Patent Grant
11419355
The present application relates to probiotic grain-based compositions comprising lactic acid-producing bacteria.
The gastrointestinal microflora plays a number of vital roles in maintaining gastrointestinal tract function and overall physiological health. The growth and metabolism of the many individual bacterial species inhabiting the gastrointestinal tract depend primarily upon the substrates available to them, most of which are derived from the diet. Since probiotics do not generally permanently colonize the host, they need to be ingested regularly for any health promoting properties to persist.
The invention is based on the discovery that lactic acid-producing bacteria, particularly Bacillus species, remain viable and retain their beneficial probiotic properties in food compositions, such as those prepared at high temperatures (e.g., 80, 90, 100, 120, or 150° C.) in boiling water. The invention describes probiotic grain-based compositions. Specifically, the invention provides an isolated Bacillus coagulans bacterium in a grain-based composition.
The invention provides compositions comprising a cooked or uncooked composition of a grain and an isolated Bacillus coagulans bacterium or spore. The grain is processed, e.g., altered from its naturally-occurring state. For example, the grain is husked, crushed, cracked, or ground. The grain is in the form of flour or a composition made from further manipulation of a grain-based flour. Exemplary grains include wheat, rice, buckwheat, barley, Kamut, corn and oats. Exemplary cooked compositions include pasta, oatmeal, and grits. Suitable pastas include egg pasta, spaghetti (solid, thin cylinders), macaroni (tubes or hollow cylinders), fusilli (spiral-shaped), lasagna (sheets), tagliatelle (flat ribbons), vermicelli (thin spaghetti), ravioli (filled pasta), spätzle and gnocchi. Other suitable pastas include penne rigate (furrowed cylinder-shaped pasta), penne lisce (smooth cylinder-shaped pasta), rotini (corkscrew-shaped pasta), and rigatoni (tube-shaped pasta).
In one aspect, the isolated Bacillus coagulans comprise between about 0.01% to about 50% by weight of the grain-based composition. Optionally, the isolated Bacillus coagulans comprise between about 0.01% and about 10% by weight of the grain-based composition. Preferably, the isolated Bacillus coagulans comprise between about 0.01% and about 0.1% by weight of the grain-based composition.
The invention also provides bacterial species including Bacillus coagulans, e.g., Bacillus coagulans hammer, preferably Bacillus coagulans hammer strain Accession No. ATCC 31284, or one or more strains derived from Bacillus coagulans hammer strain Accession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20, ATCC Designation Number PTA-6085; GBI-30 or BC30, ATCC Designation Number PTA-6086; and GBI-40, ATCC Designation Number PTA-6087; see U.S. Pat. No. 6,849,256 to Farmer).
Optionally, the isolated Bacillus coagulans is in the form of a spore. In one aspect, the Bacillus coagulans spores activate upon contacting hot liquid. Preferably, the hot liquid is water or milk. Alternatively, the isolated Bacillus coagulans is in the form of a vegetative cell. In another aspect, the isolated Bacillus coagulans is in the form of a mixture of vegetative cells and spores. Preferably, the Bacillus coagulans is predominantly in spore form, e.g., about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% spores.
The invention also provides compositions comprising a dry mix for grain-based compositions comprising a grain and an isolated Bacillus coagulans bacterium. Also provided are compositions comprising a dry mix for soup comprising a dehydrated matter and an isolated Bacillus coagulans bacterium.
Also provided are methods of making a grain-based composition comprising providing a grain-containing base mix and a liquid portion; mixing the grain-containing base mix and the liquid portion to form a batter or dough; combining an isolated Bacillus coagulans bacterium with the batter or dough; and heat processing the batter or dough to cook the grain-based composition. Suitable liquid portions include water and milk. In one aspect, the isolated Bacillus coagulans is in the form of a spore. In another aspect, the isolated Bacillus coagulans is in the form of a vegetative cell. In one aspect, the isolated Bacillus coagulans comprise between about 0.1% to about 50% by weight of the grain-based composition. Preferably, the isolated Bacillus coagulans comprise between about 1% and about 10% by weight of the grain-based composition. Most preferably, the amount of Bacillus coagulans bacteria is about 5×107 colony forming units (CFU) of bacteria per gram of food matrix.
Bacillus coagulans bacteria are included in the grain-based or soup compositions of this invention. Bacterial species include Bacillus coagulans, e.g., Bacillus coagulans hammer, preferably Bacillus coagulans hammer strain Accession No. ATCC 31284, or one or more strains derived from Bacillus coagulans hammer strain Accession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20, ATCC Designation Number PTA-6085; GBI-30 or BC30, ATCC Designation Number PTA-6086; and GBI-40, ATCC Designation Number PTA-6087; see U.S. Pat. No. 6,849,256 to Farmer).
In one aspect, the isolated Bacillus coagulans is in the form of a spore. The invention provides for the activation of Bacillus coagulans spores upon heating. Optionally, the isolated Bacillus coagulans is in the form of a vegetative cell. In another aspect, the isolated Bacillus coagulans is in the form of a mixture of vegetative cells and spores.
The Bacillus coagulans Hammer strains of the invention are non-pathogenic and generally regarded as safe for use in human nutrition (i.e., GRAS classification) by the U.S. Federal Drug Administration (FDA) and the U.S. Department of Agriculture (USDA), and by those skilled in the art. Furthermore, the Bacillus coagulans Hammer strains of the invention germinate at or below human body temperature, rendering them useful as probiotics. Many Bacillus coagulans strains outside the Hammer group have mostly industrial applications, little or no nutritional benefit, and environmental contaminants that have not been evaluated for safety. Moreover, many other non-Hammer strains of Bacillus coagulans grow optimally at temperatures that exceed human body temperature and, thus, do not germinate efficiently in the human body. Such strains are less or not suitable as probiotics for human consumption.
Cited publications are incorporated herein by reference. Both the foregoing general description and the following detailed description and examples are exemplary and explanatory only and are not restrictive of the invention as claimed.
Probiotic organisms are non-pathogenic, non-toxigenic, retain viability during storage, and survive passage through the stomach and small intestine. Non-pathogenic lactic acid-producing bacteria (i.e., “lactic acid bacteria”), such as the exemplary Bacillus coagulans, remain viable and retain their beneficial probiotic properties in grain-based and soup compositions, such as those prepared in boiling water. Specifically, the probiotic organisms described herein, e.g., Bacillus coagulans strain GBI-30 or BC30, ATCC Designation Number PTA-6086, survive the harsh manufacturing and cooking processes of the grain-based and soup compositions described below.
Probiotic Lactic Acid-Producing Bacteria
A probiotic lactic acid-producing bacteria suitable for use in the methods and compositions of the invention produces acid and is non-pathogenic. There are many suitable bacteria identified as described herein, although the invention is not limited to currently known bacterial species insofar as the purposes and objectives of the bacteria is described. The property of acid production is important to the effectiveness of the probiotic lactic acid-producing bacteria of this invention.
The invention provides using a lactic acid-producing bacteria, such as a spore-forming Bacillus species, such as B. coagulans. Preferably, the spore-forming Bacillus species of the invention is B. coagulans Hammer.
Exemplary methods and compositions are described herein using Bacillus coagulans as a probiotic. Purified and/or isolated Bacillus coagulans is particularly useful as a probiotic in grain-based or soup compositions. Probiotic B. coagulans is non-pathogenic and is generally regarded as safe (i.e., GRAS classification) by the U.S. Federal Drug Administration (FDA) and the U.S. Department of Agriculture (USDA), and by those skilled in the art.
Bacillus coagulans is a non-pathogenic gram positive spore-forming bacteria that produces L(+) lactic acid (dextrorotatory) in fermentation conditions. It has been isolated from natural sources, such as heat-treated soil samples inoculated into nutrient medium (Bergey's Manual off Systemic Bacteriology, Vol. 2, Sneath, P. H. A., et al., eds., Williams & Wilkins, Baltimore, Md., 1986). Purified B. coagulans strains have served as a source of enzymes including endonucleases (e.g., U.S. Pat. No. 5,200,336); amylase (U.S. Pat. No. 4,980,180); lactase (U.S. Pat. No. 4,323,651); and cyclo-malto-dextrin glucano-transferase (U.S. Pat. No. 5,102,800). B. coagulans has been used to produce lactic acid (U.S. Pat. No. 5,079,164). A strain of B. coagulans (referred to as L. sporogenes; Sakaguti & Nakayama (ATCC 31284)) has been combined with other lactic acid producing bacteria and B. natto to produce a fermented food product from steamed soybeans (U.S. Pat. No. 4,110,477).
Bacterial species include Bacillus coagulans, e.g., Bacillus coagulans hammer, preferably Bacillus coagulans hammer strain Accession No. ATCC 31284, or one or more strains derived from Bacillus coagulans hammer strain Accession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20, ATCC Designation Number PTA-6085; GBI-30, ATCC Designation Number PTA-6086; and GBI-40, ATCC Designation Number PTA-6087; see U.S. Pat. No. 6,849,256 to Farmer).
Bacillus coagulans was previously mis-characterized as a Lactobacillus and labeled as Lactobacillus sporogenes (See Nakamura et al. 1988. Int. J. Syst. Bacteriol. 38: 63-73). However, initial classification was incorrect because Bacillus coagulans produces spores and excretes L(+)-lactic acid through metabolism. Both of these characteristics provide key features to the utility of Bacillus coagulans. These developmental and metabolic aspects required that the bacterium be classified as a lactic acid Bacillus. In addition, it is not generally appreciated that classic Lactobacillus species are unsuitable for colonization of the gut due to their instability in the harsh (i.e., acidic) pH environment of the bile, particularly human bile. By contrast, Bacillus coagulans is able to survive and colonize the gastrointestinal tract in the bile environment and even grown in this low pH range.
Probiotic Activity of Bacillus coagulans
It is well-documented clinically that many species of bacterial, mycotic and yeast pathogens possess the ability to cause a variety of gastrointestinal disorders including, but not limited to: disruption of normal gastrointestinal biochemical function, necrosis of gastrointestinal tissues, and disruption of the bioabsorption of nutrients, and like conditions. The probiotic microorganism-containing compositions described herein inhibit these pathogens. Thus, the compositions are useful in the prophylactic or therapeutic treatment of conditions associated with infection by these aforementioned pathogens.
In one aspect, a Bacillus coagulans strain is included in the composition in the form of vegetative cells. In another aspect, the Bacillus coagulans strain is included in the composition in the form of spores. The invention also provides for including the Bacillus coagulans strain in the composition in the form of a powder, a dried cell mass, a stabilized paste, or a stabilized gel.
Because Bacillus spores are heat and pressure-resistant and can be stored as a dry powder, they are particularly useful for formulation into and manufacture of products such as the various grain-based and soup compositions described herein. A Bacillus species is well suited for the present invention, particularly species having the ability to form spores which are relatively resistant to heat and other conditions, making them ideal for storage (shelf-life) in product formulations, e.g., grain-based and soup compositions. Due to the shelf-stable properties of the Bacillus coagulans strains described herein, e.g., Bacillus coagulans strain GBI-30 or BC30, ATCC Designation Number PTA-6086, the product formulations of the invention are not confined to a refrigerator and may be stored at room temperature.
The Bacillus coagulans of the invention survives storage (shelf-life) from about 12 days to about 2 years; from about 1 month to about 18 months; from about 3 months to about 1 year; or from about 6 months to about 9 months.
Anti-Microbial Probiotic Activity
The probiotic organisms described herein, e.g., Bacillus coagulans strain GBI-30 or BC30, ATCC Designation Number PTA-6086, promote digestive health and support the immune system. The ability of Bacillus coagulans to inhibit various bacterial pathogens was quantitatively ascertained by use of an in vitro assay. This assay is part of a standardized bacterial pathogen screen (developed by the U.S. Food and Drug Administration (FDA)) and is commercially available on solid support disks (DIFCO® BACTROL® Antibiotic Disks). To perform the assay, potato-dextrose plates (DIFCO®) were initially prepared using standard procedures. The plates were then individually inoculated with the bacteria (approximately 1.5×106 CFU) to be tested so as to form a confluent bacterial bed.
Inhibition of microorganisms (e.g. gastrointestinal pathogens) by Bacillus coagulans was subsequently ascertained by placing approximately 1.8×106 CFU of Bacillus coagulans in 10 μl of broth or buffer, directly in the center of the potato-dextrose plate with one test locus being approximately 8 mm in diameter per plate. A minimum of three test loci were used for each assay. The negative control consisted of a 10 μl volume of a sterile saline solution, whereas the positive control consisted of a 1 μl volume of glutaraldehyde. The plates were then incubated for approximately about 18 hr at 30° C., at which time the zones of inhibition were measured. As designated herein, “excellent inhibition” means the zone was 10 mm or greater in diameter; and “good inhibition” means the zone was greater than 2 mm in diameter but less than 10 mm in diameter.
As expected, no “inhibition” was seen with the negative, saline control, and excellent “inhibition” (approximately 16.2 mm diameter; average of three tests) was seen with the positive, glutaraldehyde control. For the enteric microorganisms tested, the following inhibition by Bacillus coagulans was found: (i) Clostridium species—excellent inhibition; (ii) Escherichia coli—excellent inhibition; (iii) Clostridium species—excellent inhibition, where the zone of inhibition was consistently greater than 15 mm in diameter. Similarly, excellent inhibition was also seen for the opportunistic pathogens Pseudomonas aeruginosa, and Staphylococcus aureus. Pathogenic enteric bacteria which were inhibited by Bacillus coagulans activity include, but are not limited to: Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus pyogenes; Pseudomonas aeruginosa; Escherichia coli (enterohemorragic species); numerous Clostridium species (e.g., Clostridium perfingens, Clostridium botulinum, Clostridium tributrycum, Clostridium sporogenes, and the like); Gardnereia vaginails; Proponbacterium aenes; Aeromonas hydrophia; Aspergillus species; Proteus species; and Klebsiella species.
Micro-Encapsulation
In one aspect, the lactic-acid producing bacteria are incorporated into a microcapsule coating prior to addition to the grain-based composition, using any micro-encapsulation process well-known in the art. The isolated Bacillus coagulans are packaged, or encapsulated, within another material in order to protect the bacteria from the surrounding environment. The capsules of the invention range in size from one-thousandth of a millimeter to seven millimeters. The internal ingredients of the microcapsule are released from their shells in various ways, including mechanical rupture of the capsule wall, dissolution of the wall, melting of the wall and diffusion through the wall. Thus, micro-encapsulation provides additional protection to the isolated Bacillus bacterium during heat processing of the grain-based compositions of the invention. Physical methods of micro-encapsulation include pan coating, air-suspension coating, centrifugal extrusion, vibrational nozzle, and spray-drying. Chemical methods of micro-encapsulation include interfacial polymerization, in-situ polymerization, and matrix polymerization.
Alternatively, the lactic-acid producing bacteria is added to the grain-based composition without micro-encapsulation.
Probiotic Grain-Based and Soup Compositions
The invention is directed to the surprising discovery that lactic acid-producing bacteria, particularly Bacillus species, remain viable and retain their beneficial probiotic properties in grain-based and soup compositions, such as those prepared in boiling water. The compositions are prepared by combining dry matter and a liquid, e.g., water or milk. In one aspect, the composition is prepared by combining dry matter and a liquid, and heating the resulting combination. Optionally, the combination is heated (heat-processed) using applied heat, a flame, or a microwave. The grain-based or soup composition is boiled in hot water, e.g., stovetop boiling, addition of boiling water to a container, or microwaving the grain-based or soup composition along with water. Preferably, boiling water (about 100° C.) is added to a combination of grain-based composition and Bacillus coagulans bacteria.
In one aspect, at least about 5%-25% of the bacteria are viable after heating, e.g., at least about 25%-50%; at least about 50% to 75%; or at least about 75%-99% of the bacteria are viable after heating. As the recommended dietary allowances (RDA or recommended daily intake; RDI) is about 1×109 bacterium (according to EU guidelines), preferably, the grain-based or soup composition comprises at least about 1×109 viable bacteria after heating. In another aspect, the grain-based or soup composition comprises at least about 1×106 to 1×107; at least about 1×107 to 1×108; or at least about 1×108 to 1×109 viable bacteria after heating.
The compositions are formulated in many configurations, because the bacterium is present as a vegetative cell or as a spore, or both, depending on the species and form of the probiotic organism. The cells/spores are formulated in a variety of compositions suited for use in a grain-based or soup composition. In one aspect, the bacterium is present as a mixture of spores and vegetative cells. In another aspect, the bacterium is present as at least 90% spores, e.g., 95%, 98%, or 99% spores. Optionally, prior to addition to the grain-based or soup compositions of the invention, the Bacillus coagulans cells are cultured in liquid in the absence of or with limited quantities of a food source to induce sporulation. In another aspect, heat gun spray drying kills about 50%, about 75%, about 90%, about 95%, or about 99% of vegetative cells prior to addition to the grain-based or soup compositions of the invention.
Grain-based compositions, such as those described herein, are made from a variety of grains known to those skilled in the art. Suitable grains include rice, wheat, maize, barley, rye, oats, buckwheat, sorghum, millets, triticale, fonio, and quinoa. Other types of grains used to make the grain-based compositions of the invention include teff, wild rice, and durum.
Exemplary grain-based compositions include pasta, oatmeal, grits, cereal, etc. The invention provides probiotic-enhanced pasta, e.g., isolated Bacillus coagulans and pasta. Pasta (Italian for “dough”) is a generic term for Italian variants of noodles, food made from a dough of flour, water and/or eggs. Pasta is cooked in hot water/boiling water prior to consumption. The probiotic organisms described herein, e.g., Bacillus coagulans strain GBI-30 or BC30, ATCC Designation Number PTA-6086, uniquely survive the harsh manufacturing and cooking processes of the grain-based and soup compositions. In one aspect, the pasta is the primary ingredient, served with sauce or seasonings. Common varieties of pasta include tubular pasta, straight round rod pasta, ribbon pasta, micro pasta, stuffed pasta, and irregular-shaped pasta. Exemplary pastas include spaghetti (solid, thin cylinders), macaroni (tubes or hollow cylinders), fusilli (spiral-shaped), lasagna (sheets), tagliatelle (flat ribbons), vermicelli (thin spaghetti), and ravioli (filled pasta). Other suitable pastas include penne (cylinder-shaped pasta), rotini (corkscrew-shaped pasta), and rigatoni (tube-shaped pasta). In Italy, penne are produced in two variants: “penne lisce” (smooth) and “penne rigate” (furrowed), the latter having ridges on each noodle. Two other noodles, gnocchi and spätzle, are sometimes counted as pasta because they are traditional in Italy; however, their “native” distributions (and perhaps their origins) are outside Italy, and these noodles have more in common with dumplings than with typical pasta. The two basic styles of pasta are dried and fresh. Dried pasta has a firmer, denser texture when cooked and is suited to chunky, meaty or oily sauces. Fresh pasta has a softer, more absorbent texture and is suited to buttery or creamy sauces or sauces with delicate flavors. There are also variations in the ingredients used in pasta. The time for which pasta can be stored varies from days to years depending upon whether the pasta is made with egg or not, and whether it is dried or fresh.
Many ingredients are used to make pasta dough, ranging from a simple flour and water mixture, to those that call for the addition of eggs, spices and cheeses, or even squid ink to the dough. Optionally, the pasta contains a filling, e.g., cheese, vegetables, fruit, and/or meat. In one aspect, dry pasta is made from durum wheat or semolina flour. Durum flour has a yellow tinge in color. Alternatively, dry pasta is made from other types of flour (such as farina), which yields a softer product. Particular varieties of pasta may also use other grains and/or milling methods to make the flour. Some pasta varieties, such as Pizzoccheri, are made from buckwheat flour. Various types of fresh pasta include eggs (egg pasta). Gnocchi are often considered pasta dishes, although they are quite different in ingredients (mainly milled potatoes).
Also provided are probiotic grain-based compositions in the form of oatmeal with isolated Bacillus coagulans. Oatmeal is a product of ground oat groats (i.e., oat-meal, cornmeal, peasemeal, etc.) or a porridge made from this product (also called oatmeal cereal). In regions such as the United States and Canada, “oatmeal” can refer to other products made from oat groats, such as cut oats, crushed oats, and rolled oats. The groats are coarsely ground to make oatmeal, or cut into small pieces to make steel-cut oats, or steamed and rolled to make rolled oats. In the case of rolled oats (old-fashioned oats), oat groats are steamed, pressed with a roller, and dried. Rolled oats take about 15 minutes to cook. The quick-cooking rolled oats (“quick oats”) are cut into small pieces before being steamed and rolled. “Instant” oatmeal is pre-cooked and dried. Optionally, the oatmeal includes: sweetener and flavor additives. Suitable sweeteners and flavor additives include salt, white sugar, brown sugar, stevia, cinnamon, honey, jam, molasses, maple syrup, butter, chocolate, soy sauce, soy milk, milk, vinegar, condensed or evaporated milk, and cream. Various types of fruit and nuts are also often added, including: strawberries, blueberries, apples, peaches, mangos, bananas, raisins, dried cherries, dried cranberries, pecans, walnuts, and peanut butter. Oatmeal is used to make porridge, as an ingredient as in oatmeal cookies and oat cakes, or as an accent as in the topping on many oat bran breads and the coating on Caboc cheese. Oatmeal is used as a thickener in some foods such as canned chili con cane. Oatmeal is also used in some alcoholic drinks, cosmetics, soaps, external medical treatments, and is sometimes added to animal feed products.
In another aspect, the probiotic composition of the invention is grits with isolated Bacillus coagulans. Grits is an American Indian corn-based food common in the southern United States, consisting of coarsely ground corn. Traditionally the corn for grits is ground by a stone mill. The results are passed through screens, with the finer part being corn meal, and the coarser part being grits.
Also provided are probiotic instant soups including isolated Bacillus coagulans in soups that require hot water. Soup is a food that is made by combining ingredients such as meat and vegetables in stock or hot/boiling water, until the flavor is extracted, forming a broth. Traditionally, soups are classified into two broad groups: clear soups and thick soups. Thick soups are classified depending upon the type of thickening agent used: purées are vegetable soups thickened with starch; bisques are made from puréed shellfish thickened with cream; cream soups are thickened with béchamel sauce; and veloutés are thickened with eggs, butter and cream. Other ingredients commonly used to thicken soups and broths include rice, flour, and grain. Oriental-style soup mixes containing ramen noodles are marketed as an inexpensive instant lunch, requiring only hot water for preparation. Various types of soups include tomato soup, cream of mushroom soup, chicken noodle soup, vegetable beef soup, minestrone soup, leek and potato soup, lentil soup, fish soup, miso soup, pea soup, fruit soup, clam chowder, gumbo, and bisque. Many soups, such as vegetable, chicken base, potato, pasta and cheese soups, are also available in dry mix form, ready to be served by adding hot water. Dry mix soup includes dehydrated matter, e.g., dehydrated meat, such as poultry and beef, dehydrated vegetables, dehydrated herbs, dehydrated spices, dehydrated noodles, etc. A packet of dry soup stock (e.g., ramen) typically does not contain water. The instant soup is prepared by adding water first, and then heating the product for a short time (usually 3-5 minutes) or by adding hot water directly to the dry soup mix. Instant soup can also be preserved into a dry powder which can be stored in, e.g., a packet or a cup. Bacillus coagulans bacteria in the form of spray-dried powder is added prior to or subsequent to addition of the dry mix soup powder to hot water.
In one aspect, Bacillus coagulans bacteria in the form of a spray-dried powder is included in or on the surface of the probiotic grain-based composition described herein. Preferably, the isolated Bacillus coagulans is in the form of a spore. The isolated Bacillus coagulans are at least 85%, at least 90%, at least 95%, or at least 99% pure spores. Alternatively, the isolated Bacillus coagulans is in the form of a vegetative cell. In one aspect, the isolated Bacillus coagulans are at least 85%, at least 90%, or at least 95% pure vegetative cells. In another aspect, the isolated Bacillus coagulans is in the form of a mixture of vegetative cells and spores. The Bacillus coagulans mixture is 90% spores, 10% vegetative cells; 75% spores, 25% vegetative cells; 60% spores, 40% vegetative cells; 50% spores, 50% vegetative cells; 60% vegetative cells, 40% spores; 75% vegetative cells; 25% spores; or 90% vegetative cells, 10% spores.
The Bacillus and/or Bacillus coagulans isolated active agent is applied using any of a variety of known methods including, for example, applying a powder, spray-drying the probiotic onto the grain-based or dry mix soup composition, or soaking the composition in a solution containing the probiotic. Optionally, the Bacillus bacterium is added to the dough and dried into the product (e.g., pasta). Alternatively, the Bacillus bacterium is mixed with the dry mix product (e.g., oatmeal or soup) prior to boiling in water. In another aspect, Bacillus coagulans bacteria in the form of spray-dried powder is added directly to the grain-based or soup composition itself. In yet another aspect, maltodextrin along with Bacillus coagulans bacteria in the form of spray-dried powder is added directly to the grain-based or soup composition itself. Optionally, about 5×107 CFU Bacillus coagulans bacteria (per gram of food matrix) in the form of spray-dried powder along with maltodextrin is added directly to the food composition itself.
Any of a variety of methods for placing the bacterial composition into a grain-based or soup composition can be used. However, preferred methods include a “spray-dry” method in which the compositions are exposed in a low humidity chamber to an atomized mix containing a liquid composition, where the chamber is subsequently exposed to approximately 80-110° F. to dry the liquid, thereby impregnating the material of grain-based or dry mix soup composition with the components.
A typical concentration is from approximately 1×107 to 1×1012 CFU; 1×108 to 1×1011 CFU; or 1×109 to 1×1010 CFU of viable bacterium or spores/g of food matrix. Following drying, the food is ready for immediate use or for storage in a sterile package, e.g., a 3-ounce package, a 6-ounce package, a 9-ounce package, a 12-ounce package, a 15-ounce package, an 18-ounce package, or a 24-ounce package.
The active ingredients (i.e., live bacteria or extracellular components), comprise between about 0.01% to about 10%; 0.01% to about 1%; or about 0.05% to about 0.1% by weight of the probiotic grain-based or soup composition. Optionally, the isolated Bacillus coagulans comprise about 1 mg to about 10 g; about 10 mg to about 1 g; or about 25 mg to about 75 mg by weight of the probiotic composition. Most preferably, the amount of Bacillus coagulans bacteria is about 5×107 colony forming units (CFU) of bacteria per gram of food matrix.
In one aspect, the amount of bacteria is about 104 to 1014 colony forming units (CFU) of bacteria per gram of probiotic composition (i.e., vegetative cells and/or bacterial spores), preferably 105 to 1013 CFU/g of food matrix. Alternatively, the concentrations are 108 to 1013 CFU/g; 109 to 1012 CFU/g; or 1010 to 1011 CFU/g of food matrix. In one aspect, the amount of bacteria is about 1×106 CFU per gram of food matrix. The actual amount in a grain-based or soup composition will vary depending upon the amounts of composition to be dispersed into the food composition and upon routes of dispersal.
In one aspect, the invention provides for storing the grain-based or dry mix soup composition in a sterile package at room temperature prior to consumption. Alternatively, the composition is consumed immediately.
In another aspect, the composition comprises at least 85%, at least 90%, at least 95% or 100% isolated Bacillus coagulans spores.
By way of example, and not of limitation, Bacillus coagulans spores may be incorporated into any type of dry or lyophilized product which is dissolved or mixed with hot water, so long as the temperature of the Bacillus coagulans spore-containing mixture is raised to the required heat-shock temperature (i.e., 80° C. for 5 minutes) necessary for germination of the spores. The Bacillus coagulans spores may either be incorporated into the dry or lyophilized product by the manufacturer of the product or by the consumer during preparation. These dry or lyophilized products include, but are not limited to: dry mix soups, pasta, oatmeal, grits, etc. The grain-based or soup composition is subsequently boiled in hot water, e.g., stovetop boiling, addition of boiling water to a container, or microwaving the grain-based or soup composition along with water.
In one aspect, the Bacillus coagulans spores survive storage (shelf-life), i.e., retain viability or the ability to germinate at physiological conditions (e.g., ingestion), from about 12 days to about 2 years; from about 1 month to about 18 months; from about 3 months to about 1 year; or from about 6 months to about 9 months.
Bacillus coagulans Hammer bacteria (ATCC Accession No. 31284) was inoculated and grown to a cell density of about 108 to 109 cells/ml in nutrient broth containing 5 g Peptone, 3 g Meat extract, 10-30 mg MnSO4, and 1,000 ml distilled water, adjusted to pH 7.0, using a standard airlift fermentation vessel at 30° C. The range of MnSO4 acceptable for sporulation is 1 mg/l to 1 g/l. The vegetative cells can actively reproduce up to 45° C., and the spores are stable up to 90° C. After fermentation, the B. coagulans bacterial cells or spores are collected using standard methods (e.g., filtration, centrifugation) and the collected cells and spores can be lyophilized, spray-dried, air-dried or frozen. As described herein, the supernatant from the cell culture is collected and used as an extracellular agent secreted by B. coagulans.
A typical yield from the above culture is in the range of about 109 to 1010 viable spores and more typically about 100 to 150 billion cells/spores per gram before drying. Spores maintain at least 90% viability after drying when stored at room temperature for up to ten years, and thus the effective shelf life of a composition containing B. coagulans Hammer spores at room temperature is about 10 years.
A culture of dried B. coagulans spores was prepared as follows. Ten million spores were inoculated into a one liter culture containing 24 g potato dextrose broth, 10 g of enzymic-digest of poultry and fish tissue, 5 g of FOS and 10 g MnSO4. The culture was maintained for 72 hours under a high oxygen environment at 37° C. to produce culture having about 150 billion cells per gram of culture. Thereafter, the culture was filtered to remove culture medium liquid, and the bacterial pellet was resuspended in water and freeze-dried. The freeze-dried powder is then ground to a fine powder using standard good manufacturing practice (GMP).
This study was performed in order to determine the survivability rate of Bacillus coagulans spores as they pass through the stomach. Samples of Bacillus coagulans spores 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 at least 12 grams was prepared. Saline solution at pH 1 was prepared using 3N HCl (150 mls each into six 250 ml media bottles) and sterilized. Additional saline solutions with pH 2 and 3 were prepared similarly, resulting in 6 sterile 250 ml bottles, each containing 150 ml pH adjusted saline. Six sterile 250 ml media bottles each containing 150 ml normal saline solution were prepared and sterilized. Phosphate buffer (˜400 ml) was prepared at pH 7.2. Test tubes (24) were prepared and sterilized, each containing 9 ml of phosphate buffer pH 7.2. Test tubes (120) were prepared, each containing 9 ml of normal saline. GYE (glucose-yeast extract) agar medium was prepared and sterilized and cooled to 45° C. in a water bath. Samples (24) of raw material were weighed, each ˜500 milligrams (theoretically equivalent to 10 billion spores). The samples were added to media bottles at 37° C. and incubated half for 20 minutes the other half for 120 minutes. After 20 and 120 minutes incubation, respectively, the samples were mixed to uniformity and pipet 1 ml into 9 ml of sterile phosphate buffer pH 7.2. After all 12 samples from each time point were placed into test tubes containing sterile phosphate buffer, serial dilutions were made until 6 tubes had been used for each sample. The final dilution for the final two test tubes were 3×107 and 3×108, which gave a count of roughly 300 and 30 CFU, respectively. The final 2 test tubes from each sample were placed into 70° C. water bath for 30 minutes. After 30 minutes, they were cooled immediately to 45° C. Three sterile petri plates per tube were set out. 1.0 ml from the heat-treated tube was added into each petri plate, then 15 ml of sterile molten GYE Agar medium (at 45° C.) was poured into each of the petri plates and mixed thoroughly. When solidified, the plates were incubated in an inverted position for 48 hours at 40° C. The individual colonies were counted. Results were expressed as CFU per gram as shown in Table 1 below. 1.0E+10=1×1010.
The purpose of the following study was to determine the survivability rate of GBI-30 (Bacillus coagulans-30; BC30) in gnocchi (potato pasta) after cooking and pasteurization. BC30 was mixed into gnocchi (potato pasta) at the dose of 5×107 CFU/g of food matrix, spray dried, and boiled in water at 100° C. for 1 minute and 30 seconds. The potato pasta was subsequently pasteurized for 1 hour and 20 minutes at 95° C. Following pasteurization, the potato pasta was cooked at 100° C. for 1 minute and 30 seconds to simulate home cooking. The pasta was stored at 4° C. for 30 days (shelf life is 60 days), heat shocked at approximately 80° C. for about 5 minutes, and placed in GYE agar medium. The results demonstrate that approximately 1.3×107 CFU of Bacillus coagulans per gram of food matrix survived even after storage for about 30 days. Comparable results were observed after heat shock (thermiz), suggesting that after the cooking process, the pasta comprises mostly Bacillus coagulans spores and few vegetative cells. The potato pasta was packaged in a modified atmosphere. The water activity (Aw) of the composition was approximately 0.95%. The data in
This study was performed in order to determine the survivability rate of GBI-30 (Bacillus coagulans-30; BC30) in fresh egg pasta after pasteurization. BC30 was mixed into the fresh egg pasta with cheese, vegetables, and meat filling at the dose of 5×107 CFU/g of food matrix. The fresh egg pasta was subsequently spray dried and pasteurized for approximately 5 minutes at 100° C. The shelf life of the fresh egg pasta was 50 days. The pasta was packaged in a modified atmosphere. The water activity (Aw) of the composition was approximately 0.92-0.97%. The results illustrated in
The ability of GBI-30 (Bacillus coagulans-30; BC30) in oatmeal to survive heating via microwave was determined. Table 2 demonstrates that approximately 82% of the BC30 alone survived microwaving for 1 minute and 50 seconds. As shown in Table 2, approximately 79% of the initial Bacillus coagulans bacteria in oatmeal survived after microwaving for 1 minute and 50 seconds, suggesting that Bacillus coagulans retain viability in oatmeal after cooking. Table 2 shows the survival of BC30 after heat treatment under various conditions.
The probiotic Bacillus coagulans of the invention was added to dry Turtle Island soup mix in the amount indicated in Table 3. Table 3 is a chart indicating the number of colony forming units (CFU) of BC30 per serving of dry soup mix.
The purpose of the following study was to determine the survivability rate of GBI-30 (Bacillus coagulans-30; BC30) in durum wheat semolina pasta after cooking. BC30 was mixed into the durum wheat semolina pasta at the dose of about 4×108 CFU/serving (about 7×106 CFU/g of food matrix (about 30 mg of BC30); serving size is about 56 grams). The composition was extruded at 37-38° C., followed by 20 hours of drying at 50° C. Table 4 shows the survival of BC30 after manufacturing the dry pasta and after cooking the dry pasta via boiling for about 8 minutes. The results shown in Table 4 demonstrate that approximately 55% of BC30 survive the manufacturing process, while approximately 30% of BC30 survive the cooking process, indicating that Bacillus coagulans BC30 retain viability in durum wheat semolina pasta.
This application is a continuation of U.S. Ser. No. 15/487,230 filed Apr. 13, 2017, which is a continuation of U.S. Ser. No. 12/580,622 filed Oct. 16, 2009, and claims the benefit of priority to U.S. Provisional Application No. 61/106,116 filed Oct. 16, 2008. Each of these applications is incorporated herein by reference its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 3091572 | Luedemann et al. | May 1963 | A |
| 3840684 | Fazzina et al. | Oct 1974 | A |
| 4110477 | Naruse et al. | Aug 1978 | A |
| 4144346 | Heeres et al. | Mar 1979 | A |
| 4278690 | Onodera | Jul 1981 | A |
| 4321258 | Dunlap | Mar 1982 | A |
| 4323651 | Long et al. | Apr 1982 | A |
| 4695546 | Aiba et al. | Sep 1987 | A |
| 4756913 | Khorkova et al. | Jul 1988 | A |
| 4956177 | King et al. | Sep 1990 | A |
| 4980180 | Cully et al. | Dec 1990 | A |
| 5021344 | Armau et al. | Jun 1991 | A |
| 5079164 | Kirkovits et al. | Jan 1992 | A |
| 5102800 | Hirikoshi | Apr 1992 | A |
| 5176911 | Tosi et al. | Jan 1993 | A |
| 5200336 | Kong et al. | Apr 1993 | A |
| 5266315 | Taguchi et al. | Nov 1993 | A |
| 5413960 | Dobrogosz et al. | May 1995 | A |
| 5439678 | Dobrogosz et al. | Aug 1995 | A |
| 5439995 | Bailly et al. | Aug 1995 | A |
| 5531998 | Mares et al. | Jul 1996 | A |
| 5534253 | Casas et al. | Jul 1996 | A |
| 5665354 | Neyra et al. | Sep 1997 | A |
| 5785990 | Langrehr | Jul 1998 | A |
| 5814319 | Nakano | Sep 1998 | A |
| 5895672 | Cooper | Apr 1999 | A |
| 5968569 | Cavadini et al. | Oct 1999 | A |
| 6080401 | Reddy et al. | Jun 2000 | A |
| 6132710 | Panigrahi et al. | Oct 2000 | A |
| 6461607 | Farmer | Oct 2002 | B1 |
| 6531126 | Farmer | Mar 2003 | B2 |
| 6537543 | Minakawa | Mar 2003 | B1 |
| 6645506 | Farmer | Nov 2003 | B1 |
| 6706290 | Kajander et al. | Mar 2004 | B1 |
| 6716435 | Farmer et al. | Apr 2004 | B1 |
| 6723326 | Farmer | Apr 2004 | B1 |
| 6733751 | Farmer | May 2004 | B2 |
| 6811786 | Farmer et al. | Nov 2004 | B1 |
| 6835397 | Lee et al. | Dec 2004 | B2 |
| 6849256 | Farmer | Feb 2005 | B1 |
| 6905692 | Farmer | Jun 2005 | B2 |
| 7024497 | Maffezoni | Apr 2006 | B1 |
| 7025974 | Farmer et al. | Apr 2006 | B2 |
| 7048950 | Farmer | May 2006 | B2 |
| 7232571 | Farmer et al. | Jun 2007 | B2 |
| 7371407 | Farmer | May 2008 | B2 |
| 7374753 | Farmer et al. | May 2008 | B1 |
| 7507402 | Farmer et al. | Mar 2009 | B1 |
| 7541042 | Farmer | Jun 2009 | B2 |
| 7544363 | Farmer | Jun 2009 | B2 |
| 7555715 | Randall et al. | Jun 2009 | B2 |
| 7700093 | Farmer et al. | Apr 2010 | B2 |
| 7708988 | Farmer | May 2010 | B2 |
| 7713726 | Farmer | May 2010 | B2 |
| 7767203 | Farmer et al. | Aug 2010 | B2 |
| 7807151 | Farmer | Oct 2010 | B2 |
| 7807185 | Farmer | Oct 2010 | B2 |
| 7854927 | Farmer et al. | Dec 2010 | B2 |
| 8097247 | Farmer | Jan 2012 | B2 |
| 8187590 | Farmer | May 2012 | B2 |
| 8273346 | Farmer et al. | Sep 2012 | B2 |
| 8277799 | Farmer | Oct 2012 | B2 |
| 8343484 | Farmer et al. | Jan 2013 | B2 |
| 8349337 | Farmer et al. | Jan 2013 | B1 |
| 8409591 | Farmer et al. | Apr 2013 | B2 |
| 8568743 | Farmer et al. | Oct 2013 | B2 |
| 8568744 | Farmer et al. | Oct 2013 | B2 |
| 8697055 | Farmer | Apr 2014 | B2 |
| 8821854 | Farmer et al. | Sep 2014 | B2 |
| 9192659 | Farmer et al. | Nov 2015 | B2 |
| 9220736 | Farmer et al. | Dec 2015 | B2 |
| 9301982 | Lefkowitz | Apr 2016 | B2 |
| 9446111 | Farmer et al. | Sep 2016 | B2 |
| 9597286 | Lefkowitz | Mar 2017 | B2 |
| 9622502 | Farmer | Apr 2017 | B2 |
| 10321704 | Farmer | Jun 2019 | B2 |
| 20030031659 | Farmer | Feb 2003 | A1 |
| 20030138936 | Mizuguchi et al. | Jul 2003 | A1 |
| 20030185811 | Teasdale et al. | Oct 2003 | A1 |
| 20040010510 | Hotti | Jan 2004 | A1 |
| 20040071685 | Houston et al. | Apr 2004 | A1 |
| 20040161522 | Toves | Aug 2004 | A1 |
| 20040175459 | Ting | Sep 2004 | A1 |
| 20040197277 | Gonzales | Oct 2004 | A1 |
| 20050100535 | Farmer et al. | May 2005 | A1 |
| 20050153040 | Axelrod et al. | Jul 2005 | A1 |
| 20050154682 | Taylor | Jul 2005 | A1 |
| 20050202145 | Dorr et al. | Sep 2005 | A1 |
| 20050232909 | Farmer | Oct 2005 | A1 |
| 20050271643 | Sorokulova et al. | Dec 2005 | A1 |
| 20060099197 | Farmer | May 2006 | A1 |
| 20060112584 | Jones | Jun 2006 | A1 |
| 20060154350 | Kolbakov et al. | Jul 2006 | A1 |
| 20060177429 | Farmer et al. | Aug 2006 | A1 |
| 20060184538 | Randall et al. | Aug 2006 | A1 |
| 20060204633 | Moore | Sep 2006 | A1 |
| 20060234375 | Doronin et al. | Oct 2006 | A1 |
| 20070059400 | Goto et al. | Mar 2007 | A1 |
| 20070077652 | Peled et al. | Apr 2007 | A1 |
| 20080233104 | Farmer | Sep 2008 | A1 |
| 20080274153 | Farmer | Nov 2008 | A1 |
| 20080305096 | Verdegem et al. | Dec 2008 | A1 |
| 20090186057 | Farmer et al. | Jul 2009 | A1 |
| 20090186126 | Farmer et al. | Jul 2009 | A1 |
| 20090208606 | Hakansson et al. | Aug 2009 | A1 |
| 20090232941 | Farmer | Sep 2009 | A1 |
| 20100074993 | Cooreman et al. | Mar 2010 | A1 |
| 20100210000 | Farmer et al. | Aug 2010 | A1 |
| 20110020305 | Farmer | Jan 2011 | A1 |
| 20110195154 | Farmer | Aug 2011 | A1 |
| 20110274670 | Nam et al. | Nov 2011 | A1 |
| 20110274676 | Farmer et al. | Nov 2011 | A1 |
| 20120251512 | Farmer et al. | Oct 2012 | A1 |
| 20120276215 | Riordan et al. | Nov 2012 | A1 |
| 20130164398 | Farmer | Jun 2013 | A1 |
| 20130195824 | Farmer et al. | Aug 2013 | A1 |
| 20130216577 | Farmer et al. | Aug 2013 | A1 |
| 20130251695 | Farmer et al. | Sep 2013 | A1 |
| 20130344046 | Farmer et al. | Dec 2013 | A1 |
| 20140242051 | Farmer | Aug 2014 | A1 |
| 20150044317 | Farmer et al. | Feb 2015 | A1 |
| 20150313951 | Cash et al. | Nov 2015 | A1 |
| 20160213612 | Lefkowitz | Jul 2016 | A1 |
| 20160213613 | Lefkowitz | Jul 2016 | A1 |
| 20160213614 | Lefkowitz | Jul 2016 | A1 |
| 20160213615 | Lefkowitz | Jul 2016 | A1 |
| 20170000872 | Farmer et al. | Jan 2017 | A1 |
| 20170035813 | Farmer et al. | Feb 2017 | A1 |
| 20170189331 | Lefkowitz | Jul 2017 | A1 |
| 20170189457 | Farmer et al. | Jul 2017 | A1 |
| Number | Date | Country |
|---|---|---|
| 2606495 | Nov 2006 | CA |
| 1507812 | Jun 2004 | CN |
| 4132296 | Dec 1992 | DE |
| 1020123 | Jul 2000 | EP |
| 1344458 | Sep 2003 | EP |
| 1810579 | Jul 2007 | EP |
| 1040278 | Aug 1966 | GB |
| S45032905 | Oct 1970 | JP |
| S52136998 | Nov 1977 | JP |
| S5350399 | May 1978 | JP |
| 64083025 | Mar 1989 | JP |
| 4082827 | Mar 1992 | JP |
| 4158771 | Jun 1992 | JP |
| 6166626 | Jun 1994 | JP |
| 08175921 | Jul 1996 | JP |
| H09107869 | Apr 1997 | JP |
| H1084845 | Apr 1998 | JP |
| 11169145 | Jun 1999 | JP |
| 11335285 | Dec 1999 | JP |
| 2000093162 | Apr 2000 | JP |
| 2001252012 | Sep 2001 | JP |
| 2001286278 | Oct 2001 | JP |
| 2001292763 | Oct 2001 | JP |
| 2002502430 | Jan 2002 | JP |
| 2002522393 | Jul 2002 | JP |
| 2003513649 | Apr 2003 | JP |
| 2004337125 | Dec 2004 | JP |
| 2005137357 | Jun 2005 | JP |
| 2005536994 | Dec 2005 | JP |
| 2006025621 | Feb 2006 | JP |
| 2006254837 | Sep 2006 | JP |
| 2007000140 | Jan 2007 | JP |
| 2007044014 | Feb 2007 | JP |
| 2007054081 | Mar 2007 | JP |
| 2007082403 | Apr 2007 | JP |
| 2007537270 | Dec 2007 | JP |
| 2008013543 | Jan 2008 | JP |
| 4082827 | Apr 2008 | JP |
| 4158771 | Oct 2008 | JP |
| 1228974 | Mar 2005 | TW |
| 8905849 | Jun 1989 | WO |
| 9314187 | Jul 1993 | WO |
| 9400019 | Jan 1994 | WO |
| 9411492 | May 1994 | WO |
| 9611014 | Apr 1996 | WO |
| 9729762 | Aug 1997 | WO |
| 9734615 | Sep 1997 | WO |
| 9854982 | Dec 1998 | WO |
| 0007606 | Feb 2000 | WO |
| 0134168 | May 2001 | WO |
| 2004004747 | Jan 2004 | WO |
| 2004008870 | Jan 2004 | WO |
| 2005019417 | Mar 2005 | WO |
| 2005055934 | Jun 2005 | WO |
| 2005092122 | Oct 2005 | WO |
| 05110445 | Nov 2005 | WO |
| 2005117926 | Dec 2005 | WO |
| 2006007470 | Jan 2006 | WO |
| 2006090729 | Aug 2006 | WO |
| 2007012847 | Feb 2007 | WO |
| 2007058027 | May 2007 | WO |
| 2008112296 | Sep 2008 | WO |
| 09029267 | Mar 2009 | WO |
| 09051753 | Apr 2009 | WO |
| Entry |
|---|
| Tanaka et al., Antibacterial Compounds of Licorice against Upper Airway Respiratory Tract Pathogens. J Nutr Sci Vitaminol. 2001;47:270-3. |
| Balasubramaniam et al. “High-Pressure Food Processing”, Food Sci Tech Int: 14(5):413-418 (2008). |
| Christl et al., “Role of dietary sulphate in the regulation of methanogenesis in the human large intestine”, Gut, 33:12341238 (1992). |
| Daryaei. et al. “Kinetics of Bacillus coagulans spore inactivation in tomato juice by combined pressure-heat treatment” Food Control 30:168-75( 2013). |
| De Veechi et al. “Lactobacillus sporogenes or Bacillus coagulans: Misidentification or Mislabeling” 2006 International Journal of Probiotics and Prebiotics vol. 1 No. 1 pp. 3-10. |
| El-Baz. “Herbal and Floral Teas, Infusions, or Tisanes?” The Essence of Herbal and Floral Teas. New York: Universe.:1-5. (2006). |
| Hammer, 1915, Bacteriological Studies on the Coagulation of Evaporated Milk, Iowa Agric. Expt. Sta. Res. Bull. 19:119-132. |
| Johnson et al. “Inactivation of Bacillus coagulans Spores by Pressure-assisted Thermal Processing”, Oculus, vol. 1: 35-38 (2010). |
| Marsh. “Antimicrobial Strategies in the Prevention of Dental Caries.” Caries Res. 27(1993):7276. |
| Peng et al. “Thermal Inactivation Kinetics of Bacillus coagulans Spores in Tomato Juice”, Journal of food protection, vol. 75, No. 7; 1236-1242 (2012). |
| Rice et al. “Boiling and Bacillus Spores” Emerg Infect Dis. Oct. 2004; 10(10): 18871888 (available at www.ncbi.nlm.nih.gov/pmc/articles/PMC3323245/table/T1l). |
| Rogers. “Bacillus Isolates from Refrigerated Doughs, Wheat Flour and Wheat.” Cereal Chem. 55(1978):671-674. |
| Saavedra. “Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to Infants in Hospital for Prevention of Diarrhoea and Shedding of Rotavirus” Lancet. 344(1994):10461049. |
| Singh et al. “Stevia: The Herbal Sugar of 21st Century” 2005 Sugar Technology vol. 7 No. 1 pp. 17-24. |
| Wang et al. “Inactivation Kinetics and Reduction of Bacillus Coagulans Spore by the Combination of High Pressure and Moderate Heat”, Journal of Food Process Engineering 32 692-708 (2009). |
| Zimmermann et al. “Modeling the inactivation kinetics of Bacillus coagulans spores in tomato pulp from the combined effect of high pressure and moderate temperature”, LWT Food Sci and Tech, (53):107-112 (2013). |
| Akoachere et al., Antibacterial effect of Zingiber officinale and Garcinia kola on respiratory tract pathogens. East Afr Med J. Nov. 2002;79(11):588-92. |
| Al-Turki, A. Antibacterial effect of thyme, peppermint, sage, black pepper and garlic hydrosols against Bacillus subtilis and Salmonella enteritidis. Journal of Food, Agriculture & Environment 2007;5(2):92-4. |
| Barrile et al., Microflora of cocoa beans before and after roasting at 150 C. J Milk Food Technol. 1971;34(7):369-71. |
| Cornelis et al., 1982, Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli, Mol Gen Genet. 186(4):507-11 [Abstract]. |
| Ekwenye et al., Antibacterial Activity of Ginger (Zingiber officinale) Roscoe and Garlic (Allium sativum L.) Extracts on Escherichia coli and Salmonella typhi. International Journal of Molecular Medicine and Advance Science. 2005; 1(4):411-6. |
| Hattori et al., Studies on Dental Caries Prevention by Traditional Medicines (IX) Potent Antibacterial Action of Coumarin Derivatives from Licorice Roots against Streptococcus mutans. Shoyakugaku Zasshi. 1986;40(4):406-12. |
| Nanasombat et al., Antibacterial activity of crude ethanolic extracts and essential oils of spices against Salmonellae and other enterobacteria KMITL Sci. Tech. J. Dec. 2005;5(3):527-38. |
| Olaleye, M. Cytotoxicity and antibacterial activity of Methanolic extract of Hibiscus sabdariffa. Journal of Medicinal Plants Research. Aug. 2007;1(1):9-13. |
| Pattnaik et al., Antibacterial and antifungal activity often essential oils in vitro. Microbios. 1996;86(349):237-46. [Abstract]. |
| Pepe et al., 2003, Rope-Producing Strains of Bacillus spp. from Wheat Bread and Strategy for Their Control by Lactic Acid Bacteria, Applied and Environmental Microbiology 69(4):2321-29. |
| Sharaf et al., Further Study on the Antibacterial Effect of H. sabdariffa. Path Microbiol. 1996;29:120-5. |
| Tadhani et al., In Vitro Antimicrobial Activity of Stevia Rebaudiana Bertoni Leaves. Tropical Journal of Pharmaceutical Research. Jun. 2006;5(1):557-60. |
| Nakamura et al., “Taxonomic study of Bacillus coagulans Hammer 1915 with a proposal for Bacillus smithii sp. Nov.” Int'l. J. Syst. Bacteriol., 38(1):63-73 (1988). |
| Perdigon et al. “Symposium: Probiotic Bacteria for Humans: Clinical Systems for Evaluation of Effectiveness.” J. Dairy Sci. 78(1995):1597-1606. |
| Perlman et al. “Persistent Campylobacterjejuni Infections in Patients Infected with Human Immunodeficiency Virus (HIV).” Ann. Intern. Med. 108(1988):540-546. |
| Peterson. “Clinical Aspects of Campylobacterjejuni Infections in Adults.” Wes. J. Med. 161.2(1994):148-152. |
| Peterson. “Rheumatic Manifestations of Campylobacterjejuni and C. fetus Infections in Adults.” Scand. J. Rheumatol. 23(1994):167-170. |
| Piddock. “Quinolone Resistance and Campylobacter spp.” J. Antimicrob. Chemother. 36(1995):891-898. |
| Rafter. “The Role of Lactic Acid Bacteria in Colon Cancer Prevention.” Scand. J. Gastroenterol. 30(1995):497-502. |
| Reddy et al. “Inhibitory Effect of Bifidobacterium longum on Colon, Mammary, and Liver Carcinogenesis Induced by 2-Amino-3-methylimidazo[4,5-f]quinoline, a Food Mutagen.” Cancer Res. 53(1993):3914-3918. |
| Reid et al. “Is There a Role for Lactobacilli in Prevention of Urogenital and Intestinal Infections?” Clin. Microbiol. Rev. 3(1990):335-344. |
| Rogers, R. F. 1978. Bacillus isolates from refrigerated doughs, wheat flour and wheat. Cereal Chem. 55:671-674. |
| Rowland et al. “Degradation of N-Nitrosamines by Intestinal Bacteria.” Appl. Microbiol. 29(1975):7-12. |
| Rychen et al. “Effects of Three Microbial Probiotics on Postprandial Porto-Arterial Concentration Differences of Glucose, Galactose and Amino-Nitrogen in the Young Pig.” Brit. J. Nutr. 74(1995):19-26. |
| S.D. Singh and G.P. Rao “Stevia: The Herbal Sugar of 21 st Century” 2005 Sugar Technology vol. 7 No. 1 pp. 17-24. |
| Salminen et al. “Clinical Uses of Probiotics for Stabilizing the Gut Mucosal Barrier: Successful Strains and Future Challenges.” Antonie Van Leeuwenhoek. 70(1996):347-358. |
| Schiffrin et al. “Immune Modulation of Blood Leukocytes in Humans by Lactic Acid Bacteria: Criteria for Strain Selection.” Am. J. Clin. Nutr. 66(1997):5155-5205. |
| Sekine et al. “Induction and Activation of Tumoricidal Cells In Vivo and In Vitro by the Bacterial Cell Wall of Bifdobacterium infantis.” Bifidobact. Microflora. 13.2(1994):65-77. |
| Shannon. “Multiple-Antibiotic-Resistant Salmonella.” Lancet. 352(1998):490-491. |
| Shoenfeld et al. “Guillain-Barre as an Autoimmune Disease.” Int. Arch. Allergy Immunot 109(1996):318-326. |
| Smith et al. “Fluoroquinolone-Resistant Campylobacter Isolated from Humans and Poultry in Minnesota.” Int. Conf. Emerging Infect. Dis. 1998:69. |
| Sneath et al., eds. Bergey's Manual of Systematic Bacteriology. Baltimore: Williams and Wilkons. 2(1986):1117. |
| Solis-Pereyra et al. “Induction of Human Cytokines by Bacteria Used in Dairy Foods.” Nutr. Res. 13(1993):1127-1140. |
| Sorvillo et al. “Incidence of Campylobacteriosis Among Patients with AIDS in Los Angeles County.” J. Acquired Immune Defic. Syndr. 4(1991):598-602. |
| Standiford et al. “Lipoteichoic Acid Induces Secretion of Interleukin-8 From Human Blood Monocytes: A Cellular and Molecular Analysis” Infect. Immun. 62.1(1994):119-125. |
| Sussman et al. “Clinical Manifestations and Therapy of Lactobacillus Endocarditis: Report of a Case and Review of the Literature.” Rev Infect. Dis. 8.5(1986):771-776. |
| Suzuki et al. “Purification and Characterization of Bacillus coagulans Oligo-1,6-Glucosidase.” Eur. J. Biochem. 158.1 (1986):77-83. |
| Tauxe. “Epidemiology of Campylobacterjejuni Infections in the United States and Other Industrial Nations.” Campylobacterjejunk Current Status and Future Trends. Nachamkin et al., eds. Washington, DC:American Society for Microbiology. (1992):9-13. |
| The Merck Index. Rahway, NJ: Merck & Co., Inc. Windholz et al., eds. 10(1983):549. |
| Thomason et al. “Bacterial Vaginosis: Current Review With Indications for Asymptomatic Therapy.” Am. J. Obstet Gynecol. 165(1991):1210-1217. |
| Tojo et al. “The Effects of Bifidobacterium breve Administration on Campylobacter Enteritis.” Acta Paediatr. Jpn. 29(1987):160-167. |
| Winberg et al. “Pathogenesis of Urinary Tract Infection-Experimental Studies of Vaginal Resistance to Colonization.” Pediatr. Nephrol. (1993):509-514. |
| Yamazaki et al. “Protective Effect of Bifidobacterium-Monoassociation Against Lethal Activity of Escherichia colL” Bifidobacteria Micro flora. 1(1982):55-59. |
| Zhang et al. “Antimutagenicity and Binding of Lactic Acid Bacteria from a Chinese Cheese to Mutagenic Pyrolyzates.” J. Dairy ScL 73(1990):2702-2710. |
| Daryaei H. et al. Kinetics of Bacillus coagulans spore inactivation in tomato juice by combined pressure-heat treatment. J Food Prot:168-75( 2013). |
| Zimmermann et al. Modeling the inactivation kinetics of Bacillus coagulans spores in tomato pulp from the combined affect of high pressure and moderate temperature. LWT Food Sci and Tech, (53):107-112 (2013). |
| Balasubramaniam et al., “Principles and Application of High Pressure-Based Technologies in the Food Industry”, Ann. Rev. Food Sci Technol. 6:435-62. |
| El-Baz. “Herbal and Floral Teas, Infusions, or Tisanes?” The Essence of Herbal and Floral Teas. New York: iUniverse. (2006):1-5. |
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323245/table/T1/. |
| Sneath et al., eds. Bergey's Manual of Systematic Bacteriology. Baltimore: Williams and Wilkons. 2(1986):1128. |
| Hammer, “Bacterialogical Studies on the Coagulation of Evaporated Milk,” Iowa State College of Agriculture and Mechanic Arts, Research Bulletin No. 19, Jan. 1915. |
| Hammer, B. W., 1915, Bacteriological Studies on the Coagulation of Evaporated Milk, Iowa Agric. Expt. Sta. Res. Bull. 19:119-132. |
| Maruzen Shokuhin Sogo Jiten. Maruzen General Encyclopedia of Food. Mar. 25, 1998. p. 861. |
| Maruzen Shokuhin Sogo Jiten. Maruzen General Encyclopedia of Food. Mar. 25, 1998. p. 999. |
| Amaha et al. Sporulation requirements of Bacillus coagulans; var. Thermoacidurans in complex media. J Bacteriol. Jul. 1956;72(1):34-41. |
| ATCC Catalogue of Bacteria and Bacteriophages, Accession No. 31284, retrieved Aug. 11, 2009. |
| Bergey's Manual of Systematic Bacteriology. vol. 2, Sneath et al., eds. Baltimore, MD: Williams & Wilkens. (1986):1117. |
| Nakamura et al. “Taxonomic Study of Bacillus coagulans Hammer 1915 with a Proposal for Bacilius smithii sp. nov.” Int. J. Sytematic Bacteriol. 38.1(1988):63-73. |
| Sawatari et al. “Development of Fermented Instant Chinese Noodle Using Lactobacillus plantarum.” Food Microbiol. 22.6(2005):539-546. |
| Allos. “Association Between Campylobacter Infection and Guillain-Barre Syndrome.” J. Infect. Dis. 176(1997):S125-S128. |
| Baker et al. “Growth Requirements of 94 Strains of Thermophilic Bacilli.” Can. J. Microbiol. 6(1960):557-563. |
| Barefoot et al. “Antibiosis Revisited: Bacteriocins Produced by Dairy Starter Cultures.” J. Diary ScL 76(1993):2366-2379. |
| Bergey's Manual of Systematic Bacteriology, 2:1128 (1986). |
| Bergey's Manual of Systematic Bacteriology, vol. 2, Sneath et al., eds., Williams & Wilkens, Baltimore, MD, p. 1117 (1986). |
| Bernet et al. “Adhesion of Human Bifidobacterial Strains to Cultured Human Intestinal Epithelial Cells and Inhibition of Enteropathogen-Cell Interactions.” Appl. Environ. Microbiol. 59.12(1993): 4121-4128. |
| Bernet et al. “Lactobacillus acidophilus LA 1 Binds to Cultured Human Intestinal Cell Lines and Inhibits Cell Attachment and Cell Invasion by Enterovirulent Bacteria.” Gut. 35(1994):483-489. |
| Black et al. “Experimental Camplylobacteijejuni Infection in Humans.” J. Infect. Dis. 157.3(1988):472-479. |
| Blaser et al. “The Influence of Immunity on Raw Milk-Associated Campylobacter Infection.” JAMA. 257.1(1987):43-46. |
| Blaser. “Campylobacter Species.” Principles and Practice of Infectious Diseases. Mandell et al.,eds. New York Churchill Livingstone Inc. 3(1990):1649-1658. |
| Challa et al. “Bifidobacterium longum and Lactulose Suppress Azoxymethane-Induced Colonic Aberrant Crypt Foci in Rats.” Carcinogenesis. 18.3(1997):517-521. |
| Christi et al., “Role of dietary sulphate in the regulation of methanogenesis in the human large intestine”, Gut, 33:1234-1238 (1992). |
| Cometta et al. “Escherichia coli Resistant to Fluoroquinolones in Patients with Cancer and Neutropenia.” New Engl. J. Med. 330.17(1994):1240-1241. |
| Database WPI Week 198532 Thomson Scientific, London, GB; AN 1985-195034 XP002502608 & SU 1 134 151 A (Kiev Food Ind Techn Inst) (Jan. 15, 1985). |
| De Simone et al. “Effect of Bifidobacterium bifidum and Lactobacillus acidophilus on Gut Mucosa and Peripheral Blood B Lymphocytes.” Immunopharmacol. ImmunotoxicoL 14.1&2(1992):331-340. |
| E. De Veechi and L. Drago “Lactobacillus sporogenes or Bacillus coagulans: Misidentification or Mislabeling” 2006 International Journal of Probiotics and Prebiotics vol. 1 No. 1 pp. 3-10. |
| Elmer et al. “Biotherapeutic Agents. A Neglected Modality for the Treatment and Prevention of Selected Intestinal and Vaginal Infections.” JAMA. 275.11(1996):870-876. |
| English Muffins. Yakitate/Oishii Tezukurinopan. (1999):109-111. (Japanese Original and English Translation). |
| Famularo et al. “Stimulation of Immunity by Probiotics.” Probiotics 2: Applications and Practical Aspects. Fuller, ed. London: Boundary Row. (1997):133-161. |
| Fernandez et al. “Effect of Diatomaceous Earth as an Anthelmintic Treatment on Internal Parasites and Feedlot Performance of Beef Steers” Animal Science 66.3(1998): 635-641. |
| Fuller. “Probiotics in Man and Animals.” J. Appl. BacterioL 66(1989):365-378. |
| Gandhi. “Lactobacillus Sporogenes: An Advancement in Lactobacillus Therapy.” Townsend Left. Doctors Patients. 150(1996):108-110. |
| Gibson et al., Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin, Gastroenterol., 108:975-982 (1995). |
| Girardin et al. “Antimicrobial Activity of Foodbome Paenibacillus and Bacillus spp. Against Clostridium botulinum.” J. Food Protection. 65.5(2002):806-813. |
| Gorbach. “Lactic Acid Bacteria and Human Health.” Ann. Med. 22(1990):37-41. |
| Hata et al. “Meningitis Caused by Bifidobacterium in an Infant.” Pediatr. Infect. Dis. 7(1988):669-671. |
| Hill et al. “Vaginitis: Current Microbiologic and Clinical Concepts.” Can. Med. Assoc. J. 134(1986):321-331. |
| Hyronimus et al. “Coagulin, a Bacteriocin-Like Inhibitory Substance Produced by Bacillus Coagulans 14.” J. Appl. Microbiol. 85.1(1998):42-50. |
| Jacobs-Reitsma et al. “The Induction of Quinolone Resistance in Campylobacter Bacteria in Broilers by Quinolone Treatment.” Campylobacter, Helicobacters, and Related Organisms. Newell et al., eds. New YorkL Plenum Press. (1996):307-311. |
| Ketley. “Pathogenesis of Enteric Infection by Campylobacter.” Microbiol. 143(1997):5-21. |
| Kim et al. “Development of Lactobacillus-Sporogenes Resistant to Rifampicin an Antituberculosis Agent.” Korean J. Micro. 27.2(1989):155-161. (English Abstract Only). |
| Klaenhammer. “Genetics of Bacteriocins Produced by Lactic Acid Bacteria.” FEMS Microbial. Rev. 12(1993):39-85. |
| Koo et al. “Long-Term Effect of Bifidobacteria and Neosugar on Precursor Lesions of Colonic Cancer in CFI Mice.” Nutrit. Rev 16(1991):249-257. |
| Korshunov et al. “Effect of the Combined Administration of Antibiotic Resistant Bifidobacteria and the Corresponding Antibiotics on the Survival of Irradiated Mice.” Zh. MikrobioL Epidemol. ImmunobioL 5(1982):50-53. (Russian Original and English Abstract). |
| Lidbeck et al. “Lactobacilli, Anticarcinogenic Activities and Human Intestinal Microflora.” Eur. J. Cancer Prey. 1 (1992):341-353. |
| Iino et al. “A Study on the Effect of Bacillus Coagulans, a Spore-Forming Lactic Acid Bacteria, in Improving Microflora int he Intestine of a Human.” Prog. Med. 17(1997):3303-3308. (No English Translation Available). |
| Iino et al. “A Study on the Effect of Bacillus Coagulans, a Spore-Forming Lactic Acid Bacteria, in Improving the Properties of Feces.” Prog. Med. 17(1997):3299-3302. (No English Translation Available). |
| Malin et al. “Promotion of IgA Immune Response in Patients with Crohn's Disease by Oral Bacteriotherapy with Lactobacillus GG.” Ann. Nutr. Metab. 40(1996):137-145. |
| Mary El-Baz “The Essence of Herbal and Floral Teas” 2006 iUniverse pp. 1 and 2. |
| Matsumara et al. “Interferon Induction by Murine Peritoneal Macrophage Stimulated with Lactobacillus gasserL” Animal Sci. TechnoL (Jpn). 63(1992):1157-1159. |
| Metchinikoff. “Longevity in the Animal Kingdom.” Prolongation of Life. New York: The Knickerbocker Press. (1910):39-93, 132-183. |
| Mitchell. “Rearming in the Fight against Bacteria.” Lancet. 352(1998):462-463. |
| Murphy et al. “Ciprofloxacin- and Azithromycin-Resistant Campylobacter Causing Travelers Diarrhea in U.S. Troops Deployed to Thailand in 1994.” Clin. Infect. Dis. 22(1996):868-869. |
| Number | Date | Country | |
|---|---|---|---|
| 20190297924 A1 | Oct 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61106116 | Oct 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15487230 | Apr 2017 | US |
| Child | 16443823 | US | |
| Parent | 12580622 | Oct 2009 | US |
| Child | 15487230 | US |
