Intestinal polyamine producers

Abstract

The purpose of the present invention is to produce a polyamine in the intestines using an oral agent.　An enteral polyamine production agent according to the present invention comprises: a bean-derived digestion-resistant peptide comprising a protein component comprising a bean protein material and a degradation residue obtained by the degradation with a degrading enzyme; and a lactic acid bacterium capable of degrading agmatine into putrescine.

Description

[Technical field]

[0001] The present invention relates to a technique for producing polyamines in the intestine, and more particularly to a technique for producing polyamines in the intestine by orally ingesting indigestible peptides. [Background technology]

[0002] In recent years, it has been reported that polyamine intake has the effect of extending lifespan and improving brain function (see Non-Patent Document 1), and there is a demand for the development of a method for efficiently supplying polyamines to the body. Various methods for producing polyamines in the large intestine using intestinal bacteria have been proposed (see Patent Document 1 and Non-Patent Document 2).

[0003] For example, Patent Document 1 proposes an intestinal polyamine enhancer in which a component that promotes the production of polyamines by intestinal bacteria, such as alanine or arginine, is formulated into a capsule or the like so that it can be delivered undigested to the large intestine.

[0004] In response to this, the present inventors are conducting research into a method for delivering a component that promotes polyamine production to the large intestine by orally ingesting a so-called indigestible peptide. It is believed that resistant peptides reach the large intestine in an undigested state, not broken down into the numerous amino acids that make them up, and are broken down into amino acids by the proteases of a wide variety of intestinal flora, which then further utilize these amino acids. It is known that arginine is included among the amino acids that make up soybean-derived resistant peptides (see Non-Patent Document 2). In addition, Non-Patent Document 3 reports that in tests using human feces and mice, arginine, a precursor of polyamines, increases the amount of polyamines produced by intestinal bacteria. Therefore, a method was devised in which soybean-derived resistant peptides are orally ingested and delivered undigested to the large intestine, where arginine is separated from the undigested resistant peptide by intestinal resident bacteria, and this is then utilized by the intestinal resident bacteria to produce polyamines. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Unexamined Patent Application Publication No. 2015-71604 [Non-Patent Document]

[0006] [Non-Patent Document 1] R. Hirano, H. Shirasawa, S. Kurihara, Health-Promoting Effects of Dietary Polyamines. Med Sci (Basel) 9, (2021). [Non-Patent Document 2] Noriko HIGAKI, Kenji SATO, Hitoshi SUDA, Tomohiko SUZUKA, Takeo KOMORI, Tohru SAEKI, Yasushi NAKAMURA, Kozo OHTSUKI, Kimikazu IWAMI, and Ryuhei KANAMOTO. Evidence for the Existence of a Soybean Resistant Protein That Captures Bile Acid and Stimulates Its Fecal Excretion. Biosci. Biotechnol. Biochem., 70 (12), 2844-2852, (2006) [Non-Patent Document 3] R. Kibe, S. Kurihara, Y. Sakai, H. Suzuki, T. Ooga, E. Sawaki, K. Muramatsu, A. Nakamura, A. Yamashita, Y. Kitada, M. Kakeyama, Y. Benno, M. Matsumoto, Upregulation of colonic luminal polyamines produced by intestinal microbiota delays senescence in mice. Sci Rep 4, 4548 (2014). [Non-Patent Document 4] Y. Sugiyama, M. Nara, M. Sakanaka, A. Gotoh, A. Kitakata, S.Okuda, S. Kurihara, Comprehensive analysis of polyamine transport and biosynthesis in the dominant human gut bacteria: potential presence of novel polyamine metabolism and transport genes. Int J Biochem Cell Biol, (2017).

Direct Environment 5

Outdoor Configuration 6

Direct Environment 7

[0007] However, the results of Test Example 1 described later showed that the polyamine concentration in the human fecal culture medium did not increase in the majority of people when soybean-derived indigestible peptides were simply added to the human fecal culture medium. On the other hand, as shown in Test Example 2 described later, it was found that the concentration of agmatine, a reaction intermediate in the production of putrescine from arginine, increased in the human fecal culture medium of Test Example 1 in the majority of people. Furthermore, Non-Patent Document 4 reports that Enterococcus faecalis takes up agmatine in the medium to produce ATP, and releases putrescine as a by-product outside the cell. Therefore, the inventors believe that by orally ingesting bacteria with the ability to break down agmatine into putrescine together with soy-derived indigestible peptides, even people who are unable to break down the agmatine produced in the intestine into putrescine will be able to produce putrescine in the large intestine, and ultimately be able to produce polyamines such as spermidine and spermine, which use putrescine as a precursor, in the intestine. That is, the present invention aims to provide a polyamine producer comprising a legume-derived, indigestible peptide and a bacterium that decomposes the indigestible-derived agmatine into putrescine when orally ingested simultaneously with the indigestible peptide. [Means for solving the problem]

[0008] The present invention, which has been made to solve the above problems, is a food product containing bean-derived indigestible peptides consisting of the residue of a bean protein material decomposed with a protease and lactic acid bacteria having the ability to decompose agmatine into putrescine. The lactic acid bacteria include Enterococcus faecalis or Leviractobacillus brevis. It is an intestinal polyamine producer.

[0009] The intestinal polyamine production agent of the present invention contains lactic acid bacteria that decompose agmatine into putrescine together with bean-derived indigestible peptides, so that agmatine released from the indigestible peptides in the intestine can be decomposed into putrescine by the lactic acid bacteria, and putrescine, a type of polyamine, can be efficiently produced in the intestine.

[0010] The legume protein material is preferably a soy protein material, which allows more efficient production of putrescine in the intestine.

[0011] The lactic acid bacteria are preferably food-derived lactic acid bacteria, which allows the polyamine producer to be orally ingested safely.

[0012] The lactic acid bacteria is preferably Lactobacillus brevis, which can produce putrescine more efficiently in the intestine.

[0013] The lactic acid bacteria is preferably Lactobacillus brevis derived from blue cheese, which allows more putrescine to be produced in the intestine. Effect of the Invention

[0014] As described above, the intestinal polyamine producer of the present invention can efficiently produce polyamines in the intestine by orally taking it. [Brief description of the drawings]

[0015] [Figure 1] 1 is a graph showing the results of Test Example 1, which investigated the effect of adding soybean-derived indigestible peptides on the putrescine concentration in human fecal culture medium. [Diagram 2] 2 is a graph showing the average putrescine concentration in the human feces culture fluid in Test Example 1 of FIG. 1. [Diagram 3] 1 is a graph showing the results of Test Example 2, which investigated the effect of adding soybean-derived indigestible peptides on the concentration of agmatine in human feces culture medium. [Figure 4] 4 is a graph showing the average agmatine concentration in human feces culture fluid in Test Example 2 of FIG. 3. [Diagram 5] 1 is a graph showing the results of investigating the effect of a medium (Example 1) to which soybean-derived indigestible peptide and Enterococcus faecalis were simultaneously added on the putrescine concentration in human feces culture medium. [Figure 6] FIG. 6 is a graph showing the average putrescine concentration in the human fecal culture fluid in FIG. 5. [Figure 7] 1 is a graph showing the results of Comparative Example 2 and Example 1, in which the effect of adding a resistant peptide alone or adding a resistant peptide and E. faecalis simultaneously on the putrescine concentration in human fecal culture medium was examined. [Figure 8] 1 is a graph showing the results of Example 2 in which the effect of simultaneously adding a soybean-derived indigestible peptide and Levilactobacillus brevis F215 to a medium on the putrescine concentration in a human feces culture medium was investigated. [Figure 9] 9 is a graph showing the average putrescine concentration in the human fecal culture fluid in Example 2 of FIG. 8. [Figure 10]1 is a graph showing the results of Example 3 in which the effect of simultaneously adding a soybean-derived indigestible peptide and Levilactobacillus brevis ( Levilactobacillus brevis JCM1059T ) to a medium on the putrescine concentration in a human feces culture medium was investigated. [Figure 11] 11 is a graph showing the average putrescine concentration in the human feces culture medium in Example 3 of FIG. 10. [Figure 12] This is a graph showing the results of Comparative Example 2 and Example 3, in which the effect of adding soybean-derived resistant peptide alone, or the simultaneous addition of soybean-derived resistant peptide and L. brevis JCM1059T, on the putrescine concentration in human fecal culture medium was investigated. [Figure 13] FIG. 1 is a graph showing the results of Examples 4 to 6 in which the effect of simultaneously adding indigestible peptides derived from peas, chickpeas, and broad beans and Levilactobacillus brevis F215 to a medium on the putrescine concentration in a human fecal culture medium was investigated. [Figure 14] 14 is a graph showing the average putrescine concentrations in human fecal culture fluids in Examples 4 to 6 of FIG. 13.

[0016] Hereinafter, embodiments of the present invention will be described in detail, however, the present invention is not limited to the following embodiments.

[0017] (Intestinal polyamine producer) The intestinal polyamine producer according to the present invention contains bean-derived indigestible peptides consisting of residues of a bean protein material decomposed with a protease, and lactic acid bacteria having the ability to decompose agmatine into putrescine.

[0018] (beans) Examples of beans that can be used as a raw material for the bean-derived indigestible peptides used in this embodiment include legumes such as soybeans, lupin beans, alfalfa, white clover, mung beans, adzuki beans, broad beans, peas, chickpeas, kidney beans, lentils, and cowpeas. It is widely known that legumes contain arginine (see, for example, Non-Patent Documents 6 to 8). As more specific embodiments, the cases of soybeans, peas, chickpeas, and broad beans will be described below.

[0019] (soy) The type of soybean used as the raw material for the soybean-derived resistant peptide of this embodiment is not particularly limited as long as it contains arginine as a constituent amino acid after being formed into the resistant peptide, and any known soybean such as yellow soybeans, green soybeans, black soybeans, white soybeans, brown soybeans, etc. can be used as appropriate.

[0020] (peas, chickpeas, broad beans) The type of beans used as the raw material for the pea-, chickpea-, or fava bean-derived resistant peptide according to this embodiment is not particularly limited as long as it contains arginine as a constituent amino acid after being formed into the resistant peptide, and any known pea, chickpea, or fava bean can be used as appropriate.

[0021] (Soy protein material) As the pulse protein material, a soy protein material can be suitably used. The type of soy protein material is not particularly limited as long as it is in a state that can be treated with a protease to form indigestible peptides, and examples thereof include whole soybeans, defatted soybeans, isolated soy protein, concentrated soy protein, fractionated soy protein, soy milk, defatted soy milk, and various processed products thereof, with isolated soy protein being preferred because it is readily available in the market.

[0022] (Protein ingredients derived from peas, chickpeas and fava beans) As the legume protein material, a protein material derived from pea, chickpea, or broad bean can be used. As the protein material derived from pea, Pea Protein PP-CS (manufactured by Organo Food Tech Co., Ltd.) is preferably used, as the protein material derived from chickpea, ChickP S930 (manufactured by ChickP Protein Ltd.), and as the protein material derived from broad bean, Broad Bean Protein Ol Protein (registered trademark) FP-AC (Organo Food Tech Co., Ltd.) is preferably used.

[0023] (Protease) In this embodiment, the protease used for forming the legume-derived indigestible peptide may be appropriately selected from proteases classified as "metal proteases", "acid proteases", "thiol proteases", and "serine proteases", regardless of whether it is of animal, plant, or microbial origin, and preferably from proteases classified as "metal proteases", "thiol proteases", and "serine proteases". In particular, enzymes belonging to two or more different categories, or three or more different categories, may be used.

[0024] This classification of proteases is a classification method based on the type of amino acid in the active center that is commonly used in the field of enzyme science. Representative examples of each are Bacillus neutral protease, Streptomyces neutral protease, Aspergillus neutral protease, and samoase for metalloproteases, pepsin, Aspergillus acid protease, and sumithium AP for acidic proteases, bromelain and papain for thiol proteases, and trypsin, chymotrypsin, subtilisin, Streptomyces alkaline protease, alcalase, and bioprase for serine proteases. The classification of enzymes other than these can also be confirmed based on their pH of action and reactivity with inhibitors. Since the site of action on the substrate differs greatly between enzymes with different active centers, the "uncleaved residue" can be reduced and enzyme hydrolysates can be obtained efficiently. In addition, by using enzymes from different origins (origin organisms) in combination, enzyme hydrolysates can be produced even more efficiently. If proteases of the same classification have different origins, they act on different sites on the substrate protein, which results in an increased proportion of dipeptides and tripeptides. It is preferable that these proteases have low exoactivity.

[0025] (Method of forming indigestible peptides) In this embodiment, the reaction pH and reaction temperature during the protease treatment of the pulse protein material may be set according to the characteristics of the protease used, and usually the reaction pH may be near the optimum pH, and the reaction temperature may be near the optimum temperature. The reaction temperature is generally 20 to 80° C., preferably 40 to 60° C. After the reaction, the mixture is heated to a temperature (approximately 60 to 170° C.) sufficient to inactivate the enzyme, thereby inactivating the remaining enzyme activity.

[0026] The reaction solution after the protease treatment can be used as it is or after concentration, but is usually sterilized and used in the form of a dry powder by spray drying, freeze drying, or the like. Sterilization is preferably heat sterilization, and the heating temperature is preferably 110 to 170°C, more preferably 130 to 170°C. The heating time is preferably 3 to 20 seconds. The reaction solution may be adjusted to a desired pH, and precipitates or suspended matter generated during pH adjustment may be removed by centrifugation, filtration, or the like. It may also be purified with activated carbon or an adsorption resin.

[0027] (lactic acid bacteria) The lactic acid bacteria used in this embodiment are not particularly limited as long as they have the ability to decompose agmatine into putrescine in the large intestine, and any of lactic acid bacteria not derived from foods, such as those derived from intestinal bacteria such as Enterococcus faecalis, and lactic acid bacteria derived from miso, yogurt, cheese, sake, seasonings, and other fermented foods can be used. However, in terms of safe oral intake, lactic acid bacteria derived from fermented foods, such as Levilactobacillus brevis, are preferred, and in particular, Levilactobacillus brevis derived from blue cheese is preferably used.

[0028] (Determination of whether lactic acid bacteria have the ability to decompose agmatine into putrescine) Whether or not a lactic acid bacterium has the ability to degrade agmatine derived from a legume-derived indigestible peptide into putrescine is determined by adding the lactic acid bacterium to be determined, together with a legume-derived indigestible peptide prepared by the same method as in Test Example 1 described below, and human feces, to a GAM medium, and measuring the putrescine concentration by HPLC as in Test Example 1. The putrescine concentration is measured using 10 or more media prepared using feces collected from 10 or more people, and if the increase in the average concentration is determined to be statistically significant based on the P value, the lactic acid bacterium is determined to have the ability to degrade agmatine derived from a legume-derived indigestible peptide into putrescine.

[0029] (Dosage form) When orally ingesting the intestinal polyamine-producing agent according to this embodiment, the form is not particularly limited as long as it can be safely ingested orally, and any form such as food, medicine, supplement, etc. can be selected. If the form is a medicine or a supplement, known dosage forms such as tablets, powders, liquids, gel tablets, capsules, etc. can be appropriately used. When the form is food, as a known solid, gel-like, or liquid food, or the intestinal polyamine-producing agent according to this embodiment can be mixed into such food and used.

Examples

[0030] Next, the tests conducted using the examples and comparative examples according to the present invention will be described in detail. However, the present invention is not limited to the following examples.

[0031] (Test Example 1) Using a sample with only human feces added to GAM medium (Comparative Example 1) and a sample with human feces and soy-derived indigestible peptides added to GAM medium (Comparative Example 2), the concentration of putrescine produced in the culture solution was examined. However, in Comparative Example 2, due to sterilization, externally, it appears that human feces are added to the medium with soy-derived indigestible peptides added.

[0032] (Comparative Example 1) <GAM Medium> To 200 mL of Elix water, 11.8 g of GAM broth was added and stirred to completely dissolve the GAM broth. After sterilization at 120 °C in an autoclave, degassing was performed immediately.

[0033] <Formation of Human Feces Sample> Human feces collected from 5 males and 5 females, a total of 10 people aged 4 to 58 years old shown in the donor list in Table 1 were dissolved and diluted in Elix water to adjust the initial turbidity (OD600) = 0.01 to form a human feces sample.

Table 1

[0034] <Human Fecal Culture> A human fecal sample was inoculated into GAM medium and cultured at 37 °C under anaerobic conditions for 24 hours. All experimental operations were performed inside an anaerobic chamber.

[0035] <Polyamine Analysis in Culture Supernatant> After completion of this culture with the human fecal-added medium, the quantification of the polyamine concentration in the centrifuged culture supernatant was performed using high performance liquid chromatography (HPLC) according to Non-Patent Document 4.

[0036] (Comparative Example 2) <Soybean-Derived Indigestible Peptide> As a soybean protein material, isolated soy protein (FujiPro F) was dissolved in water at pH 7 to a concentration of 3%, and Bioplase (origin: Bacillus sp., serine protease, Nagase ChemteX) was added at 1% based on the weight of the protein, and allowed to act at pH 7.5 and 58 °C for 60 minutes. Next, Sumitume FP (origin: Asprgillus sp., metalloprotease, Shin Nippon Chemical Industry) was added at 1% based on the weight of the protein, and allowed to act at pH 7.5 and 58 °C for 60 minutes. After the above treatments, the reaction was stopped at 90 °C for 20 minutes, followed by centrifugation to recover the insoluble precipitate, and the solid was obtained by lyophilization. The obtained solid was dissolved (suspended) in water to a concentration of 5%, pepsin was added at 2% based on the weight of the protein and adjusted to pH 2, and treated at 37 °C for 4 hours. Pancreatin was added at 1% based on the weight of the protein and adjusted to pH 8, and treated at 37 °C for 24 hours. After the above treatments, the reaction was stopped at 90 °C for 20 minutes, followed by centrifugation to recover the insoluble precipitate, and the soybean-derived indigestible peptide was obtained by lyophilization.

[0037] <GAM + 0.5% (w / v) Soybean-Derived Indigestible Peptide Medium> To 200 mL of Elix water, 11.8 g of GAM broth and 1 g (0.5% (w / v)) of soybean-derived indigestible peptide were added and stirred to completely dissolve the GAM broth. The soybean-derived indigestible peptide was hardly dissolved and became a suspended state. This was sterilized at 120 °C in an autoclave and immediately degassed.

[0038] <Human fecal culture> The human feces sample prepared in Comparative Example 1 was inoculated into a GAM+0.5% (w / v) soybean-derived indigestible peptide medium and cultured for 24 hours under anaerobic conditions at 37° C. All experimental procedures were carried out in an anaerobic chamber.

[0039] <Analysis of putrescine in culture supernatant> After completion of the culture in the medium supplemented with human feces, the putrescine concentration in the culture supernatant obtained by centrifugation was quantified using high performance liquid chromatography (HPLC) according to Non-Patent Document 4.

[0040] <Results of Test Example 1> The results of Test Example 1 are shown in Figures 1 and 2. As shown in Figure 1, in Comparative Example 2, in which the resistant peptide was added, the proportion of feces in which the putrescine concentration was increased was only 30% of the total (3 out of 10 people, feces 3, 5, and 8 in Figure 1) compared to Comparative Example 1, in which no resistant peptide was added to the human feces culture medium. Furthermore, as shown in Figure 2, the average putrescine concentration produced in the feces of 10 people showed no statistically significant difference between Comparative Example 1, in which only feces was added to the culture medium, and Comparative Example 2, in which the resistant peptide was added in addition to feces.

[0041] (Test Example 2) Using Comparative Example 1 (GAM medium to which only human feces was added) and Comparative Example 2 (GAM medium to which human feces and soybean-derived indigestible peptides were added) used in Test Example 1, the agmatine concentration in the culture medium was measured by HPLC in the same manner as the putrescine concentration was measured in Test Example 1.

[0042] <Results of Test Example 2> The results of Test Example 2 are shown in Figures 3 and 4. As shown in Figure 3, in Comparative Example 2 in which the resistant peptide was added, the feces in which the concentration of agmatine, a reaction intermediate in the production of putrescine from arginine, was increased accounted for 70% of the total (7 out of 10 people, feces 2 to 8 in Figure 3) compared to Comparative Example 1 in which no human feces was added to the culture medium. In addition, as shown in Figure 4, the average agmatine concentration in Comparative Example 2 in which feces and the resistant peptide were added was significantly higher than that in Comparative Example 1 in which only feces was added to the culture medium.

[0043] From the results of Test Examples 1 and 2, it is considered that there are some people for whom arginine released from the soybean-derived indigestible peptide that reaches the large intestine is converted to putrescine, and others for whom it is only converted to agmatine. Therefore, it is considered possible that putrescine in the large intestine can be increased by orally ingesting bacteria that convert agmatine to putrescine together with the soybean-derived indigestible peptide.

[0044] Example 1 In a similar manner to Test Example 1, a human fecal medium was used to investigate whether putrescine increases in the culture medium by adding soybean-derived indigestible peptides and Enterococcus faecalis (E. faecalis), a bacterium that breaks down agmatine into putrescine.

[0045] <Preparation of bacterial samples> A frozen glycerol stock of E. faecalis was scraped off with a sterile toothpick in an anaerobic chamber and inoculated into GAM medium that had been dispensed into a vial. The GAM medium in the vial and the glycerol stock of the added bacteria were mixed by inverting the vial, and then placed in a sealed container together with Anaeropack (Mitsubishi Gas Chemical Company, Inc.; product number A-03) and cultured for 24 hours at 37°C. Before culturing human feces, the mixture was diluted with Elix water and adjusted to an initial turbidity (OD600) of 0.01 to prepare a bacterial sample.

[0046] <Human fecal culture> The human feces sample prepared in Comparative Example 1 and the E. faecalis bacterial sample were inoculated into the GAM+0.5% (w / v) soybean-derived indigestible peptide medium prepared in the same manner as in Comparative Example 2, and cultured at 37°C under anaerobic conditions for 24 hours. All experimental procedures were performed in an anaerobic chamber.

[0047] <Analysis of putrescine in culture supernatant> After completing the cultivation of the medium containing human feces, soybean-derived indigestible peptides, and E. faecalis, the putrescine concentration in the centrifuged culture supernatant was quantified using high performance liquid chromatography (HPLC) in the same manner as in Test Example 1.

[0048] <Results of Example 1> The results of Example 1 are shown in Figures 5 to 7. As shown in Figure 5, in the medium of Example 1 to which human feces, soybean-derived indigestible peptide, and E. faecalis were added, an increase in putrescine was observed in 50% of the feces (5 out of 10 people) compared to the medium of Comparative Example 1 to which only human feces was added. Furthermore, as shown in Figure 6, in the medium of Example 1 to which human feces, soybean-derived indigestible peptides, and E. faecalis were added, the putrescine concentration reached an average of 3.6 times that of the medium of Comparative Example 1 to which only human feces was added, and the p-value of 0.038 indicated that the increase in putrescine concentration was statistically significant. Figure 7 shows the putrescine concentrations in Comparative Example 1, in which only human feces was added, Comparative Example 2, in which human feces and a resistant peptide were added, and Example 1, in which human feces, a resistant peptide, and E. faecalis were added. It was found that the putrescine concentration increased in the feces of a total of 70% (7 out of 10 people) by adding the resistant peptide alone and by adding the resistant peptide and E. faecalis simultaneously.

[0049] Example 2 The putrescine concentration in the culture medium was measured in the same manner as in Example 1, except that the bacteria added to the medium was changed from E. faecalis to Levilactobacillus brevis obtained as described below.

[0050] <Collection of bacteria> 1-2g of blue stilton cheese was filled up to 10-20mL with sterile PBS to make a 10% (w / V) solution. Kitchen scissors sterilized by spraying 70% ethanol were used for weighing. The sample was suspended by vortexing and stirring. Next, serial dilutions of 10-2, 10-3, 10-4, and 10-5 were prepared using sterile PBS. 100 μL of the diluted solution was spread on an MRS plate and cultured anaerobically at 37°C for 120 hours. A single colony was streaked on the MRS plate and cultured anaerobically at 37°C for 48 hours. Colonies of the 40 strains were inoculated into 500 μL of MRS broth and cultured anaerobically at 37°C for 48 hours. The culture was then centrifuged (1,900 xg, 20 minutes) and the culture supernatant was subjected to a simple quantitative analysis of putrescine using a simple quantitative method (PuO-POD-4AA-TOPS method, Anal Biochem 2020 593:113607.). Among the 40 strains, a bacterium that was suggested to be a high producer of putrescine was named FB215, and the putrescine concentration in the culture supernatant was quantified by HPLC, revealing that it contained 150 μM putrescine. The 16S rRNA gene was amplified by PCR using primer 1510R (5'-ACGGYTACCTTGTTACGACTT-3') and primer 7F (5'-AGAGTTTGATYMTGGCTCAG-3') with genomic DNA extracted from the FB215 cells as a template. The DNA base sequence of the amplified fragment was analyzed by Sanger sequencing using primer 518R (5'-GTATTACCGCGGCTGCTGG-3'). The obtained sequence was subjected to BLAST analysis, and it was found to be more than 98% identical to the 16S rDNA of Levilactobacillus brevis. Therefore, this strain is hereafter referred to as Levilactobacillus brevis FB215.

[0051] <Results of Example 2> The results of Example 2 are shown in Figures 8 and 9. As shown in Figure 8, in the medium of Example 2 to which human feces, soybean-derived indigestible peptide, and L. brevis F215 were added, the putrescine concentration increased in 80% of the feces (8 out of 10 people) compared to the medium of Comparative Example 1 to which only human feces was added. Furthermore, as shown in FIG. 9 , in the medium of Example 2 to which human feces, soybean-derived indigestible peptides, and L. brevis F215 were added, the putrescine concentration increased by an average of 3.8-fold compared to the medium of Comparative Example 1 to which only human feces was added, and the p-value was 0.0377, indicating that the increase in putrescine concentration was statistically significant.

[0052] Example 3 The putrescine concentration in the culture medium was measured in the same manner as in Example 1, except that the bacteria added to the medium was changed from E. faecalis to Levilactobacillus brevis (JCM1059T).

[0053] <Results of Example 3> The results of Example 3 are shown in Figures 10 and 11. As shown in Figure 10, in the medium of Example 3 to which human feces, soybean-derived indigestible peptide, and L. brevis JCM1059T were added, the putrescine concentration increased in 40% of the feces (4 out of 10 people) compared to the medium of Comparative Example 1 to which only human feces was added. Furthermore, as shown in FIG. 11, although the p-value was 0.1212 in the medium of Example 3 to which human feces, soybean-derived indigestible peptides, and L. brevis were added, it was found that the putrescine concentration in the feces of 10 individuals tended to increase compared to the medium of Comparative Example 1 to which only human feces was added.

[0054] Figure 12 shows the putrescine concentrations in Comparative Example 1, in which only human feces was added, Comparative Example 2, in which human feces and a resistant peptide were added, and Example 3, in which human feces, a resistant peptide, and L. brevis JCM1059T were added. It was found that the putrescine concentration increased in the feces of a total of 50% (5 out of 10 people) by adding the resistant peptide alone and by adding the resistant peptide and L. brevis JCM1059T simultaneously.

[0055] Comparative Example 3 Human feces collected at the same time as the human feces used in Comparative Example 1 was stored at -80°C under anaerobic conditions, thawed, and added to GAM medium. The putrescine concentration in the culture medium was measured in the same manner as in Comparative Example 1, using the method of Test Example 1.

[0056] Example 4 The resistant peptide was the pea-derived resistant peptide described below, and the same human feces as in Comparative Example 3 was used. In the same manner as in Example 2, except for this, Levilactobacillus brevis FB215 was cultured using GAM medium, and the putrescine concentration in the culture medium was measured.

[0057] <Pea-derived indigestible peptides> The pea-derived resistant peptide was prepared in the same manner as the soybean-derived resistant peptide described above, except that the bean protein material was pea protein PP-CS (manufactured by Organo Food Tech Co., Ltd.).

[0058] Example 5 The putrescine concentration in the culture medium was measured in the same manner as in Example 4, except that the resistant peptide was the chickpea-derived resistant peptide described below.

[0059] <Chickpea-derived indigestible peptides> The chickpea-derived resistant peptide was prepared in the same manner as the soybean-derived resistant peptide described above, except that the bean protein material was ChickP S930 (manufactured by ChickP Protein Ltd.).

[0060] Example 6 The putrescine concentration in the culture medium was measured in the same manner as in Example 4, except that the resistant peptide was the broad bean-derived resistant peptide described below.

[0061] <Indigestible peptides derived from broad beans> The broad bean-derived resistant peptide was prepared in the same manner as the soybean-derived resistant peptide described above, except that the bean protein material was broad bean protein Olprotein (registered trademark) FP-AC (Organo Food Tech Co., Ltd.).

[0062] <Results of Examples 4 to 6> The results of Example 4 to Example 6 are shown in Figures 13 and 14. As shown in Figure 13, in the media of Examples 4 to 6 to which human feces, pea-, chickpea-, or fava bean-derived indigestible peptides, and Levilactobacillus brevis FB215 were added, the putrescine concentration increased in all feces (10 out of 10 people) compared to the medium of Comparative Example 3 to which only human feces was added.

[0063] 14, in the media according to Examples 4 to 6 to which human feces, pea-, chickpea-, or fava bean-derived indigestible peptides, and L. brevis FB215 were added, the putrescine concentrations in the feces of 10 people were found to be significantly increased by 6.75-fold, 6.5-fold, and 9-fold, on average, compared to the medium according to Comparative Example 3 to which only human feces was added, as shown in FIG. 14. In addition, the results of Examples 4 to 6 were found to have low P values ​​of 0.0073, 0.0158, and 0.0068, respectively, and thus were found to be statistically significant.

[0064] As described above, the intestinal polyamine producer of the present invention is not limited to the above-mentioned embodiments and examples, and for example, lactic acid bacteria not derived from food may be used as the lactic acid bacteria that decomposes agmatine into putrescine. Lactic acid bacteria derived from fermented foods other than cheese may be used, or lactic acid bacteria collected from cheese other than blue cheese may be used. The indigestible peptide may be derived from beans other than soybeans, peas, chickpeas, and broad beans.

Claims

1. a bean-derived indigestible peptide comprising a residue of a bean protein material decomposed with a protease; Lactic acid bacteria capable of decomposing agmatine into putrescine Contains The lactic acid bacteria is an intestinal polyamine producing agent, which includes Enterococcus faecalis or Leviractobacillus brevis.

2. 2. The intestinal polyamine producing agent according to claim 1, wherein the legume protein material is a soybean protein material.

3. The intestinal polyamine producing agent according to claim 1 , wherein the lactic acid bacteria is food-derived lactic acid bacteria.

4. 4. The intestinal polyamine producing agent according to claim 3, wherein the lactic acid bacterium is Leviractobacillus brevis.

5. 5. The intestinal polyamine producing agent according to claim 4, wherein the lactic acid bacterium is Leviractobacillus brevis derived from blue cheese.

6. 2. The intestinal polyamine producing agent according to claim 1, wherein the legume protein material is a protein material derived from any one of peas, chickpeas, and broad beans.

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