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WO2023196003A1 - <i />COMPOSITIONS DE BACILLUS ET LEURS UTILISATIONS - Google Patents

<i />COMPOSITIONS DE BACILLUS ET LEURS UTILISATIONS Download PDF

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Publication number
WO2023196003A1
WO2023196003A1 PCT/US2022/040239 US2022040239W WO2023196003A1 WO 2023196003 A1 WO2023196003 A1 WO 2023196003A1 US 2022040239 W US2022040239 W US 2022040239W WO 2023196003 A1 WO2023196003 A1 WO 2023196003A1
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WO
WIPO (PCT)
Prior art keywords
bacillus subtilis
strain
day
subtilis strain
food
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2022/040239
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English (en)
Inventor
Jessica SPEARS
Sean GARVEY
Sebhat GEBRECHRISTOS
Christopher Schuler
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Bio Cat Microbials LLC
Original Assignee
Bio Cat Microbials LLC
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Filing date
Publication date
Application filed by Bio Cat Microbials LLC filed Critical Bio Cat Microbials LLC
Priority to EP22936698.4A priority Critical patent/EP4504227A1/fr
Publication of WO2023196003A1 publication Critical patent/WO2023196003A1/fr
Priority to MX2024012240A priority patent/MX2024012240A/es
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
    • A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
    • A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
    • A23G3/364—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
    • A23G3/366—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G4/00—Chewing gum
    • A23G4/06—Chewing gum characterised by the composition containing organic or inorganic compounds
    • A23G4/12—Chewing gum characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
    • A23G4/123—Chewing gum characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A23K50/42—Dry feed
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A23K50/48—Moist feed
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/38—Other non-alcoholic beverages
    • A23L2/382—Other non-alcoholic beverages fermented
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C2220/00—Biochemical treatment
    • A23C2220/20—Treatment with microorganisms
    • A23C2220/202—Genetic engineering of microorganisms used in dairy technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • a unique spore-forming Bacillus species, and more particularly, a Bacillus subtilis strain identified as BS50 is provided.
  • the disclosure also relates to compositions comprising the BS50 strain, methods of making products comprising the same, and methods of using the same.
  • probiotic is generally used to refer to live microbes that provide health benefits or other positive effects when administered (e.g., by ingestion of a probiotic-containing food or dietary supplement).
  • Probiotics may benefit a host directly, e.g., by excreting compounds that interact with the host’s gastrointestinal (GI) system or by expressing useful enzymes that are absent from or insufficiently expressed by the host.
  • Probiotics may also benefit the host indirectly by interacting with other gut microbes in a manner that has a beneficial effect on the host, e.g., by displacing pathogenic bacteria. The precise nature of these interactions is often poorly understood due to the complexity of the GI system and the gut microbiota.
  • probiotic activity of a microbe can be evaluated based on the detection and measurement of its effects on a host regardless of whether the precise mechanism underlying the effects remains unknown.
  • probiotics have emerged as promising candidates for dietary supplements and therapeutics intended to promote gastrointestinal health, digestive health, immune health, and other physiological benefits.
  • Probiotic microbes have been identified across several genera, including Akkermansia, Anaerobutyricum, Bacillus, Bifidobacterium, Clostridium, Enterococcus, Faecalibacterium, Lacticaseibacillus, Lactiplantibacillus, Lactobacillus, Ligilactobacillus, Limosilactobacillus, Propionibacterium, Saccharomyces, Streptococcus, and Weizmannia.
  • probiotics suitable for human consumption in foods and beverages has been limited to fermented foods and dairy compositions (e.g., miso, tempeh, kefir, buttermilk, cheese, and yogurt), which provide an environment suitable to allow a sufficient amount of the probiotic bacteria to survive during typical storage conditions (e.g., refrigerated).
  • fermented foods and dairy compositions e.g., miso, tempeh, kefir, buttermilk, cheese, and yogurt
  • typical storage conditions e.g., refrigerated
  • dietary supplements e.g., tablets, sachets, and other delivery vehicles
  • concerns related to survivability, durability, and stability limit the widespread use of many probiotics.
  • the present disclosure relates to a novel, spore-forming Bacillus species, and more particularly, to Bacillus subtilis strain BS50, a sample of which has been deposited under ATCC Accession Number PTA-127287. Bacillus subtilis strain BS50 is capable of surviving extended exposure to high temperatures and stable when stored for prolonged periods (e.g., as a spore).
  • compositions, supplements and other delivery vehicles comprising the BS50 strain are disclosed.
  • compositions, such as food products, beverage products, and dietary supplements comprising the BS50 strain are provided.
  • a food product comprising BS50 cells and/or spores is provided.
  • the food product may be probiotic (e.g., comprising BS50 in an amount effective to provide a health benefit or other beneficial effect when administered to a human or animal).
  • the food product is a grain product, bread, cookie, cracker, bar, dairy product, or confection.
  • the BS50 may be present at a concentration of at least 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 2 x 10 9 colony-forming units (CFUs)/gram, or an amount of at least 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 2 x 10 9 CFUs per food product or serving of the food product.
  • CFUs colony-forming units
  • the food product may comprise flour, and/or at least one other probiotic, such as a species of Weizmannia genus (formerly Bacillus), and Lactobacillaceae family of the Bacillota phylum (formerly Firmicutes), and species from the Bifidobacterium genus of the Actinomycetota phylum (formerly Actinobacteria).
  • a species of Weizmannia genus (formerly Bacillus)
  • Lactobacillaceae family of the Bacillota phylum formerly Firmicutes
  • species from the Bifidobacterium genus of the Actinomycetota phylum previously Actinobacteria
  • the disclosure provides beverages (e.g., juice, dairy product, non- dairy milk alternative, legume-based milk alternative, nut-based milk alternative, carbonated water, soda, tea, kombucha, kefir, coffee, sports drink, energy drink, or alcoholic beverage) comprising BS50 at a concentration of at least 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 2 x 10 9 CFUs/gram.
  • the beverage may comprise at least one other probiotic, such as a strain from the genus Lactobacillus or Bifidobacterium.
  • the beverage may also comprise one or more of the following additives: natural sweeteners (e.g., cane sugar, corn syrup, sucrose, maltodextrin, agave syrup or powder, stevia and stevia leaf derivatives, monk fruit powder or extract, etc.), artificial sweeteners (e.g., sucralose, acesulfame potassium, aspartame, etc.), soluble fiber (e.g., pectin, inulin, beta-glucans, fructo-oligosaccharides, galacto-oligosaccharides, xylo- oligosaccharides, arabino-xylooligosaccharides, psyllium, wheat dextrin, polydextrose, carboxymethylcellulose, guar gum and guar gum derivatives, oat powder and other oat derivatives, chickpea powder and other chickpea derivatives, pea powder and other pea derivatives, etc.), insoluble fiber (e.g.
  • the disclosure provides dietary supplements (e.g., a powder, tablet, pill, sachet, capsule, or suspension) comprising BS50.
  • the BS50 may be present at a concentration of at least 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 2 x 10 9 , or 1 x 10 10 CFUs/gram.
  • the dietary supplement may comprise at least one other probiotic, such as a strain from the genus Lactobacillus or Bifidobacterium.
  • the supplement may include spray-dried BS50 spores.
  • the dietary supplement may also comprise one or more of the following additives: natural sweeteners (e.g., cane sugar, corn syrup, sucrose, maltodextrin, agave syrup or powder, stevia and stevia leaf derivatives, monk fruit powder or extract, etc.), artificial sweeteners (e.g., sucralose, acesulfame potassium, aspartame, etc.), soluble fiber (e.g., pectin, inulin, beta-glucans, fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, arabino-xylooligosaccharides, psyllium, wheat dextrin, polydextrose, carboxymethylcellulose, guar gum and guar gum derivatives, oat powder and other oat derivatives, chickpea powder and other chickpea derivatives, pea powder and other pea derivatives, etc.), insoluble fiber (e.g
  • the disclosure provides pet food compositions comprising BS50. These compositions may be generally formulated similarly to the disclosed food products and beverages.
  • the pet food may be a dry mixture or a wet mixture. In some aspects, it may comprise one or more fatty acids, free amino acids, or protein, and optionally one or more additional probiotics.
  • the disclosure provides methods of making and using compositions comprising BS50. For example, methods of using BS50 to provide a health benefit or other beneficial effect to a human or animal are disclosed.
  • the methods are directed to supporting digestive health, supporting gastrointestinal health, and/or reducing gastrointestinal symptoms (e.g., one or more of abdominal bloating, flatulence, burping, stomach rumbling, diarrhea, constipation, loose stool, or firm stool).
  • the methods are directed to supporting immune health and/or supporting healthy blood levels of anti-inflammatory cytokines (e.g., interleukin-10 (IL-10)) and/or decreasing the number of pathogenic bacteria across the intestine.
  • the B. subtilis strain BS50 may be administered in an aerosol, for example by way of a nasal spray, for instance for administration to the respiratory tract.
  • the B. subtilis strain BS50 may be administered in an aerosol, for example by way of a nasal spray, for instance for administration to the respiratory tract.
  • the B. subtilis strain BS50 may be administered in an aerosol, for example by way of a nasal spray, for instance for administration to the respiratory tract.
  • the B. subtilis strain BS50 may be administered in an aerosol, for example
  • subtilis strain BS50 may be administered topically, for example by way of a lotion, cream, for instance for administration to the skin.
  • the B. subtilis strain BS50 may be in a lyophilized or spray dried form.
  • the B. subtilis strain BS50 may be heat treated, filtered to remove viable cells, or both.
  • FIG.1 is a graph illustrating the durability of B. subtilis strain BS50 at 70, 80, 90, and 100 ⁇ C.
  • FIG. 2 is a graph illustrating the durability of B. subtilis strain BS50 vs.
  • FIG.3 is a graph illustrating the survivability of B. subtilis strain BS50 at room temperature.
  • FIG.4A depicts the survivability of B. subtilis strain BS50 at acidic pH levels over time.
  • FIG.4B depicts the survivability of B. subtilis BS50 at alkaline pH levels.
  • FIG.5 depicts the survivability of B. subtills strain BS50 in tea steeped at increasing temperatures.
  • FIG. 6 illustrates the compatibility between B. subtilis strain BS50 and other probiotics.
  • FIG.7 is a graph illustrating B. subtilis strain BS50 pathogen inhibition activity.
  • the present disclosure relates to a unique spore-forming Bacillus species, and more particularly, to a B. subtilis BS50 strain which has been deposited as ATCC Accession Number PTA-127287. Compositions comprising B. subtilis BS50 and methods of making and using the same are also provided.
  • the microbiota, or ecosystem of microbes and all microbial strains therein, of a typical human gastrointestinal tract is understood to contain approximately 10 14 cells, comprising several hundred different bacterial species. Interactions between the gut microbiota and the host’s gastrointestinal, digestive, and immune systems are believed to play a fundamental role in influencing physiological and homeostatic functions of the host.
  • the gut microbiota may be considered to function as a separate organ. Disruption of the complex interplay between the host’s systems and the gut microbiota can lead to the development of diseases and negative physiological effects. For example, pathogenic bacteria may displace commensal microbes—microbes living in harmony within its host—or explicitly beneficial gut microbes, resulting in inflammation and/or other gastrointestinal diseases, infections, or disorders. Similarly, a host’s gut microbiota may lack a commensal microbe or beneficial microbe, whether due to natural processes or due to exposure to an antibiotic.
  • the host may be administered a probiotic food composition, dietary supplement, or other suitable vehicle comprising the probiotic in a quantity sufficient to support colonization and/or maintenance of beneficial and commensal microbes, or otherwise shift the imbalanced microbiota toward a more usual, healthy microbiota.
  • a probiotic food composition, dietary supplement, or other suitable vehicle comprising the probiotic in a quantity sufficient to support colonization and/or maintenance of beneficial and commensal microbes, or otherwise shift the imbalanced microbiota toward a more usual, healthy microbiota.
  • current probiotics are limited to a relatively narrow range of foods, beverages, and dietary supplement dosage forms.
  • probiotics of the Lactobacillaceae family and Bifidobacterium genus may be unsuitable for foods, beverages, and dietary supplements exposed to high temperatures or varying pH conditions.
  • many types of food matrices e.g., baked goods, powdered supplements, heat-treated beverages, shelf-stable beverages
  • the B. subtilis BS50 strain disclosed herein addresses these and other shortcomings.
  • probiotic compositions having excellent survivability across a wide range of temperatures and pH levels are provided.
  • the BS50 strain is a particularly well- suited probiotic for foods, such as baked goods, and other food products or dietary supplements exposed to high temperatures during production or storage.
  • the BS50 strain is also useful as, among other things, a cleaning product or other products used for reduction of deleterious microbes where temperature, pH and/or chemicals may present a concern for other less hardy microbes.
  • the BS50 strain is a gram-positive, spore forming, rod-shaped facultative anaerobe. Initial characterization assays indicate that BS50 retains the ability to express antimicrobial compounds (e.g., bacillibactin, bacilysin, fengycin, or surfactin.) and digestive enzymes (e.g., amylase, cellulase, lipase, protease, etc.) to varying extents.
  • antimicrobial compounds e.g., bacillibactin, bacilysin, fengycin, or surfactin.
  • digestive enzymes e.g., amylase, cellulase, lipase, protease, etc.
  • the BS50 strain is typically manufactured in pure culture and then spray-dried.
  • the BS50 strain described herein was originally isolated from composted soil from Gallatin County, Montana, USA and has subsequently been produced in a laboratory and industrial fermentation facility (BIO-CAT Microbials, LLC, Shakopee, Minnesota, USA). Whole genome sequencing and subsequent bioinformatic analysis have shown that the BS50 strain has at least 98.5% similarity to other Bacillus subtilis strains found in the food supply.
  • B. subtilis is not considered pathogenic or toxigenic to humans, animals, or plants and, for example, at least five B. subtilis strains have been classified by the U.S.
  • FIG.3 illustrates that > 95% of the spray dried BS50 spores remain viable when stored at room temperature (25 o C with 65% relative humidity) for at least 9 months.
  • the BS50 strain is highly tolerant to a wide variety of pH levels over time. As a result, the BS50 strain is a good candidate for use in consumer products, such as food, dietary supplement, oral and/or nasal spray, topical skin, and cleaning products, capable of withstanding high temperatures, exposure to room temperature for long periods of time, and/or pH changes.
  • compositions comprising BS50 cells, spores, or a combination thereof capable of surviving exposure to heat and/or long periods of time at room temperature (e.g., at least 24 months).
  • compositions e.g., food and beverage products, dietary supplements
  • compositions comprising BS50 cells, spores, extract, and/or heat-treated forms of either in an amount effective to provide a health benefit to a consumer of a food product, beverage or supplement.
  • compositions may comprise mutants or derivatives of BS50 having the identifying characteristics of BS50. All mutants or derivatives are isolated without the use of genetic engineering.
  • BS50, its mutants, or its derivative cells, spores, or a combination thereof are produced and harvested by humans in a laboratory or industrial setting.
  • the composition comprising BS50 cells and/or spores is a food product, such as a baked good.
  • exemplary baked goods include, but are not limited to, muffins, breads, waffles, cakes, biscuits, cookies, pies, tarts, pastries, candy/energy bars, granola, cereal, crackers.
  • the composition includes any baked good that comprises flour, or which is prepared by baking (e.g., by exposure to dry heat).
  • Other baked goods that may serve as a vehicle for the BS50 include pizza, pasta, corn or potato chips, dehydrated fruits or vegetables.
  • the BS50 may be included in a beverage composition, whether as vegetative cells, spores, or a combination thereof.
  • the beverage is a hot beverage (e.g., tea, coffee), while in others it is a shelf-stable or cold beverage (e.g., carbonated water, juice, soda, tea, coffee, kefir, kombucha).
  • BS50 spores and/or cells may be added to the beverage during processing by a manufacturer, or by an end user (e.g., by a consumer adding a dry mixture comprising BS50 spores and optionally other nutrients to a water or another liquid to prepare a probiotic meal replacement beverage).
  • the beverage product comprises BS50 and one or more of the following additives: natural sweeteners (e.g., cane sugar, corn syrup, sucrose, maltodextrin, agave syrup or powder, stevia and stevia leaf derivatives, monk fruit powder or extract, etc.), artificial sweeteners (e.g., sucralose, acesulfame potassium, aspartame, etc.), soluble fiber (e.g., pectin, inulin, beta-glucans, fructo-oligosaccharides, galacto- oligosaccharides, xylo-oligosaccharides, arabino-xylooligosaccharides, psyllium, wheat dextrin, polydextrose, carboxymethylcellulose, guar gum and guar gum derivatives, oat powder and other oat derivatives, chickpea powder and other chickpea derivatives, pea powder and other pea derivatives, etc.), insoluble fiber (e
  • BS50 cells or spores may be added to the numerous beverages currently sold or prepared for human consumption.
  • the BS50 strain and its derivatives may be included in a dietary supplement, whether as vegetative cells, spores, extracts, or a combination thereof.
  • the dietary supplement may be a powder, tablet, pill, sachet, capsule, or suspension.
  • Exemplary dietary supplements include products that may be added to foods or drinks, such as protein powders.
  • the dietary supplement comprises BS50 and one or more of the following additives: natural sweeteners (e.g., cane sugar, corn syrup, sucrose, maltodextrin, agave syrup or powder, stevia and stevia leaf derivatives, monk fruit powder or extract, etc.), artificial sweeteners (e.g., sucralose, acesulfame potassium, aspartame, etc.), soluble fiber (e.g., pectin, inulin, beta- glucans, fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, arabino- xylooligosaccharides, psyllium, wheat dextrin, polydextrose, carboxymethylcellulose, guar gum and guar gum derivatives, oat powder and other oat derivatives, chickpea powder and other chickpea derivatives, pea powder and other pea derivatives, etc.),
  • the dietary supplement is a composition, such as a capsule, comprising BS50 that can be taken with or without food or drink.
  • the BS50 cells and/or spores may comprise between about 0.001% to about 10% by weight of the food product (e.g., a baked good, granola bar), supplement, or beverage.
  • the BS50 cells and/or spores may comprise between about 0.01% and about 10% by weight of the food product. Heating and processing will affect the amount or concentration of BS50 is a final product.
  • the amount or concentration of BS50 cells and/or spores present in a baked good will depend on both the number of colony-forming units applied to the pre-baked composition and parameters related to the baking step (e.g., time, temperature, moisture levels).
  • the BS50 cells and/or spores may comprise at least about 0.001%, 0.01%, 0.1%, 1%, or 10% by weight of the food product, or a range between about 0.001% to about 0.01%, about 0.01% to about 0.1%, about 1% to about 10%, about 10% to about 20%, or ⁇ 20% by weight of the food product.
  • the amount of BS50 cells and/or spores may comprise a minimum and/or a maximum percentage amount selected from any of the aforementioned ranges.
  • the composition may comprise a mixture or batter for preparing a food product that will be baked (e.g., bread, muffins), fried, or otherwise heated, wherein the mixture comprises BS50 cells and/or spores.
  • composition may be formulated such that a given percentage (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) of the BS50 cells and/or spores present in a given amount or volume of the starting mixture or batter remain viable in the final baked, fried, or otherwise heated food product.
  • a given percentage e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%
  • the composition may be formulated such that about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or ⁇ 90% of the BS50 cells and/or spores present in a given amount or volume of the starting mixture or batter remain viable in the final baked, fried, or otherwise heated food product.
  • the percentage of viable cells may be a range that includes a minimum and/or a maximum percentage amount selected from any of the aforementioned ranges.
  • Baked goods and baking mixes including all ready-to-eat and ready-to-bake products, flours, and mixes requiring preparation before serving.
  • Beverages alcoholic, including malt beverages, wines, distilled liquors, and cocktail mix.
  • Beverages and beverage bases nonalcoholic, including only special or spiced teas, soft drinks, coffee substitutes, and fruit and vegetable flavored gelatin drinks.
  • Breakfast cereals including ready-to-eat and instant and regular hot cereals.
  • Cheeses including curd and whey cheeses, cream, natural, grating, processed, spread, dip, and miscellaneous cheeses.
  • Chewing gum including all forms. Coffee and tea, including regular, decaffeinated, and instant types. Condiments and relishes, including plain seasoning sauces and spreads, olives, pickles, and relishes, but not spices or herbs. Confections and frostings, including candy and flavored frostings, marshmallows, baking chocolate, and brown, lump, rock, maple, powdered, and raw sugars. Dairy product analogs, including nondairy milk, frozen or liquid creamers, toppings, and other nondairy products. Fats and oils, including margarine, dressings for salads, butter, salad oils, shortenings and cooking oils.
  • Fresh fruit juices including only raw fruits, citrus, melons, and berries, and home prepared “ades” and punches made therefrom.
  • Frozen dairy desserts and mixes including ice cream, ice milks, sherbets, and other frozen dairy desserts and specialties.
  • Fruit and water ices including all frozen fruit and water ices.
  • Gelatins, puddings, and fillings including flavored gelatin desserts, puddings, custards, parfaits, pie fillings, and gelatin base salads.
  • Grain products and pastas including macaroni and noodle products, rice dishes, and frozen multicourse meals, without meat or vegetables.
  • Hard candy and cough drops including all hard type candies.
  • Jams and jellies commercial, including only commercially processed jams, jellies, fruit butters, preserves, and sweet spreads.
  • Milk, whole and skim including only whole, lowfat, and skim fluid milks.
  • Milk products including flavored milks and milk drinks, dry milks, toppings, snack dips, spreads, weight control milk beverages, and other milk origin products.
  • Nuts and nut products including whole or shelled tree nuts, peanuts, coconut, and nut and peanut spreads.
  • Plant protein products including the National Academy of Sciences/ National Research Council “reconstituted vegetable protein” category, and meat, poultry, and fish substitutes, analogs, and extender products made from plant proteins.
  • Processed fruits and fruit juices including all commercially processed fruits, citrus, berries, and mixtures; salads, juices and juice punches, concentrates, dilutions, “ades”, and drink substitutes made therefrom.
  • Processed vegetables and vegetable juices including all commercially processed vegetables, vegetable dishes, frozen multicourse vegetable meals, and vegetable juices and blends.
  • Snack foods including chips, pretzels, and other novelty snacks.
  • Soft candy including candy bars, chocolates, fudge, mints, and other chewy or nougat candies.
  • Soups and soup mixes including commercially prepared meat, fish, poultry, vegetable, and combination soups and soup mixes.
  • Sugar substitutes including granulated, liquid, and tablet sugar substitutes.
  • Sweet sauces, toppings, and syrups including chocolate, berry, fruit, corn syrup, and maple sweet sauces and toppings.
  • probiotic compositions e.g., food products, beverages, or dietary supplements
  • BS50 cells and/or endospores that remain shelf stable for long periods of time, such as 4 months, 6 months, 12 months, 18 months, 24 months, 30 months, or more than 30 months at, for example, room temperature.
  • compositions may be formulated to increase or decrease stability (e.g., by varying moisture levels). In select aspects, the compositions are formulated to retain a particular percentage of viable cells after a given amount of time stored at room temperature (e.g., at least 50%, 60%, 70%, 80%, or 90%).
  • the food or dietary supplement comprising the BS50 cells and/or spores may be a spray-dried product (e.g., wherein either the entire product or the BS50 cells and/or spores have been subjected to a spray drying process).
  • Spray drying is a method of producing a dry powder from a liquid or slurry by rapidly drying with a hot gas, and is a preferred method of drying many thermally-sensitive materials, such as foods and pharmaceuticals.
  • Spray drying of the BS50 cells and/or spores may be used to further enhance the survivability of the BS50 in the delivery vehicle.
  • a spray drying step during processing may generate a dry mixture for a food product that displays a higher degree of stability at room temperature than a comparable mixture lacking this spray drying step.
  • Various methods of spray drying are known in the art to be suitable for bacteria and may be used or adapted for use with BS50 cells.
  • spray drying protocols may include carbohydrates, such as polysaccharides or polyols, that enhance preservation by preventing crystallization during the drying step.
  • methods known in the art allow for spray drying of bacteria in the presence of inactive agents, such as plasticizers and glidants, so as to produce a particle that provides controlled release after ingestion.
  • inactive agents such as plasticizers and glidants
  • the BS50 cells and/or spores disclosed herein may be spray dried by any methods known in the art suitable for bacteria, particularly methods suitable for B. subtilis.
  • the BS50 cells, spores, and/or supernatants may be lyophilized.
  • Lyophilization or freeze drying is a method of drying by first freezing a liquid or slurry then removing water from the frozen product. Water removal takes place under a vacuum so solid water leaves the product as a vapor. Because lyophilization does not require heat, this method of drying can be useful to preserve, for example, heat-sensitive vitamins or metabolites produced by the BS50 strain effectively concentrating the beneficial products of BS50 beyond anything found in nature.
  • the BS50 is heat treated, filtered to remove viable cells, or both.
  • Heating and/or filtering are methods of removing viable cells or spores from the BS50 preparation by using high heat (e.g., greater than 100 o C) and/or sterilizing filtration (e.g., 0.2- micron filter) to preserve the beneficial products produced by BS50 without any remaining live component. These cell free preparations are useful for administration to humans and/or animals whereby the beneficial products of the B. subtilis strain BS50 are desired but viable cells are not.
  • the amount of BS50 cells and/or spores added to a food product, beverage, or dietary supplement may be varied to ensure that a desired number of viable cells remain in the product administered to an end user.
  • This amount may be selected to ensure that the amount present is sufficient to provide a given benefit to the user, such as a reduction in gastrointestinal symptoms. This amount may be selected to ensure that the amount present is sufficient to provide a given benefit to the user, such as an improvement in immune health.
  • the amount may also be varied based upon an expected administration regimen (e.g., a dietary supplement comprising BS50 may be marketed for daily use). Daily use may include a once-daily, twice- daily, or several times daily. In alternative aspects, bidiurnal, once-weekly, twice-weekly and other weekly or longer regimens are possible.
  • concentration of BS50 in a given food product, beverage, or dietary supplement may also be varied, for example, to provide an amount effective to achieve a given health benefit.
  • concentration of BS50 in the food product, beverage, or dietary supplement is about 10 2 to 10 10 CFUs of BS50 per gram.
  • concentration may comprise 10 4 to 10 8 CFU/g, or 10 6 to 10 7 CFU/g.
  • the concentration may comprise 1 x 10 9 to 1 x 10 10 CFU/g or 1 x 10 9 to 1 x 10 11 CFU/g.
  • the amount or concentration of BS50 may be determined on a per unit basis (e.g., up to 1 ⁇ 10 9 CFU or 5 x 10 9 CFU per serving).
  • the concentration may be measured on a per food product, beverage product, or dietary supplement basis.
  • the amount of BS50 is determined on a daily or weekly basis, such as 1-10 x 10 9 CFUs/day, or 1-2 x 10 10 CFUs/week.
  • the daily intake level for BS50 may be approximately 1 x 10 9 to 1 x 10 10 CFUs of BS50/day, though the amount may vary within that range based upon the particular application and intended effect (e.g., 5 x 10 9 CFU/day).
  • Dietary supplements may be formulated to include an amount of BS50 CFUs sufficient to achieve any of these daily intake amounts when administered per instructions or expected use by a consumer (e.g., a twice-daily supplement may comprise 5 x 10 9 CFUs per serving in order to reach a recommended daily intake of 1 x 10 10 ). Amounts will vary depending on whether the supplement is once-daily, twice-daily, etc.
  • the product When administered as a food product, in some aspects the product may be formulated to satisfy a recommended daily intake of up to 2 x 10 9 CFUs.
  • a food or beverage product expected to be consumed at a rate of two servings per day may be formulated to comprise up to 1 x 10 9 CFUs per serving.
  • the formulation of a food product may be designed to provide a suitable concentration of BS50 per gram or unit of volume.
  • the product may be formulated to include approximately to 2 x 10 8 CFUs/gram.
  • Other formulations may take into account a higher or lower expected number of servings or amount consumed per day, or based on the particular application.
  • the BS50 may be included at approximately 1 x 10 9 or 2 x 10 9 CFUs per gram.
  • a food product, beverage, dietary supplement or other vehicle for administering BS50 to a human or animal may comprise at least one bacterial cell or spore having genomic DNA that is at least 95%, 96%, 97%, 98%, or 99% identical to the genomic DNA of the Bacillus subtilis BS50 which has been deposited under ATCC Accession Number PTA-127287.
  • the BS50 may be in the form of a vegetative cell, a spore, a heat-killed version of either, or a heat-killed version of both.
  • the bacterial cell, spore, or heat-killed BS50 possesses one or more of the various functional characteristics described herein as a characteristic of BS50 (e.g., the capability to withstand high temperatures, exposure to room temperature for long periods of time, and/or pH changes). It is understood that in some aspects, these bacterial cells, spores, or heat-killed cells or spores may display any of the survivability or stability characteristics of BS50 described herein.
  • bacterial cells spores, or heat-killed cells or spores may be added to a food product, beverage, supplement, pet food or other vehicle for providing BS50 to a human or pet in any of the amounts of concentrations described herein for BS50, or in other such amounts or concentrations as desired for a given application.
  • the disclosure provides a composition comprising the isolated BS50 strain, or a cell extract, supernatant, spores, or cell material derived from the isolated strain.
  • any of the compositions described herein may include suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
  • any of the compositions described herein may include magnesium stearate, titanium dioxide, stearic acid, cellulose, silicon dioxide, maize (e.g., waxy maize) maltodextrin, microcrystalline cellulose, calcium carbonate, corn starch, maize maltodextrin, tapioca maltodextrin, tapioca dextrin, agenamalt non gmo maltodextrin, calcium chloride, flour salt, potassium sorbate, pretzel salt, sodium benzoate, sodium borate, sodium sulfate (food grade), rice bran extract, and/or potato maltodextrin.
  • maize e.g., waxy maize
  • maltodextrin e.g., microcrystalline cellulose
  • calcium carbonate e.g., corn starch
  • maize maltodextrin e.g., tapioca maltodextrin, tapioca dextrin
  • any of the compositions may include additives such as anti-caking agents, anti-oxidation agents, bulking agents, and/or protectants.
  • additives include polysaccharides (e.g., starches, maltodextrins, methylcelluloses, gums, chitosan and/or inulins), protein sources (e.g., skim-milk powder and/or sweet-whey powder), peptides, sugars (e.g., lactose, trehalose, sucrose and/or dextrose), lipids (e.g., lecithin, vegetable oils and/or mineral oils), salts (e.g., sodium chloride, sodium carbonate, calcium carbonate, chalk, limestone, magnesium carbonate, sodium phosphate, calcium phosphate, magnesium phosphate and/or sodium citrate), and/or silicates (e.g., clays, in particular beolite clay, amorphous silica, fumed/precipitated silicas), and/or silicates
  • compositions comprising B. subtilis strain BS50 may be formulated for nasal or topical administration.
  • methods of formulating compositions for nasal administration are disclosed in U.S. Pub. No. 2022/0047627, incorporated herein by reference in its entirety.
  • Methods of Preparing Food Products, Beverages, and Supplements Comprising BS50 Cells and/or Spores [0050] BS50 cells, spores, or heat-killed cells or spores may be added to a variety of food products, beverages, and dietary supplements.
  • BS50 cells and/or spores may be applied to most food products, beverages and dietary supplements in their current form, or with minor modifications.
  • BS50 spores may remain viable for at least 24 months at room temperature, (e.g., without refrigeration), making BS50 spores particularly well-suited for products with a long shelf life.
  • Processing conditions may need to be varied based on the type of food product (e.g., the amount of BS50 cells, spores, or heat-killed cells or spores added to a pre-processed or pre- baked composition may need to be increased in order to ensure sufficient colony-forming units in the end product).
  • Other parameters that may be adjusted include moisture levels, temperature, and pH conditions. For example, if a composition comprising BS50 spores is to be baked at a higher temperature or for a longer time, survivability may be enhanced by increasing the moisture level of the pre-baked composition.
  • Each of the aforementioned parameters may be varied in order to suit a desired application of the methods and compositions disclosed herein.
  • the composition is prepared by at least one step involving the application of a high temperature for a short or sustained period of time.
  • the composition may be baked, boiled, or fried.
  • BS50 spores, cells, or a mixture thereof are included in the composition prior to the application of a high temperature.
  • the BS50 cells may be dispersed in a dry mixture, a batter, or a liquid component that is then baked or mixed with additional components prior to baking.
  • the composition is heated by baking to at least 300, 325, 350, 375, 400, or 425°F for at least 10, 15, 20, 25, or 30 minutes.
  • the composition is a muffin, pancake, bread, cake, biscuit, pancake, or waffle mix comprising BS50 spores or heat killed spores.
  • the BS50 cells and/or spores are pre-mixed into the mixture prior to purchase, while in others the BS50 cells and/or spores may be provided as a separate component in a kit, to be mixed into the composition prior to heating by an end user.
  • the composition is a composition, such as a pancake comprising BS50 cells, spores, or heat-killed cells or spores, is cooked on a frying pan or griddle.
  • the composition is cooked by heating to 375°F (190.6°C) for at least 2-3 minutes per side.
  • the composition is formulated to retain at least 50% viability of the BS50 cells and/or spores when the mixture is heated to 375°F (190.6°C) for at least 2-3 minutes, twice (e.g., by cooking on each side).
  • the composition is heated by boiling or steeping BS50 cells, spores, or heat-killed cells or spores in a hot solvent such as water (e.g., a tea bag comprising ground tea leaves, BS50 spores, and optionally other ingredients).
  • compositions, food product, beverage, or dietary supplement composition may comprise BS50 cells, spores, or heat-killed cells or spores according to any of the aspects disclosed herein, in addition to at least one other probiotic.
  • the at least one other probiotic is a probiotic bacterium (e.g., a Lactobacillus species such as L. acidophilus).
  • the bacterium is an Akkermansia, Anaerobutyricum, Bifidobacterium, Clostridium, Enterococcus, Faecalibacterium, Lacticaseibacillus, Lactiplantibacillus, Lactobacillus, Ligilactobacillus, Limosilactobacillus, Propionibacterium, Saccharomyces, or Streptococcus bacterial or fungal strain.
  • the at least other probiotic is a second strain from the genus Bacillus. In some aspects, at least two probiotic strains are present. However, additional compositions featuring multiple probiotics are also contemplated.
  • combination products may comprise refrigerated or non-refrigerated dairy (e.g., yogurt, milk, cheese), and non-dairy products (e.g., a soda, energy drink, or sports drink), fermented products, etc.
  • the one or more additional probiotics may be present in a composition, food product, beverage, or dietary supplement composition in particular combinations or ratios that provide improved health benefits or other beneficial effects resulting from administration to a human or animal.
  • two strains that each promote gastrointestinal health or a reduction in negative gastrointestinal symptoms, and/or promote immune health may be combined in a single composition in a ratio that provides a greater benefit that administration of the same amount of each probiotic separately and/or at different times.
  • BS50 is compatible with several other probiotics via streak plate assays and thus may display synergistic effects when paired with these or other members of the Lactobacillus or Bifidobacterium genera, or other probiotics.
  • the amounts, ratios and combinations of probiotics may be varied to achieve different outcomes or efficacy levels.
  • the parameters of the composition may be adjusted to provide an environment conducive to survival of both the BS50 cells and/or spores, and the one or more additional probiotics.
  • compositions featuring a Lactobacillus or Bifidobacterium may be prepared at a lower temperature suitable for these probiotics. While the BS50 strain is particularly well-suited at surviving high temperatures, compositions according to the present disclosure may be prepared at any suitable temperature (e.g., without a heating step), depending on the intended use for the composition and its components. Pet Food Products, Beverages, and Supplements Comprising BS50 Cells and/or Spores [0059] Compositions comprising BS50 formulated for animal consumption are also provided. While the present disclosure has thus far described compositions suitable for a human, there exists an analogous need in the art for new probiotics for animals (e.g., pets or livestock).
  • the composition comprises a wet pet food comprising BS50 cells, spores, or heat-killed cells or spores.
  • the composition comprises a dry pet food comprising BS50 spores.
  • the composition may be a cat or dog food product, such as a bone.
  • the pet food composition may be coated with the BS50 cells and/or spores, e.g., as an outer layer applied to dry pet food after the individual pieces have been formed, or mixed into the pet food prior to shaping.
  • the composition is a liquid or dietary supplement comprising BS50 cells and/or spores (e.g., which is added to food or water in a dog bowl).
  • the composition may comprise one or more of the following: protein, an amino acid, a plasticizer, a vitamin, and any other components known to be useful for promoting pet health and/or improving flavor.
  • a pet food product, beverage, supplement or other vehicle for providing BS50 to a pet may be formulated to include BS50 cells and/or spores in any amounts or ranges described herein (e.g., any of the amounts and/or ranges described above in the context of food products).
  • compositions comprising BS50 cells, spores, or heat- killed cells or spores to individuals are also provided.
  • the disclosure provides methods of treating or reducing gastrointestinal symptoms in a human subject.
  • the gastrointestinal symptoms comprise abdominal bloating, flatulence, burping, stomach rumbling, diarrhea, constipation, loose stool, or firm stool.
  • the disclosure provides methods of promoting immune health in a human subject.
  • immune health is determined by measuring blood levels of the anti-inflammatory cytokine IL-10.
  • IL- 10 is a robust anti-inflammatory and immunosuppressive cytokine protein produced and secreted by many different types of immune cells, and in particular, monocytes, macrophages, regulatory T cells, and T helper type 2 cells.
  • IL-10 plays a role in dampening inflammatory responses, e.g., by inhibiting the release of pro-inflammatory cytokines (Ref: 2008 Couper).
  • immune health is determined by measuring levels of pathogenic bacteria in stool samples.
  • the disclosure provides for methods of improving GI tolerance, improving GI health, improving GI comfort, improving digestive health, supporting GI tolerance, supporting GI health, supporting GI comfort, supporting digestive health, enhancing GI tolerance, enhancing GI health, enhancing GI comfort, enhancing digestive health, optimizing GI tolerance, optimizing GI health, optimizing GI comfort, optimizing digestive health, promoting GI tolerance, promoting GI health, promoting GI comfort, and/or promoting digestive health by administering any of the compositions comprising BS50 cells and/or spores to individuals described herein.
  • the disclosure provides for methods of reducing GI symptoms, reducing digestive symptoms, reducing GI discomfort, reducing abdominal discomfort, reducing burping, reducing abdominal bloating, reducing flatulence, alleviating GI symptoms, alleviating digestive symptoms, alleviating GI discomfort, alleviating abdominal discomfort, alleviating burping, alleviating abdominal bloating, alleviating flatulence, helping with occasional gas and bloating, helping with occasional flatulence, and/or helping with occasional burping by administering any of the compositions comprising BS50 cells and/or spores to individuals described herein.
  • “reducing” means reducing the severity or frequency of the condition (e.g., GI symptoms).
  • the disclosure provides for methods of preventing GI symptoms, preventing digestive symptoms, preventing GI discomfort, preventing abdominal discomfort, preventing burping, preventing abdominal bloating, preventing flatulence, treating GI symptoms, treating digestive symptoms, treating GI discomfort, treating abdominal discomfort, treating burping, treating abdominal bloating, and/or treating flatulence by administering any of the compositions comprising BS50 cells and/or spores to individuals described herein.
  • GI can be used interchangeably with gut.
  • the disclosure provides for methods of supporting immune health, supporting immunity, improving immune health, improving immunity, promoting immune health, and/or promoting immunity by administering any of the compositions comprising BS50 cells and/or spores to individuals described herein.
  • the disclosure provides for methods of reducing inflammation, alleviating inflammation, helping with inflammation, preventing inflammation, and/or treating inflammation by administering any of the compositions comprising BS50 cells and/or spores to individuals described herein.
  • “reducing” means reducing the severity or frequency of the condition (e.g., GI symptoms).
  • the methods comprise administering a composition (e.g., a food product, dietary supplement, or other vehicle) comprising at least 100, 150, 200, 250, 300, 350, or 400 mg of BS50 spores, to a patient on a daily basis.
  • a composition e.g., a food product, dietary supplement, or other vehicle
  • the BS50 may be administered once-daily, twice-daily (or more frequently). In other aspects it may be once- weekly, twice-weekly, etc.
  • the method comprises administering the composition at least once daily for 1, 2, 3, or 4 or more consecutive weeks, at least 6 months, at least 12 months, or other regimens that may be suitable to provide a desired effect or health benefit.
  • the composition may be administered in any suitable format or vehicle (e.g., as a capsule, tablet, suspension, etc.).
  • the BS50 may be administered to a human once per day as a capsule, tablet, suspension or other dosage form comprising 2 x 10 9 CFU of BS50.
  • Other amounts and formulations may be developed to suit the particular dosage regimen and amount necessary for a given effect. For example, if administered twice-daily, each dosage form may be formulated to comprise 1 x 10 9 CFU of BS50.
  • Liquid dosage forms may be formulated to provide similar amounts (e.g., 2 x 10 9 CFU of BS50) when administered. In some cases, more or less BS50 may need to be administered (e.g.
  • BS50 may be administered in an aerosol, for example by way of a nasal spray, for instance for administration to the respiratory tract.
  • BS50 may be administered topically, for example by way of a lotion, cream, for instance for administration to the skin.
  • the BS50 cells, spores, or heat-killed cells or spores composition may be administered to achieve some or all of these effects and may be any of the other food product, beverage, or dietary supplement compositions disclosed herein, provided in an amount and frequency sufficient to achieve the desired effect.
  • Similar methods may be employed to improve the health of animals, including house pets (e.g., cats, dogs) as well as farm animals (e.g., livestock).
  • BS50 cells, spores, or heat-killed cells or spores may be administered to an animal according to a regimen similar to that used for humans, as discussed above.
  • compositions for Inhibiting Microbial Pathogens may be used to inhibit the growth of pathogenic microbes (e.g., harmful bacteria).
  • compositions comprising BS50 may be useful to inhibit the growth of Escherichia species (e.g., E.
  • compositions comprising BS50 have been tested using cross streak assays against multiple pathogenic bacteria, as described in Example 6. The results show that BS50 displays the ability to inhibit pathogenic members of at least several bacterial genera. These results suggest an additional basis for BS50’s its probiotic effects. As such, compositions comprising BS50, as described herein, may be prepared and used as an antimicrobial treatment.
  • compositions may be administered to an animal or human, or applied to a surface or area in order to inhibit the growth of a pathogenic bacteria.
  • Antimicrobial compositions for administration to a human or animal may be delivered as part of a supplement, food product, beverage. In some aspects, it may be delivered via a tablet, capsule, spray (e.g., nasal spray) or suspension as described herein.
  • Antimicrobial compositions suitable for application to a surface or area may comprise a liquid, dry mixture, powder or any other vehicle suitable for administering bacterial cells or spores.
  • inhibitory compositions may comprise a combination of BS50 with one or more other probiotics or other microbes known to display antimicrobial effects.
  • a combination may include BS50 and a second non-pathogenic bacteria known to inhibit one or more pathogenic microbes (e.g., MRSA).
  • the combinations may be formulated and/or selected to provide an additive or synergistic antimicrobial effects against one or more pathogenic bacteria.
  • the pathogenic bacteria is a species selected from one of the following genera: Staphylococcus (e.g., S. aureus, MRSA S. aureus), Listeria (e.g., L. monocytogenes), Salmonella (e.g., S. heidelberg), Streptococcus (e.g., S. agalactiae) Escherichia (e.g., E.
  • Bacillus subtilis strain identified as BS50 was deposited under the terms of the Budapest Treaty on March 29, 2022 with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia 20110-2209, U.S.A., under accession number PTA- PTA-127287. [0076] The following non-limiting examples are provided to further illustrate the embodiments disclosed herein.
  • Example #1 – BS50 survival during high heat conditions [0077] To determine BS50 spore tolerance to high heat, spray dried BS50 spores were suspended in Butterfield’s Buffer and mixed with a high shear mixing (i.e., kitchen blender). Following mixing, the suspended spores were serially diluted via 10-fold dilution such that the last dilution would deliver approximately 100 colony forming units (CFU) when 100uL was spread plated evenly on the surface of tryptic soy agar (TSA). The final dilution was split into 1mL aliquots in 1.5mL microcentrifuge tubes.
  • CFU colony forming units
  • Tubes were heated to 70, 80, 90, and 100°C and held at that temperature in a dry bath for up to three hours. At each time point (5, 10, 15, 20, 30, 45, 60, 90, 120, 150, and 180 minutes), a tube was removed and spread plated on triplicate TSA plates. Plates were inverted and incubated at 35°C overnight (18-24 hours). [0078] After incubation, colonies were counted and recorded using InterScience Scan 500 colony counting software. The number of colonies per plate were multiplied by the total dilution factor to determine CFU/mL Percent survival was determined by comparing CFU/mL at each time point to the CFU/mL at time zero at room temperature (20-23°C).
  • BS50 was stable for at least 2 hours at 70 and 80°C with greater than 80% viability. Remarkably, BS50 was greater than 85% viable after 30 minutes at 90°C. This durability at 90°C is notable even for spore formers as shown by the comparison in FIG. 2 of BS50 spores and other Bacillus subtilis spores. BS50 spores have a heat tolerance profile that makes them suitable for many higher heat food and beverage processes and compositions.
  • Example #2 – BS50 production of digestive enzymes Bacillus strains are known to produce a variety of extracellular hydrolytic enzymes.
  • agar-based screens were used to determine the relative amount of enzyme activity (amylase, protease, lipase, and cellulase) produced by 36 different Bacillus subtilis strains including BS50. Enzyme specific substrates were incorporated into agar-based media. Media was sterilized, cooled, and poured into sterile empty Petri plates. Once solidified, the surface of the agar media was inoculated with 10uL of an enzyme control or a Bacillus subtilis strain colony suspension equivalent to a 0.5 McFarland standard.
  • Bacillus subtilis strains generally have the ability to produce amylase, protease, lipase, and cellulase but not all strains produce all enzymes and the level of activity also differs between strains.
  • BS50 has greater amylase (2.0 vs 1.8), protease (3.0 vs 1.9), lipase (2.0 vs 1.3), and cellulase (3.0 vs 2.4) activity than the average activity of 35 other Bacillus subtilis strains. Notably, BS50 has as much protease activity as the enzyme control.
  • Example #3 – BS50 in baked goods [0082] A series of baking experiments were conducted to evaluate how well BS50 survives the baking process. BS50 survivability was assessed by comparing the number of BS50 spores in baked muffins and cookies to the amount of BS50 added to the respective batter.
  • boxed muffin mix (ingredients including enriched bleached flour (wheat flour, malted barley flour, niacin, reduced iron, thiamin mononitrate, riboflavin, folic acid), sugar, food starch-modified, natural flavor, salt, dextrose, arabic gum, soybean oil, leavening (baking soda, sodium aluminum phosphate), tapioca starch, xanthan gum, citric acid) was prepared per the manufacturer directions with the exception of the addition of BS50. BS50 powder was added to the dry muffin mix to achieve 2 x 10 9 CFU per muffin.
  • enriched bleached flour wheat flour, malted barley flour, niacin, reduced iron, thiamin mononitrate, riboflavin, folic acid
  • sugar food starch-modified, natural flavor, salt, dextrose, arabic gum, soybean oil, leavening (baking soda, sodium aluminum phosphate), tapioca starch, xant
  • the wet ingredients consisting of 2/3 cup water, 1/2 cup vegetable oil, and 2 eggs were then hand mixed then added to the dry ingredients containing BS50 per manufacturer directions. Finally, the box mix pre-packaged blueberry fruit was incorporated into the batter. The mixed batter was weighed into foil baking cups to ensure each muffin was 40 ⁇ 1.0 grams before baking. Remaining batter was used to enumerate and confirm, in triplicate, the amount of BS50 in the batter prior to baking. [0083] The muffins were baked per the manufacturer’s recommended guidelines for baking time and temperature for baking 12 standard size muffins (18-22 minutes at 400°F (204.4°C)). After baking for 18 minutes, the muffins were allowed to cool for at least one hour.
  • each muffin was added to a blending jar containing 180 mL Butterfield’s buffer and then homogenized by high-speed blending for 2 minutes with a standard kitchen and bar blender.
  • the homogenized muffins samples were serially diluted (10- fold dilutions), plated on the surface of TSA plates using a spiral plater (EasySprial Pro, InterScience), and incubated at 35°C overnight. After incubation, colonies were counted and recorded using InterScience Scan 500 colony counting software. The number of colonies per plate were multiplied by the total dilution factor to determine CFU/g.
  • Percent survival was determined by comparing CFU/g post baking to target. Data are presented as the average of at least two trials. [0085] Based on the target dose of 2 billion CFU per muffin, 100% of BS50 spores survived the batter mixing process exemplified by the enumeration of unbaked wet batter.63% of the spores survived the baking process and remained viable in the fully baked muffins.
  • Cookie dough was weighed to achieve 23 ⁇ 0.5 g per cookie, dispensed onto baking sheet trays and baked at 350°F (176.6°C) for 12-14 minutes until golden brown. The baked cookies were allowed to cool for 15 minutes before determining post-baking weight. [0087] After baking, each cookie was added to a blending jar containing 180 mL Butterfield’s buffer and then homogenized by high-speed blending for 2 minutes with a standard kitchen and bar blender. The homogenized cookie samples were serially diluted (10- fold dilutions), plated on the surface of TSA plates using a spiral plater (EasySprial Pro, InterScience), and incubated at 35°C overnight.
  • Example #4 – Hot Beverage To evaluate BS50 stability in hot beverage matrices, BS50 spore preparation was added to store bought pekoe cut black tea teabags to achieve 2 billion CFU per teabag. Per the manufacturer instructions, each teabag was steeped in 8 oz (227 g) of water for 3 minutes. The teabags were steeped in hot water held at 90 °C, 95 °C or 100 °C. After steeping for 3 minutes, the resulting hot tea was serially diluted (10-fold dilutions), plated on the surface of TSA plates using a spiral plater (EasySprial Pro, InterScience), and incubated at 35°C overnight.
  • the resulting coffee was serially diluted (10- fold dilutions), plated on the surface of TSA plates using a spiral plater (EasySprial Pro, InterScience), and incubated at 35°C overnight. After incubation, colonies were counted and recorded using InterScience Scan 500 colony counting software. The number of colonies per plate were multiplied by the total dilution factor to determine CFU/g. Percent survival was determined by comparing CFU/mL in the coffee to the target dose. Data is presented as the average of at least two trials. [0092] More than 95% of the BS50 spores passed through the coffee grounds and coffee filter into the brewed coffee.
  • Bifidobacterium spp. Bostobacterium animalis, Bifidobacterium adolescentis, and Bifidobacterium bifidum
  • Lacticaseibacillus rhamnosus formerly Lactobacillus rhamnosus
  • Lactiplantibacillus plantarum formerly Lactobacillus plantarum
  • Lactobacillus acidophilus were individually streaked onto the surface of the agar perpendicular to the BS50 center streak without touching the center streak.
  • the perpendicular streaks were streaked starting near the BS50 center streak moving towards the edge of the plate.
  • the cross-streak plates were placed into an anaerobic chamber with anaerobic sachets (BD GasPak EZ Container Systems) and incubated at 35 ⁇ 2°C for 48 hours. The plates were inspected for signs of growth inhibition near the streak junction. The degree of inhibition of the LAB strains was measured using calipers to measure the distance (mm) between the center streak and the beginning of growth of the perpendicular.
  • B. subtilis BS50 was compatible with 5 of the 6 LAB probiotic strains tested as shown by the lack of growth inhibition (i.e., no clearing zones) in FIG.6.
  • BS50 does appear to be somewhat inhibitory towards Bifidobacterium bifidum given the 6.75 mm clearing zone between the BS50 center streak and the growth of the B. bifidum cross streak. Other similar Bacillus strains also appear to inhibit to varying degrees B. bifidum (BCM data not shown).
  • BCM data not shown BCM data not shown.
  • the BS50 compatibility with multiple LAB probiotics provides options for compositions comprising one or more additional LAB probiotics.
  • Example #6 – Cross Streak Assays to Evaluate BS50’s Potential to Inhibit the Growth of Pathogenic Bacteria [0095] To conduct this assay, isolated colonies of BS50 were suspended in sterile water to a 0.5 McFarland standard.
  • Each test organism Esscherichia coli, Streptococcus agalactiae, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella enterica Salmonella typhimurium, Salmonella heidelberg, Pasteurella multocida, Bordetella bronchiseptica, and Listeria monocytogenes
  • each inoculum was streaked perpendicular up to, but not touching, the BS50 streak.
  • Each cross- streaked test organism was streaked onto 3 separate BS50 test plates to allow for measurement of inhibition in triplicate. All cross-streaked plates were incubated at 35°C for 24 hours to allow test organisms to grow. [0097] Zones of inhibition were then measured in millimeters from the edge of growth of the center BS50 streak to the beginning of growth of the test organism using calipers. The average value from the triplicate measure was determined. The results of this assay shown in FIG.
  • Example #7 – BS50 Stability in a Gummy Preparation [0098] Pectin- and gelatin-based gummy preparations are a popular format for dietary supplements. Gummy-based vitamin, calcium and probiotic supplements are commercially available. However, some agents and in particular probiotic agents, are incompatible with gummy preparations and/or manufacturing procedures used to create such preparations.
  • gelatin-based, unflavored gummies containing 2 billion CFU per gummy of BS50 were prepared and sealed in a clear plastic bottle or Alu pouch.
  • 4 gummies (approx. 20 grams) were hydrated in 180mL of Butterfield’s buffer and heated to 60 o C for 5 minutes to aid in the dissolving process. Following the heating, gummy suspension was blended for 90 seconds using a standard kitchen blender to obtain a homogeneous gummy solution. After mixing, a 10-fold dilution series was completed and the final dilution as plated on triplicate TSA. The TSA plates were incubated inverted at 35 o C for 18-24 hours.
  • subtilis probiotics have been demonstrated to modulate several functions and characteristics of the human gastrointestinal tract, including macronutrient digestion, gut motility, inflammation, microbiota composition, short chain fatty acid profile, and pathogen profile.
  • BS50 secretes digestive enzymes and candidate gut microbiota-modulating molecules which may help support digestion, support GI health, and support immune health.
  • BS50 also showed robust heat resistance and pH tolerance, which predicts strain survival across the wide pH range of the human gut.
  • GITQ daily gastrointestinal tolerance questionnaire
  • Visit 1 At Visit 1 (Day -7), subjects arrived at the clinic in a fasting state (12 ⁇ 2 h). After subjects provided voluntary informed consent, subjects underwent medical history, prior and current medication/supplement use, inclusion and exclusion criteria assessments. Additionally, height, body weight, and vital signs were measured and BMI was calculated. Blood samples were collected for chemistry and hematology analyses. Subjects were dispensed a paper 3-day Diet Record with instructions to record all foods and beverages consumed during 3 days (1 weekend day and 2 weekdays) prior to Visit 2 (Day 0). Subjects were also dispensed a paper GITQ with instructions to rate the severity of daily GI symptoms occurring from the morning of Day -7 through the morning of Day -0, just before their Visit 2.
  • the GITQ contained a series of questions regarding the presence and severity of GI symptoms occurring during the past 24 h. Therefore, subjects reported GI symptoms for Day -7 on Day -6, for Day -6 on Day -5, for Day -5 on Day -4, etc. Individual components included the severity of GI symptoms including flatulence, abdominal distention/bloating, burping, borborygmus/stomach rumbling, abdominal cramping, reflux (heartburn), nausea, and vomiting, and these were ranked on a 4- point scale ranging from none (score 0) to severe (score 3). GITQ is hereby incorporated by reference and has been previously described in a number of well-designed, peer-reviewed, and published clinical trials (See Boler, B. M., et al.
  • Subjects were instructed to report GI symptoms daily for the 6 weeks of study product consumption using the electronic GITQ whereby the link to the questionnaire was emailed to subjects daily. Subjects were instructed to start filling in their first GITQ ⁇ 24 h after the consumption of their first product and before the consumption of the second study product. For example, if study product consumption occurred at 12 pm on Day 0 with lunch, the first GITQ should be completed the afternoon (12 pm or later) of Day 1 before Day 1’s study product consumption to enable subjects to record any events occurring from 12 pm on Day 0 to 12 pm on Day 1. The final GITQ was completed the morning of Day 42, immediately prior to Visit 3. Subjects were also dispensed a paper Study Product Log with instructions to record the date and time of each study product consumption event.
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • IL-10 interleukin-10
  • TNF- ⁇ tumor necrosis factor- alpha
  • the proportion of subjects with an improvement in each individual GI symptom was compared between groups with a chi-square test; if the cell count was ⁇ 5, a Fisher’s exact test was used.
  • the 3-item weekly composite score was analyzed with a repeated measures model where the within subject correlation was modeled with an AR(1) covariance structure.
  • the model contained fixed effect terms for product, week, product ⁇ week interaction, sex, and BMI group. Contrast/estimate statements were used to estimate the difference (95% confidence interval) between products in the change from baseline to each follow-up time point. Additionally, the within product change from baseline to each follow-up time point was estimated along with a 95% confidence interval.
  • Blood inflammation markers were compared between products with the analysis of covariance (ANCOVA) approach.
  • the change from baseline was used as the outcome and adjusted for baseline, product, sex, and BMI group.
  • the estimated product effect from an ANCOVA model for the change score is equivalent to a model of just Day 42 values.
  • model assumptions were violated and the rank transformation was used.
  • the Wilcoxon rank sum test and the stratified Wilcoxon test was also used to evaluate differences between products at Day 0, Day 42, and the change from Day 0 to Day 42.
  • Table A reports the subject characteristics at screening. Based on returned unused study product, the mean compliance was 101.0%.
  • Table B shows the proportion of subjects demonstrating an improvement in the 7-day composite score for 3 items: flatulence, bloating, and burping. Improvement was defined as having a decrease of at least 2 in composite score without any increase in scores for the individual symptoms measured the 7 days prior to Day 42 compared to the 7 days prior to Day 0.
  • a Improvement was defined as having a decrease of at least 2 in the composite score without any increase in scores for the individual symptoms measured the 7 days prior to Day 42 as compared to the 7 days prior to Day 0.
  • bDoes not adjust for sex and body mass index groups This is defined as the primary analysis in the Statistical Analysis Plan.
  • c The proportion of subjects that had an improvement in the 7-day 3-item total composite score were compared between groups with the chi-square test.
  • d The odds of observing an improvement in symptoms were modeled with logistic regression and adjusted for sex and body mass index group.
  • n sample size
  • OR odds ratio
  • CI confidence interval
  • aImprovement was defined as showing flatulence score decrease of at least 1 and no improvement was defined as no change in score or showing a score increase (indicating worsening of symptom) prior to Day 42 as compared to the 7 days prior to Day 0.
  • the per protocol population comprised a reduced number of subjects due to removal of 2 subjects that deviated from one or more aspects of the study protocol (i.e., end of study visit out of window).

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Abstract

L'invention concerne une espèce de Bacillus formant des spores, et plus particulièrement, une souche de Bacillus subtilis identifiée sous l'appellation BS50. L'invention concerne également des compositions comprenant la souche BS50, des procédés de fabrication de produits les comprenant, et des procédés d'utilisation de celles-ci.
PCT/US2022/040239 2022-04-07 2022-08-12 <i />COMPOSITIONS DE BACILLUS ET LEURS UTILISATIONS Ceased WO2023196003A1 (fr)

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WO2025090080A1 (fr) * 2023-10-25 2025-05-01 KBS Research, LLC Compléments à base de plantes et leurs procédés d'utilisation et de fabrication
WO2025093415A1 (fr) 2023-10-30 2025-05-08 Novozymes A/S Composition probiotique à base de spores acides
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Publication number Priority date Publication date Assignee Title
WO2025090080A1 (fr) * 2023-10-25 2025-05-01 KBS Research, LLC Compléments à base de plantes et leurs procédés d'utilisation et de fabrication
WO2025093415A1 (fr) 2023-10-30 2025-05-08 Novozymes A/S Composition probiotique à base de spores acides
EP4559470A1 (fr) * 2023-11-22 2025-05-28 State Research Institute Centre for Innovative Medicine Utilisation de lysat de bacillus subtilis pour l'immunomodulation des cellules du système respiratoire
CN118649223A (zh) * 2024-08-20 2024-09-17 北京一品堂医药科技有限公司 一种调节胃肠道功能提高免疫的枯草芽孢杆菌de111制剂及其制备方法
CN118649223B (zh) * 2024-08-20 2024-10-25 北京一品堂医药科技有限公司 一种调节胃肠道功能提高免疫的枯草芽孢杆菌de111制剂及其制备方法

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