A combination of prebiotics and probiotics that produce butyric acid for synergistic effect.

Abstract

Provided is a synergistic combination of butyric-acid-producing prebiotics and probiotics, which is used in the fields of foods, dietary supplements and drugs. Specifically, the present invention relates to a composition containing probiotics and prebiotics, a food, dietary supplement or pharmaceutical preparation comprising the composition, and the use of the composition for alleviating and improving a disease or a discomfort, or reducing the occurrence of a disease or a discomfort, wherein the disease or discomfort is related to insufficient butyric acid in the intestine, or to insufficient butyric-acid-producing microorganisms in the intestine. The present invention further relates to a method for promoting Bifidobacterium lactis HN019 to produce butyric acid, the method comprising using a galactooligosaccharide and isomerized lactose as carbon sources for the cultivation of Bifidobacterium lactis HN019, wherein the weight ratio of the galactooligosaccharide to isomerized lactose is 1:1 to 4:1.

Description

Butyrate-producing prebiotic probiotic synergistic combination

[0001] This application claims priority to the Chinese patent application No. 201911115800.7, filed on November 15, 2019, and entitled "Butyrate-producing prebiotic probiotic synergistic combination", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of food, dietary supplements and pharmaceuticals, in particular to a composition comprising probiotics and prebiotics, a food, dietary supplement or pharmaceutical preparation comprising the composition, and the use of the composition for alleviating, ameliorating or reducing the occurrence of a disease or discomfort associated with butyrate deficiency in the gut or with butyrate-producing microorganism deficiency in the gut. The present application also provides a method for promoting the production of butyrate by Bifidobacterium lactis HN019, comprising using galactooligosaccharide and isomerized lactose as the carbon source for the culture of Bifidobacterium lactis HN019, wherein the weight ratio of galactooligosaccharide to isomerized lactose is 1:1-4:1. BACKGROUND

[0003] The intestinal system of the human body contains a large number of bacteria, the total amount of which is more than 100 times that of human cells. These bacteria constitute the intestinal micro-ecosystem of the human body. More and more studies have shown that the intestinal micro-ecosystem is closely related to the health status of the human body, and the intestinal micro-ecosystem in the early life has an important influence on the health of a lifetime (Sommer F, Bäckhed F. Nat Rev Microbiol. 2013; 11(4): 227-38). F.Nat Rev Microbiol.2013;11(4):227-38).

[0004] Previous studies have shown that different feeding methods have an important influence on the early intestinal micro-ecosystem of infants. The proportion of Bifidobacterium and Lactobacillus in the feces of formula-fed infants is significantly lower than that of breast-fed infants. By adding probiotics and prebiotics to infant formula, the intestinal micro-ecosystem of infants can be adjusted, so that the intestinal micro-ecosystem of formula-fed infants is more similar to that of breast-fed infants, especially the proportion of Bifidobacterium (Adlerberth I, Wold AE. Acta Paediatr. 2009; 98: 229-38).

[0005] Prebiotics are a class of indigestible carbohydrates that can interact with intestinal flora in the host's intestinal tract to produce substances or effects that are beneficial to the host (Gibson GR et al. Nat Rev Gastroenterol Hepatol. 2017; 14(8): 491-502). The prebiotics currently studied more include: galactooligosaccharides, fructooligosaccharides, inulin, isomerized lactose, polydextrose, oligomeric xylo-lose, etc.

[0006] Galactooligosaccharide (GOS) is a functional oligosaccharide with natural properties, and its molecular structure is generally one or more galactose groups connected to a galactose group or a glucose group. Animal milk contains a small amount of GOS, and human breast milk contains a slightly higher amount, which plays an important physiological role in the establishment of microbial flora in the intestines of animals and nursing infants.

[0007] Isomerized lactose (LOS) is the earliest discovered prebiotic (Ackerman DL et al. Carbohydr Res. 2017; 437: 16-27), and before the concept of "prebiotics" was introduced, isomerized lactose was called "bifidus factor", which has a good promoting effect on the growth of Bifidobacterium. Isomerized lactose is a disaccharide with many physicochemical advantages such as low sweetness, acid resistance, and good thermal stability. Isomerized lactose can have the effects of prebiotics, laxative, and detoxification at different doses. Watson's in vitro experimental results show that isomerized lactose has a good promoting effect on the growth of most Bifidobacterium and Lactobacillus (Hill C et al. Nat Rev Gastroenterol Hepatol. 2014; 11(8): 506-514). In the scientific opinion on the substantiation of health claims related to isomerized lactose and reduction of potential pathogenic micro-organisms and of the length of gut transit time, according to Directive 2006 / 54 / EC, Article 13(1), of the European Food Safety Authority (EFSA), it is claimed that isomerized lactose helps to reduce potential pathogenic micro-organisms and shorten the length of gut transit time. Isomerized lactose is metabolized by probiotics to produce short-chain fatty acids such as lactic acid and butyric acid, which are beneficial to the growth and metabolism of intestinal epithelial cells, and can inhibit the growth of harmful bacteria in the intestine by reducing the pH value (Dhiren Pranami et al. Drugs Ther Perspect. 2017; 33: 228-233).

[0008] The definition of probiotics by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) is: by taking an appropriate amount of live bacteria that can effectively affect the health of the consumer (Hill C et al. Nat Rev Gastroenterol Hepatol. 2014; 1(8): 506-514). Bifidobacterium lactis HN019 (Sanders ME et al. J. Clin. Gastroenterol. 2006; 40(9): 776-783) is a probiotic strain isolated from dairy products, which has strong survival ability in the intestinal tract and can effectively promote intestinal peristalsis. It is the lowest concentration of lactic acid bacteria / bifidobacteria strains published so far that can significantly shorten the intestinal transit time. Clinical studies have shown that HN019 can promote nutrient absorption in children and improve immunity in the elderly.

[0009] It has been a research hotspot to optimize the combination of probiotics and prebiotics in infant formula to make the intestinal microecological composition of formula-fed infants closer to that of breastfed infants.

[0010] SUMMARY

[0011] Carbohydrates that cannot be directly utilized by the human body can be fermented by intestinal bacteria to produce short-chain fatty acids (SCFAs), mainly including lactic acid (i.e., 2-hydroxypropanoic acid), acetic acid, propionic acid, and butyric acid. These short-chain fatty acids can have a positive impact on the health of the human body.

[0012] The present inventors have surprisingly found that a combination of galactooligosaccharides and isomerized lactose in a specific ratio can synergistically promote the production of butyric acid by Bifidobacterium lactis HN019, and can be used to reduce the occurrence of diseases or discomforts related to butyric acid deficiency or insufficient butyric acid-producing microorganisms in the intestinal tract, or to relieve, improve diseases or discomforts related to butyric acid deficiency or insufficient butyric acid-producing microorganisms in the intestinal tract, thereby providing the following invention:

[0013] In one aspect, the present application provides a composition comprising galactooligosaccharides, isomerized lactose, and Bifidobacterium lactis HN019, wherein the weight ratio of galactooligosaccharides to isomerized lactose is 1:1-4:1.

[0014] In one aspect, the present application provides a food or dietary supplement comprising the composition. In certain embodiments, the composition, food or dietary supplement of the present application is used to relieve, improve diseases or discomforts, or to reduce the occurrence of diseases or discomforts, which are related to butyric acid deficiency in the intestinal tract, or related to insufficient butyric acid-producing microorganisms in the intestinal tract.

[0015] In one aspect, the present application provides a pharmaceutical preparation comprising the composition of the present application and one or more pharmaceutically acceptable carriers.

[0016] In one aspect, the present application provides a method of alleviating, ameliorating or reducing the incidence of a disease or a disorder associated with butyric acid deficiency in the gut, or associated with butyrate-producing microorganism deficiency in the gut, comprising administering to an individual in need thereof the composition, food or dietary supplement of the present application.

[0017] In yet another aspect, the present application provides use of the composition of the present application for the manufacture of a food, a dietary supplement or a medicament for use in alleviating, ameliorating or reducing the incidence of a disease or a disorder associated with butyric acid deficiency in the gut, or associated with butyrate-producing microorganism deficiency in the gut.

[0018] The present application also provides a method for promoting Bifidobacterium lactis HN019 to produce butyric acid, comprising culturing Bifidobacterium lactis HN019 with galacto- oligosaccharide and isomerized lactose as carbon source, wherein the weight ratio of galacto- oligosaccharide to isomerized lactose is 1:1-4:1.

[0019] The present application also provides a composition for promoting Bifidobacterium lactis HN019 to produce butyric acid, comprising galacto-oligosaccharide and isomerized lactose, wherein the weight ratio of galacto-oligosaccharide to isomerized lactose is 1:1-4:1.

[0020] DETAILED DESCRIPTION

[0021] Compositions comprising prebiotics and probiotics

[0022] In one aspect, the present application provides a composition comprising galacto- oligosaccharide, isomerized lactose and Bifidobacterium lactis HN019, wherein the weight ratio of galacto-oligosaccharide to isomerized lactose is 1:1-4:1 (e.g. 1:1-2:1, 2:1-3:1 or 3:1-4:1, the unit of the weight ratio can be g:g).

[0023] In certain embodiments, the composition comprises about 10 6 Cfu / 100g- about 10 12 Cfu / 100g (e.g. about 10 6 Cfu / 100g- about 10 7 Cfu / 100g, about 10 7 Cfu / 100g- about 10 8 Cfu / 100g, about 10 8 Cfu / 100g- about 109 Cfu / 100g, about 10 9 Cfu / 100g - about 10 10 Cfu / 100g, about 10 10 Cfu / 100g - about 10 11 Cfu / 100g or about 10 11 Cfu / 100g - about 10 12 Cfu / 100g) of Bifidobacterium lactis HN019.

[0024] In the composition of the present application, the galactooligosaccharides (GOS) can be β-galactooligosaccharides and / or α-galactooligosaccharides. The structure of the galactooligosaccharides can be represented by the following formula: [Galactose] m - glucose, or represented by the following formula: [Galactose] n wherein m and n are integers from 1 to 20, preferably m and n are each independently selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0025] An exemplary structure of the galactooligosaccharides is that 1-7 galactosyl groups are mainly connected to the galactosyl side of lactose by β-1,4-glycosidic bonds. In an exemplary structure of the galactooligosaccharides, the glucose group is connected to the galactosyl group by β-1,4-glycosidic bonds, and adjacent galactosyl groups are connected by β-1,3, β-1,4 and β-1,6-glycosidic bonds, wherein the connection by β-1,4-glycosidic bonds is predominant. Common galactooligosaccharides mainly include galactooligosaccharide triose, galactooligosaccharide tetraose, and a small amount of galactooligosaccharide pentaose, galactooligosaccharide hexaose, etc. In addition, the β-D-galactosidase of some microorganisms can catalyze the glucose and galactose produced by the hydrolysis of the substrate lactose to synthesize galactooligosaccharide disaccharides connected by β-1,3 and β-1,6-glycosidic bonds, which are different from the structure of lactose. An exemplary molecular structure of the galactooligosaccharides is shown below:

[0026]

[0027] Commercially produced galactooligosaccharides are usually a mixture of glucose, galactose, lactose, galactooligosaccharide disaccharide, galactooligosaccharide triose, galactooligosaccharide tetraose, galactooligosaccharide pentaose, galactooligosaccharide hexaose, galactooligosaccharide heptaose, and / or galactooligosaccharide octaose. Therefore, in the present application, "galactooligosaccharides" or "GOS" can also refer to a mixture of galactooligosaccharides with different chain lengths (e.g. a mixture comprising glucose, galactose, lactose, galactooligosaccharide disaccharide, galactooligosaccharide triose, galactooligosaccharide tetraose, galactooligosaccharide pentaose, galactooligosaccharide hexaose, galactooligosaccharide heptaose, and / or galactooligosaccharide octaose).

[0028] In certain embodiments, the galactooligosaccharide used in the present application is galactooligosaccharide meeting the quality requirements in the Announcement on Approving New Resource Foods such as Galactooligosaccharide issued by the Ministry of Health in 2008. In certain embodiments, the galactooligosaccharide is a colorless transparent or light yellow sugar syrup, wherein the content of galactooligosaccharide (galactooligosaccharide disaccharide to galactooligosaccharide octasaccharide) is not less than 57.0% on a dry basis, the content of anhydrous lactose is not higher than 23.0%, the content of anhydrous glucose is not higher than 22.0%, and the dry matter is 74.0%-76.0%; the pH is 2.8-3.8.

[0029] The galactooligosaccharide used in the present application can be lactose-derived galactooligosaccharide. In certain embodiments, the galactooligosaccharide is obtained by using lactose in cow's milk as raw material, catalyzing hydrolysis of galactoside bond by β-galactosidase to generate galactose and glucose, and transferring the hydrolyzed galactoside to lactose molecules by transgalactosylation to generate galactooligosaccharide.

[0030] In the composition of the present application, isomerized lactose is also known as lactulose, lactoketose, 4-β-D-galactoside-D-fructose, 4-O-β-D-galactopyranosyl-4-D-fructopyranose, and its use form can be solid or liquid. In certain embodiments, isomerized lactose is used in the present application in the form of a solid powder, such as a solid product meeting the GB 1886176-2016 National Food Safety Standard, wherein the content of isomerized lactose is not less than 97%. In certain embodiments, isomerized lactose is used in the present application in the form of a liquid, such as isomerized lactose liquid meeting the GB 1886176-2016 National Food Safety Standard, wherein the content of isomerized lactose is not less than 50%.

[0031] In the composition of the present application, Bifidobacterium lactis HN019, also known as Bifidobacterium animalis subsp. lactis HN019, is a gram-positive, non-motile, non-spore-forming, irregular rod-shaped anaerobic bacterium with an optimal growth temperature of 37-41°C, which can ferment various sugars but not starch, and can grow in milk or milk-containing medium, and is a commercially available strain that has been publicly used.

[0032] In the present application, Bifidobacterium lactis HN019 can be provided as live cells or non-live cells, and can be in various forms, such as freeze-dried cells, wet cells, culture broth, yogurt, or combinations thereof. In certain embodiments, freeze-dried cells are preferred. Bifidobacterium lactis HN019 can be obtained commercially.

[0033] The composition of the present application can be in the form of a liquid, semi-liquid, powder or other form. In certain embodiments, the composition further comprises necessary excipients or dispersing agents.

[0034] In certain embodiments, the composition comprises at least 50%, for example at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% by weight of the composition of oligofructose and isomerized lactose. In certain embodiments, the composition consists of oligofructose, isomerized lactose and Bifidobacterium lactis HN019.

[0035] The composition of the present application can be included in a food product as a synbiotic, synbiotic combination or synbiotic mixture or as a dietary supplement alone. The synbiotic, synbiotic combination or synbiotic mixture all refer to the combination of probiotic and prebiotic which can enhance the growth and proliferation of the probiotic as well as the survival of the probiotic in the host's intestinal system, thereby providing health benefits to the host.

[0036] Food products, dietary supplements and uses

[0037] In another aspect, the present application provides a food product comprising the composition of the present application.

[0038] The food product of the present application can be a liquid food product, a semi-liquid food product, a solid food product or a semi-solid food product.

[0039] In certain embodiments, the food product of the present application is a formula, for example an infant formula (e.g. infant formula, older infant formula, young children's formula, infant formula for special medical purposes).

[0040] In the present application, "formula" means a nutritional composition designed for infants, young children, children, adults or a combination thereof, which contains sufficient macronutrients (e.g. protein, carbohydrate, lipids, vitamins, minerals and electrolytes) to be a sole, primary or supplemental source of nutrition.

[0041] In the present application, "infant" means a human up to about 12 months of age, including an infant of 0 to about 4 months of age, an infant of about 4 to about 8 months of age, an infant of about 8 to about 12 months of age.

[0042] In the present application, "older infant" means a human of about 6 to about 12 months of age.

[0043] In the present application, "young children" means a human of more than 12 months of age and less than 3 years of age.

[0044] In the present application, "children" means people from 3 years old to 12 years old.

[0045] In the present application, "infant formula" means a human milk substitute or replacement which is nutritionally complete and which provides all required nutrients in amounts appropriate to the stage of development, to meet the unique requirements of infants from birth to 6 months of age.

[0046] In the present application, "follow-on formula" means a formula which is nutritionally complete and which provides all required nutrients in amounts appropriate to the stage of development, to meet the unique requirements of infants from 7 months to 12 months of age, and which can be used as a human milk substitute or replacement.

[0047] In the present application, "young child formula" means a formula which is nutritionally complete and which provides all required nutrients in amounts appropriate to the stage of development, to meet the unique requirements of young children from 13 months to 36 months of age, and which can be used as a human milk substitute or replacement.

[0048] In the present application, "special medical purpose infant formula" means a formula which is nutritionally complete and which provides all required nutrients in amounts appropriate to the stage of development, to meet the unique requirements of infants from 0 months to 6 months of age with special medical conditions, and which can be used as a human milk substitute or replacement.

[0049] In certain embodiments, in addition to the composition of the present application, the infant or baby formula (e.g. infant formula, follow-on formula, young child formula or special medical purpose infant formula) further comprises the following ingredients: protein (e.g. milk protein, soy protein) as an energy source, fat (e.g. linoleic acid, alpha-linolenic acid) and carbohydrate (e.g. lactose, lactose and glucose polymers), vitamins (e.g. vitamin A, vitamin D, vitamin K, vitamin Bl, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, vitamin C, biotin, choline), minerals (e.g. sodium, potassium, copper, magnesium, iron, zinc, manganese, calcium, phosphorus, iodine, chlorine, selenium), and optionally, the infant or baby formula can further comprise inositol, taurine, L-carnitine, docosahexaenoic acid, eicosapentaenoic acid or any combination thereof.

[0050] In certain embodiments, the formula of the present application is a liquid or powder product which is produced and processed only by physical methods.

[0051] In certain embodiments, the food of the present application is an infant or baby complementary food.

[0052] In certain embodiments, the food product of the present application is a dairy product (e.g., 1st stage formula, 2nd stage formula, 3rd stage formula, fermented milk), a chocolate or chocolate product, a cereal product (e.g., dry cereal mix, cereal bar, porridge product), or a pet food.

[0053] In another aspect, the present application provides a dietary supplement comprising the composition of the present application.

[0054] The dietary supplement of the present application can be in the form of a pill, capsule, tablet, granule, liquid preparation, or other forms.

[0055] In addition to the composition, the food product or dietary supplement of the present application can optionally add a nutritional ingredient selected from the group consisting of protein, carbohydrate, lipid, mineral, vitamin, amino acid, unsaturated fatty acid, polyphenol, phytosterol, or any mixture thereof.

[0056] The components of the composition of the present application can be mixed into other components of the food product or dietary supplement, sequentially, together, or as a premix, by conventional processing techniques.

[0057] In certain embodiments, the composition, food product, or dietary supplement of the present application is used to alleviate, ameliorate, or reduce the occurrence of a disease or discomfort associated with insufficient butyric acid in the intestinal tract, or insufficient butyric acid-producing microorganisms in the intestinal tract.

[0058] In the present application, "insufficient butyric acid in the intestinal tract" or "insufficient butyric acid-producing microorganisms in the intestinal tract" means that the content of butyric acid or the number of butyric acid-producing microorganisms in the intestinal tract is lower compared to a population without the associated disease or discomfort.

[0059] Butyric acid has a sour smell and is difficult to accept directly orally. The composition, food product, dietary supplement, or pharmaceutical preparation of the present application can be used as a substitute for butyric acid to reduce or alleviate, ameliorate a disease or discomfort associated with insufficient butyric acid in the intestinal tract, or insufficient butyric acid-producing microorganisms in the intestinal tract, and can be safely and conveniently used in the population including infants and young children.

[0060] In one aspect, the present application provides the use of the composition of the present application for the preparation of a food product, dietary supplement, or medicament for alleviating, ameliorating, or reducing the occurrence of a disease or discomfort associated with insufficient butyric acid in the intestinal tract, or insufficient butyric acid-producing microorganisms in the intestinal tract.

[0061] The present application also provides a pharmaceutical preparation comprising the composition of the present application and one or more pharmaceutically acceptable carriers.

[0062] The pharmaceutical preparation of the present application can be in any pharmaceutically acceptable dosage form. Preferably, the pharmaceutical preparation of the present application is an oral preparation, such as an oral solid preparation, e.g., a tablet, a capsule, a pill, a granule, etc., or an oral liquid preparation, e.g., an oral solution, an oral suspension, a syrup, etc. The oral preparation can further comprise suitable fillers, binders, disintegrants, lubricants, etc.

[0063] An "effective amount" as used herein means an amount sufficient to achieve or at least partially achieve a desired effect. For example, a "prophylactically effective amount" refers to an amount effective, at the dosage administered, to prevent, retard or delay the onset of a disease or condition; a "therapeutically effective amount" refers to an amount effective, at the dosage administered, to cure or at least partially arrest the disease or condition in an individual already suffering from the disease or condition. Determining such effective amounts is well within the capability of those skilled in the art, e.g., given the teachings of the specification. For example, the amount effective for therapeutic uses will depend on the severity of the disease or condition to be treated, the general state of the patient's own immune system, the general state of the patient, e.g., age, weight, and sex, the mode of administration of the drug, and other therapies to be administered to the patient.

[0064] In certain embodiments, the pharmaceutical preparation is used to alleviate, ameliorate, or reduce the occurrence of a disease or condition associated with a deficiency of butyric acid in the gut, or a deficiency of butyrate-producing microorganisms in the gut.

[0065] In one aspect, the present application provides a method of alleviating, ameliorating, or reducing the occurrence of a disease or condition associated with a deficiency of butyric acid in the gut, or a deficiency of butyrate-producing microorganisms in the gut, the method comprising administering to an individual in need thereof a composition, food, dietary supplement, or pharmaceutical preparation of the present application.

[0066] In the present application, an "individual" includes a human or a non-human animal. Exemplary human individuals include a human individual (referred to as a patient) suffering from a disease or condition, e.g., a disease or condition described herein, or a normal individual. In the present application, a "non-human animal" includes all vertebrates, e.g., non-mammals (e.g., birds, amphibians, reptiles) and mammals, e.g., non-human primates, farm animals, and / or domestic animals (e.g., sheep, dog, cat, cow, pig, etc.).

[0067] In certain embodiments, the individual is an infant, a toddler, a child, or an adult (e.g., an adult over 60 years old).

[0068] In one aspect, the present application provides a composition, food, dietary supplement, or pharmaceutical preparation described above for use in alleviating, ameliorating, or reducing the occurrence of a disease or condition associated with a deficiency of butyric acid in the gut, or a deficiency of butyrate-producing microorganisms in the gut.

[0069] Methods of promoting butyrate production by Bifidobacterium lactis HN019

[0070] The present application also provides a method of promoting butyrate production by Bifidobacterium lactis HN019, comprising culturing Bifidobacterium lactis HN019 with galacto- oligosaccharide and isomerized lactose as carbon sources, wherein the weight ratio of galacto- oligosaccharide to isomerized lactose is 1:1-4:1 (e.g. 1:1-2:1, 2:1-3:1 or 3:1-4:1).

[0071] In certain embodiments, the method is used to promote butyrate production by Bifidobacterium lactis HN019 in vitro, e.g. to promote butyrate production by Bifidobacterium lactis HN019 under in vitro culture conditions.

[0072] In certain embodiments, the method is performed in vitro, comprising the steps of:

[0073] Step 1: providing a culture medium suitable for growth of Bifidobacterium lactis HN019, the culture medium comprising galacto-oligosaccharide and isomerized lactose as carbon sources;

[0074] Step 2: inoculating Bifidobacterium lactis HN019 into the culture medium of step 1 and culturing under anaerobic conditions suitable for growth of Bifidobacterium lactis HN019.

[0075] In certain embodiments, the culture medium in step 1 is MRS medium with addition of cysteine. In certain embodiments, the amount of cysteine added is 50-500 mg / L, e.g. 50-100 mg / L, 100-200 mg / L, 200-300 mg / L, 300-400 mg / L or 400-500 mg / L.

[0076] In certain embodiments, the total amount of galacto-oligosaccharide and isomerized lactose added in the culture medium of step 1 is 0.1%-10%, e.g. 0.1%-1%, 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9% or 9%-10%.

[0077] In certain embodiments, the conditions in step 2 are anaerobic conditions at 37-41 °C.

[0078] In certain embodiments, the method is used to promote butyrate production by Bifidobacterium lactis HN019 in a subject.

[0079] The method can be used for non-preventive or therapeutic purposes, or for preventive or therapeutic purposes.

[0080] In certain embodiments, the method is used to promote butyrate production by Bifidobacterium lactis HN019 in a subject, and for non-preventive or therapeutic purposes.

[0081] The present application also provides a composition for promoting Bifidobacterium lactis HN019 to produce butyric acid, the composition comprising galacto-oligosaccharide and isomerized lactose, wherein the weight ratio of galacto-oligosaccharide and isomerized lactose is 1:1-4:1 (for example 1:1-2:1, 2:1-3:1 or 3:1-4:1). Beneficial effects

[0082] The composition comprising galacto-oligosaccharide and isomerized lactose of the present application can effectively promote Bifidobacterium lactis HN019 to produce butyric acid. The use of the composition comprising galacto-oligosaccharide, isomerized lactose and Bifidobacterium lactis HN019 in human body can improve the intestinal micro-ecology of human body, reduce or relieve, improve the diseases or discomforts related to the deficiency of butyric acid in the intestine or the deficiency of butyric acid-producing microorganisms in the intestine. DETAILED DESCRIPTION

[0083] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application.

[0084] Example 1 Growth test of Bifidobacterium lactis HN019

[0085] Bifidobacterium lactis HN019 was obtained from Danisco. It was grown in de Man Rogosa Sharpe (MRS) broth (Difco, Detroit, MD) supplemented with 0.5 g / L L-cysteine and incubated anaerobically at 37°C for 24 h. Subsequently, the culture was subcultured twice on semi-synthetic MRS (sMRS) medium supplemented with 0.5 g / L L-cysteine and 1% (w / v) filter-sterilized glucose as the sole carbohydrate source.

[0086] After the second subculture, the culture was prepared for use as inoculum for the growth test described below. The various carbon source stock solutions were sterilized using a 0.22 micron filter and used at a 1% final concentration.

[0087] HN019 was inoculated at an OD600 reading of 0.1 and 1% of galacto-oligosaccharide (GOS), 1% of isomerized lactose (LOS) and 1% of GOS / LOS combination were added to the MRS + 0.05% cysteine medium, respectively, and the contents of lactic acid, acetic acid, propionic acid and butyric acid in the above-mentioned various combined culture solutions at 12 hours were determined by high performance liquid chromatography (HPLC).

[0088] As shown in Table 1, the butyric acid production of GOS / LOS 1:1 and 4:1 as carbon source was significantly higher than that of GOS or LOS as carbon source alone; the concentrations of other organic acids and total organic acids of GOS / LOS combination group were between those of GOS group and LOS group.

[0089] Table 1 Organic acid content in culture solution

[0090] Lactic acid Acetic acid Propionic acid Butyric acid Total organic acids LOS 0.034 ± 0.002 0.238 ± 0.002 ND ND 0.272 ± 0.004 GOS 0.374 ± 0.007 1.081 ± 0.014 ND 0.084 ± 0.004 1.539 ± 0.017 GOS / LOS 1:1 0.216 ± 0.019 0.650 ± 0.080 ND 0.248 ± 0.012*# 1.115 ± 0.111 GOS / LOS 4:1 0.176 ± 0.071 0.658 ± 0.002 ND 0.248 ± 0.009*# 1.082 ± 0.078 GOS / LOS 9:1 0.109 ± 0.003 0.699 ± 0.023 ND ND 0.807 ± 0.019

[0091] *: vs LOS, p < 0.05; #: vs GOS, p < 0.05

[0092] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those skilled in the art, and it is impossible to enumerate all the embodiments. Any obvious changes or variations derived from the technical solutions of the present application are still within the protection scope of the present application.

Claims

DEPCT6611 / 07 / 25651. Composition consisting of galactooligosaccharide, lactulose, and Bifidobacterium lactis HN019 in a weight ratio of galactooligosaccharide to lactulose of 1:1-4:1; ideally, the composition should contain 10⁶ Cfu / 100g - 10⁻¹² Cfu / 100g of Bifidobacterium lactis HN019; ideally, the composition should also include an exepiant or dispersing agent; ideally, in the composition, galactooligosaccharide and lactulose together constitute at least 50% by weight.

1. The food contains the components according to claim 1; where appropriate, the food is liquid, semi-liquid, solid or semi-solid; where appropriate, the food is a formula such as infant formula or infant foods supplement; where appropriate, the food is a dairy product, chocolate or chocolate products or grain product. 3.

4. Dietary supplements containing the components of claim 1; where appropriate, the dosage of the dietary supplement is selected from the group of pills, capsules, tablets, granules and liquid preparations; where appropriate, the dietary supplement also contains nutritional ingredients selected from the group of proteins, carbohydrates, lipids, minerals, vitamins, amino acids, unsaturated fatty acids, polyphenols, phytosterols, and mixtures thereof.

6. Use of the components under claim 1 for the preparation of food or dietary supplements where the food or dietary supplements are used to alleviate, cure, or reduce the occurrence of disease or discomfort, and the disease or discomfort is associated with insufficient butyric acid in the intestines, or with insufficient butyric acid-producing microorganisms in the intestines.A method for supporting the production of butyric acid by Bifidobacterium lactis HN019 in combination with the use of galactooligosaccharides and lactulose as carbon sources for culturing Bifidobacterium lactis HN019 where the weight ratio of galactooligosaccharides to lactulose is 1:1-4:1; appropriately, the method was used to support the production of butyric acid by Bifidobacterium lactis HN019 in vitro.

8. The method according to claim 7 where the method was performed in vitro and in laboratory settings. The process involves the following steps: Step 1: Preparation of a suitable culture medium for the growth of Bifidobacterium lactis HN019, consisting of galactooligosaccharides and lactulose as a carbon source; and Step 2: Inoculating Bifidobacterium lactis HN019 into the culture medium from Step 1 and cultivating it under anaerobic conditions suitable for its growth.Composition for supporting the production of butyric acid by Bifidobacterium lactis HN019, consisting of galacto-oligosaccharides and lactulose, where the weight ratio of galacto-oligosaccharides to lactulose is 1:1-4:1-----------------------------------------------------------1. Composition consisting of galacto-oligosaccharides, lactulose, and Bifidobacterium lactis HN019, where the weight ratio of galacto-oligosaccharides to lactulose is 1:1-4:1 The lactulose ratio is 1:1-4:1; ideally, the composition should include 10'Cfu / 100g - 10'Cfu / 100g of Bifidobacterium lactis HN019; ideally, the composition should also include excipients or dispersants; ideally, the composition should include at least 50% by weight of the components in galacto-oligosaccharide and lactulose; ideally, the composition should consist of galacto-oligosaccharide, lactulose and Bifidobacterium lactis HN0192.Foods under Reputation 1 are those in which the food is used to alleviate, ameliorate, or reduce the occurrence of disease or discomfort, and the disease or discomfort is associated with insufficient butyric acid in the intestines, or with insufficient butyric acid-producing microorganisms in the intestines.

3. Foods under Reputation 2 are those in which the food is used to alleviate, ameliorate, or reduce the occurrence of disease or discomfort, and the disease or discomfort is associated with insufficient butyric acid production in the intestines. 4.Dietary supplements containing the components according to claim 1: where appropriate, the dosage of the dietary supplement is selected from the group of pills, capsules, tablets, granules and liquid preparations; where appropriate, the dietary supplement also contains nutritional ingredients selected from the group of proteins, carbohydrates, lipids, minerals, vitamins, amino acids, unsaturated fatty acids, polyphenols, phytosterols, and mixtures thereof. Dietary supplements according to claim 4 where the dietary supplement is used to alleviate, cure or treat a disease or discomfort, or to reduce the occurrence of a disease or discomfort, and the disease or discomfort is associated with insufficient butyric acid in the intestines, or with insufficient butyric acid-producing microorganisms in the intestines.The use of the components under Claim 1 for the preparation of food or dietary supplements where the food or dietary supplement is used to alleviate, cure, treat or reduce the occurrence of disease or discomfort, and the disease or discomfort is associated with insufficient butyric acid in the intestines, or with microorganisms that produce insufficient butyric acid in the intestines.

7. A method for supporting the production of butyric acid by Bifidobacterium lactis HN019 comprising the use of galacto-oligosaccharide and lactulose as a carbon source for culturing Bifidobacterium lactis HN019 where the weight ratio of galacto-oligosaccharide to lactulose is 1:14:1; appropriately, the method is used to support the production of butyric acid by Bifidobacterium lactis HN019 in vitro.8.The method pursuant to claim 7, where the procedure is performed in vitro and consists of the following steps: Step 1: Preparation of a culture medium suitable for the growth of Bifadobacterium lactis HN019, and the culture medium consists of galacto-oligosaccharide and lactulos as a carbon source; and Step 2: Inoculating Bjfdobacterium lactis HN019 into the culture medium of step 2.

1. Cultivating Biidobacterium lactis HN0199 under anaerobic conditions is ideal for its growth. The components supporting the production of butyric acid are galactose-containing Biidobacterium lactis HN0199, which consists of galacto-oligosaccharides and lactulose, with a weight ratio of galacto-oligosaccharides to lactulose of 1:1-4.