Food for improving the intestinal milieu

Abstract

To provide a food for promoting proliferation of lactic acid bacteria and the like in the intestinal tract and improving the intestinal environment. The present invention provides a food for improving the intestinal environment, which is a combination of (1) γ-polyglutamic acid or a composition containing γ-polyglutamic acid and (2) an oligosaccharide or a composition containing an oligosaccharide.

Description

Technical Field

[0001] This invention relates to foods that promote the proliferation of lactic acid bacteria and bifidobacteria in the intestine and improve the intestinal environment. Background Technology

[0002] Promoting the proliferation of beneficial bacteria such as lactobacilli and bifidobacteria in the gut and improving the gut environment (normalization of gut microbiota) is important for the prevention of various diseases and the maintenance of health.

[0003] Patent document 1 describes the laxative effect of γ-polyglutamic acid. Non-patent document 1 describes the lactic acid bacteria-increasing effect of γ-polyglutamic acid. It is known that oligosaccharides are not easily digested and absorbed in the stomach and small intestine, but become food for beneficial bacteria such as lactic acid bacteria and bifidobacteria in the large intestine. However, it is not known that the combined use of γ-polyglutamic acid and oligosaccharides significantly increases the number of lactic acid bacteria and bifidobacteria compared to using them alone.

[0004] Existing technical documents Patent documents Patent Document 1: Japanese Patent Application Publication No. 2007-22982 Non-patent literature Non-licensed reference 1: J. Microbiol. Biotechnol. (2017), 27(2), 412-415. Summary of the Invention

[0005] The technical problem that the invention aims to solve The subject of this invention is to provide food that promotes the proliferation of lactic acid bacteria and bifidobacteria in the intestine and improves the intestinal environment.

[0006] Technical solutions adopted to solve technical problems After careful investigation to solve the aforementioned problems, the inventors discovered that the combined use of γ-polyglutamic acid and oligosaccharides significantly increased the number of lactic acid bacteria and bifidobacteria compared to using them alone. Based on the above findings, the inventors further investigated and completed the present invention.

[0007] That is, the present invention provides the following: [1] A food for improving the intestinal environment, the food being composed of the following (1) and (2): (1) γ-polyglutamic acid or a composition containing γ-polyglutamic acid, (2) Oligosaccharides or compositions containing oligosaccharides; [2] The food according to [1] above, wherein the composition containing oligosaccharides is in liquid form; [3] According to the food described in [2] above, the composition containing oligosaccharides in liquid form is a liquid preparation containing oligosaccharides; [4] According to the food described in [2] above, the oligosaccharide-containing composition in liquid form is an oligosaccharide-containing beverage.

[0008] The effects of the invention According to the present invention, by supplying γ-polyglutamic acid and oligosaccharides in combination, a significantly superior effect on promoting the proliferation of lactic acid bacteria and bifidobacteria is achieved compared with the use of them separately, thus providing a food with the function of improving the intestinal environment. Attached Figure Description

[0009] Figure 1 The results of Experiment 1 (the experiment of adding γ-polyglutamic acid and fructooligosaccharides to human fecal suspension) are expressed as mean ± standard error, n=6. Figure 2 The results of Experiment Example 2 (the experiment of adding γ-polyglutamic acid and various oligosaccharides to human fecal suspension) are shown (mean ± standard error, n=3). Figure 3 The results of Experiment 3 (experiment with administration of γ-polyglutamic acid and fructooligosaccharides to pigs) are represented as mean ± standard deviation, n=3. Figure 4 The results of Experiment 4 (the experiment of administering γ-polyglutamic acid and fructooligosaccharides to pigs) are shown (mean ± standard deviation, n=6, *: P<0.05, Student's t test). Detailed Implementation

[0010] The present invention will now be described in detail.

[0011] The food for improving the intestinal environment of the present invention is composed of (1) γ-polyglutamic acid or a composition containing γ-polyglutamic acid and (2) oligosaccharides or a composition containing oligosaccharides.

[0012] The food of the present invention is a food combining (1) and (2) in combination.

[0013] In the food products of the present invention, (1) and (2) can be formulated simultaneously and contained in the same formulation (food), or (1) and (2) can be formulated separately and ingested simultaneously or at intervals via the same or different routes. That is, the food products of the present invention include: food products containing (1) and (2) in one formulation, and food products containing (1) and (2) formulated separately and used in combination.

[0014] In this instruction manual, "food" is a broad term encompassing substances that can be ingested orally (excluding pharmaceuticals). It includes not only so-called "food" but also beverages, health supplements, functional foods (such as foods for specific health purposes and foods with functional labels), supplements, etc.

[0015] In this instruction manual, "improvement of the intestinal environment" refers to a relative increase in the number of lactobacilli and / or bifidobacteria present in the lower digestive tract. A relative increase means promoting the dominance of lactobacilli and / or bifidobacteria in the intestinal flora. The dominance of lactobacilli and bifidobacteria in the intestinal flora can be confirmed by measuring the amount of lactobacilli and bifidobacteria in feces using quantitative PCR or similar methods.

[0016] In this instruction manual, "lower digestive tract" refers to the ileum, cecum, colon, and rectum.

[0017] (1) γ-polyglutamic acid or a composition containing γ-polyglutamic acid In this invention, as (1), γ-polyglutamic acid can be used directly or in the form of a composition containing γ-polyglutamic acid.

[0018] In this invention, the preferred γ-polyglutamic acid is a polymer (polymer) consisting of a mixture of D-glutamic acid and L-glutamic acid in a ratio of about 8:2 and about 30 to about 5000 of them.

[0019] γ-polyglutamic acid is also known as polyglutamic acid.

[0020] γ-polyglutamic acid can also be used in commercially available products (such as Calteic manufactured by Ajinomoto Co., Ltd.).

[0021] The γ-polyglutamic acid-containing compositions of this invention are not particularly limited in form and may include, for example, powders, granules (including fine granules), tablets, hard capsules, soft capsules, liquid formulations (e.g., solutions, suspensions, emulsions), beverages, jellies, puddings, yogurts, candies, chewing gum, etc. These compositions can be manufactured by known methods. For example, γ-polyglutamic acid can be mixed with a carrier (e.g., excipients, binders, disintegrants, lubricants, solvents) and manufactured as powders, granules, tablets, capsules, liquid formulations, etc., using methods known in the fields of food or pharmaceutical formulations. Furthermore, γ-polyglutamic acid can also be added to or mixed into foods and beverages (e.g., water, soft drinks) for manufacturing.

[0022] (2) Oligosaccharides or compositions containing oligosaccharides In this invention, as (2), oligosaccharides can be used directly or in the form of a composition containing oligosaccharides.

[0023] In this invention, examples of oligosaccharides include coffee bean mannan oligosaccharides, lactulose oligosaccharides, galactooligosaccharides, fructooligosaccharides, soybean oligosaccharides, xylooligosaccharides, and isomaltooligosaccharides, with fructooligosaccharides, coffee bean mannan oligosaccharides, isomaltooligosaccharides, and galactooligosaccharides being preferred. One type of oligosaccharide or a combination of two or more types can be used.

[0024] The oligosaccharide-containing compositions of this invention are not particularly limited in form, and may include, for example, powders, granules (including fine granules), tablets, hard capsules, soft capsules, liquid formulations (e.g., solutions, suspensions, emulsions, liquid flavorings (e.g., liquid sweeteners)), beverages, jellies, puddings, yogurts, candies, chewing gum, etc. Preferably, they are in liquid form (e.g., liquid formulations (e.g., solutions, suspensions, emulsions, liquid flavorings (e.g., liquid sweeteners)) and beverages).

[0025] These compositions can be manufactured using known methods. For example, oligosaccharides can be mixed with a carrier (e.g., excipients, binders, disintegrants, lubricants, solvents) and manufactured into powders, granules, tablets, capsules, liquid formulations, etc., using methods known in the fields of food or pharmaceutical preparations. Furthermore, oligosaccharides can be added to or mixed into foods and beverages (e.g., water, soft drinks) to manufacture them. Additionally, liquid sweeteners containing oligosaccharides can be added to or mixed into foods and beverages to manufacture them.

[0026] As described above, the food products of the present invention include: food products containing (1) and (2) above in a single formulation, and food products that separately formulate (1) and (2) above and use them in combination.

[0027] Examples of foods that combine (1) and (2) are formulated separately. Examples include (1) granules, tablets, hard capsules or soft capsules containing γ-polyglutamic acid, and (2) beverages containing oligosaccharides.

[0028] In this invention, (1) and (2) can be ingested simultaneously or at intervals via the same or different pathways. Specifically, for example, capsules (e.g., hard capsules, soft capsules), granules, tablets, etc., containing γ-polyglutamic acid can be ingested together with beverages containing oligosaccharides.

[0029] Foods that incorporate (1) and (2) into a single formulation are not particularly limited in form and may include, for example, powders, granules (including fine granules), tablets, hard capsules, soft capsules, liquid formulations (e.g., solutions, suspensions, emulsions, liquid flavorings (e.g., liquid sweeteners)), beverages, jellies, puddings, yogurts, candies, chewing gum, etc. These foods can be manufactured by known methods.

[0030] Regarding the intake of γ-polyglutamic acid in the food for improving the intestinal environment according to the present invention, the usual intake for adults (60 kg) is 0.1 to 100 g per day, preferably 0.1 to 50 g, and more preferably 1 to 20 g.

[0031] Regarding the intake of oligosaccharides in the food for improving the intestinal environment according to the present invention, the usual intake for adults (60 kg) is 0.1 to 100 g per day, preferably 0.1 to 50 g, and more preferably 1 to 20 g.

[0032] In the food of the present invention for improving the intestinal environment, the weight ratio of oligosaccharide to γ-polyglutamic acid (oligosaccharide:γ-polyglutamic acid) is preferably 1:0.05-20, more preferably 1:0.1-10, further preferably 1:0.1-5, even more preferably 1:0.1-3, and particularly preferably 1:0.2-2.

[0033] Humans and other animals (e.g., livestock, poultry, laboratory animals, and other mammals, birds) can safely consume the food of this invention. As a form for animals other than humans to consume, it can be added to animal feed.

[0034] Other embodiments of the present invention include: “a commercial package comprising: a food comprising (1) γ-polyglutamic acid or a composition containing γ-polyglutamic acid and (2) oligosaccharides or a composition containing oligosaccharides, and a description relating to its use in improving the intestinal environment.”; “a food labeled for improving the intestinal environment, comprising (1) γ-polyglutamic acid or a composition containing γ-polyglutamic acid and (2) oligosaccharides or a composition containing oligosaccharides.”; “a food for enhancing the intestinal environment improvement effect, comprising (1) γ-polyglutamic acid or a composition containing γ-polyglutamic acid and (2) oligosaccharides or a composition containing oligosaccharides.”; and “a food for enhancing the intestinal environment improvement effect of oligosaccharides or compositions containing oligosaccharides, wherein the food is γ-polyglutamic acid or a composition containing γ-polyglutamic acid.” "γ-polyglutamic acid or a composition containing γ-polyglutamic acid", "oligosaccharide or a composition containing oligosaccharide", "improvement of intestinal environment", "intake of γ-polyglutamic acid", "intake of oligosaccharide" are the same as the examples and definitions given for the foods described above for improving the intestinal environment. Example

[0035] The present invention will now be described in more detail based on experimental examples, embodiments, comparative examples, and reference examples, but the present invention is not limited to these examples.

[0036] The abbreviations are as follows: γ-PGA: γ-polyglutamic acid (Poly-γ-Glutamic Acid) FOS: Fructooligosaccharides PBS: Phosphate-buffered saline CMOS: Coffee bean mannan oligosaccharides IMOS: Isomaltooligosaccharide GOS: Galacto-oligosaccharides.

[0037] [Experimental Example 1] Experiment on adding γ-PGA and fructooligosaccharides to human fecal suspension PBS solutions of the examples and comparative examples shown in Table 1 were prepared as culture media for fecal culture. PBS-only was used as a negative control. γ-PGA was polyglutamic acid "Caltec" manufactured by Ajinomoto Co., Ltd., and FOS was fructooligosaccharide manufactured by Wako Pure Chemical Industries, Ltd.

[0038] Then, 10 ml of the solution was dispensed into each glass vial, sterilized in an autoclave, and anaerobically treated. The vials were then sealed with butyl rubber stoppers. The fecal suspension was prepared as follows: approximately 10 g of feces was collected from six healthy individuals (3 men and 3 women) aged 20–40 years and suspended in 80 ml of anaerobic PBS. 0.5 ml of this fecal suspension was added to each vial, and the vials were placed in an incubator at 37°C and anaerobically cultured for 4 days with inverted mixing once daily. After culture, intestinal bacterial pellets were obtained from the total culture volume by centrifugation (8,000 × g, 5 min). DNA was extracted from the pellet using ISOFECAL for BeadsBeat (manufactured by NIPPON GENE, Japan), and quantitative PCR was performed using GeneAce SYBR qPCR Mix α (manufactured by NIPPON GENE, Japan). Quantitative PCR was performed under the following conditions: 45 cycles of 95°C for 10 minutes, followed by 30 seconds at 95°C and 60°C for 60 seconds. Primers used for quantitative PCR to detect lactic acid bacteria (mainly Lactobacillus spp.) are described below. LAB-F: 5'-AGCAGTAGGGAATCTTCCA-3' (SEQ ID NO: 1) (Appl EnvironMicrobiol. 67(6):2578-2585, 2001) LAB-R: 5'-CACCGCTACACATGGAG-3' (SEQ ID NO: 2) (Appl Environ Microbiol. 68(1):114-123, 2002).

[0039] [Table 1] .

[0040] For the results obtained by quantitative PCR, the relative amount when PBS (negative control) alone is set to 1 is calculated, and the results are normalized using z-score (Z score).

[0041] The results are shown in Figure 1 Data are expressed as mean z-score ± standard error (n=6). Significant differences were determined by Dunnet's test with a positive control as a reference (*: P < 0.05).

[0042] As a result, no proliferation of lactic acid bacteria was observed when γ-PGA was added alone, but a more significant concentration-dependent proliferation of lactic acid bacteria was observed when γ-PGA was added together with 0.5% FOS than when FOS was added alone.

[0043] As described above, in this invention, a synergistic effect exceeding the additive effect was observed in the proliferation of lactic acid bacteria through the combination of γ-PGA and oligosaccharides.

[0044] [Experimental Example 2] Experiment on adding γ-PGA and various oligosaccharides to human fecal suspension PBS solutions of the examples and comparative examples shown in Table 2 were prepared as culture media for fecal culture. PBS alone was used as a negative control. CMOS used coffee bean mannan oligosaccharide manufactured by Ajinomoto AGF Co., Ltd., IMOS used isomaltooligosaccharide manufactured by Wako Pure Chemical Industries Co., Ltd., and GOS used galactooligosaccharide manufactured by Wako Pure Chemical Industries Co., Ltd.

[0045] Stool culture and lactic acid bacteria detection were performed using the same methods as in Experiment 1.

[0046] [Table 2] .

[0047] For the results obtained by quantitative PCR, the relative amount when PBS (negative control) alone is set to 1 is calculated, and the results are normalized using z-score.

[0048] The results are shown in Figure 2 Data are expressed as mean z-score ± standard error (n=3). Significant differences were determined by Dunnet's test with a positive control as a reference (*: P < 0.05).

[0049] As a result, all oligosaccharides, when combined with γ-PGA, showed a more significant increase in lactic acid bacteria populations than when oligosaccharides were added alone.

[0050] As described above, in this invention, regardless of the type of oligosaccharide, a synergistic effect exceeding the additive effect was observed in the proliferation of lactic acid bacteria through combination with γ-PGA.

[0051] [Experimental Example 3] Experiment on pigs given γ-PGA and FOS Six 2-3 month old male pigs (HI-COOP SPF pigs, LWD) were purchased from the Japan National Agricultural Cooperative Association and divided into two experimental groups, each containing three pigs to ensure uniform average weight. The pigs were housed at 3-pig / pen for four weeks. In each experimental group, pigs were fed a restricted diet of 400g / head of MIRAI CEX (Japan National Agricultural Cooperative Association) twice daily. The test substances were administered to each experimental group according to the dosages and methods shown in Table 3. γ-PGA was polyglutamic acid "Kalteic" manufactured by Ajinomoto Co., Ltd., and FOS was fructooligosaccharide "Meioligo P granules" manufactured by Meiji Food Materials Co., Ltd. Four weeks after administration, cecums were collected, and the lactic acid bacteria in the cecal contents were examined using quantitative PCR in the same manner as in Experiments 1 and 2.

[0052] [Table 3] .

[0053] The results are shown in Figure 3 Data are expressed as relative values ​​(mean ± standard deviation, n=3) with the DNA copy number from the lactic acid bacteria of Comparative Example 6 set to 1.

[0054] The result was that the combination of γ-PGA and FOS confirmed that it increased the number of lactic acid bacteria in the pig gut by about two times on average compared with FOS intake alone.

[0055] [Experimental Example 4] Experiment on pigs given γ-PGA and FOS Twenty-four 2-3 month old male pigs (HI-COOP SPF pigs, LWD) were purchased from the Japan National Agricultural Cooperative Association and divided into four experimental groups, each containing six pigs to ensure uniform average weight. Pigs were housed at a rate of three per pen for four weeks. Each experimental group was fed a restricted diet of 400g / head of pig MIRAI CEX (Japan National Agricultural Cooperative Association) twice daily. The test substances were administered to each experimental group according to the dosages and methods shown in Table 4. γ-PGA was polyglutamic acid "Kalteic" manufactured by Ajinomoto Co., Ltd., and FOS was fructooligosaccharide "Meioligo P granules" manufactured by Meiji Food Materials Co., Ltd. Four weeks after administration, cecums were collected, and quantitative PCR was used to examine the presence of Bifidobacteria in the cecal contents. Quantitative PCR was performed under the following conditions: 95°C for 10 minutes, followed by 45 cycles of 95°C for 30 seconds and 60°C for 60 seconds. The primers used for quantitative PCR detection of Bifidobacteria are described below. g-Bifid-F:5'-CTCCTGGAAACGGGTGG-3' (SEQ ID NO: 3) g-Bifid-R: 5'-GGTGTTTCTCCCGATATCTACA-3' (SEQ ID NO: 4).

[0056] [Table 4] .

[0057] The results are shown in Figure 4 Data are expressed as relative values ​​(mean ± standard deviation, n=6) with the DNA copy number from Bifidobacterium from Comparative Example 7 set to 1.

[0058] The results showed that the combination of γ-PGA and FOS confirmed an effect of approximately 10-fold, on average, proliferation of Bifidobacteria in the pig gut compared to FOS intake alone. In particular, an even more significant increase in Bifidobacteria was observed when supplementing with γ-PGA at half the amount of FOS intake compared to FOS intake alone.

[0059] This application is based on Japanese Special Application No. 2017-195311 filed in Japan, the contents of which are fully contained in this specification. sequence list <110> Ajinomoto Co., Ltd. <120> Foods for improving the gut environment <130> 092805 <150> JP 2017-195311 <151> 2017-10-05 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 1 agcagtaggg aatcttcca 19 <210> 2 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 2 caccgctaca catggag 17 <210> 3 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 3 ctcctggaaa cgggtgg 17 <210> 4 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 4 ggtgttcttc ccgatatcta ca 22

Claims

1. A food for improving the intestinal environment, the food being composed of a combination of γ-polyglutamic acid and oligosaccharides. in, γ-Polyglutamic acid is a polymer composed of D-glutamic acid and L-glutamic acid in an 8:2 ratio, and containing 30 to 5000 glutamic acid molecules. Oligosaccharides are selected from one or more of the following: fructooligosaccharides, coffee bean mannan oligosaccharides, isomaltooligosaccharides, and galactooligosaccharides. The weight ratio of oligosaccharide to γ-polyglutamic acid, i.e., oligosaccharide:γ-polyglutamic acid, is 1:0.1 to 3.

2. The food product according to claim 1, wherein, Oligosaccharides exist in liquid form.

3. The food product according to claim 2, wherein, Oligosaccharides exist in the form of liquid preparations containing oligosaccharides.

4. The food product according to claim 2, wherein, Oligosaccharides exist in the form of beverages containing oligosaccharides.

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