Composition for improving intestinal bacterial 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

Composition for improving intestinal flora

[0001] The present invention relates to a composition for improving the intestinal microflora. Specifically, the present invention relates to a composition that improves the intestinal microflora by promoting the growth of bacteria of the genus Parabacteroides, particularly by increasing the occupancy rate of bacteria of the genus Parabacteroides in the intestinal microflora.

[0002] The intestinal flora provides nutrients to the host, maintaining its health and playing an important role in intestinal homeostasis and the host's immune response. Many studies have shown a link between an imbalance in the intestinal flora and diseases such as obesity, type 2 diabetes, and colitis, and it is now widely recognized 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 syndrome, can lead to various diseases and poor health. 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 reside, is believed to have three distinct barrier functions. The first is an environmental barrier involving the intestinal microbiota (gut flora), which functions to eliminate highly pathogenic bacteria by maintaining a healthy intestinal microbiota. The second is a physical barrier that prevents the entry of harmful substances by tightly sealing the connections between cells that make up the intestinal tract. It also forms a mucus layer on the cell surface, preventing external bacteria and various substances from directly coming into contact with intestinal cells. The third is a biological barrier, which is believed to function through antimicrobial peptides and immune-regulating cells.

[0004] Although the intestine is equipped with numerous defense mechanisms, various factors in daily life, such as lifestyle habits (e.g., high-fat diets, high-sugar diets, alcohol consumption, food additives, and medications) and stress, can weaken the intestinal barrier function. For example, in a colon with impaired barrier function, an excessive immune response can cause inflammation of the intestine itself, leading to the development of inflammatory bowel diseases (Crohn's disease and ulcerative colitis). Furthermore, a weakened intestinal barrier function not only leads to the onset of intestinal inflammation, but also allows harmful substances that would normally be eliminated by the intestine to enter the body and be transported throughout the body via the bloodstream, causing inflammation there. This is 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.

[0005] One approach to improving various symptoms associated with impaired intestinal barrier function is to improve the intestinal microbiota. For example, Non-Patent Documents 1 to 3 report that Parabacteroides bacteria in the intestinal tract 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 microbiota improves intestinal barrier function, and that improved intestinal barrier function suppresses inflammation and has the associated effect of improving insulin resistance and weight gain. Furthermore, Non-Patent Document 2 describes that P. distasonis, a Parabacteroides species, exerts an obesity-suppressing effect through the production of succinic acid and secondary bile acids in the intestine, and that one of the mechanisms of this obesity suppression is that secondary bile acids metabolized in the intestine by P. distasonis improve intestinal barrier function via farnesoid X receptor (FXR). Furthermore, Non-Patent Documents 4 and 5 show that there is a negative correlation between pathologies such as multiple sclerosis and fatty liver and Parabacteroides bacteria in the intestinal tract.

[0006] Recently, melanoidins, browning products formed by the heat reaction (Maillard reaction) between amino compounds (proteins, peptides, amino acids, etc.) and carbonyl compounds (sugar and fat breakdown products, etc.), have been reported to have various useful physiological functions (Non-Patent Document 6). Specifically, melanoidins are irregular polymers with physicochemical properties similar to dietary fiber. Therefore, melanoidins are known to have dietary fiber-like effects such as cholesterol-lowering and lactic acid bacteria-increasing effects, as well as glucose tolerance-improving effects, insulin secretion-promoting effects, angiotensin I-converting enzyme activity inhibition, lipase activity inhibition, antioxidant effects, active oxygen scavenging effects, and suppression of the production of carcinogenic nitrosamines (Non-Patent Document 6). Furthermore, research on melanoidins derived from various foods has shown that some melanoidins are widely used by intestinal microorganisms, increasing the production of short-chain fatty acids and promoting the growth of Bifidobacterium and Faecalibacterium (Non-Patent Document 7).

[0007] 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) 2;26(1):222-235)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)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) 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) Riyo Miura, "Physiological functions of melanoidins," Journal of the Brewing Society of Japan, Vol. 97 (2002) No. 4, p.253-256Bioactivity of food melanoidins is mediated by gut microbiota (S. Perez-Burillo, et al. Food Chemistry 316 (2020)) Naoko Ikuta et al., "Evaluation of the impact of dietary fiber using the cultured human gut microbiota model KUHIMM," New Food Industry 2018 Vol. 60, No. 5, pp. 37-43 Akira Osawa, "The relationship between digestive diseases and gut microbiota - clinical significance and impact on treatment -" "2. Development and use of human gut models," Progress in Medicine Vol. 42, No. 3, 2022, pp. 215-220 "Bile acids produced by gut bacteria related to healthy longevity: A new bile acid biosynthetic pathway increased 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) 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.

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

[0009] In recent years, numerous studies have been conducted using in vitro tests utilizing the Kobe University Human Integrative Microbiota Model (KUHIMM), a cultured human intestinal microbiota model developed at Kobe University, to examine the clinical relevance of various foods and the effects of these foods on the intestinal microbiota. These studies have provided valuable insights into the safety and functionality of food ingredients mediated by intestinal bacteria (Non-Patent Documents 8 and 9).

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

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

[0012] (I-2) The composition for improving intestinal flora according to (I-1), which increases the proportion of Parabacteroides bacteria in the intestinal flora. This composition is also a composition for increasing the proportion of Parabacteroides bacteria in the intestinal flora, which is used to improve the intestinal flora of a mammal. Therefore, the invention can also be rephrased as "a composition for increasing the proportion 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 to inhibit or improve the decline of intestinal barrier function. This invention can be rephrased as "a composition for inhibiting or improving the decline of 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 a whey melanoidin-containing composition (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 intestinal flora is promoting the growth of Parabacteroides bacteria in the intestinal flora or increasing the occupancy rate of Parabacteroides bacteria in the intestinal flora. (II-3) The use according to (II-2), wherein the Parabacteroides bacteria 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 whey melanoidin-containing composition 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) A whey melanoidin or 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 intestinal flora is by promoting the growth of Parabacteroides bacteria in the intestinal flora or increasing 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 bacteria 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.

[0018] According to the present invention, the proliferation of Parabacteroides bacteria in the intestinal flora of mammals, particularly humans, can be promoted, thereby increasing their occupancy rate. That is, 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, improvement of the intestinal flora effectively inhibits or improves the decline of intestinal barrier function, and can be expected to have a preventive or ameliorative effect on diseases associated with the decline of the intestinal flora or intestinal barrier function.

[0019] In Experimental Example 1, the occupancy rate (%) of Parabacteroides bacteria in the whey melanoidin-supplemented (test) group (n=4) was measured over the 24-hour incubation period using a human intestinal model. In the figure, an asterisk (*) indicates a significant difference (p<0.05) compared to the occupancy rate at the start of incubation (0 h). In the boxplot 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.

[0020] The composition for improving intestinal bacterial flora 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 a "whey-containing composition"). It is a mixture of irregular polymeric compounds produced by the amino-carbonyl reaction (Maillard reaction) 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.

[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 8 kDa or more that can be fractionated at least 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 the water-soluble component (whey) remaining after removing fat, casein, fat-soluble vitamins, and the like from milk. However, whey may contain fat, casein, and / or fat-soluble vitamins that were not completely removed. The whey used as a raw material can be undiluted whey (sweet whey, acid whey, etc.), as well as its concentrate, dried product (whey powder, etc.), and frozen product. Desalted whey, whey protein concentrate (WPC), protein-enriched whey powder, and whey protein purified product (WPI) can also be used. Examples of whey include cheese whey and rennet whey (also known as sweet whey), which are obtained as by-products in the production of natural cheese and rennet casein, and casein whey, acid whey, and quark whey, which are obtained in the production of fermented milk and quark.

[0025] Generally, whey contains 10 to 15% by mass of protein and 70 to 85% by mass of lactose, calculated on a solid content basis. Because whey has a relatively high lactose content relative to its 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 by reducing the lactose content. Preferably, a method of reducing the lactose content is employed.

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

[0027] In addition, in this specification, the content of a component in a target substance (e.g., a whey-containing composition) in terms of solid content (also referred to as "solid content equivalent amount") means the value obtained by converting the proportion of the component contained in the target substance into solid content. Specifically, when the total amount of solid content (dry mass) of the target substance is taken as 100 mass%, it is expressed as the proportion (mass %) of the component in the total amount of solid content.

[0028] The mass and concentration of solids contained in the object (solids concentration) can be determined by the atmospheric pressure heating and drying method (sand mixing method). Specifically, after determining a constant weight with sea sand and a glass rod placed in a weighing dish, the test sample and distilled water are added and 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 between before and after drying. The mass of solids contained in the test sample (before drying) can be determined from the moisture content thus measured, and the solids concentration can then be calculated.

[0029] The ratio of the total protein content to the lactose content in the whey-containing composition is not limited, but the ratio of the lactose content to 10 parts by mass of the total protein content can be 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 term "protein" refers primarily to proteins contained in whey, including whey proteins, specifically α-lactalbumin, β-lactoglobulin, immunoglobulins, and lactoferrin. The amino acids constituting whey proteins 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. Casein, a milk protein, may remain in whey. Therefore, casein may be contained in the whey-containing composition to the extent that it does not impair the effects of the present invention.

[0031] Methods for reducing the lactose content in a whey-containing composition include methods for removing or decomposing lactose. A preferred method is a method for selectively decomposing lactose contained in a whey-containing composition. Examples of such methods include, but are not limited to, treating a whey-containing composition with a lactase (e.g., lactase). Lactase is an enzyme that decomposes lactose into glucose and galactose. Therefore, a whey-containing composition that has been subjected to a lactose degradation treatment (lactase treatment) using lactase contains glucose and galactose as reducing sugars in addition to lactose. Although not limited to, it is preferable to decompose and reduce the lactose in the whey-containing composition by approximately 10 to 20% by mass using such a lactose degradation treatment.

[0032] The whey-containing composition may contain lipids, glucose, galactose, amino acids, vitamins, and / or minerals as components other than protein and lactose, as long as the production of whey melanoidin, which is the subject of the present invention, is not impaired. The lipids may include residues from whey that were not completely removed during whey preparation, as well as any lipid-containing raw material, regardless of origin, such as raw milk, milk or dairy products (e.g., concentrated milk, reconstituted milk, whole milk powder, cream, butter, butter oil), vegetable oils (e.g., safflower oil, corn oil, cottonseed oil, sesame oil, rice oil, olive oil), and processed vegetable oil products (e.g., margarine, shortening, fast spread). Milk-derived lipids are preferred. When lipids are contained in the whey-containing composition, the lipid content is not limited, but can range from 10 to 50 parts by weight per 10 parts by weight of the total protein content in the whey-containing composition. The amount 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 solids concentration of the whey-containing composition to be heat-treated is not limited, but a lower concentration is preferable for efficient production of whey melanoidin by the Maillard reaction. The solids concentration can be adjusted appropriately depending on the form of the intestinal microflora-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 solids concentration of the whey-containing composition to be heat-treated to 10% by mass or higher in order to ensure efficiency. The solids concentration 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. To efficiently perform 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, although this is not a limitation. Furthermore, although not a limitation, the heat treatment is preferably carried out under atmospheric pressure rather than reduced pressure to promote the Maillard reaction. The heat treatment may be carried out as long as whey melanoidin is produced. Although not particularly limited, it is preferable to heat and concentrate the whey-containing composition so that the solids concentration of the whey-containing composition after the heat treatment is 55% by mass or higher. The solids concentration of the whey-containing composition after heat concentration can be appropriately set depending on the final form of the composition to be produced. For example, although not limited, the solids concentrations of the present composition in a paste form (semi-fluid, unformed), soft solid form, and hard solid form are approximately 55 to 74% by mass, approximately 75 to 79% by mass, and approximately 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 composition only needs to contain whey melanoidin as its 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. Furthermore, the heat-treated product can be subjected to various purification processes, such as membrane treatment, to remove unreacted residues (e.g., lactose) and by-products from the amino-carbonyl reaction, and then used.

[0036] Whey melanoidin preferably has a molecular weight cutoff of 8 kDa or more. Therefore, the membrane treatment may be any treatment that utilizes molecular weight differences to remove low-molecular-weight compounds and purify the whey melanoidin (molecular weight fractionation purification treatment), such as dialysis using a semipermeable membrane or ultrafiltration membrane treatment. Dialysis and ultrafiltration membrane treatment may be performed independently or in combination. A semipermeable membrane made of regenerated cellulose with a molecular weight cutoff (MWCO) of 6 to 8 kDa is suitable for dialysis. Furthermore, any membrane with a nominal molecular weight cutoff (NMWL) in the range of 3 to 10 kDa may be used for ultrafiltration membrane treatment, such as polyethersulfone (PES), cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, etc., which can be selected and used. Fractionation by such membrane treatment allows for the purification and recovery of whey melanoidin (a collection of high-molecular-weight compounds) with a molecular weight in the range of 8 kDa or more.

[0037] In order to more efficiently and effectively carry out molecular weight fractionation purification by membrane treatment, a treatment may be performed in advance to decompose, assimilate, or remove components other than whey melanoidin contained in the heat-treated whey-containing composition prior to the membrane treatment. The decomposition treatment may be any treatment that can selectively decompose lactose contained as an unreacted residue in the heat-treated product, such as hydrolysis using a lactase or other lactase-degrading enzyme. Furthermore, subsequent treatment with a microorganism such as yeast can assimilate the monosaccharides remaining in the heat-treated product and the monosaccharides produced by lactose degradation. Furthermore, the assimilated substances thus produced can be efficiently removed by subsequent membrane purification.

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

[0039] The whey melanoidin content of the present composition can also be expressed as a "glycine-melanoidin equivalent" converted into the glycine-melanoidin content. The method for determining the glycine-melanoidin equivalent is as detailed in Example 1(3) below, and can be carried out according to the description therein. When the whey melanoidin content in the present composition is expressed 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 as the 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 the present composition has the effect of increasing the proportion of Parabacteroides bacteria in the human intestinal flora can be evaluated by an in vitro test using a culture-based human intestinal flora model (human intestinal model) that mimics the human intestinal flora, such as the human intestinal model (KUHIMM) developed at Kobe University (Japan). The evaluation method using the KUHIMM in vitro test is described in detail in the experimental examples below. In this in vitro test, if anaerobically culturing a diluted fecal solution (test medium) containing a whey melanoidin-containing composition promotes the growth of Parabacteroides bacteria, and an increase in the proportion (occupancy) of Parabacteroides bacteria in the total number of bacteria is observed, the composition can be determined to be the present composition containing an effective amount of whey melanoidin.

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

[0042] In the present invention, promoting the growth of Parabacteroides bacteria includes increasing the proportion of Parabacteroides bacteria in the intestinal flora. Here, "prevalence" refers to the proportion of the bacteria relative to the total bacteria detected in the intestinal flora, and can also be referred to as "occupancy rate." The promotion of growth may occur simultaneously with an increase or decrease in the proportion of bacteria other than Parabacteroides bacteria in the intestinal flora, so long as the proportion of Parabacteroides bacteria increases. In other words, this promotion includes not only cases where the proportion of Parabacteroides bacteria increases because the growth of Parabacteroides bacteria is higher than the growth of other bacteria, but also cases where the proportion of Parabacteroides bacteria increases due to a relative increase in the amount of Parabacteroides bacteria caused by a decrease in other bacteria.

[0043] The increase in the occupancy rate of Parabacteroides bacteria in the intestinal bacterial 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, when the occupancy rate before application of the present composition is set to 100, the value is more than 100, preferably 110 or more, more preferably 150 or more.

[0044] Such analysis of the bacterial composition of the intestinal flora (bacterial flora analysis) generally involves shotgun metagenomic analysis targeting the entire genome of the bacterial flora, and 16S rRNA metagenomic analysis targeting the 16S rRNA gene. 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 performed by amplicon sequencing, which involves extracting DNA from a sample containing various bacteria, amplifying specific regions (the third and fourth variable regions) of the bacterial 16S rRNA gene by PCR, and analyzing the resulting PCR amplification product (amplicon). This amplicon sequencing can be performed using a next-generation sequencer (Miseq: manufactured by Illumina). The vast amount of sequence data obtained by sequencing is then analyzed by computer. The bacterial flora composition analysis (16S amplicon analysis) can be performed using amplicon sequence data analysis software (e.g., QIIME2) according to its manual. QIIME2 is an open-source analysis pipeline that compiles the software necessary for amplicon sequence analysis (https: / / qiime2.org / ).

[0046] Thus, by analyzing the composition of the intestinal flora, 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 determined.

[0047] Parabacteroides bacteria, i.e., bacteria belonging to Parabacteroides, may be any bacteria present in the intestinal flora of mammals, preferably humans, and include 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 (Non-Patent Document 10), which was isolated from the feces of centenarians and confirmed to have the ability to produce a secondary bile acid called isoalloLCA (isoallolithocholic acid); Parabacteroides merdae (Non-Patent Document 11), which may prevent obesity-related cardiovascular disease; and Parabacteroides johnsonii.

[0048] The composition is used to improve the composition of the intestinal microbiota so as to increase the proportion (occupancy) of Parabacteroides bacteria in the intestinal microbiota. This improvement of the intestinal microbiota makes it possible to maintain or promote health in response to the increase in Parabacteroides bacteria. The maintenance or promotion of health includes maintaining or promoting health by preventing and / or ameliorating a disease or pathological condition. The target disease or pathological condition may be any disease or pathological condition that can be prevented or ameliorated by the proliferation of Parabacteroides bacteria in the intestinal microbiota, including, but not limited to, a decrease or failure of the intestinal barrier, a disease or pathological condition resulting therefrom (e.g., inflammation, glucose metabolism disorders, obesity, hepatic steatosis, allergic diseases, and intestinal barrier syndromes such as depression), and cancer.

[0049] The target subject (recipient or recipient) of the present composition is a mammal, preferably a human. The subject may be an adult, a child, an infant, a newborn (including a low birth weight infant), or the like. The gender is not particularly limited. The dosage (intake) can be appropriately determined depending on the species, age, gender, condition, and the like of the target mammal. The dosage (intake) can be appropriately set and adjusted within the range of 0.1 to 10 g / day in terms of the dosage (intake) of whey melanoidin, without any limitation.

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

[0051] When the composition is prepared as an oral composition, it is preferably in the form of a food or beverage composition, a quasi-drug composition, or a pharmaceutical composition. Food or beverage compositions include nutritional compositions. Such nutritional compositions include enteral nutrients that are 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, granules, cream, gel, sol, liquid, and suspension 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 plant 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 described as 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 considered a prebiotic. Therefore, food and beverage compositions containing whey melanoidin can be referred to as prebiotic foods and beverages (prebiotic foods and beverages). Food and beverage compositions covered by the present invention include, in addition to the above-mentioned labeling, food and beverage compositions (e.g., functional foods, foods for specified health uses, etc.) that are permitted to label the proliferation of Parabacteroides bacteria, the effects associated with said proliferation, and their uses (uses related to health promotion). In addition, one embodiment of food and beverage compositions includes feed compositions, which are foods and beverages for non-human mammals such as pets (dogs, cats, rabbits, etc.), laboratory 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, for 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. For parenteral administration, it can be formulated into a form for tube administration such as an enterostomy tube.

[0055] In addition to the whey melanoidin described above, ingredients commonly used in formulations, such as excipients, pH adjusters, colorants, and flavoring agents, can be used when formulating the formulation. It is also possible to use other medicinal ingredients, or other pharmaceuticals, such as known or future ingredients that promote the growth of Parabacteroides bacteria or prevent or improve specific diseases or pathological conditions. Furthermore, formulation can be carried out by known methods as appropriate depending on the dosage form.

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

[0057] The present invention will be described below 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. Unless otherwise specified, the following experiments were carried out at room temperature (25±5°C) and atmospheric pressure. Unless otherwise specified, "%" and "parts" in the following descriptions mean "% by mass" and "parts by mass," respectively.

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

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

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

[0061] Ultrafiltration membrane: Nominal molecular weight cutoff (NMWL) 3-10 kDa, Omega membrane, manufactured by Pall. Ultrafiltration membrane (UP20): NMWL 200 KDa, manufactured by Advance Tokyo. Ultrafiltration membrane (UK10): NMWL 10 KDa, 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 for synthesizing glycine-melanoidin] 1 M D-glucose, 1 M glycine, and 0.1 M NaHCO ３A mixture consisting of these was heated at 90°C for 72 hours, and the resulting brown solution was 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 exceeding 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 lyophilized.

[0063] (2) Production method A whey-containing liquid was prepared by mixing the raw materials in the proportions shown in Table 1. The whey-containing liquid (solid concentration 72%) contained 100% of the protein content (9% in terms of solid content), 42% in terms of lactose (58% in terms of solid content), and 21% in terms of lipid content (29% in terms of solid content).

[0064]

[0065] Lactase was added to the resulting whey-containing liquid, and the mixture 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 mixture was then heated and concentrated in a continuous heating vacuum cooker (manufactured by Mihama Co., Ltd.) at 20 to 60 rpm, a rotation radius of 0.175 m, and stirring at 0.37 to 1.1 m / s under conditions of a steam pressure of 0.1 to 0.2 MPa and a heating temperature of 100 to 110°C, yielding a brown heat-treated whey concentrate (solids concentration 90%).

[0066] The resulting brown heat-treated whey concentrate was diluted with Milli-Q water to a solids concentration of 20%, and 1% lactase was added. The mixture was then allowed to stand at 40°C for at least 1 hour to completely hydrolyze the lactose contained therein into glucose and galactose. 1% yeast powder was added to the hydrolyzed heat-treated whey concentrate and allowed to stand at room temperature until carbon dioxide production ceased, allowing the glucose and galactose contained therein to be assimilated. The resulting product was centrifuged to recover the intermediate layer. The resulting intermediate layer was subjected to solid-liquid separation using an ultrafiltration membrane, and the solids were dissolved and recovered with Milli-Q water. The recovered product was then sealed in a dialysis membrane tube and dialyzed against approximately 30 times the volume of Milli-Q water at 5°C for 48 hours to remove components with a molecular weight of less than 8 kD. This series of processes was intended to further reduce and remove the disaccharide (lactose) contained in the heat-treated whey concentrate prepared above. The aqueous solution containing components with a molecular weight cutoff of 8 kD or more (whey melanoidin-containing aqueous solution) in the tube was collected, then pre-frozen at -40°C and freeze-dried to prepare brown powdered whey melanoidin.

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

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

[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 heat-treated whey concentrate was 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 a semi-solid 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-solid medium for GAM glycolysis was used after filtering to remove the agar from the components contained therein, adjusting the pH to 6.5 with 1N sodium hydroxide, and autoclaving (115°C for 15 minutes). The feces used were from healthy adults, and were frozen and stored in a state suspended in a protective agent for anaerobic bacteria from the time of fecal collection until the preparation of the fecal dilution solution (test medium).

[0072] The test medium was prepared by filtering a nitrogen and carbon dioxide mixed gas (N ２ :CO ２The mixture was kept in anaerobic conditions by aeration (15 mL / min) at 37°C for at least 8 hours.

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

[0074] Before adding whey melanoidin, human feces was added to a concentration of 0.01%, and the mixture was pre-cultured for 24 hours at 37°C with stirring at 100 rpm while maintaining anaerobic conditions by aeration using 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. Anaerobic conditions were maintained by aeration using the mixed gas, and the culture was carried out for 24 hours at 37°C with stirring at 100 rpm (test group: n=4). A control group (n=4) was also cultured for 24 hours using the test medium prepared above without adding whey melanoidin. Cultures were collected over time from the start of culture for each of the test group and control group, and subjected to the intestinal flora evaluation described below.

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

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

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

Claims

1. An oral or enteral composition comprising whey melanoidin, The whey melanoidin has a molecular weight cutoff of 8 kDa or more and is obtained by heat-treating a whey-containing composition at 100° C. or more, The whey-containing composition has a lactose content of 45 to 61 parts by mass per 10 parts by mass of total protein content. 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. An oral or enteral composition as described in claim 1, which is a composition for improving intestinal flora that promotes the growth 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. A method for producing whey melanoidin, comprising the steps of: heat-treating a whey-containing composition having a lactose content of 45 to 61 parts by mass per 10 parts by mass of total protein content at 100°C or higher to obtain a collection of polymeric compounds having a molecular weight of 8 kDa or more.