Improvement of insoluble fiber fermentability and short-chain fatty acid production by Bifidobacterium longum

Abstract

The present invention relates to a Bifidobacterium longum probiotic for use in improving the fermentability of plant fiber in the gastrointestinal tract of a subject, the plant fiber having an insoluble fraction of 40-80% (w / w).

Description

[Technical Field]

[0001] Insoluble complex dietary fibers are poorly fermentable in the human intestine because their structure makes them very difficult to utilize by the gut microbiota, which greatly reduces their beneficial physiological effects.

[0002] Soluble indigestible oligosaccharides, which are commonly used to increase the dietary fiber content in food products, can cause gastrointestinal discomfort due to their rapid fermentation rate in the intestine. Furthermore, they are not natural products and therefore are not readily acceptable to consumers.

[0003] There is a clear need to improve the fermentability of complex dietary fibers in the human intestine to increase their beneficial physiological effects without causing associated gastrointestinal discomfort.

[0004] [Summary of the Invention] Combining complex dietary fiber derived from peas with probiotics, particularly Bifidobacterium longum NCC3001 (ATCC BAA-999, hereafter referred to as B. longum NCC3001), resulted in a surprising improvement in the fermentability of the fiber. When combined with probiotics, commensal microorganisms present in the gut ferment the fiber, resulting in increased production of short-chain fatty acids such as acetate and propionate, exerting beneficial effects on the host. These effects include immune and appetite regulation, as well as protection against colon cancer. Furthermore, specific fermentation results indicate that this fiber and probiotic combination unexpectedly does not cause intestinal discomfort due to excessive gas production.

[0005] In a first aspect, the present invention provides the use of Bifidobacterium probiotics to improve the fermentability of plant fibre, wherein the plant fibre has an insoluble fraction of 40 to 80% (w / w).

[0006] In a second aspect, the present invention provides a composition comprising an effective amount of fiber from a plant of the Leguminoseae family and a Bifidobacterium probiotic, wherein the plant fiber has an insoluble fraction of 40-80% (w / w).

[0007] In a third aspect, the present invention provides a composition comprising an effective amount of legume fiber and Bifidobacterium probiotic, wherein the plant fiber has an insoluble fraction of 40-80% (w / w), and the Bifidobacterium is obtained by a process comprising: a. fermenting the Bifidobacterium in a bacterial growth medium; and b. recovering the cultivated Bifidobacterium probiotic.

[0008] In a fourth aspect, the present invention provides a composition as described herein for use as a medical food. [Brief explanation of the drawings]

[0009] [Figure 1] Schematic of a calibrated SHIME® system with a reactor that continuously simulates gastric and small intestinal conditions under both fed and fasted conditions. [Figure 2] pH profile of incubation mimicking conditions in the upper GIT. Samples were taken at the beginning of gastric incubation (ST0), the end of gastric incubation (ST2), the beginning of small intestinal incubation (SI0), the beginning of the absorption phase of small intestinal incubation (SI0.4), and the end of small intestinal incubation (SI4). [Figure 3] Mean changes in concentrations (mM) of (A) acetate, (B) propionate, (C) butyrate, and (D) total SCFAs during 48 h of colonic incubation. (*), (**), (***), and (****) represent statistically significant differences between samples indicated by connecting lines; p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. [Figure 4]Mean change in gas pressure within the apical space during 0-48 hours of colonic incubation. (*), (**), (***), and (****) represent statistically significant differences between samples indicated by connecting lines, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. [Figure 5] Mean change in ammonia concentration (mM) during 48 h colonic incubation. (*), (**), (***), and (****) represent statistically significant differences between samples indicated by connecting lines, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. [Figure 6A] Mean absolute levels (log counts / mL) of (A) Bifidobacteriaceae, (B) Bifidobacterium longum-associated OTU6, (C) Bifidobacterium pseudocatenulatum-associated OTU3, and (D) Bifidobacterium bifidum-associated OTU8 over 48 h of colonic incubation. Shading intensity correlates with absolute abundance. (*), (**), (***), and (****) indicated by a connecting line represent statistically significant differences between samples. (*), (**), (***), and (****) without a connecting line represent statistically significant differences between samples and controls, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. [Figure 6B] Mean absolute levels (log counts / mL) of (A) Bifidobacteriaceae, (B) Bifidobacterium longum-associated OTU6, (C) Bifidobacterium pseudocatenulatum-associated OTU3, and (D) Bifidobacterium bifidum-associated OTU8 over 48 h of colonic incubation. Shading intensity correlates with absolute abundance. (*), (**), (***), and (****) indicated by a connecting line represent statistically significant differences between samples. (*), (**), (***), and (****) without a connecting line represent statistically significant differences between samples and controls, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. [Figure 6C] Mean absolute levels (log counts / mL) of (A) Bifidobacteriaceae, (B) Bifidobacterium longum-associated OTU6, (C) Bifidobacterium pseudocatenulatum-associated OTU3, and (D) Bifidobacterium bifidum-associated OTU8 over 48 h of colonic incubation. Shading intensity correlates with absolute abundance. (*), (**), (***), and (****) indicated by a connecting line represent statistically significant differences between samples. (*), (**), (***), and (****) without a connecting line represent statistically significant differences between samples and controls, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. [Figure 6D] Mean absolute levels (log counts / mL) of (A) Bifidobacteriaceae, (B) Bifidobacterium longum-associated OTU6, (C) Bifidobacterium pseudocatenulatum-associated OTU3, and (D) Bifidobacterium bifidum-associated OTU8 over 48 h of colonic incubation. Shading intensity correlates with absolute abundance. (*), (**), (***), and (****) indicated by a connecting line represent statistically significant differences between samples. (*), (**), (***), and (****) without a connecting line represent statistically significant differences between samples and controls, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. [Figure 7]Effect of colonic batch samples on (A) IL6 and (B) IL10 secretion. After 24 hours of apical pretreatment with colonic batch samples, cytokine levels were measured 6 hours after basolateral LPS treatment of Caco-2 / THP1-Blue™ co-cultures. The red dotted line corresponds to the LPS+ control experiment. Data are expressed as mean ± SEM. (*) indicates a statistically significant difference between treated and control samples or between the indicated treatments. (*) = p<0.05 (*) = p<0.01 (A) Effect of probiotic 3 as a synbiotic on cytokine production. Fiber 3 = fiber mixture (fiber 1 as described herein), fiber 4 = pea fiber, probiotic 3 = B. longum; synbiotic 5 = fiber 3 + probiotic 3; synbiotic 6 = fiber 4 + probiotic 3. [Figure 8] Effect of colonic batch samples on (A) TNF-α and (B) IL-8 secretion. After 24 h of apical pretreatment with colonic batch samples, cytokine levels were measured 6 h after LPS treatment of the basolateral side of Caco-2 / THP1-Blue™ co-cultures. The red dotted line corresponds to the LPS+ control experiment. Data are expressed as mean ± SEM. (*) indicates a statistically significant difference between treated and control samples or between the indicated treatments. (*) = p<0.05 (**) = p<0.01 (A) Effect of probiotic 3 as a synbiotic on cytokine production. Fiber 3 = fiber mixture (as described herein), fiber 4 = pea fiber; probiotic 3 = B. longum; synbiotic 5 = fiber 3 + probiotic 3; synbiotic 6 = fiber 4 + probiotic 3.

[0010] Problems that the invention aims to solve The present invention broadly relates to Bifidobacterium longum probiotics.

[0011] Specifically, the present invention relates to the use of Bifidobacterium longum subsp. longum to improve the fermentability of plant fiber, the plant fiber having an insoluble fraction of 40-80% (w / w). In one embodiment, the present invention relates to the use of Bifidobacterium longum subsp. longum to improve the in vivo fermentability of plant fiber in a human subject.

[0012] In some embodiments, the probiotic Bifidobacterium longum subsp. longum is preconditioned by growing it in the presence of fiber.

[0013] In some embodiments, the probiotic Bifidobacterium longum subsp. longum is preconditioned by growing it in vitro in the presence of fiber.

[0014] In some embodiments, the probiotic is B. longum NCC3001 (ATCC BAA-999).

[0015] In some embodiments, the probiotic B. longum NCC3001 is preconditioned by growing it in vitro in the presence of fiber.

[0016] Grass fiber, such as corn fiber, having a high insoluble fraction can be used. In some embodiments, the grass fiber has an insoluble fraction of 70-80% (w / w). In some embodiments, the grass fiber is corn fiber. In some embodiments, the corn fiber is a corn fiber blend comprising about 45% (w / w) corn fiber.

[0017] In some embodiments, the probiotic B. longum NCC3001 is preconditioned by growing it in vitro in the presence of corn fiber.

[0018] Legume fiber, such as pea cell wall fiber, having a low insoluble fraction can be used. In some embodiments, the legume fiber has an insoluble fraction of 40-50% (w / w). In some embodiments, the legume fiber is pea cell wall fiber.

[0019] In some embodiments, the probiotic Bifidobacterium longum subsp. longum is preconditioned by growing it in vitro in the presence of fiber.

[0020] In some embodiments, the probiotic is B. longum NCC3001 (ATCC BAA-999).

[0021] In some embodiments, the probiotic B. longum NCC3001 is preconditioned by growing it in vitro in the presence of pea cell wall fiber.

[0022] The vegetable fibers may be a combination of grass and legume fibers.

[0023] In one embodiment, improved fermentability of the plant fiber corresponds, for example, to reduced gas formation in the gastrointestinal tract of a subject.

[0024] In one embodiment, the improved fermentability of the plant fiber corresponds, for example, to a reduction in gas formation in the gastrointestinal tract of the subject, and the probiotic is B. longum NCC3001 (ATCC BAA-999), which is preconditioned by growing in vitro in the presence of pea cell wall fiber.

[0025] In one embodiment, the improved fermentability of the plant fiber corresponds, for example, to increased production of total short-chain fatty acids in the gastrointestinal tract of the subject. In one embodiment, the improved fermentability of the plant fiber corresponds, for example, to increased production of acetate in the gastrointestinal tract of the subject. In one embodiment, the improved fermentability of the plant fiber corresponds, for example, to increased production of propionate in the gastrointestinal tract of the subject.

[0026] In one embodiment, the improved fermentability of the plant fiber corresponds, for example, to an increased production of total short-chain fatty acids in the gastrointestinal tract of the subject, and the probiotic is B. longum NCC3001 (ATCC BAA-999), which is preconditioned by growing in vitro in the presence of pea cell wall fiber.

[0027] In one embodiment, increased production of short chain fatty acids corresponds to, for example, increased production of IL6 and IL10 in the subject's gastrointestinal tract.

[0028] In one embodiment, increased production of IL6 and IL10 in the human gastrointestinal tract corresponds to an improved anti-inflammatory state. IL6 has a positive effect on intestinal epithelial regeneration and wound healing. IL6 is involved in the induction of cells that play an important role in host defense against extracellular microorganisms in mucosal tissues.

[0029] IL-10 is an anti-inflammatory cytokine that can suppress several innate and adaptive immune cell types and is involved in restoring immune homeostasis.

[0030] It is believed that beneficial effects can be obtained by fermenting the plant fibres, particularly those from grasses such as corn fibre, with Bifidobacterium.

[0031] The present invention further relates to a composition comprising an effective amount of grass and / or legume fiber and Bifidobacterium probiotics, wherein the plant fiber has an insoluble fraction of 40-80% (w / w), and the Bifidobacterium probiotics have been obtained by a process comprising: a. fermenting Bifidobacterium in a bacterial growth medium; and b. recovering the cultured Bifidobacterium probiotics.

[0032] In some embodiments, the plant fiber is grass fiber, such as corn fiber.

[0033] In some embodiments, the plant fiber is a legume fiber, such as pea cell wall fiber.

[0034] In one embodiment, the composition is a food, a medical food, a tube feeding, or a dietary supplement.

[0035] In one embodiment, the food product is selected from milk, yogurt, curd, cheese, fermented milk, fermented milk-based products, rice-based products, milk-based powders, infant formula, and pet food.

[0036] In one embodiment, the composition is a pharmaceutical composition, which comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0037] definition As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. Thus, for example, reference to "a bacterial strain" or "the bacterial strain" includes two or more bacterial strains.

[0038] The words "comprise," "comprises," and "comprising" are to be construed as inclusive and not exclusive. Similarly, the words "include," "including," and "or" should all be construed as inclusive unless such a construction is clearly prevented from the context.

[0039] However, compositions disclosed herein may not include elements not specifically disclosed. Accordingly, disclosure of embodiments presented using the term "comprising" includes disclosure of embodiments "consisting essentially of" and "consisting of" the specified components. Similarly, methods disclosed herein may be absent any step not specifically disclosed herein. Accordingly, disclosure of embodiments using the term "comprising" includes disclosure of embodiments "consisting essentially of" and "consisting of" the specified steps.

[0040] The term "and / or" when used in the context of "X and / or Y" should be interpreted as "X," or "Y," or "X and Y." As used herein, the terms "example" and "such as," particularly when followed by a listing of terms, are merely exemplary and illustrative and should not be considered exclusive or comprehensive. Unless otherwise stated, any embodiment disclosed herein can be combined with any other embodiment disclosed herein.

[0041] As used herein, "about" and "approximately" are understood to refer to numbers within a numerical range, e.g., within -10% to +10% of the referenced number, preferably within -5% to +5% of the referenced number, more preferably within -1% to +1% of the referenced number, and most preferably within -0.1% to +0.1% of the referenced number.

[0042] Furthermore, all numerical ranges herein should be understood to include all integers, whole numbers, or fractions within that range. The term "between" includes the endpoints of the particular range. Furthermore, these numerical ranges should be interpreted as supporting claims directed to any number or subset of numbers within that range. For example, a disclosure of 1 to 10 should be interpreted as supporting ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0043] Although the term "subject" is often used herein in reference to humans, the present disclosure is not limited to humans. Accordingly, the terms "individual" and "patient" refer to any animal, mammal, that may benefit from treatment.

[0044] As used herein, an "effective amount" is an amount that prevents a deficiency, treats a disorder, condition, or disease in a subject, or more generally, alleviates symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to the subject.

[0045] The terms "treatment" and "treating" include any effect that results in the improvement of a symptom or disease (disorder), such as alleviating / ameliorating, alleviating, controlling, or eliminating a symptom or disease (disorder). The term does not necessarily imply that a subject is treated to the point of complete cure. Non-limiting examples of "treating" or "treatment" of a symptom or disease (disorder) include: (1) inhibiting the symptom or disease (disorder), i.e., arresting the development of the symptom or disease (disorder) or its clinical symptoms, and (2) alleviating the symptom or disease (disorder), i.e., causing temporary or permanent relief of the symptom or disease (disorder) or its clinical symptoms. Treatment may be patient-related or physician-related.

[0046] The term "prevention" or "preventing" means not causing clinical symptoms of the referenced condition or disease (disorder) in an individual, or reducing the risk of their development. An individual may be exposed to or susceptible to the condition or disease (disorder), but has not yet experienced or exhibited symptoms of the condition or disease (disorder). The terms "condition" and "disease (disorder)" refer to any disease, condition, symptom, or manifestation.

[0047] As used herein, the relative terms "optimize" or "optimise" mean to improve, increase, or enhance.

[0048] The terms "food," "food product," and "food composition" refer to a product or composition intended for consumption by an individual, such as a human, that provides at least one nutrient to such an individual. A food product can be, for example, a nutritionally complete formula (e.g., infant formula or clinical nutrition product), a dairy product, a drink powder, a dried soup, a dietary supplement, a meal replacement, a nutritional bar, a cereal, a confectionery product, or a complete and balanced pet food, e.g., a dry pet food composition or a wet pet food composition.

[0049] The term "pet food" or "pet food composition" refers to any food composition intended for consumption by a pet. The term "pet" refers to any animal that can benefit from or enjoy the compositions provided by the present disclosure. For example, a pet may be an avian, bovine, canine, equine, feline, caprine, olivian, murine, ovine, or porcine animal, although a pet may be any suitable animal. In one aspect, a pet may be a companion animal. Thus, the term "cat food composition" refers to any food composition intended for consumption by cats, and the term "dog food composition" refers to any composition intended for consumption by dogs.

[0050] The term "complete and balanced," when referring to a food composition, means a food composition that contains all known necessary nutrients in appropriate amounts and proportions, based on the recommendations of recognized authorities in the field of animal nutrition, and thus can serve as the sole source of dietary intake to sustain life or promote production without the need for additional nutritional supplements. Nutritionally balanced pet food and animal diet compositions are known and widely available in the art, such as complete and balanced food compositions formulated according to standards established by the Association of American Feed Control Officials (AAFCO).

[0051] The term "companion animal" means a dog or a cat.

[0052] "Wet pet food" refers to pet food with a moisture content of about 50% to about 90%, and in one embodiment, about 70% to about 90%. "Dry pet food" refers to pet food with a moisture content of less than about 20%, and in one embodiment, less than about 15%, and in a specific embodiment, less than about 10%. "Semi-moist food" refers to pet food with a moisture content of about 20% to about 50%, and in one embodiment, about 25% to about 35%.

[0053] In some embodiments, the pet food composition can include protein. The protein can be a crude protein source and can include vegetable proteins such as soybean meal, soy protein concentrate, corn gluten meal, wheat gluten, cottonseed, and peanut meal, or animal proteins such as casein, albumin, and meat protein. Examples of meat proteins useful herein include beef, pork, lamb, horse, poultry, fish, and mixtures thereof. In one embodiment, the food composition can include protein in an amount from about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or even 60% to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or even 70%, including various subranges therein. In one aspect, the protein can be from about 30% to about 55% of the food composition.

[0054] In some embodiments, the pet food composition can include carbohydrates. Generally, any type of carbohydrate can be used in the food composition. Examples of suitable carbohydrates include grains or cereals such as rice, corn, millet, sorghum, alfalfa, barley, soybeans, canola, oats, wheat, rye, triticale, and mixtures thereof. The composition can also optionally include other ingredients such as dried whey and other dairy by-products. In one embodiment, carbohydrates comprise about 5% to about 10% of the food composition. In another embodiment, carbohydrates comprise about 10% to about 15% of the food composition. In other aspects, carbohydrates can be present in an amount from about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or even 40% to about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or even 50%.

[0055] In some embodiments, the pet food composition can include fat. Examples of suitable fats include animal fats and vegetable fats. In one aspect, the fat source can be an animal fat source, such as tallow or poultry fat. Vegetable oils, such as corn oil, sunflower oil, safflower oil, grapeseed oil, soybean oil, olive oil, and other oils rich in monounsaturated and polyunsaturated fatty acids, can also be used. In one embodiment, the food composition can include fat in an amount from about 5%, 10%, 15%, 20%, 25%, 30%, or even 35% to about 10%, 15%, 20%, 25%, 30%, 35%, or even 40%, including various subranges within these amounts. In one aspect, the fat comprises about 25% to about 35% of the food composition.

[0056] The pet food composition can be administered periodically or intermittently as needed and desired. In one aspect, the pet food composition can be administered to the animal periodically. In one aspect, administration can be at least once a week. In certain embodiments, more frequent administration or consumption can be performed, such as two or three times a week. In one aspect, the administration regimen can include at least one daily consumption.

[0057] In broad terms, a pet food composition can be suitable for consumption as a meal, meal ingredient, snack, dietary supplement, or treat by animals, including companion animals such as dogs and cats. Such compositions can include complete foods intended to provide the necessary dietary requirements of the animal. Examples of such pet food compositions include, but are not limited to, dry foods, wet foods, semi-moist foods, drinks, bars, pre-cooked frozen meals, pre-cooked preserved meals, and pre-cooked refrigerated meals.

[0058] As discussed herein, pet food compositions may be administered to animals alone, as a complete and balanced diet, as a nutritional supplement, or in combination with dietary supplements, vitamins, and / or other nutritionally beneficial agents well known to those skilled in the art as part of an overall animal health and wellness program. The compositions of the present invention may also be useful as veterinary therapeutic products. Accordingly, the compositions may optionally contain carriers, diluents, or additives, the suitability of which for the intended use will be well known to those skilled in the art.

[0059] The term "prebiotic" refers to a substrate selectively utilized by host microorganisms that confers a health benefit (Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics, Nature Reviews Gastroenterology & Hepatology, 2017, 14, 491-502).

[0060] The term "probiotic" refers to live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (FAO / WHO, 2002). Microbial cells are broadly bacteria or yeasts.

[0061] The term "synbiotic" refers to a nutritional composition or food supplement that combines both probiotic(s) and prebiotic(s), in which the prebiotic(s) selectively enhance the probiotic(s) (see DeVrese and Schrezenmeir, Probiotics, prebiotics and synbiotics in food biotechnology, Springer Berlin Heidelberg, pp. 1-66). The term "synbiotic effect," as used herein, refers to the increased beneficial health effects of the synbiotic compared to the effects of the probiotic alone.

[0062] The compositions of the present disclosure, including many of the embodiments described herein, may comprise, consist of, or consist essentially of, in addition to the essential elements and limitations described herein, any additional or optional ingredients, components, or limitations described herein or otherwise useful in dietary therapy. DETAILED DESCRIPTION OF THE INVENTION

[0063] Uses of Bifidobacterium The present invention relates generally to the use of Bifidobacterium probiotics to improve the fermentability of plant fibers.

[0064] In one embodiment, the present invention relates to the use of Bifidobacterium longum subsp. longum to improve the in vivo fermentability of plant fiber in a human subject.

[0065] Specifically, the present invention relates to the use of Bifidobacterium longum probiotics to improve the fermentability of plant fibres, the plant fibres having an insoluble fraction of more than 40% (w / w), preferably 40-80% (w / w).

[0066] The improvement of the fermentability of plant fibres by Bifidobacterium longum is associated with a reduction in gas formation in the gastrointestinal tract of a subject, preferably a human subject.

[0067] Improved fermentability of plant fiber also corresponds to increased production of total short-chain fatty acids in the gastrointestinal tract of a subject, preferably a human subject.Improved fermentability of plant fiber may also correspond to decreased proteolytic activity of the gut microbiota in the gastrointestinal tract of a subject, preferably a human subject.

[0068] The short chain fatty acid may be acetate, or propionate, or a combination of acetate and propionate.

[0069] Increased production of short-chain fatty acids corresponds to increased IL production in the gastrointestinal tract of a subject. In particular, increased production of short-chain fatty acids corresponds to increased production of IL6 and IL10 in the gastrointestinal tract of a subject, preferably a human subject. In particular, increased production of short-chain fatty acids, especially acetate and propionate, corresponds to increased production of IL6 and IL10 in the gastrointestinal tract of a human subject.

[0070] In one embodiment, increased IL production in the gastrointestinal tract corresponds to an improved anti-inflammatory state in a subject. In particular, increased production of IL6 and IL10 in the gastrointestinal tract corresponds to an improved anti-inflammatory state in a subject, preferably a human subject.

[0071] How to improve fermentation The present invention further relates to a method for improving the fermentability of plant fiber, comprising growing Bifidobacterium longum in a culture medium, characterized in that the culture medium contains grass and / or legume fiber, or a combination thereof. Preferably, the Bifidobacterium longum is Bifidobacterium longum subsp. longum.

[0072] composition The present invention further relates to a composition comprising an effective amount of vegetable fiber and Bifidobacterium probiotics, wherein the vegetable fiber has an insoluble fraction of 40-80% (w / w).

[0073] Specifically, the present invention relates to a composition comprising an effective amount of grass and / or legume fiber and Bifidobacterium probiotics, wherein the plant fiber has an insoluble fraction of 40-80% (w / w).

[0074] Specifically, the present invention relates to a composition comprising an effective amount of grass and / or legume fiber and Bifidobacterium probiotics, wherein the plant fiber has an insoluble fraction of 40-80% (w / w).

[0075] Specifically, the present invention relates to a composition comprising an effective amount of grass and / or legume fiber and Bifidobacterium probiotics, the plant fiber having an insoluble fraction of 40-80% (w / w), the Bifidobacterium probiotics being obtained by a process comprising the steps of: a. fermenting Bifidobacterium in a probiotic bacterial growth medium; and b. recovering the cultured Bifidobacterium probiotics.

[0076] In one embodiment, the bacterial growth medium comprises grass and / or legume fiber.

[0077] The compositions of the present invention may be in the form of a food, a medical food, a tube feeding, a nutritional composition, or a dietary supplement. The term "dietary supplement" refers to a product intended to supplement the normal diet of a subject.

[0078] In one embodiment, the food product is selected from milk, yogurt, curd, cheese, fermented milk, fermented milk-based products, rice-based products, milk-based powders, infant formula, and pet food.

[0079] The composition may be in the form of a medical food. As used herein, the term "medical food" refers to a food product specially formulated for the dietary management of a medical disease or condition. A medical food may be administered under medical supervision. A medical food may be for oral intake or tube feeding.

[0080] The composition may be in the form of tube feeding. The term "tube feeding" refers to a product intended to introduce nutrition directly into the gastrointestinal tract of a subject through a feeding tube. Tube feeding may be administered, for example, through a feeding tube placed through the subject's nose (such as a nasogastric tube, a nasoduodenal tube, or a nasojejunal tube), or through a feeding tube placed directly in the subject's abdomen (such as a gastrostomy feeding tube, a gastrojejunostomy feeding tube, or a jejunal feeding tube).

[0081] The composition may be in the form of a pharmaceutical composition and may comprise one or more suitable pharmaceutically acceptable carriers, diluents and / or excipients.

[0082] Examples of such suitable excipients for the compositions described herein can be found in "Handbook of Pharmaceutical Excipients", 2nd Edition (1994), edited by A Wade and PJ Weller.

[0083] Acceptable carriers or diluents for therapeutic use are known in the pharmaceutical art and are described, for example, in "Remington's Pharmaceutical Sciences", Mack Publishing Co. (AR Gennaro edit. 1985).

[0084] Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include ethanol, glycerol, and water.

[0085] The choice of pharmaceutical carrier, excipient, or diluent may be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient, or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and / or solubilizing agent(s).

[0086] Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, fluid lactose, β-lactose, and the like, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.

[0087] Preservatives, stabilizers, dyes, and even flavoring agents may be included in the composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used.

[0088] Nutritionally acceptable carriers, diluents, and additives include those used as standards in the food industry and suitable for human or animal consumption. Typical nutritionally acceptable carriers, diluents, and additives are well known to those skilled in the art.

[0089] The composition can be in the form of tablets, dragees, lozenges, capsules, gelcaps, powders, granules, solutions, emulsions, suspensions, coated particles, spray-dried particles, or pills.

[0090] It will be apparent to one skilled in the art that the ideal dosage will vary depending on, for example, the subject being treated, its health condition, sex, age, or weight, and the route of administration. As a result, the dosage ideally used may vary but can be readily determined by one skilled in the art.

[0091] Generally, however, the compositions of the present invention contain Bifidobacterium longum subsp. longum in a daily dose of 10 6 ~10 10 cfu, and / or 10 6 ~10 10 The compositions of the present invention preferably contain Bifidobacterium longum subsp. longum in an amount of 10 cells per gram of dry weight of the composition. 6 ~10 11 cfu, and / or 10 6 ~10 11 It may be contained in individual cells.

[0092] Grass fiber The grass fiber may be derived from corn, barley, oats, rice, rye, sorghum, wheat, or millet. Preferably, the plant fiber is derived from corn. The plant fiber may be produced as a milling by-product, for example, in the form of a fiber mixture.

[0093] When the plant fiber is grass fiber, the insoluble fraction is preferably 70 to 80% (w / w).

[0094] The corn fiber can be in the form of a corn fiber blend. The mixture may contain 30-60% (w / w), or 35-55% (w / w), or 40-50% (w / w), or about 45% (w / w) dietary fiber, and / or 20-50% (w / w), or 30-40% (w / w), or about 33% (w / w) corn bran, and / or 1-10% (w / w) wheat flour, preferably whole wheat wheat flour, preferably about 5.3% (w / w) whole wheat wheat flour, and / or 1-10% (w / w) dextrin, preferably resistant dextrin, preferably about 5% resistant dextrin, and / or 1-5% (w / w) gum, preferably guar gum, preferably about 0.8% guar gum, and / or 1-5% (w / w) carboxymethylcellulose, preferably about 0.5% (w / w) carboxymethylcellulose.

[0095] In some embodiments, the grass fiber comprises corn fiber, preferably about 45% (w / w) corn fiber, hi some embodiments, the grass fiber further comprises about 5% (w / w) resistant dextrin, about 2.5% (w / w) wheat bran, about 1% (w / w) guar gum, and about 0.5% (w / w) carboxymethyl cellulose.

[0096] Bifidobacterium probiotics can be preconditioned with grass fiber, for example, B. longum NCC3001 can be preconditioned by growing it in the presence of corn fiber.

[0097] Legume fiber The plant fiber may be legume fiber. When the plant fiber is legume fiber, the insoluble fraction is preferably 40 to 50% (w / w). The Legume family includes many important agricultural and food plants, such as Pisum sativum (pea), Glycine max (soybean), Phaseolus (kidney bean), Cicer arietinum (chickpea), Medicago sativa (alfalfa), Arachis hypogaea (peanut), Ceratonia silicate (carob), and Glycyrrhiza glabra (licorice). Preferably, the legume fiber is pea fiber. Preferably, the pea fiber is pea cell wall fiber.

[0098] Bifidobacterium probiotics can be preconditioned with legume fiber, for example, B. longum NCC3001 can be preconditioned by growing it in the presence of pea cell wall fiber.

[0099] Bifidobacterium Probiotics The Bifidobacterium probiotic of the present invention may be Bifidobacterium longum, Bifidobacterium animalis subsp. lactis, or Bifidobacterium breve. Most preferably, it is Bifidobacterium longum, such as B. longum subsp. longum, B. longum subsp. infantis, or B. longum subsp. suis, preferably B. longum subsp. longum. B. longum subsp. longum can be selected from Bifidobacterium longum ATCC BAA-999, Bifidobacterium longum ATCC 15707, and Bifidobacterium longum CNCM I-2618. Most preferably, it is Bifidobacterium longum ATCC BAA-999 (NC3001).

[0100] B. longum ATCC BAA-999 is also known as NCC3001 and is commercially available from specialized suppliers, for example, Morinaga Milk Industry Co., Ltd. (Japan) under the trade name BB536. The term "B. longum ATCC BAA-999" includes such bacteria, parts of such bacteria, and / or growth media that have been fermented by such bacteria.

[0101] B. longum ATCC BAA-999 has been deposited and is publicly available on June 7, 2004, by Tomoko Yaeshima (Morinaga Milk Industry Co., Ltd., Higashihara, Zama City, Kanagawa Prefecture, Japan) at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209.

[0102] B. longum ATCC BAA-999 can be cultivated by any suitable method. B. longum ATCC BAA-999 can be added to the composition in any technically suitable form, for example, freeze-dried or spray-dried form.

[0103] The ATCC15707 strain was deposited before 1990 and is commercially available. The CNCMI-2618 strain was deposited by Nestec SA (Avenue Nestle 55, 1800 Vevey, Switzerland) on January 29, 2001. Nestec SA was subsequently merged with Société des Produits Nestle SA, and therefore, Société des Produits Nestle SA is Nestec SA's successor in title pursuant to Article 2(ix) of the Budapest Treaty.

[0104] ATCC refers to the American Type Culture Collection, 10801 University Blvd., Manassas, Virginia 20110-2209, USA. CNCM refers to the Collection nationale de cultures de micro-organismes (Institut Pasteur, 28, rue du Dr Roux, F-75724 Paris Cedex 15, France).

[0105] The Bifidobacterium probiotics of the present invention are live probiotic bacteria. Bacteria are considered "live" if they are capable of growing under controlled culture conditions and forming colonies or suspensions, or if the microbial metabolic activity and / or membrane integrity can be confirmed using methods known to those skilled in the art, such as, for example, flow cytometry.

[0106] food products The present invention further relates to a food product comprising the composition described herein. The food product may be a cereal bar, biscuit, yogurt, powdered drink, etc.

[0107] Use as a medical food The present invention further relates to a composition or food product described herein for use as a medical food for preventing or treating a condition or disease in a subject. In some embodiments, the composition is for use as a medical food for weight management, irritable bowel syndrome, chronic enteropathy, and / or atopic dermatitis in a subject. In some embodiments, the composition is for use as a medical food for irritable bowel syndrome or chronic enteropathy in a subject. In some embodiments, the medical food is for preventing or treating irritable bowel syndrome. In some embodiments, the medical food is for preventing or treating chronic bowel disease. In some embodiments, the medical food is for preventing or treating atopic dermatitis. In some embodiments, the medical food is for preventing or treating inflammation. In some embodiments, the medical food is for preventing or treating colon cancer. [Example]

[0108] Example 1: Simulation of the upper GIT and short-term colonic incubation B. longum subsp. longum NCC3001, also known as BL999, was used in this in vitro study. A total of 2.0E8 CFU was fed to the system. Pea cell wall fiber (obtained commercially) and a corn fiber blend (obtained from CPW, a breakfast cereal manufacturer, also known as Fiber 1) were used as fibers and fed to the system at a concentration of 22 g / L each. The corn fiber blend (Fiber 1) contained 45% dietary fiber and included 33.5% corn bran, 5.3% whole wheat flour, 5% resistant dextrin, and 0.8% guar gum. Each synbiotic combination, as well as the probiotic and prebiotic components alone, were tested in triplicate.

[0109] The calibrated SHIME® system was used to test the viability of probiotics in the upper GIT (stomach and small intestine). For in vivo upper GIT testing, the InfoGest consensus method and a calibrated protocol with a dynamic pH profile were used to conduct the test. One reactor was first subjected to gastric conditions and then to small intestinal conditions, mimicking both fed and fasted conditions (Figure 1).

[0110] Incubation began with feeding the probiotic and prebiotic samples with 7 g / L fructose at 37°C for 2 hours with mixing. During this time, the pH decreased from 5.5 to 2.5 (Figure 2). A special gastric suspension (pepsin, phosphatidylcholine, fructose, and additional nutrient medium) was then added to simulate the gastric phase. After 5 minutes, standardized pancreatic juice containing porcine pancreatin and 10 mM bovine bile extract was added to simulate the duodenal phase. After 20 minutes, the reactor contents were transferred to a dialysis membrane (regenerated cellulose, 3.5 kDa), which was immersed in a four-fold volume of a pH 7 solution containing bicarbonate at the same concentration as the membrane content. Dialysis was performed for 3.67 hours to simulate the jejunal and ileal incubation.

[0111] Short-term colonic incubations for gut microbial metabolism analysis were performed using representative colonic media containing host- and diet-derived compounds and the colonic microbiota of a single healthy adult human donor. Upper GIT suspensions (containing the unabsorbed fraction of fructose) were incorporated into the colonic incubations in triplicate, with no added fermentable carbohydrates, except for samples with prebiotic fiber. Controls included upper GIT suspensions (without added probiotics or prebiotics) containing only the unabsorbed fraction of fructose in gastric juice. Incubations were performed anaerobically at 37°C and pH 6.5 for 48 hours.

[0112] Example 2: Improvement of short-chain fatty acid production After 48 h of colonic incubation, short-chain fatty acids (SCFAs) produced by the microbiota were measured using gas chromatography with flame ionization detection (GC-FID).

[0113] Increased acetate production was observed when NCC3001 was administered with prebiotic fiber, especially with the corn bran mixture (Figure 3A). It is unclear whether acetate production is due to NCC3001 itself or other members of the microbiota present in the system. It is likely that B. longum can produce large amounts of acetate in the presence of complex fiber through the bifid shunt. Overall, the high acetate concentrations indicate not only the increased metabolic activity of B. longum when co-administered with the corn bran mixture, but also increased fiber utilization by the probiotic and other colonic microbiota members.

[0114] Propionate production also increased with synbiotic incubation compared to that with fiber alone (Figure 3B). Furthermore, preconditioning NCC3001 with pea fiber also resulted in significantly higher propionate production than without preconditioning (data not shown). Because B. longum does not produce propionate, this result suggests that propionate production was ultimately due to the increased metabolic activity of other colonic bacteria and the increased utilization of the corn fiber mixture or pea fiber, ultimately resulting from cross-feeding interactions. The increase in acetate and propionate was also reflected in the change in total SCFA concentration; i.e., the synbiotic formulation improved SCFA production, especially when the corn bran mixture was co-added with NCC3001 (Figure 3D).

[0115] Example 3: Improving Fiber Tolerance Consumption of dietary fiber that is not digested by the human body often promotes increased gas production by the colonic microbiota, leading to bloating and flatulence. In this study, changes in gas pressure within the apical space of colonic incubations were recorded from 0 to 48 hours. Co-addition of the corn fiber mixture with NCC3001 resulted in a promising reduction in gas pressure, but was not statistically significantly different from the gas pressure observed in incubations with the corn fiber mixture alone (Figure 4). Co-addition of pre-conditioned NCC3001 with pea fiber resulted in a significant reduction in gas production in colonic incubations, indicating that pea fiber is well tolerated.

[0116] Example 4: Reduction of proteolytic fermentation by pea fiber Ammonium production at the end of colonic incubation was quantified to provide an indication of overall proteolytic fermentation occurring in the system. When NCC3001 was co-added with pea fiber, statistically significantly less ammonium was produced compared to experiments performed with pea fiber alone or with the corn bran mixture (Figure 5). This production may suggest a synergistic effect on the reduction of proteolytic fermentation when B. longum and pea fiber were co-administered.

[0117] The colonic microbiota was determined using live / dead flow cytometry coupled to microbiome profiling using 16S-specific Illumina 18. Live / dead flow cytometry was performed on a BD FACSVerse by preparing samples at 10-fold serial dilutions in phosphate-buffered saline. Viable, nonviable, and total cell counts in culture samples were assessed by staining with SYTO24 and propidium iodide. Bifidobacteriaceae growth was induced after 48 hours of colonic incubation, particularly when pea fiber was added together with NCC3001 (Figure 6A). This increase was not only due to the increased presence of NCC3001 and other B. longum subspecies (Figure 6B), but also due to an increased presence of B. pseudocatenulatum (a key acetate producer) (Figure 6C) and an increase in B. bifidum (Figure 6D).

[0118] The previously observed strong stimulation of propionate production (Figure 3B) may have been due to stimulation of OTU4, associated with Phascolarctobacterium succinatutens, a known succinate-transforming, propionate-producing gut microorganism of the phylum Firmicutes. Succinate was likely derived from OTU2, associated with Bacteroides thetaiotaomicron, OTU5, associated with Bacteroides stercoris, and OTU12, associated with Bacteroides vulgatus (Table 1).

[0119] Table 1. Mean absolute levels (log counts / mL) of OTUs (associated with a given species) after 48 h of colonic incubation. Shading intensity correlates with absolute abundance, normalized within each OTU at each time point (within each row). Statistically significant differences between control and specific treatments within each OTU and time point are indicated in bold (p<0.05).

[0120] [Table 1]

[0121] Preselected B. longum NCC3001 strains were tested in an advanced simulated gastrointestinal system (SHIME®) with a corn bran mixture (Fiber 1) or pea fiber, demonstrating synbiotic behavior. Results indicated increased viability and metabolic activity of NCC3001 during upper GIT incubation when fiber was added. During colonic incubation, the addition of both probiotics and prebiotics increased SCFA production, particularly acetate and propionate, compared with administration of fiber alone. Furthermore, colonic incubation of preconditioned NCC3001 with pea fiber reduced overall gas tension, suggesting increased fiber tolerance. The synbiotic NCC3001 and pea fiber also significantly increased the abundance of several species from the families Bifidobacteriaceae, Bacteroidetes, and Acidaminococcaceae. Longer-term SHIME studies are planned to verify the viability and health benefits of the probiotic after 4 weeks of GIT incubation with daily probiotic addition. Furthermore, the specific quantification of B. longum subsp. longum NCC3001 in colonic cultures is currently being analyzed by qPCR, with the aim of identifying the specific abundance of this strain relative to total Bifidobacterium OTUs and thus confirming the viability of the probiotic in the colon.

[0122] Example 5: Evaluation of the effects of probiotic and synbiotic products on inflammation after passage through the entire GIT using SHIME® technology The effects of Bifidobacterium longum and its synbiotic combination (preconditioned and unpreconditioned) on gut wall function during colonic fermentation were examined in terms of modulating "leaky gut" under inflammatory conditions. B. longum was formulated with a fiber mixture and pea fiber. Endpoints were correlated with immune markers (pro- and anti-inflammatory cytokines and chemokines) in an in vitro Caco-2 / THP1 co-culture model. Colon samples were selected from a phase 1 study to allow evaluation of the effects induced by the products and their fermentation-derived metabolites produced by the gut microbiota.

[0123] After 24 hours of co-culture of apical Caco-2 / THP1-Blue™ with colonic batch samples, the basolateral supernatant was discarded and the cells were stimulated with LPS. After 6 hours of stimulation, the basolateral supernatant was collected to measure cytokines and chemokines secreted into the medium.

[0124] Under natural conditions, synbiotic 6 (pea fiber + B. longum) tended to increase IL-6 secretion compared to treatment with fiber 4 (pea fiber) or probiotic 3 (B. longum) (Figure 7A). Natural synbiotic 6 significantly increased IL-10 secretion compared to probiotic 3 (Figure 7B). This level also tended to be higher compared to each fiber. Synbiotic 6 reduced TNF-α secretion compared to treatment with fiber 4 (Figure 8A). Natural synbiotic 6 tended to reduce IL-8 secretion compared to probiotic treatment alone; however, levels were still higher compared to treatment with fiber 4 alone (Figure 8B).

Claims

1. 1. A composition comprising an effective amount of legume fiber and Bifidobacterium longum subsp. longum probiotics, The composition is used as a medical food, the legume fiber is pea cell wall fiber and has an insoluble fraction of 40-50% (w / w) of the total plant fiber; The Bifidobacterium longum subsp. longum a. fermenting the Bifidobacterium in a bacterial growth medium comprising the pea cell wall fiber; and b. recovering the cultured Bifidobacterium probiotic.

2. The composition described in claim 1, wherein the probiotic is Bifidobacterium longum NCC3001.

3. A composition as described in claim 1 or 2 for use as a medical food to reduce gas formation in the gastrointestinal tract of a subject.

4. A composition described in claim 1 or 2 for use as a medical food to increase the production of total short-chain fatty acids in the gastrointestinal tract of a subject.

5. The composition described in claim 4, wherein the short-chain fatty acid is acetate and / or propionate.

6. A composition according to claim 1 or 2 for use as a medical food for increasing the production of IL6 and IL10 in the gastrointestinal tract.

7. A composition described in claim 1 or 2 for use as a medical food for improving anti-inflammatory conditions.

8. 3. The composition of claim 1 or 2 for use as a medical food for irritable bowel syndrome or chronic enteropathy in a subject.

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