Composition for improving intestinal flora

Abstract

The present invention provides a composition that improves intestinal bacterial flora by promoting the growth of Parabacteroides bacteria. The composition is characterized by containing whey melanoidin.

Description

[Technical field]

[0001] The present invention relates to a composition for improving intestinal flora. In particular, the present invention relates to a composition for improving intestinal flora by promoting the growth of Parabacteroides bacteria, in particular by increasing the occupancy rate of Parabacteroides bacteria in the intestinal flora. [Background technology]

[0002] The intestinal flora provides nutrients to the host to maintain its health, and plays an important role in intestinal homeostasis and the host's immune response. Many studies have shown a relationship between an imbalance in the intestinal flora and diseases such as obesity, type 2 diabetes, and colitis, and it is becoming widely known that maintaining a balance in the intestinal flora is important for maintaining and improving health.

[0003] In recent years, it has become clear that intestinal barrier dysfunction, known as leaky gut, can lead to various diseases and poor health (Leaky Gut Syndrome). The term leaky gut refers to an intestine in which holes form in the intestinal mucosa, allowing foreign substances (bacteria, viruses, proteins, etc.) to leak into the bloodstream. The intestine, where approximately 70% of immune cells are said to be located, is thought to have three different barrier functions. The first is an environmental barrier involving the intestinal flora, which functions to eliminate highly pathogenic bacteria by maintaining a healthy intestinal flora. The second is a physical barrier that prevents the intrusion of harmful substances by tightly closing the connections between the cells that make up the intestinal tract, and forms a mucus layer on the surface of the cells to prevent bacteria and various substances from coming into direct contact with intestinal cells. The remaining barrier is a biological barrier, which is believed to function through antimicrobial peptides and cells that control immunity.

[0004] Although the intestine is equipped with many defense systems, the barrier function of the intestine is weakened by various factors in daily life, such as lifestyle habits (e.g., high-fat diet, high-sugar diet, drinking alcohol, food additives, taking medicines, etc.) and stress. For example, in a large intestine with impaired barrier function, the intestine itself may become inflamed due to an excessive immune response, leading to the development of inflammatory bowel disease (Crohn's disease and ulcerative colitis). In addition, a decrease in the barrier function of the intestine is not limited to the onset of such intestinal inflammation, but is also thought to be related to the onset and progression of lifestyle-related diseases such as obesity, diabetes, and liver disease, as well as vascular disorders, cancer, allergies, Parkinson's disease, and depression, as well as the onset and progression of such diseases.

[0005] One of the measures to improve the various symptoms associated with such a decrease in intestinal barrier function is improvement of the intestinal bacterial flora. For example, Non-Patent Documents 1 to 3 report that Parabacteroides bacteria in the intestine improve insulin resistance and obesity through anti-inflammatory effects, and suppress tumor formation. Specifically, Non-Patent Document 1 describes that increasing the proportion of Parabacteroides bacteria, specifically P. goldsteinii, in the intestinal flora improves intestinal barrier function, and that the improvement of intestinal barrier function suppresses inflammation and has an effect of improving associated insulin resistance and weight gain. Non-Patent Document 2 describes that P. distasonis, which belongs to Parabacteroides, exerts an effect of suppressing obesity through the production of succinic acid and secondary bile acids in the intestine, and that as one of the mechanisms of this obesity suppression, secondary bile acids metabolized in the intestine by P. distasonis improves intestinal barrier function through farnesoid X receptor (FXR). Non-Patent Documents 4 and 5 also show that there is a negative correlation between pathologies such as multiple sclerosis and fatty liver and Parabacteroides bacteria in the intestine.

[0006] In recent years, it has been reported that melanoidins, which are browning products produced by the heat reaction (Maillard reaction) between amino compounds (proteins, peptides, amino acids, etc.) and carbonyl compounds (decomposition products of sugars and fats, etc.), have various useful physiological functions (Non-Patent Document 6). Specifically, melanoidins are irregular polymeric compounds with physicochemical properties similar to those of dietary fiber, and therefore have dietary fiber-like effects such as cholesterol-lowering effects and lactic acid bacteria-increasing effects, as well as glucose tolerance-improving effects, insulin secretion-promoting effects, angiotensin I-converting enzyme activity-inhibiting effects, lipase activity-inhibiting effects, antioxidant effects, active oxygen scavenging effects, and effects of suppressing the production of nitrosamines, which are carcinogens (Non-Patent Document 6). In addition, research on melanoidins derived from various foods has reported that some melanoidins are widely used by intestinal microorganisms, increase the production of short-chain fatty acids, and promote the proliferation of Bifidobacterium, Faecalibacterium, etc. (Non-Patent Document 7). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Gut commensal Parabacteroides goldsteinii plays a predominant role in the anti-obesity effects of polysaccharides isolated from Hirsutella sinensis (Gut. 2019 Feb;68(2):248-262) [Non-Patent Document 2] Parabacteolides distasonis alleviates obesity and metabolic dysfunctions via production of succinate and secondary bile acids (Cell Rep. 2019 Jan 2;26(1):222-235) [Non-Patent Document 3] Parabacteroides distasonis attenuates toll-like receptor 4 signaling and Akt activation and blocks coon tumor formation in high-fat diet-fed azoxymethane-treated mice.(Koh et al., Int. J. Cancer 143, 1797-1805) [Non-Patent Document 4] Gut bacteria from multiple sclerosis patients modulate human T cells and worsening symptoms in mouse models.(Cekanaviciute et al., Proc. Natl. Acad. Sci. USA 114, 10713-10718) [Non-Patent Document 5] Gut microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients unveiled by an integrated meta-omics-based approach.(Del Chierico et al, Hepatology 65, 451-464) [Non-Patent Document 6] Riyo Miura, "Physiological Function of Melanoidin", Journal of the Brewing Society of Japan, Vol. 97 (2002) No. 4, pp. 253-256 [Non-Patent Document 7] Bioactivity of food melanoidins is mediated by gut microbiota (S. Perez-Burillo, et al. Food Chemistry 316 (2020) ) [Non-Patent Document 8] Naoko Ikuta et al., "Evaluation of the effects of dietary fiber using the cultured human intestinal microbiota model KUHIMM," New Food Industry 2018 Vol. 60, No.5, P.37-43 [Non-Patent Document 9] Akira Osawa, "The relationship between digestive diseases and intestinal microbiota - clinical significance and impact on treatment -" "2. Development and use of human intestinal models", Progress in Medicine Vol. 42, No.3, 2022, P.215-220 [Non-Patent Document 10] "Bile acids produced by intestinal bacteria related to healthy longevity: A new bile acid biosynthetic pathway that increases in the microbiome of centenarians" Keio University School of Medicine and RIKEN press release, July 30, 2021 (https: / / www.keio.ac.jp / ja / press-releases / files / 2021 / 7 / 30 / 210730-1.pdf) [Non-Patent Document 11] Gut Parabacteroides merdae protects against cardiovascular damage by enhancing branched-chain amino acid catabolism | Nature Metabolism 2022 Oct;4(10):1271-1286. doi: 10.1038 / s42255-022-00649-y. Epub 2022 Oct 17. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a composition for improving the intestinal flora. More specifically, an object of the present invention is to provide a composition that improves the intestinal flora by promoting the growth of Parabacteroides bacteria, in particular by increasing the occupancy rate of Parabacteroides bacteria in the intestinal flora. [Means for solving the problem]

[0009] In recent years, many studies have been conducted on the relevance to clinical practice and the effects of various foods on the intestinal microbiota using in vitro tests using a cultured human intestinal microbiota model (KUHIMM) developed at Kobe University. These studies have also provided a wealth of useful information on the safety and functionality of food ingredients mediated by intestinal bacteria (Non-Patent Documents 8 and 9).

[0010] In order to solve the above problems, the present inventors have conducted extensive research using the cultured human intestinal flora model (hereinafter also simply referred to as "human intestinal model") and have found that a composition containing melanoidin (whey melanoidin) obtained from whey has a growth promoting effect on Parabacteroides bacteria and can increase the occupancy rate of Parabacteroides bacteria in mammalian intestinal flora. Therefore, it is believed that such a composition can be effectively used to improve the intestinal flora by increasing the occupancy rate of Parabacteroides bacteria, and to maintain or inhibit the decline of the intestinal barrier function.

[0011] The present invention was completed based on these findings and includes the following embodiments. (I) Composition for improving intestinal flora (I-1) A composition for improving intestinal flora that promotes the proliferation of Parabacteroides bacteria, characterized by containing whey melanoidin. The composition is also a composition for promoting the proliferation of Parabacteroides bacteria used to improve the intestinal flora of a mammal. Therefore, the invention can be rephrased as "a composition for promoting the proliferation of Parabacteroides bacteria used to improve the intestinal flora, characterized by containing whey melanoidin."

[0012] (I-2) A composition for improving intestinal flora according to (I-1), which increases the occupancy rate of Parabacteroides bacteria in intestinal flora. The composition is also a composition for increasing the occupancy rate of Parabacteroides bacteria in the intestinal flora, which is used to improve the intestinal flora of a mammal. Therefore, the invention can be rephrased as "a composition for increasing the occupancy rate of Parabacteroides bacteria in the intestinal flora, which is used to improve the intestinal flora, characterized by containing whey melanoidin."

[0013] (I-3) The composition for improving intestinal flora according to (I-1) or (I-2), wherein the Parabacteroides bacterium is at least one species selected from the group consisting of Parabacteroides goldsteinii, Parabacteroides distasonis, Parabacteroides goldsteinii, Parabacteroides merdae, and Parabacteroides johnsonii.

[0014] (I-4) A composition for improving intestinal flora, according to any one of (I-1) to (I-3), which is used for inhibiting or improving a decline in intestinal barrier function. This invention can be rephrased as "a composition for inhibiting or improving a decline in intestinal barrier function, characterized by containing whey melanoidin."

[0015] (I-5) The composition for improving intestinal flora according to any one of (I-1) to (I-4), wherein the whey melanoidin has a molecular weight cutoff of 8 kDa or more.

[0016] (II) Use of whey melanoidin or whey melanoidin-containing composition for (II-1) Use of whey melanoidin or a whey melanoidin-containing composition for preparing a composition for improving intestinal flora. (II-2) The use according to (II-1), wherein the improvement of the intestinal flora is promotion of the proliferation of Parabacteroides bacteria in the intestinal flora or an increase in the occupancy rate of Parabacteroides bacteria in the intestinal flora. (II-3) The use according to (II-2), wherein the Parabacteroides bacterium is at least one species selected from the group consisting of Parabacteroides goldsteinii, Parabacteroides distasonis, Parabacteroides goldsteinii, Parabacteroides merdae, and Parabacteroides johnsonii. (II-4) The use according to any one of (II-1) to (II-3), wherein the composition for improving intestinal flora is a composition for inhibiting or improving a decline in intestinal barrier function. This method can be rephrased as "use of whey melanoidin or a composition containing whey melanoidin for preparing a composition for inhibiting or improving a decline in intestinal barrier function."

[0017] (III) Uses of whey melanoidin or whey melanoidin-containing composition (III-1) Whey melanoidin or a whey melanoidin-containing composition for use in improving intestinal flora. (III-2) The whey melanoidin or whey melanoidin-containing composition according to (III-1), wherein the improvement of the intestinal flora is promotion of the growth of Parabacteroides bacteria in the intestinal flora or an increase in the occupancy rate of Parabacteroides bacteria in the intestinal flora. (III-3) The whey melanoidin or whey melanoidin-containing composition according to (III-1) or (III-2), wherein the Parabacteroides genus bacterium is at least one species selected from the group consisting of Parabacteroides goldsteinii, Parabacteroides distasonis, Parabacteroides goldsteinii, Parabacteroides merdae, and Parabacteroides johnsonii. (III-4) Whey melanoidin or a whey melanoidin-containing composition for use in inhibiting or improving the decline of intestinal barrier function. Effect of the Invention

[0018] According to the present invention, the proliferation of Parabacteroides bacteria in the intestinal flora of mammals, particularly humans, can be promoted, thereby increasing the occupancy rate. In other words, according to the present invention, the proliferation of Parabacteroides bacteria in the large intestine of a mammal can be promoted, thereby improving the intestinal flora. Furthermore, according to the present invention, the improvement of the intestinal flora is effective in suppressing or improving the decline of the intestinal barrier function, and it can be expected to have a preventive or ameliorative effect on diseases related to the decline of the intestinal flora or the intestinal barrier function. [Brief description of the drawings]

[0019] [Figure 1] In Experimental Example 1, using a human intestinal model, the occupancy rate (%) of Parabacteroides bacteria in the whey melanoidin-added group (test group) (n=4) was measured over the culture period (24 hours) and the results are shown below. In the figure, "*" indicates that there is a significant difference (p < 0.05) compared to the occupancy rate at the start of culture (0 h). In the box plot in Figure 1, the top of the vertical line indicates the maximum value, the bottom indicates the minimum value, the box indicates the interquartile range, and the horizontal line within the box indicates the median. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The intestinal flora-improving composition of the present invention (hereinafter also referred to as "the composition") is characterized by containing whey melanoidin.

[0021] (1) Whey melanoidin and its preparation Whey melanoidin is a brown substance produced by heat-treating a composition containing whey as a raw material (hereinafter referred to as the "whey-containing composition"). It is a mixture of irregular polymeric compounds produced by the combination of amino compounds (proteins, peptides, amino acids, etc.) and carbonyl compounds (reducing sugars, fat decomposition products, etc.) present in the whey-containing composition to be heat-treated through the amino-carbonyl reaction (Maillard reaction).

[0022] The whey melanoidin targeted by the present invention is, but is not limited to, a collection of polymeric compounds with a molecular weight of at least 8 kDa that can be fractionated by an ultrafiltration membrane and / or a dialysis membrane.

[0023] The whey-containing composition used to prepare whey melanoidin may be any composition that uses whey as a raw material and contains at least a protein, which is an amino compound, and a reducing sugar as a carbonyl compound bonded thereto.

[0024] Whey is a water-soluble component (whey) that remains when fat, casein, fat-soluble vitamins, etc. are removed from milk. However, whey may contain fat, casein, and / or fat-soluble vitamins that have not been completely removed. As the whey used as a raw material, in addition to whey concentrate (sweet whey, acid whey, etc.), its concentrate, its dried product (whey powder, etc.), and its frozen product can be used. In addition, desalted whey, whey protein concentrate (WPC), protein concentrated whey powder, and whey protein purified product (WPI) can also be used. In addition, whey includes, for example, cheese whey and rennet whey (also called sweet whey) obtained as a by-product when producing natural cheese and rennet casein, and casein whey, acid whey, and quark whey obtained when producing fermented milk and quark.

[0025] Generally, whey contains 10-15% by mass of protein and 70-85% by mass of lactose calculated as solid content. As whey has a relatively high lactose content relative to the total protein content, the total protein content and lactose content in the whey-containing composition can be adjusted by supplementing the protein content by adding whey protein separately or reducing the lactose content. Preferably, a method of reducing the lactose content is adopted.

[0026] In this specification, the term "solid content" refers to the components excluding water. As used herein, the term "solids concentration" refers to the proportion of solids contained in a subject (e.g., a whey-containing composition), and is expressed as the proportion (mass %) of solids in the total amount when the total amount (wet mass) of the subject is taken as 100 mass %.

[0027] In this specification, the content of a component in a target object (e.g., a whey-containing composition) calculated as solid content (also referred to as "solid content equivalent") means the ratio of the component contained in the target object calculated as solid content. Specifically, when the total amount (dry mass) of solid content of the target object is taken as 100 mass%, it is expressed as the ratio (mass%) of the component to the total amount of solid content.

[0028] The mass and concentration of solids contained in the target object (solids concentration) can be determined by the normal pressure heating and drying method (sand mixing method). Specifically, after a constant weight is determined with sea sand and a glass rod placed in a weighing dish, the test sample and distilled water are added and the dish is dried in a dryer at 99°C for 4 hours. The moisture content of the test sample is then determined from the difference in weight before and after drying. From the moisture content thus measured, the mass of solids contained in the test sample (before drying) can be determined, and the solids concentration can then be calculated.

[0029] The ratio of the total protein content and lactose content in the whey-containing composition is not limited, but the ratio of the lactose content per 10 parts by mass of the total protein content is in the range of 45 to 61 parts by mass, preferably 48 to 58 parts by mass, and more preferably 50 to 56 parts by mass.

[0030] Here, the protein can mainly include proteins contained in whey, and these include whey proteins, specifically α-lactalbumin, β-lactoglobulin, immunoglobulin and lactoferrin. The amino acids constituting whey proteins can include, in descending order of abundance, glutamic acid, aspartic acid, leucine, lysine, threonine, isoleucine, valine, proline, alanine, serine, phenylalanine, tyrosine, arginine, methionine, cystine, histidine, glycine and tryptophan. In addition, casein, which is a milk protein, may remain in whey. Therefore, casein may be contained in the whey-containing composition as long as it does not interfere with the effects of the present invention.

[0031] A method for reducing the lactose content in a whey-containing composition includes a method for removing or decomposing lactose. A method for selectively decomposing lactose contained in a whey-containing composition is preferred. Although not limited thereto, a method for treating a whey-containing composition with a lactase (e.g., lactase) can be mentioned. Lactase is an enzyme that decomposes lactose into glucose and galactose. Therefore, a whey-containing composition that has been subjected to lactose decomposition treatment (lactase treatment) using lactase contains glucose and galactose as reducing sugars in addition to lactose. Although not limited thereto, it is preferred that lactose in the whey-containing composition is decomposed and reduced by about 10 to 20% by mass by such lactose decomposition treatment.

[0032] As long as the production of whey melanoidin, which is the subject of the present invention, is not impaired, the whey-containing composition may contain lipids, glucose, galactose, amino acids, vitamins, and / or minerals as components other than protein and lactose. The lipid may be any lipid-containing raw material, regardless of origin, such as residual matter in whey that could not be completely removed during whey preparation, raw milk, milk or dairy products (concentrated milk, reconstituted milk, whole milk powder, cream, butter, butter oil, etc.), vegetable oils and fats (safflower oil, corn oil, cottonseed oil, sesame oil, rice oil, olive oil, etc.), and processed products made from vegetable oils (margarine, shortening, fast spread, etc.). Preferably, the lipid is derived from milk. In addition, the lipid content in the whey-containing composition is not limited, but may be in the range of 10 to 50 parts by mass relative to 10 parts by mass of the total protein content in the whey-containing composition. It is preferably 20 to 40 parts by mass, and more preferably 30 to 35 parts by mass.

[0033] Whey melanoidin can be prepared by heat treating such a whey-containing composition at a temperature of 100°C or higher. The solid content concentration of the whey-containing composition to be subjected to the heat treatment is not limited, but is preferably low in order to efficiently produce whey melanoidin by the Maillard reaction. The solid content concentration can be appropriately adjusted depending on the shape of the intestinal flora-improving composition to be finally produced. For example, when producing a semi-solid composition such as a paste or a solid composition such as a block, it is preferable to adjust the solid content concentration of the whey-containing composition to be subjected to the heat treatment to 10% by mass or more in terms of efficiency. It is preferably 50 to 70% by mass, more preferably 70 to 75% by mass.

[0034] The heat treatment can be carried out by treating the whey-containing composition at a temperature of 100°C or higher, preferably 105 to 120°C. Although not limited thereto, in order to efficiently carry out the heat treatment, it is preferable to carry out the heat treatment while stirring so that the entire whey-containing composition is heated as evenly as possible. In addition, although not limited, the heat treatment is preferably performed under atmospheric pressure, not under reduced pressure, so as to promote the Maillard reaction. The heat treatment may be performed as long as whey melanoidin can be produced, and although not particularly limited, it is preferable to heat and concentrate the whey-containing composition so that the solid content concentration of the whey-containing composition after the heat treatment is 55% by mass or more. The solid content concentration of the whey-containing composition after the heat concentration can be appropriately set according to the shape of the present composition to be finally produced. For example, although not limited, the solid content concentrations of the present composition in a paste form (semi-liquid, non-formed form), soft solid form, and hard solid form are about 55 to 74% by mass, about 75 to 79% by mass, and about 80 to 90% by mass, respectively, and these can be used as a reference.

[0035] By carrying out such heat treatment, melanoidin is produced by the amino-carbonyl reaction (Maillard reaction), and the resulting heat-treated whey-containing composition contains whey melanoidin. The present composition only needs to contain whey melanoidin as an active ingredient, and the heat-treated product containing whey melanoidin can be used as is, or a concentrated version of the heat-treated product can be used. The heat-treated product can also be used after being subjected to various purification processes such as membrane treatment to remove unreacted residues (e.g., lactose, etc.) and by-products of the amino-carbonyl reaction.

[0036] Whey melanoidin preferably has a molecular weight cutoff of 8 kDa or more. Therefore, the membrane treatment may be any treatment (molecular weight fractionation purification treatment) that utilizes the difference in molecular weight to remove low molecular weight compounds and the like to purify the whey melanoidin, and examples thereof include dialysis treatment using a semipermeable membrane and ultrafiltration membrane treatment. The dialysis treatment and the ultrafiltration membrane treatment may be performed alone, but they can also be performed in combination. For the dialysis treatment, a semipermeable membrane made of regenerated cellulose with a molecular weight cutoff (MWCO) of 6 to 8 kDa can be suitably used. For the ultrafiltration membrane treatment, any membrane with a nominal molecular weight cutoff (NMWL) of 3 to 10 kDa can be used, and for example, a membrane made of polyethersulfone (PES), cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, etc. can be arbitrarily selected and used. By fractionation by such membrane treatment, whey melanoidin (aggregate of polymer compounds) with a molecular weight in the range of 8 kDa or more can be purified and recovered.

[0037] In order to carry out the molecular weight fractionation purification treatment by membrane treatment more efficiently and effectively, a treatment may be carried out in advance to decompose, assimilate, or remove components other than whey melanoidin contained in the heat-treated whey-containing composition before the membrane treatment. Among these, the decomposition treatment may be any treatment that can selectively decompose lactose contained as an unreacted residue in the heat-treated product, and examples of such treatments include hydrolysis treatment using lactase or other lactase-degrading enzymes. In addition, by subsequently treating the heat-treated product with a microorganism such as yeast, monosaccharides remaining in the heat-treated product and monosaccharides generated by lactose decomposition can be assimilated. In addition, the assimilated substances thus generated can be efficiently removed by subsequent purification treatment by membrane treatment.

[0038] (2) The composition and its preparation The present composition is characterized by containing the above-mentioned whey melanoidin. The content of whey melanoidin in the present composition may be within a range in which the composition has the effect of increasing the occupancy rate of Parabacteroides bacteria in the human intestinal flora, and may be appropriately selected and prepared within the range of 10 to 100% by mass.

[0039] The whey melanoidin content of the present composition can also be expressed as the "glycine-melanoidin equivalent" converted into the glycine-melanoidin content. The method for determining the glycine-melanoidin equivalent is as described in detail in Example 1(3) below, and can be carried out according to the description therein. When expressing the whey melanoidin content in the present composition based on this glycine-melanoidin equivalent, it is not limited, and can be appropriately selected from the range of, for example, 1 mg to 900 mg in terms of glycine-melanoidin equivalent per 1 g of the present composition. These ranges can be appropriately selected from the ranges of, for example, 1 to 500 mg, 1 to 100 mg, 1 to 50 mg, and 1 to 10 mg.

[0040] Whether or not the composition has the effect of increasing the occupancy rate of Parabacteroides bacteria in the human intestinal flora can be evaluated by an in vitro test using a cultured human intestinal flora model (human intestinal model) that mimics the human intestinal flora, for example, the human intestinal model (KUHIMM) developed by Kobe University (Japan). The evaluation method by the in vitro test using the KUHIMM will be described in detail in the experimental examples described below. If this in vitro test shows that anaerobically culturing a diluted fecal solution (test medium) to which a composition containing whey melanoidin has been added promotes the growth of Parabacteroides bacteria and increases the proportion (occupancy rate) of Parabacteroides bacteria in the total number of bacteria, then the composition can be determined to be the composition containing an effective amount of whey melanoidin.

[0041] In the present invention, the term "intestinal flora" refers to the flora living in the intestines of an organism and flora similar thereto. The organism is preferably a mammal, more preferably a human. The flora similar to the flora living in the intestines of an organism is a culture model of intestinal flora, such as the human intestinal tract model described above. When the proliferation of Parabacteroides bacteria is promoted and an increase in the number of Parabacteroides bacteria is observed in the culture model of intestinal flora, it is observed that the same phenomenon (effect) occurs in the flora living in the intestines of an organism.

[0042] In the present invention, the promotion of the proliferation of Parabacteroides bacteria includes increasing the proportion of Parabacteroides bacteria in the intestinal flora. Here, the "proportion" refers to the proportion of the total bacteria detected in the intestinal flora, and can also be referred to as "occupancy rate". The proliferation promotion may simultaneously increase or decrease the proportion of bacteria other than Parabacteroides bacteria in the intestinal flora, so long as the occupancy rate of Parabacteroides bacteria increases. In other words, it includes not only the case where the proliferation of Parabacteroides bacteria is higher than the proliferation of other bacteria, thereby increasing the occupancy rate of Parabacteroides bacteria, but also the case where the amount of Parabacteroides bacteria increases relatively due to the decrease of other bacteria, thereby increasing the occupancy rate of Parabacteroides bacteria.

[0043] The increase in the occupancy rate of Parabacteroides bacteria in the intestinal flora means an increase relative to the occupancy rate before application of the present composition. The degree of increase is not particularly limited as long as an increase is observed, but includes, for example, a case where the occupancy rate before application of the present composition is set to 100, and the value is more than 100, preferably 110 or more, more preferably 150 or more.

[0044] In the analysis of the composition of bacteria in such intestinal flora (bacterial flora analysis), shotgun metagenomic analysis targeting the whole genome of the bacterial flora and 16S rRNA metagenomic analysis targeting the 16S rRNA gene are generally used. In the present invention, the latter 16S rRNA metagenomic analysis is preferably used because it can be performed relatively easily.

[0045] The 16S rRNA metagenomic analysis can be carried out by amplicon sequencing, which involves extracting DNA from a sample containing various bacteria, amplifying a specific region (third to fourth variable regions) of the bacterial 16S rRNA gene by PCR, and analyzing the resulting PCR amplification product (amplicon). The amplicon sequencing can be carried out using a next-generation sequencer (Miseq: manufactured by Illumina). Next, the vast amount of base sequence data obtained by sequencing is analyzed by computer. The bacterial flora composition analysis (16S amplicon analysis) can be carried out using amplicon sequence data analysis software (e.g., QIIME2) according to the manual. QIIME2 is an open source analysis pipeline that compiles software necessary for amplicon sequence analysis (https: / / qiime2.org / ).

[0046] By analyzing the composition of the intestinal flora in this way, the total number of bacteria in the intestinal flora and the number of Parabacteroides bacteria can be calculated, and then the proportion of Parabacteroides bacteria in the total number of bacteria in the intestinal flora can be calculated.

[0047] The genus Parabacteroides, that is, bacteria belonging to Parabacteroides, may be any bacteria present in the intestinal flora of mammals, preferably humans, and includes Parabacteroides goldsteinii (Non-Patent Document 1) and Parabacteroides distasonis (Non-Patent Documents 2 and 3), which have been reported to improve intestinal barrier function; Parabacteroides goldsteinii, which was isolated from the feces of centenarians and confirmed to have the ability to produce a secondary bile acid called isoalloLCA (isoallolithocholic acid) (Non-Patent Document 10); Parabacteroides merdae, which may prevent cardiovascular diseases associated with obesity (Non-Patent Document 11); and Parabacteroides johnsonii.

[0048] The composition is used to improve the composition of the intestinal flora so that the proportion (occupancy) of Parabacteroides bacteria in the intestinal flora increases. By improving the intestinal flora in this way, it becomes possible to maintain or promote health in response to an increase in the number of Parabacteroides bacteria. The maintenance or promotion of health includes maintaining or promoting health by preventing and / or improving a disease or pathology. The target disease or pathology may be a disease or pathology that can be prevented or improved by the proliferation of Parabacteroides bacteria in the intestinal flora, and includes, but is not limited to, a decrease or failure of the intestinal barrier, a disease or pathology derived therefrom (for example, inflammation, glucose metabolism disorder, obesity, hepatic steatosis, allergic disease, and intestinal barrier syndrome such as depression), and cancer.

[0049] The subject (recipient, ingestor) of the present composition is a mammal, preferably a human. The subject may be any of adults, children, infants, and newborns (including low birth weight infants). The gender is not particularly limited. The dosage (intake amount) can be appropriately set according to the type, age, gender, condition, etc. of the target mammal. Although not limited, the dosage (intake amount) can be appropriately set and adjusted within the range of 0.1 to 10 g / day in terms of whey melanoidin dosage (intake amount).

[0050] The composition is preferably administered (ingested) orally, and parenterally includes enteral administration.

[0051] When the composition is prepared as an oral composition, it is preferable that the composition is prepared as a food or drink composition, a quasi-drug composition, a pharmaceutical composition, etc. The food or drink composition includes a nutritional composition. The nutritional composition includes an enteral nutrient that is administered into the intestine through a tube such as an enterostomy. The form of these oral compositions is not particularly limited, and can be arbitrarily selected from conventional forms such as solid, powder, granule, cream, gel, sol, liquid, suspension, etc., depending on the type, administration form, and purpose.

[0052] In addition to the whey melanoidin described above, the food and beverage composition may contain, to the extent that its action and effect are not impaired, animal and vegetable proteins; fats and oils derived from animals and plants; carbohydrates such as sugars, starch, and dietary fiber; vitamins; minerals; and other seasonings such as sweeteners, acidulants, and spices, stabilizers, and coloring agents that are normally added to foods and beverages.

[0053] Whey melanoidin can be said to be "a food ingredient that has a beneficial effect on the host and improves the host's health by enhancing the proliferation of Parabacteroides bacteria in the intestinal flora," and in this sense can also be positioned as a prebiotic. Therefore, a food and drink composition containing whey melanoidin can be said to be a food and drink containing prebiotics (prebiotic food and drink). The food and drink compositions that are the subject of the present invention include food and drink compositions (e.g., functional food, food for specified health use, etc.) that are permitted to display, in addition to the above, a description regarding the proliferation of Parabacteroides bacteria, the effects associated with said proliferation, and its uses (uses related to health promotion). In addition, one aspect of the food and beverage composition includes a feed composition, which is food and beverage for mammals other than humans, such as pets (dogs, cats, rabbits, etc.), test animals (mice, rats, rabbits, monkeys, etc.), and livestock (cows, horses, etc.).

[0054] When the present composition is prepared as a quasi-drug composition or a pharmaceutical composition, in the case of oral administration, it can be formulated into solid preparations such as powders, granules, tablets, and capsules, or liquid preparations such as solutions, syrups, suspensions, and emulsions, etc. In addition, in the case of parenteral administration, it can be formulated into a form of a tube-administered preparation such as an enterofasmia.

[0055] In the formulation, in addition to the whey melanoidin described above, ingredients such as excipients, pH adjusters, colorants, and flavorings that are usually used in formulations can be used. It is also possible to use other medicinal ingredients, or ingredients that have a known or future-discovered effect of promoting the growth of Parabacteroides bacteria or a preventive or ameliorative effect on a specific disease or pathology, and other medicines in combination. In addition, formulation can be carried out by a known method as appropriate depending on the dosage form.

[0056] As described above, in this specification, the terms "comprise" and "contain" include the meanings of "consisting of" and "consisting essentially of". EXAMPLES

[0057] In the following, the present invention will be described using experimental examples to aid in understanding the configuration and effects of the present invention. However, the present invention is not limited by these experimental examples. The following experiments were carried out at room temperature (25±5°C) and atmospheric pressure conditions unless otherwise specified. In the following, "%" means "% by mass" and "parts" means "parts by mass" unless otherwise specified.

[0058] Example 1: Production of whey melanoidin (1) Raw materials Whey powder: Dried whey powder (100% whey), total solids 99%, fat content 2%, lactose content 80%, protein content 12% (manufactured by Meiji Co., Ltd.).

[0059] Cream: Total solids 52%, fat content 47%, lactose content 3%, protein content 2% (manufactured by Meiji Co., Ltd.). In addition, cream falls under the provision of the Ministerial Ordinance on the Compositional Standards of Milk and Dairy Products (Milk Ordinance), which states that "cream is obtained by removing all ingredients other than milk fat from raw milk, cow's milk or special cow's milk."

[0060] Lactase: Saphera (registered trademark) 2600L (manufactured by Novozymes Japan Co., Ltd.). Yeast: Made by Saccharomyces cerevisiae LESAFFRE.

[0061] Ultrafiltration membrane: Nominal molecular weight cutoff (NMWL) 3-10 kDa, Omega membrane, Pall. Ultrafiltration membrane (UP20): NMWL200KDa, manufactured by Advance Tokyo. Ultrafiltration membrane (UK10): NMWL10KDa, manufactured by Advance Tokyo. Dialysis membrane: Molecular weight cutoff (MWCO): 6-8 kDa, semi-permeable membrane made of regenerated cellulose, manufactured by SPECTRUM.

[0062] Melanoidin preparation: Glycine-melanoidin synthesized by the following method was used. [Method of synthesizing glycine-melanoidin] A mixture of 1M D-glucose, 1M glycine, and 0.1M NaHCO3 was heated at 90°C for 72 hours to obtain a brown solution, which was then adjusted to pH 2.5 and allowed to stand. The resulting precipitate was collected by centrifugation and redissolved in water at pH 8. This procedure was repeated three times. The final solution was filtered through an ultrafiltration membrane (UP20) to remove fractions over 200 kDa. Filtration using an ultrafiltration membrane (UK10) was repeated to further remove fractions below 10 kDa, and the fraction between 10 kDa and 200 kDa was freeze-dried.

[0063] (2) Manufacturing method A whey-containing liquid was prepared by mixing the ingredients in the ratios shown in Table 1. The whey-containing liquid (solid concentration 72%) contained 100% protein (9% solids), lactose (58% solids), and lipid (21% solids).

[0064] [Table 1]

[0065] Lactase was added to the obtained whey-containing liquid, and the liquid was kept at 50°C for 10 to 20 minutes to decompose 10 to 20% of the total lactose contained in the whey-containing liquid (100%).The liquid was then heated and concentrated in a continuous heating vacuum cooker (manufactured by Mihama Co., Ltd.) under conditions of a steam pressure of 0.1 to 0.2 MPa and a heating temperature of 100 to 110°C while stirring at 20 to 60 rpm, a rotation radius of 0.175 m, and 0.37 to 1.1 m / s, to obtain a brownish whey heat-treated concentrate (solid concentration 90%).

[0066] The brown whey heat-treated concentrate obtained was diluted with Milli-Q water to a solid content of 20%, and lactase was added at 1%. The mixture was left to stand at 40°C for 1 hour or more to completely hydrolyze the lactose contained therein into glucose and galactose. Yeast powder was added at 1% to the hydrolyzed whey heat-treated concentrate, and the mixture was left to stand at room temperature until carbon dioxide was no longer produced, and the glucose and galactose contained therein were assimilated. The resulting product was centrifuged to recover the intermediate layer. The intermediate layer obtained was subjected to an ultrafiltration membrane for solid-liquid separation, and the solids were dissolved and recovered with Milli-Q water. The recovered product was then sealed in a tube made of a dialysis membrane, and dialyzed at 5°C for 48 hours against Milli-Q water with a volume about 30 times that of the recovered product, to remove components with a molecular weight of less than 8 kD. This series of treatments is intended to further reduce and remove the disaccharides (lactose) contained in the whey heat-treated concentrate prepared above. The aqueous solution containing components with a molecular weight cutoff of 8 kD or more (whey melanoidin-containing aqueous solution) was collected from the tube, then pre-frozen at -40°C and freeze-dried to prepare brown powdered whey melanoidin.

[0067] (3) Whey melanoidin concentration (glycine-melanoidin equivalent) of heat-treated whey concentrate The glycine-melanoidin content of the whey heat-treated concentrate prepared above was measured by the following method. This content was defined as the "glycine-melanoidin equivalent" and was used as an index reflecting the whey melanoidin concentration in the whey heat-treated concentrate.

[0068] [Measurement method] (i) 40 mL of hot water is added to 10 g of the heat-treated whey concentrate (solid content concentration: 90%) prepared by the above method, and the mixture is stirred for 1 hour while maintaining the temperature at 80°C. (ii) The obtained extract is cooled to 4°C and then allowed to stand at 4°C for 16 hours. (iii) After standing, the mixture is centrifuged (10,000×g, 15 minutes, 4° C.) and the supernatant is collected. (iv) The recovered supernatant is filtered through filter paper, and the filtrate is collected and used as a whey melanoidin extract. (v) The absorbance (400 nm) of the whey melanoidin extract is measured using an absorption spectrophotometer. (vi) For melanoidin-containing aqueous solutions of various concentrations prepared using a melanoidin standard, the absorbance (400 nm) corresponding to each concentration is measured to obtain a calibration curve, which is used to calculate the glycine-melanoidin content of the whey melanoidin extract.

[0069] As a result, the glycine-melanoidin content (glycine-melanoidin equivalent) of the whey melanoidin extract was 800 μg / mL. Based on this, the amount of glycine-melanoidin (glycine-melanoidin equivalent) contained in 1 g of the whey heat-treated concentrate is 4 mg.

[0070] Experimental Example 1: Evaluation of the prebiotic effect of whey melanoidin (1) Preparation of test medium Feces from healthy adults were suspended in semi-liquid medium for GAM glycolysis "Nissui" (manufactured by Nissui Pharmaceutical Co., Ltd.) to prepare a fecal dilution solution, which was used as the test medium.

[0071] The semi-liquid medium for GAM sugar decomposition was used after removing the agar from the components contained therein by filtration, adjusting the pH to 6.5 with 1N sodium hydroxide, and autoclaving (at 115°C for 15 minutes). The feces used were from healthy adults, and were frozen and stored in a state suspended in an anaerobic bacteria protective agent from the time of feces collection until the preparation of the fecal dilution solution (test medium).

[0072] The prepared test medium was adjusted to anaerobic conditions by aeration (15 mL / min) with a nitrogen and carbon dioxide mixed gas (N2:CO2 = 80:20) sterilized by filtration through a PTFE membrane with a pore size of 0.2 μm for at least 8 hours at 37°C.

[0073] (2)Culture The culture was carried out using a cultured human intestinal microbiota model (KUHIMM) (see Non-Patent Documents 8 and 9). Specifically, a single-batch culture device that mimics the human large intestine using a multichannel incubator was created as a culture-based human intestinal microbiota model. The device is an anaerobic culture device equipped with multiple small-capacity culture tanks, and is configured to use human feces as a starter and to culture multiple samples simultaneously in a relatively short period of time. The pH can be monitored over time, and the analysis results of metabolic products in the sampled culture solution can be considered in relation to the microbiota.

[0074] Before adding whey melanoidin, human feces was added to a concentration of 0.01%, and pre-culture was performed for 24 hours at 37°C with stirring at 100 rpm while maintaining anaerobic conditions by aeration with the mixed gas.

[0075] Whey melanoidin (Example 1) was added to the pre-cultured culture vessel to a concentration of 1%, and the pH was adjusted to 6.5 or higher using 1N sodium hydroxide, and anaerobic conditions were maintained by aeration using the mixed gas, while stirring at 100 rpm at 37°C for 24 hours (test group: n=4). In addition, as a control group (n=4), only the test medium prepared above was cultured in the same manner for 24 hours without adding whey melanoidin. Culture samples were collected from each of the test and control groups over time from the start of culture and were used for the intestinal flora evaluation described below.

[0076] (3) Evaluation of the intestinal flora using cultures Culture samples collected over time were subjected to next-generation sequencing to analyze the occupancy rate of various intestinal bacteria in the human intestinal flora. Specifically, DNA was extracted and purified from the collected culture using the Maxwell RSC PureFood GMO & Authentication Kit (Promega), and amplicon sequencing of the V3-V4 region was performed using the next-generation sequencer MiSeq (Illumina). The obtained fastq file was analyzed using the microbial flora analysis software QIIME2 (https: / / qiime2.org / ) from the amplicon (PCR amplified product) of the 16S rRNA gene (16S rDNA), a phylogenetic classification marker of bacteria, to analyze the microbial community structure (16S amplicon analysis), and the occupancy rate of various enterobacteria was calculated.

[0077] The analysis confirmed that in the test area where whey melanoidin was added to the test medium, unlike the control area, the growth of bacteria belonging to Parabacteroides was promoted during cultivation, and the proportion of Parabacteroides bacteria in the bacterial flora increased significantly. FIG. 1 shows the relationship between the incubation time (horizontal axis) and the proportion (occupancy rate %) of Parabacteroides genus bacteria in the bacterial flora (vertical axis) for the test groups.

[0078] From these results, it is considered that whey melanoidin or a composition containing it can promote the proliferation of Parabacteroides bacteria in the intestinal flora and increase the proportion of Parabacteroides bacteria in the intestinal flora, thereby improving the intestinal flora. Therefore, whey melanoidin or a composition containing it is useful as an active ingredient of foods, beverages, and medicines for preventing (suppressing) health disorders related to the intestinal flora and / or the intestinal barrier function and promoting health.

Claims

1. An oral or enteral composition comprising whey melanoidin, The whey melanoidin is a brown substance having a molecular weight cutoff of 8 kDa or more, which is obtained by subjecting a whey-containing composition to a heat concentration treatment at 100° C. or more and 120° C. or less so that the solid content concentration becomes 75 to 90% by mass, The whey-containing composition has a lactose content of 48 to 58 parts by mass relative to 10 parts by mass of total protein content, and a solid content concentration of 70 to 75% by mass. The oral or enteral composition.

2. The oral or enteral composition according to claim 1, which increases the occupancy rate of Parabacteroides bacteria in the intestinal flora.

3. The oral or enteral composition according to claim 2, wherein the Parabacteroides bacterium is at least one species selected from the group consisting of Parabacteroides goldsteinii, Parabacteroides distasonis, Parabacteroides goldsteinii, Parabacteroides merdae, and Parabacteroides johnsonii.

4. The oral or enteral composition according to claim 1, which is a composition for improving intestinal flora that promotes the proliferation of bacteria of the genus Parabacteroides.

5. The oral or enteral composition according to claim 1 , which is a composition for inhibiting or improving a decline in intestinal barrier function.

6. The method for producing whey melanoidin includes a step of subjecting a whey-containing composition having a lactose content of 48 to 58 parts by mass relative to 10 parts by mass of total protein content and a solid content of 70 to 75% by mass to a heating and concentration treatment at 100° C. or higher and 120° C. or lower so that the solid content becomes 75 to 90% by mass, thereby obtaining an aggregate of polymer compounds having a molecular weight of 8 kDa or more.

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