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WO2021081362A1 - Compositions symbiotiques pour microbiote intestinal - Google Patents

Compositions symbiotiques pour microbiote intestinal Download PDF

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Publication number
WO2021081362A1
WO2021081362A1 PCT/US2020/057111 US2020057111W WO2021081362A1 WO 2021081362 A1 WO2021081362 A1 WO 2021081362A1 US 2020057111 W US2020057111 W US 2020057111W WO 2021081362 A1 WO2021081362 A1 WO 2021081362A1
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Prior art keywords
juice
synbiotic composition
synbiotic
probiotic
composition according
Prior art date
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English (en)
Inventor
Howard F. ROBINS
A. Reza Kamarei
Michael W. KOSCHITZ
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Newgen 27 LLC
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Newgen 27 LLC
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    • 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
    • A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof containing fruit or vegetable juices
    • 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
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • 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
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • 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
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Definitions

  • the present disclosure relates to synbiotic compositions comprising probiotic bacterial strains with a juice-based medium that when combined exhibit synergistic behavior.
  • the synergistic compositions may ameliorate, repair and/or rehabilitate the gut microbiome to improve a user’s immune response before and/or after experiencing gut infections such as those caused after antibiotic associated diarrhea, Clostridium difficile
  • the microbiome is defined as a “community of microbes.”
  • the human microbiota consists of the 10-100 trillion microbial cells harbored by each person, primarily bacteria in the gut.
  • a person’s good gut bacteria is also responsible for producing vitamin B12, butyrate and vitamin K2, crowding out bad microbes, creating enzymes that destroy harmful bacteria, and stimulating secretion of IgA and regulatory T cells, which support immune function.
  • a person’s diet plays a big part in establishing gut health and supporting the microbiome’s good bacteria.
  • a person’s gut health can impact how the body extracts and uses nutrients from a person’s diet.
  • people had plenty of probiotics in their diets from eating fresh foods from good soil and by fermenting foods to keep them from spoiling.
  • probiotics means live microorganisms having a beneficial effect on the health of hosts such as humans, animals, or the like, or a component thereof, and it has been known that the probiotics provide a beneficial effect, for example, maintenance of a balance in intestinal microflora, to the host taking the probiotics.
  • probiotics in our gut have been proven effective in supporting immune function, reducing inflammation, promoting healthy digestion, as well as maintaining beautiful skin, especially when combined with prebiotics.
  • Today because of refrigeration and agricultural practices like soaking our foods with chlorine, much of our food contains little to no probiotics as a result of sanitation.
  • IBS inflammatory bowel disease
  • IBD inflammatory bowel disease
  • Crohn's Disease ulcerative colitis
  • nosocomial infection colonic polyps
  • constipation constipation and other digestive irregularities
  • food allergies diarrhea
  • certain cancers such as colorectal cancer, as well as even effecting a person’s mood and mental wellbeing.
  • Probiotic supplements are a very important part of a person’s dietary supplement regimen to improve overall health. More importantly, the role of any medicinal or immunotherapeutic intervention must be to counter-balance the loss of optimum gut function. Many of the current medicinal approaches are imperfect for improving gastrointestinal health and are even harmful to the gut microbiome.
  • Clostridium Difficile Infection causes life-threatening diarrhea which is usually a side-effect of taking antibiotics.
  • Clostridium difficile (“C. difficile ” or “C. diff’) is a Gram-positive, spore-forming, anaerobic, motile bacterium which produces two types of toxins, enterotoxin A and cytotoxin B.
  • C. diff can easily spread from person to person and is considered a major health threat.
  • the present disclosure provides a synbiotic composition containing a plurality of strains of probiotic bacteria and methods of making the composition.
  • the synbiotic composition comprises a beverage medium which may include fruit and vegetable juices and optionally herbs that provides a synergistic benefit to the host or user.
  • the present disclosure is directed to the probiotic bacterial strains of the genus Bifidobacterium, in embodiments the species Bifidobacterium breve,
  • Lactobacillus salivarius Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus brevis, and mixtures thereof.
  • the optimal synbiotic composition of the present disclosure includes five (5) strains of Bifidobacteria probiotic bacteria and ten (10) strains of Lactobacilli probiotic bacteria. In embodiments, the five strains of the genus
  • Bifidobacteria probiotic bacteria may include Bifidobacterium lactis BI-07,
  • the ten strains of the genus Lactobacilli may include Lactobacillus plantarum LP-115,
  • Lactobacillus paracasei LPC-37 Lactobacillus casei LC-11, Lactobacillus salivarius LS-
  • Lactobacillus rhamnosus LR-32 Lactobacillus acidophilus LA-14, Lactobacillus brevis LBR-35, Lactobacillus gasseri LG-36, Lactobacillus reuteri 1 El, Lactobacillus bulgaricus LB-87, and mixtures thereof.
  • the composition may also include probiotic strains of the genus Bifidobacterium such as B. adolescentis, B. thermophilum and others known in the art now and any species found in the future.
  • the composition may also include probiotic strains of the genus Lactobacillus such as Lactobacilli johnsonii,
  • Lactobacilli lactis and others known in the art now and any species found in the future.
  • the synbiotic composition of the present disclosure may include
  • the liquid medium or carrier may include the sterilized juices of at least one fruit, vegetable and/or herb or spice.
  • the liquid medium or carrier may comprise at least one of the following: mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, and lemon juice, all of the juices being organic without pesticides, chemical or additives.
  • the optimum liquid carrier may include a blend of hundred (100) percent organic green vegetable and fruit juices comprising mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, lemon juice, and mixtures thereof.
  • the synbiotic composition of the present disclosure comprises from 1 to about 100 billion Colony Forming Units (CFUs) of pre-hydrated, actively living probiotics in a 2-ounce bottle of a juice beverage at the time of manufacture.
  • composition of the present disclosure may replenish the human gut microbiome with special bacteria referred to as “Smart bacteria” (SB). It is believed that these “Smart bacteria” may change the genetic composition of the gut to create a healthier environment by attaching to the wall of the gut where they will live and thrive helping to repair damage to it caused by unhealthy biomes that have been ingested and caused to overgrow in numbers such as Candida albicans (yeast) and harmful bacteria.
  • SB Special bacteria
  • the present synbiotic composition may inhibit the growth of undesirable bacteria and pathogens and/ or help ameliorate serious medical problems caused by the use of antibiotics, i.e., Clostridium difficile infection (CDI), a worldwide medical problem.
  • CDI Clostridium difficile infection
  • the specific choice of probiotics in the synbiotic compositions of the present disclosure release bioactive compounds into the specific liquid medium or carrier disclosed herein that may inhibit growth of C. diff.
  • a bioactive compound released in the medium may be lactic acid which may reduce the pH of the gut environment to an acidic range unfavorable for spore germination and/or growth of C. diff, playing a decisive inhibitory role against growth of C. diff and potentially other possible diseases which can arise from an unhealthy or unbalanced microbiome.
  • FIG. 1 illustrates an enlarged image of the Gram-positive, spore-forming, anaerobic, motile bacterium Clostridium difficile (“C. diff”) cells under a microscope
  • FIG. 2 illustrates the synbiotic composition pre-centrifugation during a study as described in the Examples
  • FIG. 3 illustrates the synbiotic composition shown in FIG. 2 postcentrifugation/microfiltration during a study as described in the Examples;
  • FIG. 4 illustrates the presence of C. diff. colony forming units during a study as described in the Examples.
  • FIG. 5 illustrates the absence of C. diff. colony forming units during a study as described in the Examples when synbiotic composition’s supernatant is used for serial dilution.
  • probiotics live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” This means that if the microorganisms are not “live”, they cannot be considered a probiotic and if their amount is not adequate enough to confer a health benefit, they cannot be considered a probiotic either.
  • NTP Next Generation Probiotics
  • SB Smart Bacteria
  • HGT Horizontal Gene Transfer
  • NGP SB’s were configured to survive the stomach acids and digestive enzyme. Also, to ensure that they are “living” and fostering at the time of ingestion, an appropriate environment and media, would be required meeting all the above-mentioned needs including the range of acidity needed for survival and growth, such as the sterilized liquid medium presently disclosed herein.
  • the NGP SB’s that were specifically chosen in the present disclosure were required to be compatible with each other so as not to alter or interfere with each other’s growth and benefits.
  • the SB or probiotics of the present disclosure also needed to be in a sufficient number of Colony Forming Units (CFU) to repopulate the colon and attack or prevent CDI or any other gut initiated disease effectively, or alternatively maintain optimal gut health in those without a gut-related disease.
  • CFU Colony Forming Units
  • the term “synbiotics” is defined as a mixture comprising live microorganisms and substrates selectively utilized by host microorganisms that confers a health benefit on the host.
  • ‘host’ microorganisms comprise both autochthonous
  • synbiotics resident or colonizing the host
  • allochthonous microorganisms either of which can be targets for a user of the substrate contained in the synbiotic.
  • Two subsets of synbiotics may be defined as complementary and synergistic.
  • a ‘synergistic synbiotic’ is a synbiotic in which the substrate is designed to be selectively utilized by the co-administered microorganism(s).
  • a ‘complementary synbiotic’ is a synbiotic composed of a probiotic combined with a prebiotic, which is designed to target autochthonous microorganisms.
  • the specifically chosen probiotics i.e., the NGP SB
  • the sterilized liquid medium or carrier of the NGP SB probiotics presently disclosed form a synbiotic relationship with a synergistic benefit on the user.
  • Bacteria of the genus Bifidobacterium are known to promote a healthy microbiome. Bifidobacteria may lead to increased levels of lactic and acetic acids, which decreases the pH in the digestive tract, inhibiting the growth of harmful bacteria such as
  • Clostridium perfringens Clostridium difficile and certain pathogenic Escherichia coli.
  • Bacteria of the Lactobacillus genus are also known to promote a healthy microbiome and help prevent or alleviate many disorders and diseases including antibiotic-induced imbalances in gastrointestinal microflora, hypercholesterolemia, vaginal infections, E. coli infection and depressed inununity.
  • the synbiotic composition of the present disclosure may include probiotic bacterial strains of Bifidobacterium, in embodiments the species B. breve, B. animalis
  • the synbiotic composition of the present disclosure may include Bifidobacterium lactis BI-07, Bifidobacterium breve BB-03, Bifidobacterium bifidum BB-06, Bifidobacterium longum BL-05, Bifidobacterium infantis BI-26, and mixtures thereof.
  • Probiotic bacteria of the genus Lactobacillus may also be used, in embodiments the species L. acidophilus, L. casei, L. rhamnosus, L, paracasei, L.
  • the synbiotic composition of the present disclosure may include Lactobacillus plantarum LP-115, Lactobacillus paracasei LPC-
  • the composition may comprise bacteria from multiple species.
  • Suitable strains are available commercially by Dupont Nutrition and Biosciences.
  • the synbiotic composition of the present disclosure may include five strains of
  • Bifidobacterium and ten strains of Lactobacillus in embodiments, Bifidobacterium lactis
  • Lactobacillus plantarum LP-115 Lactobacillus paracasei LPC-37, Lactobacillus casei LC-11, Lactobacillus salivarius LS-33, Lactobacillus rhamnosus LR-32, Lactobacillus acidophilus LA-14, Lactobacillus brevis LBR-35, Lactobacillus gasseri LC-36,
  • Lactobacillus reuteri 1 El Lactobacillus bulgaricus LB-87, and mixtures thereof
  • the synbiotic compositions of the present disclosure may include the 15 strains in an optimum carrier such as an excipient or sterilized liquid medium forming a drinkable beverage with a desirable shelf-life and while maintaining bacteria viability without refrigeration or cooling of the beverage.
  • an optimum carrier such as an excipient or sterilized liquid medium forming a drinkable beverage with a desirable shelf-life and while maintaining bacteria viability without refrigeration or cooling of the beverage.
  • the sterilized liquid medium or carrier may include at least one of or a portion of a plant selected from fruits, vegetables, herbs, spices, seeds, other suitable plant substances, or a mixture thereof More specifically, the selected portion of a plant may be in a solid or in a liquid form (e.g. a vegetable juice or a fruit juice) or a combination thereof.
  • Examples of the plant in fruit form are apple, orange, lemon, lime, banana, watermelon, papaya, mango, pineapple, blueberry, raspberry, strawberry, cherry, peach, cucumber, grape, coconut and any other fruits or juices or extracts produced therefrom, some which may be already known in the art.
  • Examples of the plant in vegetable form may include kale, spinach, broccoli, asparagus, cauliflower, lettuce, mushroom, celery and any other vegetables or juices or extracts produced therefrom, some which may be already known in the art.
  • Examples of the plant in herb form may include basil, mint, oregano, ginger, black pepper, parsley, garlic, onion, fennel, celery seed, bay leaf, lemon grass, rosemary, sage, chives and any other herbs or juices or extracts produced therefrom, some which may be already known in the art.
  • Examples of the plant in spice form may include cardamom, cinnamon, allspice, cloves, nutmeg, pepper, turmeric, ginger, mace, saffron, vanilla, cumin, dill seed and other like fleshy spice products. It is worth mentioning that in embodiments, seeds may include fruits, roots, barks, or other plant substances primarily used for flavoring, coloring or preserving food
  • the sterilized liquid medium or carrier may comprise at least one of the following: mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, and lemon juice, all of the juices being organic without pesticides, chemical or additives.
  • the optimum sterilized liquid carrier may include a blend of hundred (100) percent organic green vegetable and fruit juices comprising mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, and lemon juice.
  • the sterilized beverage or liquid medium may alternatively be a tea-based beverage with natural sweeteners added to the beverage such as organic cane sugar, honey, stevia, monk fruit, erythritol, or any other natural sweetener known in the art.
  • natural sweeteners added to the beverage such as organic cane sugar, honey, stevia, monk fruit, erythritol, or any other natural sweetener known in the art.
  • Viable bacteria which may be included in the beverage, generally are presented in numbers which are often reported as CFU, or colony forming units.
  • CFU colony forming units.
  • One colony is formed by a single viable bacterium when the bacteria are plated at a suitable dilution for single colony formation. This is a standard technique known to microbiologists.
  • the amount is expressed as the number of CFU in a liquid measure such as milliliters (ml) or fluid ounces (fl. oz).
  • ml milliliters
  • fl. oz fluid ounces
  • U.S. regulation 21 CFR 101.9(b)(5)(viii) defines a fluid ounce as exactly 30 ml.
  • Sufficient numbers of viable bacteria may be necessary to obtain the beneficial effects of the probiotic bacteria. Often bacteria are packaged at a certain level of viable bacteria; however, before consumption, the levels may decrease preventing the consumer from acquiring a beneficial dose of bacteria. Indeed, the
  • NCCAM National Center for Complementary and Alterative Medicine
  • the synbiotic composition of the present disclosure comprises from 1 to about 100 billion CFUs of pre-hydrated, actively living probiotics in a 2-ounce bottle of a sterilized beverage at the time of manufacture. In selected embodiments, the synbiotic composition comprises from about 20 to about 30 billion CFUs, in embodiments, about 27 Billion CFUs of pre-hydrated, actively living probiotics in a 2- ounce bottle of a beverage at the time of manufacture. It is contemplated that as the product ages the bacteria may continue to grow and form additional colonies providing the ambient temperature and shelf life is not exceeded.
  • synbiotic composition of the present disclosure may comprise prebiotics, such compositions also known as a complementary synbiotic.
  • ‘complementary synbiotic’ is a synbiotic composed of a probiotic combined with a prebiotic, which is designed to target autochthonous microorganisms. Minimum criteria for the existing probiotic and prebiotic must be met for both components of a complementary synbiotic.
  • Prebiotics are substances that may not be digestible for some humans. More specifically, some human’s gastrointestinal tract cannot process or digest prebiotics.
  • fructans such as inulin and fructooligosaccharides (FOS); galacto-oligosaccharides (GOS) and lactulose.
  • Fructooligosaccharides are not hydrolyzed in the small intestine and instead pass through into the large intestine where they preferentially support the growth of probiotic strains of
  • fructooligosaccharides do not support the growth of undesirable bacteria such as species of bacteroides, Clostridia, and fusobacteria .
  • the synbiotic composition or beverage may include selected types of probiotics (optionally used in products for human consumption) which may include inulin, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), lactulose and arabinoxylan plant fiber extract.
  • probiotics may include inulin, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), lactulose and arabinoxylan plant fiber extract.
  • FOS fructooligosaccharides
  • GOS galacto-oligosaccharides
  • lactulose arabinoxylan plant fiber extract
  • Inulin is a naturally occurring soluble fiber composed of a mixture of oligomers of varying degrees of polymerizations. Inulins are mainly comprised of fructose units and typically have a terminal glucose. Plant inulins generally contain between 2 to 140 fructose units. Inulin can be obtained from a variety of sources including Jerusalem artichoke, dahlia, onion, garlic and chicory tubers. Lactulose is a synthetic sugar, which does not occur naturally. The disaccharide lactulose (galacto-fructose) is synthesized from lactose (galacto-glucose) by isomerization of glucose to fructose.
  • Galacto- oligosaccharides can also be synthesized from lactose; for example, by using ⁇ - galactosidase enzymes purified from Lactobacillus reuteri L103 as a catalyst.
  • Fructooligosaccharides may be prepared by any of several methods known in the art. For example, fructooligosaccharides can be extracted from natural substances.
  • Fructooligosaccharides occur in many kinds of plants including dahlias, chicory, onions, garlic, shallots, wheat rye, artichokes and tomatoes. Fructooligosaccharides may also be produced enzymatically through chemical techniques. Fructooligosaccharides are particularly well-known for use in promoting the growth of Bifidobacterium species.
  • Fructooligosaccharides are typically linear chains of fructose bound to a terminal glucose.
  • the fructooligosaccharides can be a mixture of short chain polymers.
  • the length of the fructose chain also called the degree of polymerization or DP, can be from about 2 to about 5.
  • the fructose chain length varies from 2 to 4.
  • Such short-chain fructooligosaccharides may also be referred to as GF2 (1-kestose), GF 3 (nystose), and
  • GF4 ⁇ - ⁇ -fructofuranosyl nystose
  • Suitable commercially available fructooligosaccharides may be used, for example, Nutraflora® by GTC Nutrition
  • the synbiotic composition may also include any vitamin or mineral known in the art.
  • vitamins A, B, C, D, and/or E may be included in specified amounts to enhance the benefits of the composition.
  • the synbiotic composition may also include an active ingredient such as polyunsaturated fatty acids, omega-3 fatty acids, omega-6 fatty acids, omega-9 fatty acids, conjugated fatty acids, coenzyme Q-containing active ingredients, oil soluble vitamins other than a vitamin E, alpha lipoic acid, medium chain triglycerides, carotenoid-containing active ingredients and phytochemicals and cannabinoids.
  • an active ingredient such as polyunsaturated fatty acids, omega-3 fatty acids, omega-6 fatty acids, omega-9 fatty acids, conjugated fatty acids, coenzyme Q-containing active ingredients, oil soluble vitamins other than a vitamin E, alpha lipoic acid, medium chain triglycerides, carotenoid-containing active ingredients and phytochemicals and cannabinoids.
  • the preselected bacteria which may be included in the synbiotic composition may be prepared in a variety of ways known in the art, including, for example, the bacteria may be grown without casein, providing a completely dairy-free bacterial preparation.
  • the selected bacteria may be stored via known methods of storage or methods specifically designed to maintain cultures alive such as refrigeration, freezing, or freeze-drying without diminishing viability below a selected level (the selected level may be a level selected from temperate, volume, time, or freshness). Still during manufacturing, the selected bacteria may be added to the beverage while in the same state as they were stored, such as while frozen, freeze-dried, or refrigerated. Optionally, the selected bacteria may be thawed prior to adding to the beverage. The selected bacteria may be frozen after growth and maintained in a frozen state until they are added to the beverage.
  • the resulting beverage comprising the synbiotic composition of the present disclosure may be a shelf stable beverage.
  • the shelf stable beverage may be kept or have an expected lifetime of about three months at room temperature (about 68- 72 degrees Fahrenheit), and/or about four months at refrigerated temperature (about 35- 40 degrees Fahrenheit).
  • the synbiotic composition of the present disclosure may compete with and exclude all harmful bacteria and yeasts e.g., Clostridium difficile and Candida albicans. Further, the synbiotic composition may aid in bringing a user’s gut- environment to optimal balance. Moreover, the synbiotic composition may help to reestablish a healthy gut and normalize digestion and absorption of nutrients. [0052] Further aspects of the invention relate to methods of manufacturing and storage associated with the synbiotic composition while maintaining a high number of viable bacteria. In an exemplary embodiment, to manufacture the synbiotic composition, in a sanitary environment, bacteria, plant, and /or synbiotic products (such as the ones noted above) are combined to form the beverage.
  • bacteria, plant, and /or synbiotic products such as the ones noted above
  • At least one strain from the genus Bifidobacterium and at least one strain from the genus Lactobacillus which may be frozen, freeze-dried, or refrigerated in powder form are mixed together to uniformity and then combined with a sterilized liquid medium or carrier, i.e., the organic fruit/vegetable juice mixture described herein.
  • the synbiotic composition of the present disclosure may include five strains of Bifidobacterium and ten strains of Lactobacillus, in embodiments, Bifidobacterium lactis BI-07, Bifidobacterium breve BB-03,
  • Lactobacillus paracasei LPC-37 Lactobacillus casei LC-11, Lactobacillus salivarius LS-
  • the preselected 15 strains are combined and blended for a selected time, of from about 20 minutes to about 40 minutes, and further stored in a selected environment, i.e., an environment including temperature control means. In embodiments, the preselected 15 strains are blended for about 30 minutes and placed in a refrigerator at a temperature of about 35 degrees to about 40 degrees Celsius.
  • the probiotic blend may include a specific concentration or strain of bacteria. More specifically, the specific concentration of each strain of probiotic may be from about 0.5% to about 15%, in embodiments from about
  • the synbiotic composition may include less than 5 grams of the preselected probiotic blend (which can be a mixture of probiotics in powder forms) per liter of sterilized juice. In selected embodiments, the synbiotic composition may include about 2 grams to about 3 grams of a combination or mixture per liter of sterilized juice.
  • the liquid medium or carrier may include any of the above noted plants. In selected embodiments, the synbiotic composition may include organic fruit and juice blend of the present disclosure. In embodiments, the liquid medium or carrier may comprise at least one of the following: mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, and lemon juice, all of the juices being organic without pesticides, chemicals or additives.
  • the optimum liquid carrier may include a blend of hundred (100) percent sterilized organic green vegetable and fruit juices comprising mint juice, cucumber juice, apple juice, lettuce juice, kale juice, celery juice, and lemon juice which may be mixed with a blend of preselected probiotics for about 20 minutes to about 30 minutes until reaching a uniform texture for the resulting beverage.
  • the synbiotic composition may include a stock of the preselected probiotic blend already mixed together and the preselected sterilized organic fruit/juice blend.
  • the stock of mixed probiotic blend and the sterilized organic juice may be stored in a refrigerated environment, and at some later time, additional organic juice may be added in a kettle, with the stock mixture and blended for an additional 20 minutes to about 30 minutes or until the stock reaches a uniform consistency.
  • the synbiotic composition may include about 20 billion CFUs to about 30 billion CFUs.
  • the synbiotic composition may include 27 Billion CFUs of pre-hydrated, and actively living probiotics in about 2-ounces of the composition or beverage at the time of manufacture.
  • the resulting beverage of the present disclosure has a substantially pleasant taste. More specifically, the resulting beverage may be slightly sweet, the sweetness generally coming from the fruit juices masking the less desirable taste of the vegetable juices.
  • compositions of the present disclosure may be effective in supporting immune function, reducing inflammation, promoting healthy digestion, as well as maintaining beautiful skin, and promoting an overall healthier microbiome.
  • compositions of the present disclosure may replenish the human gut microbiome with special bacteria referred to as “Smart bacteria” (SB). It is believed that these “Smart bacteria” may change the genetic composition of the gut to create a healthier environment by attaching to the wall of the gut where they will live and thrive helping to repair damage to it caused by unhealthy biomes that have been ingested and caused to overgrow in numbers such as Candida albicans (yeast) and harmful bacteria.
  • SB Special bacteria
  • these “Smart bacteria” may change the genetic composition of the gut to create a healthier environment by attaching to the wall of the gut where they will live and thrive helping to repair damage to it caused by unhealthy biomes that have been ingested and caused to overgrow in numbers such as Candida albicans (yeast) and harmful bacteria.
  • the present synbiotic composition may inhibit the growth of undesirable bacteria and pathogens and/ or help ameliorate serious medical problems caused by the use of antibiotics, i.e., Clostridium difficile infection (CDI), a worldwide medical problem.
  • CDI Clos
  • the specific choice of probiotics in the synbiotic compositions of the present disclosure release bioactive compounds into the specific liquid medium or carrier disclosed herein that inhibits growth of C. diff.
  • a bioactive compound released in the medium may be lactic acid which may reduce the pH of the gut environment to an acidic range unfavorable for spore germination and/or growth of C. diff, playing a decisive inhibitory role against growth C. diff and potentially other possible diseases which can arise from an unhealthy or unbalanced microbiome.
  • the presence of lactic acid in the synbiotic composition of the present disclosure and upon addition of the preselected probiotic strains to the liquid medium of the present disclosure, i.e., the fruit and vegetable juice blend, can potentially reduce the pH of the environment to an acidic range unfavorable for spore germination and/or growth of pathogens such as C. difficile.
  • the preselected probiotic strains i.e., the fruit and vegetable juice blend
  • the presence of lactic acid in the synbiotic composition of the present disclosure can potentially reduce the pH of the environment to an acidic range unfavorable for spore germination and/or growth of pathogens such as C. difficile.
  • pathogens such as C. difficile
  • probiotics in capsules and tablets are not actively living.
  • Probiotics in capsules and tablets are generally noted “alive” at the time of manufacture however these probiotics are in a so-called state of “suspended animation”, hoping to come back to life when hydrated in the highly acidic environment of the stomach.
  • the following Examples are noted to illustrate exemplary embodiments associated with the present disclosure. Some or all the examples are intended to be illustrative only and are not intended to limit the scope of the present disclosure.
  • quantities, ratios, percentages, and/or values are generally a weight measurement unless otherwise indicated.
  • the synbiotic composition of the present disclosure may include a preselected powder probiotic blend and a sterilized organic juice product which can be prepared as follows.
  • a user checks if a certified HEPA filter or an equivalent thereof is installed and functioning in the room where the composition is to be prepared.
  • a working surface is required to aid in the preparation of the composition that is disinfected and clean such as a bench which may have been treated with ethanolic paper.
  • a mixer is required such as a KitchenAid 5-Quart (4.7) liter tilt-head stand mixer or the like with at least 275 horsepower.
  • the mixer may include additional accessories such as a stainless-steel bowl, 6-wire whip and plastic lid, opening devices, scissors and spoon which are completely cleaned and disinfected.
  • probiotics are generally stored in a closed, sealed container free of contaminants.
  • the user opened the probiotic container and extracted a selected amount of the pre-selected freeze-dried probiotic in powder form.
  • the probiotics used may be in liquid form.
  • the stirring option of the mixer was turned on and the rate was gradually increased. Occasionally, the mixer was stopped and the blend was mixed with a long spoon. The mixture was stirred in the bowl with the mixer for about 30 min or until a desired uniform mixture was formed.
  • the resultant probiotic blend was transferred into a clean moisture-proof container.
  • the container was capped tightly, labeled and dated.
  • the probiotic blend was kept in the refrigerator at approximately 4 degrees Celsius until ready to mix with a sterilized liquid medium or carrier.
  • the organic fruit/vegetable juice comprised the ingredients listed in Table 2:
  • Each two (2) fluid ounce serving of the resulting beverage composition provides about 27 billion CFUs at the time of manufacturing. Every batch produced is tested by an independent, FDA-registered accredited microbiology lab for safety and to confirm the absence of pathogens.
  • Clostridial agar slant, broth tubes, and two sheep blood agar plates Clostridial agar slant, broth tubes, and two sheep blood agar plates.
  • the slant and broth tubes and one sheep blood agar plate were incubated anaerobically at 36-38°C for 48 ⁇ 2 hours.
  • the second sheep blood agar plate was incubated aerobically at 36-38°C for 48 ⁇ 2 hours and served as the contamination check. Following incubation, the agar plates and tube were observed for colony growth and turbidity.
  • the sheep blood agar plate incubated anaerobically was biochemically identified using the VITEK ANC card, i.e, a test card kit for identifying anaerobic bacteria, to verify purity.
  • Inoculum Preparation A well isolated colony from the sheep blood agar plate was used to inoculate a reinforced Clostridial broth, which was then incubated anaerobically at 36-38°C for 18 to 20 hours. Following incubation, the culture was stored overnight at 3 ⁇ 1 °C.
  • Biosciences was prepared for the synbiotic composition.
  • Six, individually packaged, intact 2-ounce glasses with the synbiotic composition of the present disclosure were composited into a sterile 500 mL glass bottle under a laminar flow hood. This bottle was mixed by gentle swirling and stored at ambient temperature (20-25°C) until testing. At that time, the composited product was homogenized. Then 10 mL was pipetted into a test tube for a before (FIG. 2) and after comparison photo with the centrifuged product.
  • the supernatant liquid was filtered through a 0.22 pm filter to ensure that the supernatant liquid was free of cells. A 10 mL portion was pipetted into a test tube and photographed (FIG. 3). The filtered sample was used as the test product sample and the 9 mL diluent tubes for its ten-fold serial dilution.
  • the inoculated control was analyzed with sterile 0.1% peptone buffer.
  • Test Procedure The test procedure was performed according to the
  • Control Test Procedure The inoculum used for the sample test procedure was vortexed for 5 seconds. 1 mL of the inoculum was transferred to a 9 mL tube of 0.1% peptone buffer and vortexed for 5 seconds. This served as the 10 -1 dilution tube. 1 mL from the 10 -1 dilution tube was transferred in duplicate into sterile petri dishes. With the same pipet, 1 mL was transferred into a second 9 mL tube containing the 0.1% peptone buffer for the 10 -2 dilution tube and vortexed for 5 seconds. 1 mL from the 10 -2 dilution tube was transferred in duplicate into sterile petri dishes.
  • Clostridial agar was added to the petri dishes and gently swirled to evenly disperse inoculum throughout the agar. After solidification, 15 mL of reinforced Clostridial agar was overlaid onto each plate. The plates were incubated anaerobically in an upright position at 35-37°C for 18-24 hours.
  • Bifidobacteria and Lactobacilli have functioned in a complementary, additive and possibly synergistic way to completely inhibit growth of C. difficile.
  • the chosen probiotics have released some bioactive compounds into the sterilized organic liquid medium or fruit and vegetable juice that has inhibited growth of C. difficile.
  • One such compound could possibly be lactic acid which can reduce the pH of the environment to an acidic range unfavorable for spore germination and/or growth of C. difficile.
  • the bioactive compounds secreted from the chosen 15 probiotic strains into the sterilized organic vegetable and fruit juice has played a decisive inhibitory role against growth of C. difficile.

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Abstract

La présente invention concerne des compositions symbiotiques, comprenant une pluralité de souches présélectionnées de bactéries probiotiques associées à une substance liquide à base de jus de fruits et de légumes organiques stérilisée qui peut être utilisées pour la santé du côlon et améliorer la fonction immunitaire globale et la santé intestinale. L'invention concerne également des procédés pour préparer le mélange probiotique et la substance liquide à base de jus pour former la composition symbiotique. Grâce à ces procédés, il est possible d'obtenir une durée de conservation souhaitable tout en maintenant la viabilité bactérienne.
PCT/US2020/057111 2019-10-25 2020-10-23 Compositions symbiotiques pour microbiote intestinal Ceased WO2021081362A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115119915A (zh) * 2022-06-27 2022-09-30 南方医科大学南方医院 一种改善炎症性肠病炎症程度的发酵果蔬汁及其复合发酵菌
CN115119915B (zh) * 2022-06-27 2023-04-25 南方医科大学南方医院 一种发酵果蔬汁
WO2024107835A3 (fr) * 2022-11-15 2024-06-20 Newgen 27, Llc D/B/A Doctor's Biome Formulations probiotiques et leurs procédés d'utilisation

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