homogeneous sample of raw material Bacillus coagulans of Example 4 at least 12 grams was prepared. Saline Solution at pH 1 was prepared using 3N HCl (150 mls each into six 250 ml media Bacillus coagulans Retain Viability in Gnocchi (Po bottles) and sterilized. Additional saline solutions with pH 2 tato Pasta) and 3 were prepared similarly, resulting in 6 sterile 250 ml 0056. The purpose of the following study was to determine bottles, each containing 150 ml pH adjusted saline. Six sterile the survivability rate of GBI-30 (Bacillus coagulans-30; 250 ml media bottles each containing 150 ml normal saline BC') in gnocchi (potato pasta) after cooking and pasteuriza solution were prepared and sterilized. Phosphate buffer (~400 tion. BC was mixed into gnocchi (potato pasta) at the dose ml) was prepared at pH 7.2. Test tubes (24) were prepared and of 5x107 CFU/g of food matrix, spray dried, and boiled in sterilized, each containing 9 ml of phosphate buffer pH 7.2. water at 100° C. for 1 minute and 30 seconds. The potato pasta Test tubes (120) were prepared, each containing 9 ml of was Subsequently pasteurized for 1 hour and 20 minutes at normal saline. GYE (glucose-yeast extract) agar medium was 95° C. Following pasteurization, the potato pasta was cooked prepared and sterilized and cooled to 45° C. in a water bath. at 100° C. for 1 minute and 30 seconds to simulate home Samples (24) of raw material were weighed, each ~500 mil cooking. The pasta was stored at 4°C. for 30 days (shelf life ligrams (theoretically equivalent to 10 billion spores). The is 60 days), heat shocked at approximately 80°C. for about 5 samples were added to media bottles at 37° C. and incubated minutes, and placed in GYE agar medium. The results dem half for 20 minutes the other half for 120 minutes. After 20 onstrate that approximately 1.3x10 CFU of Bacillus coagul and 120 minutes incubation, respectively, the samples were lans per gram of food matrix Survived even after storage for mixed to uniformity and pipet 1 ml into 9 ml of sterile phos about 30 days. Comparable results were observed after heat phate buffer pH 7.2. After all 12 samples from each time point shock (thermiz), Suggesting that after the cooking process, were placed into test tubes containing sterile phosphate the pasta comprises mostly Bacillus coagulans spores and buffer, serial dilutions were made until 6 tubes had been used few vegetative cells. The potato pasta was packaged in a for each sample. The final dilution for the final two test tubes modified atmosphere. The water activity (A) of the compo were 3x107 and 3x10, which gave a count of roughly 300 and sition was approximately 0.95%. The data in FIG. 1 show that 30 CFU, respectively. The final 2 test tubes from each sample after pre-boiling, pasteurization, and cooking, the amount of were placed into 70° C. water bath for 30 minutes. After 30 BC in the potato pasta was 3x10 CFU/g of food matrix, minutes, they were cooled immediately to 45°C. Three sterile Suggesting that the approximate daily dose of probiotics in petri plates per tube were set out. 1.0 ml from the heat-treated 100 g of gnocchi is about 1.3x10 CFU or 100% RDA (1 tube was added into each petri plate, then 15 ml of sterile billion viable cells is the recommended dose in EU guide molten GYEAgar medium (at 45°C.) was poured into each of lines). the petri plates and mixed thoroughly. When solidified, the plates were incubated in an inverted position for 48 hours at Example 5 40° C. The individual colonies were counted. Results were expressed as CFU per gram as shown in Table 1 below. Bacillus coagulans Retain Viability in Fresh Egg 1.OE+10=1x10'. Pasta 0057 This study was performed in order to determine the TABLE 1. survivability rate of GBI-30 (Bacillus coagulans-30; BC') in 20 Minutes 120Minutes fresh egg pasta after pasteurization. BC was mixed into the Incubation Incubation fresh egg pasta with cheese, vegetables, and meat filling at the Spore Count, Spore Count, dose of 5x107 CFU/g of food matrix. The fresh egg pasta was Sample CFU gram CFU gram Subsequently spray dried and pasteurized for approximately 5 Normal Saline - A 1.90E-10 188E-10 minutes at 100°C. The shelf life of the fresh egg pasta was 50 Normal Saline - B 2.12E-10 2.00E-10 days. The pasta was packaged in a modified atmosphere. The Normal Saline - C 1.64E-10 2.06E-10 water activity (A) of the composition was approximately 0.92-0.97%. The results illustrated in FIG. 2 demonstrate that US 2010/021 0000 A1 Aug. 19, 2010 approximately 2x10 CFU of BC' survived the pasteuriza Survive the cooking process, indicating that Bacillus coagul tion described above, indicating that Bacillus coagulans lans BC" retain viability in durum wheat semolina pasta. retain viability in the fresh egg pasta. TABLE 4 Example 6 Sample CFU Sample Total % of Survival of Heat-Activated Bacillus coagulans Description Spec Size Count Spec 0058. The ability of GBI-30 (Bacillus coagulans-30; Dry pasta (uncooked) 4 x 10 56 g 2.2 x 10 55% BC') in oatmeal to survive heating via microwave was deter Dry pasta (cooked) 4 x 10 56 g 1.2 x 10 30% mined. Table 2 demonstrates that approximately 82% of the BC alone survived microwaving for 1 minute and 50 sec onds. As shown in Table 2, approximately 79% of the initial What is claimed is: Bacillus coagulans bacteria in oatmeal Survived after micro 1. A composition comprising a processed grain and an waving for 1 minute and 50 seconds, suggesting that Bacillus isolated Bacillus coagulans bacterium or spore. coagulans retain viability in oatmeal after cooking Table 2 2. The composition of claim 1, wherein said composition is shows the survival of BC30 after heat treatment under various selected from the group consisting of pasta, oatmeal, and conditions. grits.

TABLE 2

Sample % of Sample Description Testing Method CFU/Spec Size Total Count Spec A: BC30, heat activation Total Plate Count 119 OOOOOOOO 1 g 119 OOOOOOOO 100% B: BC30, microwave Total Plate Count 119 OOOOOOOO 1 g 97SOOOOOOO 82% C: BC30 + oatmeal, microwave Total Plate Count 119 OOOOOOOO 1 g 93SOOOOOOO 79% A: BC301 g + 10 ml water, 75 C30 min, cooled to 45 C, serial dilution, total plate count, B: BC301 g + 250 ml water, microwave 1 min 50 second, serial dilution, total plate count. C: BC301 g + 1 serving of oatmeal (35.6 g) + 250 ml water, microwave 1 min 50 second, serial dilution, total plate count,

Example 7 3. The composition of claim 2, wherein said pasta is Selected from the group consisting of spaghetti, lasagna, egg Bacillus coagulans in Dry Turtle Island Soup Mix pasta, spätzle, penne rigate, rotini, rigatoni, and gnocchi. 0059. The probiotic Bacillus coagulans of the invention 4. The composition of claim 1, wherein said isolated Bacil was added to dry Turtle Island soup mix in the amount indi lus coagulans comprise between 0.01% and 10% by weight cated in Table 3. Table 3 is a chart indicating the number of of said cooked composition. colony forming units (CFU) of BC'per serving of dry soup 5. The composition of claim 1, wherein said isolated Bacil 1X. lus coagulans is Bacillus coagulans hammer StrainAccession No. ATCC 31284. TABLE 3 6. The composition of claim 1, wherein said isolated Bacil lus coagulans is selected from the group consisting of GBI-30 Sample Weight? Amount CFU strain (ATCC Designation Number PTA-6086), GBI-20 Description serving of BC 30 Serving strain (ATCC Designation Number PTA-6085), and GBI-40 1 Dry Soup Mix 38.2 gram 0.02 gram 2.81 x 108 strain (ATCC Designation Number PTA-6087). Dry Soup Mix 38.3 gram 0.02 gram 2.26 x 10 7. The composition of claim 1, wherein said isolated Bacil 3 Dry Soup Mix 39.1 gram 0.5 gram 7.05 x 109 lus coagulans is in the form of a spore. 8. The composition of claim 7, wherein said Bacillus coagulans spores activate upon contacting hot liquid. Example 8 9. The composition of claim 8, wherein said hot liquid is Bacillus coagulans Retain Viability in Durum Wheat water or milk. Semolina Pasta 10. The composition of claim 1, wherein said isolated Bacillus coagulans is in the form of a vegetative cell. 0060. The purpose of the following study was to determine 11. The composition of claim 1, wherein said isolated the survivability rate of GBI-30 (Bacillus coagulans-30; Bacillus coagulans is in the form of a mixture of vegetative BC') indurum wheat semolina pasta after cooking BC was cells and spores. mixed into the durum wheat Semolina pasta at the dose of about 4x10 CFU/serving (about 7x10 CFU/g of food matrix 12. A composition comprising a dry mix for grain-based (about 30 mg of BC); serving size is about 56 grams). The compositions comprising a grain and an isolated Bacillus composition was extruded at 37-38°C., followed by 20 hours coagulans bacterium. of drying at 50° C. Table 4 shows the survival of BC after 13. A composition comprising a dry mix for soup compris manufacturing the dry pasta and after cooking the dry pasta ing a dehydrated matter and an isolated Bacillus coagulans via boiling for about 8 minutes. The results shown in Table 4 bacterium. demonstrate that approximately 55% of BC' survive the 14. A method of making a grain-based composition com manufacturing process, while approximately 30% of BC' prising: US 2010/021 0000 A1 Aug. 19, 2010

providing a grain-containing base mix and a liquid portion; 17. The method of claim 14, wherein said isolated Bacillus coagulans is selected from the group consisting of GBI-30 mixing said grain-containing base mix and said liquid por strain (ATCC Designation Number PTA-6086), GBI-20 tion to form a batter or dough; strain (ATCC Designation Number PTA-6085), and GBI-40 combining an isolated Bacillus coagulans bacterium with strain (ATCC Designation Number PTA-6087). said batter or dough; and 18. The method of claim 14, wherein said isolated Bacillus heat processing said batter or dough to cook said grain coagulans is in the form of a spore. based composition. 19. The method of claim 14, wherein said isolated Bacillus coagulans is in the form of a vegetative cell. 15. The method of claim 14, wherein said liquid portion is 20. The method of claim 14, wherein said isolated Bacillus selected from the group consisting of water and milk. coagulans comprise between 1% and 10% by weight of said 16. The method of claim 14, wherein said isolated Bacillus grain-based composition. coagulans is Bacillus coagulans hammer strain Accession No. ATCC 31284. c c c c c