DPP-4 Inhibitory Composition

Abstract

To provide a novel production method for a composition containing a high proportion of DPP-4 (dipeptidyl peptidase-4) inhibitory peptides.SOLUTION: A method for producing a DPP-4 inhibitory peptide includes the steps of: preparing a fermentation mix that contains protein or raw materials containing protein, and lactic acid bacteria having low activity of PepX (an exopeptidase to decompose a peptide having a proline at the second from the N-terminal); and fermenting the fermentation mix with the lactic acid bacteria to obtain a DPP-4 inhibitory peptide.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a DPP-4 inhibitor, comprising the steps of preparing a fermentation mix containing a protein and a lactic acid bacterium having low PepX activity, and fermenting the fermentation mix with the lactic acid bacterium to obtain a DPP-4 inhibitor peptide, and to feed, pharmaceuticals, and foods and beverages prepared by the method. The present invention also relates to lactic acid bacteria used in the method for producing the DPP-4 inhibitor. The present invention also relates to a method for producing a fermented food and beverage. [Background technology]

[0002] Dipeptidyl peptidase-4 (DPP-4) is an enzyme that recognizes the second alanine or proline from the N-terminus of proteins and peptides, and cleaves the dipeptide at the N-terminus. GLP-1 and GIP, which are important hormones for regulating blood glucose after meals, are quickly degraded by DPP-4 and lose their physiological activity. Inhibiting DPP-4 suppresses the degradation of GLP-1 and GIP. Since inhibiting DPP-4 can suppress the rise in blood glucose after meals, DPP-4 inhibitors have been marketed as diabetes treatment drugs.

[0003] DPP-4 inhibitors have been reported not only as medicines such as diabetes treatment drugs, but also as foods. For example, Patent Document 1 shows that fermented adzuki beans and kidney beans have DPP-4 inhibitory activity, Patent Document 2 shows that fermented soybeans have DPP-4 inhibitory activity, and Patent Document 3 shows that cheese has DPP-4 inhibitory activity. Unlike medicines, DPP-4 inhibitors have few side effects and can be taken on a daily basis, so they are expected to have a preventive effect against diseases such as diabetes.

[0004] Many of the active ingredients reported in foods that exhibit DPP-4 inhibitory activity are peptides that exhibit competitive inhibitory action. For example, Patent Document 3 lists various cheese-derived peptides that inhibit DPP-4 activity, all of which have a structure with proline or alanine at the second position from the N-terminus. These peptides are recognized by DPP-4, and are therefore considered to have an effect of competitively inhibiting the degradation of GLP-1 and GIP by DPP-4. In other words, if a method for producing a food that contains a high content of competitive inhibitor peptides with a structure with proline or alanine at the second position from the N-terminus is established, it will be possible to produce a diabetes prevention food.

[0005] Fermentation has been used since ancient times as a process for increasing various peptides in foods at low cost and with high efficiency. Lactic acid bacteria are one of the most common bacteria used in fermentation. Lactic acid bacteria are widely used in the production of fermented dairy products such as cheese and fermented milk, and meat products such as raw ham, and produce unique peptides and amino acids during the fermentation process. These peptides and amino acids are produced by proteases and peptidases possessed by lactic acid bacteria, and it is known that the types and activity levels of enzymes possessed by different species and strains of lactic acid bacteria differ. Therefore, in order to produce fermented foods with high peptide content, an approach has been taken to select lactic acid bacteria with high protease activity and peptidase activity (Non-Patent Document 1).

[0006] On the other hand, there are enzymes with undesirable activities for the purpose of producing foods with a high content of specific peptides. For example, an enzyme called PepX possessed by lactic acid bacteria is an exopeptidase that degrades peptides having a structure with proline at the second position from the N-terminus. Since PepX degrades the above-mentioned DPP-4 competitive inhibitory peptides, there is a problem that a strain with high PepX activity reduces the DPP-4 inhibitory activity of fermented foods. That is, even if a DPP-4 competitive inhibitory peptide is produced by fermentation, there is a problem that the produced DPP-4 competitive inhibitory peptide is degraded by the PepX activity possessed by the lactic acid bacteria strain. However, as described in Non-Patent Document 1, in the past, strains with high peptidase activity including PepX were sometimes selected as strains with good fermentation performance. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 5916387 [Patent Document 2] Patent No. 5984512 [Patent Document 3] Patent No. 4915833 [Non-patent literature]

[0008] [Non-Patent Document 1] Journal of Dairy Research(1995), 62, 601-610 Summary of the Invention [Problem to be solved by the invention]

[0009] The main object of the present invention is to provide a novel method for producing a DPP-4 inhibitor peptide. [Means for solving the problem]

[0010] As a result of intensive research aimed at solving the above-mentioned problems, the inventors discovered lactic acid bacteria with extremely low PepX activity, and found that peptides with DPP-4 inhibitory activity can be obtained highly efficiently by fermenting proteins using these lactic acid bacteria.

[0011] Specifically, the present invention is as follows. <1> A method for producing a DPP-4 inhibitor peptide, comprising the steps of: A step of preparing a fermentation mix containing a protein or a raw material containing a protein and a lactic acid bacterium having low PepX activity; and a step of fermenting the fermentation mix with the lactic acid bacteria to obtain a DPP-4 inhibitory peptide. <2> The PepX activity of the crude enzyme of lactic acid bacteria is 1000 RLU / min / μg protein or less. <1> A method for producing the DPP-4 inhibitor peptide described in claim 1. <3> The lactic acid bacteria having low PepX activity are characterized in that when a protein or a raw material containing a protein is fermented, the water-soluble fraction (1 mg protein / ml) inhibits DPP-4 activity by 50% or more, compared to a control sample to which pure water has been added. <1> or <2> A method for producing the DPP-4 inhibitor peptide described in claim 1. <4> The lactic acid bacterium having low PepX activity is at least one selected from the group consisting of the genus Lactobacillus and the genus Lactococcus. <1> ~ <3> 2. A method for producing a DPP-4 inhibitor peptide according to claim 1 , <5> The lactic acid bacteria belonging to the genus Lactobacillus or Lactococcus are at least one selected from the group consisting of Lactobacillus fermentum, Lactobacillus brevis, and Lactococcus lactis. <4> A method for producing the DPP-4 inhibitor peptide described in claim 1. <6> The lactic acid bacteria belonging to the genus Lactobacillus or Lactococcus are at least one selected from the group consisting of Lactobacillus fermentum SBT1859 (NITE P-02996), Lactobacillus brevis SBT10966 (NITE P-03245), and Lactococcus lactis subsp. cremoris SBT11373 (NITE P-03246). <4> A method for producing the DPP-4 inhibitor peptide described in claim 1. <7> Food additive grade endo-protease or peptidase was used in combination. <1> ~ <6> A method for producing the DPP-4 inhibitor peptide according to any one of claims 1 to 4. <8> <1> ~ <7> A feed containing a DPP-4 inhibitory peptide, a pharmaceutical product containing a DPP-4 inhibitory peptide, or a food or drink containing a DPP-4 inhibitory peptide, which is prepared by the method according to any one of the above. <9> New lactic acid bacteria Lactobacillus brevis SBT10966(NITE P-03245) <10> New lactic acid bacteria Lactococcus lactis subsp. cremoris SBT11373(NITE P-03246) <11> A method for producing a fermented food or drink, comprising: A step of preparing a fermentation mix containing a protein or a raw material containing a protein and a lactic acid bacterium having low PepX activity; fermenting the fermentation mix with the lactic acid bacteria to obtain a fermented food or drink; Including, A method for producing a fermented food or beverage, characterized in that the PepX activity of the crude enzyme of the lactic acid bacteria is 1000 RLU / min / μg protein or less. Effect of the Invention

[0012] According to the present invention, peptides having DPP-4 inhibitory activity can be produced efficiently. [Brief description of the drawings]

[0013] [Figure 1] This graph shows the chemiluminescence intensity when a PepX substrate, which emits chemiluminescence upon decomposition, was mixed with crude enzymes from lactic acid bacteria and allowed to react for 30 minutes, divided by the time (minutes) and the weight of crude enzyme protein (μg protein). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] (Protein used as raw material) In the manufacturing method of the present invention, the raw material for the fermentation mix preparation step may be either animal-derived or plant-derived protein. Examples of such raw materials include meat, seafood, dairy products, soybeans, wheat, etc., and among them, dairy products, soybeans, or processed products thereof, which are widely consumed and suitable for the growth of safe lactic acid bacteria, are preferred. As the dairy product, any milk commonly used in food manufacturing may be used, and examples thereof include whole milk, skimmed milk preparation, reconstituted milk, concentrated milk, buttermilk, cream, skimmed milk powder, milk protein, soybean milk, etc., or mixtures thereof.

[0015] (Lactic acid bacteria belonging to the genus Lactobacillus or Lactococcus) In the fermentation mix preparation step in the production method of the present invention, it is preferable to use lactic acid bacteria whose crude enzyme X-Pro dipeptidase activity (PepX activity) is 1000 RLU / min / μg protein or less. As the lactic acid bacteria, lactic acid bacteria classified into the genus Lactobacillus or Lactococcus are preferable. In this specification, the term "crude enzyme" refers to an extract obtained by crushing lactic acid bacteria and centrifuging them. Specifically, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus delbrueckii subsp. bulgaricusdelbrueckii, Lactobacillus delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus Examples of Lactobacillus lactis include, but are not limited to, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. lactis. In the fermentation mix preparation step in the production method of the present invention, it is preferable to use, as the lactic acid bacteria belonging to the genus Lactobacillus or Lactococcus, Lactobacillus fermentum SBT1859 (NITE P-02996), Lactobacillus brevis SBT10966 (NITE P-03245), or Lactococcus lactis subsp. cremoris SBT11373 (NITE P-03246). The above strains are available from the Patent Microorganisms Depository Center, Biotechnology Center, National Institute of Technology and Evaluation (Kisarazu, Chiba, Japan).

[0016] (Preparation of bacteria belonging to the genus Lactobacillus or Lactococcus) The lactic acid bacteria used in the fermentation step in the production method of the present invention may be cultured according to a conventional method for culturing each bacterium, and a desired amount may be prepared. An example of the preparation is shown below. Lactic acid bacteria belonging to the genus Lactobacillus are cultured using MRS medium (Difco), and lactic acid bacteria belonging to the genus Lactococcus are cultured using M17 medium (Difco), and the resulting culture is collected by centrifugation to obtain bacterial cells. The obtained bacterial cells may be used as they are, or the bacterial cells may be subjected to a concentration, drying, or freeze-drying treatment. The bacterial cells may be disrupted using a disrupting device such as a bead shocker, ultrasonic disrupter, or French press, and used as they are, or the disrupted bacterial cells may be centrifuged and the crude enzyme extract of the supernatant may be used. In the production method of the present invention, the viable bacteria in the fermentation mix before culture is 1 × 10 4 cfu / g ~1×10 10 cfu / g, 1×10 5 cfu / g ~1×10 9 cfu / g, or 1 x 10 6 cfu / g1×10 8 When a crude enzyme extract is used, the crude enzyme extract prepared according to the procedure of the Examples can be adjusted to 0.01% by mass to 20% by mass, 0.05% by mass to 10% by mass, 0.05% by mass to 10% by mass, or 0.1% by mass to 5% by mass in the fermentation mix before culture.

[0017] (Method for evaluating PepX activity) The method described in the Examples, i.e., DPPIV-Glo TM The evaluation can be performed using a method that uses Protease Assay (Promega). Specifically, the method is as follows. (1) The bacterial powder was dissolved in a buffer solution (50 mM HEPES, 15 mM CaCl2 ) and then ultrasonically disrupted on ice using a SONIFIER SFX150HH (BRANSON) (on time 5 sec, off time 5 sec, total 30 min, Amplitude 30%). (2) After disruption, centrifuge at 15,000 × g for 10 minutes at 4 °C and recover the supernatant. (3) Produce DPPIV-Glo according to the kit instructions. TM Prepare the reagent. (4) DPPIV-Glo was added to each well of a 384-well white plate (Perkin Elmer). TM 25μl of Reagent, buffer (50mM HEPES, 15mM CaCl 2 ) or add 20 μl of Example Product 1, mix well by pipetting, and incubate in the dark at room temperature for 30 minutes. (5) After incubation, the luminescence intensity (RLU) is measured at 1000 msec using a VICTOR Nivo Multimode Microplate Reader (Perkin Elmer). (6) Protein concentration was measured using Pierce TM Measure using the BCA Protein Assay Kit. (7) RLU is corrected for reaction time (30 min), reaction volume (45 μl), and protein concentration (mg / ml) and calculated as PepX activity units (RLU / min / μg protein).

[0018] As used herein, "low PepX activity" preferably means that when the PepX activity of the crude enzyme from lactic acid bacteria is evaluated by the above steps (1) to (7), the PepX activity units (RLU / min / μg protein) is 1000 or less. The PepX activity of the crude enzyme from lactic acid bacteria used in the fermentation mix preparation step in the production method of the present specification is preferably 600 RLU / min / μg protein or less, more preferably 300 RLU / min / μg protein or less.

[0019] (How to use) Examples of foods and beverages obtainable by the production method of the present invention include fermented milk foods, fermented soy milk foods, pickles, natto, alcoholic beverages, and the like.

[0020] In the method of the present invention, in addition to the bacteria with low PepX activity, endoprotease or peptidase that does not recognize the structure having proline or alanine at the second position from the N-terminus may be used in combination. These enzymes do not degrade DPP-4 inhibitor peptides, so the effect of the present invention can be further improved. Examples of the enzymes that can be used in combination with the bacteria with low PepX activity include food additive grade pepsin, trypsin, chymotrypsin, etc.

[0021] Even when multiple types of bacteria with low PepX activity are inoculated into a raw material containing dairy products and fermented, conditions suitable for the growth of the bacteria used and the enzyme reaction, etc., may be set. Generally, fermentation conditions are 10 to 42°C for 1 to 3 days. The food obtained in this manner is a fermented milk food containing DPP-4 inhibitor peptide. By using raw materials containing dairy products, fermentation can be performed well and the fermentation time can be shortened. The resulting fermented milk food has a very good flavor and aroma. Examples of dairy foods include cheese, yogurt, and soy milk.

[0022] In the production method of the present invention, when a protein or a raw material containing a protein is fermented with the lactic acid bacterium having low PepX activity, the water-soluble fraction (1 mg protein / ml) inhibits DPP-4 activity, preferably by more than 50%, more preferably by more than 60%, compared to a control sample to which pure water has been added.

[0023] As the pharmaceutical product obtained by the method of the present invention, for example, the obtained DPP-4 inhibitor peptide-containing material may be used as it is, or the dried powder may be used as the active ingredient. There is no particular limitation on the drying method, but the freeze-drying method, which can suppress the deterioration of the ingredients, is preferable. These powders can be mixed with a suitable excipient such as lactose and formulated into powders, tablets, pills, capsules, syrups, etc.

[0024] Furthermore, as for the feed obtained by the method of the present invention, for example, the obtained DPP-4 inhibitor peptide-containing material may be blended into any feed, or may be added to the raw materials during the production process. Furthermore, the food and drink obtained by the method of the present invention can also be used as a functional food, a food for specified health uses, a food with nutrient function claims, or a cosmetic food.

[0025] The present invention will be described in more detail below with reference to Test Examples, Comparative Examples, and Examples, but the present invention should not be construed as being limited to these Examples, etc. Unless otherwise specified, % indicates % by mass.

[0026] [Test Example 1] In order to select lactic acid bacteria with low PepX activity, the following evaluation was carried out. Each test bacterium in the following (1) was inoculated into MRS medium (Difco) for the genus Lactobacillus and M17 medium (Difco) for the genus Lactococcus, and static culture was performed at 30 to 37°C for 16 to 24 hours. The culture was washed twice with physiological saline and once with sterile water to obtain washed bacterial cells. The washed bacterial cells were freeze-dried to obtain bacterial cell powder. The bacterial cell powder was diluted to 1 mg / ml with a buffer solution (50 mM HEPES, 15 mM CaCl 2 ) and then ultrasonically disrupted on ice using a SONIFIER SFX150HH (BRANSON) (on time 5 sec, off time 5 sec, total 30 min, Amplitude 30%). After disruption, the cells were centrifuged at 15,000 × g for 10 min at 4°C, and the supernatant was collected. The collected supernatant was used as the lactic acid bacteria crude enzyme extract. (1) Test bacteria Lactobacillus helveticus JCM1120 T , Lactobacillus helveticus A strain, Lactobacillus fermentum SBT1859 (NITE P-02996), Lactobacillus fermentum B strain, Lactobacillus brevis SBT10966 (NITE P-03245) Lactobacillus brevis C strain, Lactococcus lactis subsp. cremoris SBT11373 (NITE P-03246), Lactococcus lactis subsp. lactis D strain, and Lactococcus lactis subsp. lactis E strain were used as test bacteria. The above strains are available from the Patent Microorganisms Depository Center, Biotechnology Center, National Institute of Technology and Evaluation (Kisarazu, Chiba, Japan).

[0027] The PepX activity of the lactic acid bacteria crude enzyme extract was enhanced by DPPIV-Glo, which contains a substrate with a proline at the second position from the N-terminus (Gly-Pro-Aminoluciferine). TM The assay was performed using the Protease Assay (Promega). TM Reagent was prepared. DPPIV-Glo was added to each well of a 384-well white plate (Perkin Elmer). TM 25μl of Reagent, buffer (50mM HEPES, 15mM CaCl 2 ) or lactic acid bacteria crude enzyme extract (20 μl) was added, mixed well by pipetting, and incubated in the dark at room temperature for 30 minutes. After incubation, the luminescence intensity (RLU) was measured at 1000 msec using a VICTOR Nivo Multimode Microplate Reader (Perkin Elmer). The protein concentration in the lactic acid bacteria crude enzyme extract was measured using the Pierce TM Measurement was performed using the BCA Protein Assay Kit. The unit of PepX activity of the lactic acid bacteria crude enzyme extract was calculated as RLU / min / μg protein by correcting the RLU for the reaction time (30 min), reaction solution volume (45 μl), and protein concentration (mg / ml).

[0028] The PepX activity of the lactic acid bacteria crude enzyme extracts is shown in Figure 1. PepX activity varies depending on the species and strain of bacteria. Lactobacillus helveticus JCM1120, which is known to have high PepX activity, T The activity of Lactobacillus fermentum SBT1859, Lactobacillus brevis SBT10966, and Lactococcus lactis subsp. cremoris SBT11373 was less than 1000 RLU / min / μg protein. The activity of Lactobacillus fermentum SBT1859 was 221 RLU / min / μg protein, that of Lactobacillus brevis SBT10966 was 231 RLU / min / μg protein, and that of Lactococcus lactis subsp. cremoris SBT11373 was 542 RLU / min / μg protein. That is, Lactobacillus fermentum SBT1859, Lactobacillus brevis SBT10966 and Lactococcus lactis subsp. cremoris SBT11373 were found to be bacteria with low PepX activity.

[0029] Comparative Example 1 Lactobacillus helveticus JCM1120 is a bacterium with high PepX activity. T Fermented milk A was produced using the Lactobacillus helveticus JCM1120 prepared in Test Example 1. That is, 10% skim milk was sterilized at 85°C for 20 seconds and then cooled to 37°C. T 1% crude enzyme extract or 1×10 live bacteria 7cfu / g and reacted at 37°C for 72 hours. The enzyme reaction was then stopped by heating at 80°C for 30 minutes. The supernatant, from which the precipitate was removed by centrifugation, was filtered through filter paper (ADVANTEC) and an ultrafiltration filter (Millipore) to obtain a permeate. The protein concentration of the resulting permeate was measured, and it was diluted with ultrapure water to 1 mg protein / ml, and the DPP4-inhibitory activity was measured.

[0030] DPP-4 activity was determined by DPPIV-Glo TM The assay was performed using the Protease Assay (Promega). TM Reagent was prepared. Human recombinant DPP-4 (Bio Vision) was thawed on ice and diluted to 10 ng / ml with PBS(-). DPPIV-Glo was placed in each well of a 384-well white plate (Perkin Elmer). TM 25 μl of the reagent and 20 μl of the sample or skim milk medium diluted with ultrapure water to 1 mg protein / ml were added, mixed well by pipetting, and incubated in the dark at room temperature for 30 minutes. After incubation, the luminescence intensity (RLU) was measured at 1000 msec using a VICTOR Nivo Multimode Microplate Reader (Perkin Elmer). The luminescence intensity was corrected by the measurement value of the blank (well with PBS(-) added instead of DPP-4) that does not contain DPP-4, and then shown as a relative value when the value of the control (well with ultrapure water added instead of the sample) was set as 100%. EXAMPLES

[0031] Fermented milk B was produced using Lactobacillus fermentum SBT1859 (NITE P-02996) as a bacterium with low PepX activity. That is, 10% skim milk was sterilized at 85°C for 20 seconds and then cooled to 37°C. Then, 1% of the crude enzyme extract of Lactobacillus fermentum SBT1859 (NITE P-02996) prepared in Test Example 1 or 1 x 10 live bacteria was added. 7 cfu / g and reacted at 37°C for 72 hours. The enzyme reaction was then stopped by heating at 80°C for 30 minutes. The supernatant, from which the precipitate was removed by centrifugation, was filtered through filter paper (ADVANTEC) and an ultrafiltration filter (Millipore) to obtain a permeate. The protein concentration of the obtained permeate was measured, and the solution was diluted with ultrapure water to 1 mg protein / ml, and the DPP4-inhibitory activity was measured in the same manner as in Comparative Example 1. The results are shown in Table 1.

[0032] [Table 1]

[0033] From the results shown in Table 1, Lactobacillus helveticus JCM1120 with high PepX activity T Compared with fermented milk A produced with Lactobacillus fermentum SBT1859 (NITE P-02996), which has low PepX activity, fermented milk B produced with Lactobacillus fermentum SBT1859 (NITE P-02996) was shown to strongly inhibit DPP-4 activity. Therefore, it was demonstrated that DPP-4 inhibitor peptides can be efficiently produced by fermenting with lactic acid bacteria with low PepX activity.

[0034] Comparative Example 2 Gouda cheese was produced using lactic acid bacteria with high PepX activity. 100 kg of raw milk (fat content 3.5%) was heat-sterilized at 75°C for 15 seconds, and then Lactobacillus helveticus JCM1120, which has high PepX activity, was added. TA mixed starter containing 1% was added, fermented at 31°C, and rennet, a milk-clotting enzyme, was added. After cutting, the whey was removed while stirring, and 80°C hot water was gradually added so that the final temperature was 37°C. After stirring for another 30 minutes, the resulting curd was allowed to settle and pressed with a steel plate. When the curd solidified, the whey was removed, and the curd was coated with wax through the process of molding, squeezing, and salting, and aged for 12 months. After aging, the cheese was cut into small pieces, 10 times the amount of water was added, crushed and suspended in a homogenizer, and the supernatant, from which the precipitate was removed by centrifugation, was filtered with filter paper (ADVANTEC) and an ultrafiltration filter (Millipore) to obtain a permeate. The protein concentration of the obtained permeate was measured, and it was diluted with ultrapure water to 1 mg protein / ml, and DPP4-inhibitory activity was measured in the same manner as in Comparative Example 1. EXAMPLES

[0035] Gouda cheese was produced using lactic acid bacteria with low PepX activity. 100 kg of raw milk (fat content 3.5%) was heat-sterilized at 75°C for 15 seconds, and then 1% of a mixed starter containing Lactobacillus fermentum SBT1859 (NITE P-02996) with low PepX activity was added. Fermentation was performed at 31°C, and rennet, a milk-clotting enzyme, was added. After cutting, whey was removed while stirring, and hot water at 80°C was gradually added so that the final temperature was 37°C. After stirring for another 30 minutes, the resulting curd was allowed to settle and pressed with a steel plate. When the curd solidified, the whey was removed, and it was molded, pressed, salted, coated with wax, and aged for 12 months. After ripening, the cheese was cut into small pieces, 10 times the amount of water was added, and the cheese was crushed and suspended in a homogenizer. The precipitate was removed by centrifugation, and the supernatant was filtered through a filter paper (ADVANTEC) and an ultrafiltration filter (Millipore) to obtain a permeate. The protein concentration of the obtained permeate was measured, and the permeate was diluted with ultrapure water to 1 mg protein / ml, and the DPP4-inhibitory activity was measured in the same manner as in Comparative Example 1. As a result, compared to the Gouda cheese produced in Comparative Example 2, the Gouda cheese produced with Lactobacillus fermentum SBT1859 (NITE P-02996), which has low PepX activity, strongly inhibited DPP-4 activity. EXAMPLES

[0036] Soy milk was produced using lactic acid bacteria with low PepX activity. After soaking the raw soybeans, Lactobacillus fermentum SBT1859 (NITE P-02996), which has low PepX activity, was added. The raw materials were then ground, heated, and pressed to produce soy milk. Note that the raw materials other than the microorganisms used in this method, the amounts used, and the temperature and time in each process were the same as those used in the production of regular soy milk. [Industrial Applicability]

[0037] According to the present invention, a method for producing a DPP-4 inhibitory peptide can be provided, which comprises the steps of preparing a fermentation mix containing a protein and lactic acid bacteria having low PepX activity, and fermenting the fermentation mix with the lactic acid bacteria to obtain a DPP-4 inhibitory peptide.

[0038] [Reference to deposited biological material] (1) SBT1859 (a) The name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation Patent Microorganism Deposit Center 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818) Date of deposit of biological material in a depository institution in the Republic of Korea June 26, 2020 (original deposit date) The accession number given to the deposit by the depository institution in question NITE P-02996 (2) SBT10966 (a) The name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation Patent Microorganism Deposit Center 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818) Date of deposit of biological material in a depository institution in the Republic of Korea July 14, 2020 (original deposit date) The accession number given to the deposit by the depository institution in question NITE P-03245 (3)SBT11373 (a) The name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation Patent Microorganism Deposit Center 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818) Date of deposit of biological material in a depository institution in the Republic of Korea July 14, 2020 (original deposit date) The accession number given to the deposit by the depository institution in question NITE P-03246

Claims

1. Preparing a fermentation mix containing a protein or a raw material containing a protein and a lactic acid bacterium having low PepX activity; fermenting the fermentation mix with the lactic acid bacteria to obtain a DPP-4 inhibitor peptide; A method for producing a DPP-4 inhibitor peptide, comprising: The PepX activity of the crude enzyme of the lactic acid bacteria is 1000 RLU / min / μg protein or less; The method for producing a DPP-4 inhibitor peptide is characterized in that the lactic acid bacterium is at least one selected from the group consisting of Lactobacillus fermentum and Lactobacillus brevis.

2. The method for producing the DPP-4 inhibitor peptide described in claim 1, characterized in that when a protein or a raw material containing a protein is fermented with the lactic acid bacterium having low PepX activity, the water-soluble fraction (1 mg protein / ml) inhibits DPP-4 activity by 50% or more compared to a control sample to which pure water has been added.

3. The method for producing the DPP-4 inhibitor peptide according to claim 1 or 2, characterized in that the lactic acid bacteria belonging to the genus Lactobacillus are at least one selected from the group consisting of Lactobacillus fermentum SBT1859 (NITE P-029 96) and Lactobacillus brevis SBT10966 (NITE P-03245).

4. A method for producing the DPP-4 inhibitor peptide according to any one of claims 1 to 3, in which a food additive grade endoprotease or peptidase is used in combination.

5. Novel lactic acid bacterium Lactococcus lactis subsp. cremoris SBT11373 (NITE P-03246).

6. Preparing a fermentation mix containing a protein or a raw material containing a protein and a lactic acid bacterium having low PepX activity; fermenting the fermentation mix with the lactic acid bacteria to obtain a fermented food or drink; Including, A method for producing a fermented food or drink, characterized in that the PepX activity of the crude enzyme of the lactic acid bacteria is 1000 RLU / min / μg protein or less, and the lactic acid bacteria is at least one selected from the group consisting of Lactobacillus fermentum and Lactobacillus brevis.

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