A polypeptide, a casein hydrolyzed powder containing the polypeptide and application of the polypeptide in promoting proliferation of lactic acid bacteria

Abstract

The application discloses a kind of polypeptide and casein hydrolyzed powder containing the polypeptide and application in promoting lactic acid bacteria proliferation, belong to food microbiological technology field.The polypeptide disclosed in the application has the amino acid sequence of KEGIHAQ.The polypeptide can significantly improve the acid resistance of lactic acid bacteria, and when 0.003-5g / L is added, the viable count of lactic acid bacteria can be increased by 1.2-3 times.The short peptide can be prepared by casein powder hydrolysis, and the casein hydrolysate obtained by hydrolysis and its freeze-dried powder containing the peptide segment also have the effect of promoting lactic acid bacteria proliferation, and are suitable for actual industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food microbiology, and more particularly to a kind of polypeptide and casein hydrolyzed powder containing the polypeptide and application in promoting lactic acid bacteria proliferation. BACKGROUND

[0002] Lactic acid and other organic acids are the main metabolic products of lactic acid bacteria during cultivation, and their accumulation can cause acid stress to lactic acid bacteria, leading to a significant decrease in strain viability. In addition, low pH and the presence of organic acids are key factors that inhibit the batch cultivation of lactic acid bacteria. Providing nutrients and growth factors required for cell growth is an effective measure to promote cell proliferation. Effective strategies for improving lactic acid bacteria performance under acid stress conditions include adding protective substances such as amino acids such as arginine and glutamic acid, sugars such as trehalose, and active peptides such as glutathione. Currently, methods to improve lactic acid bacteria cultivation density mainly involve optimizing the culture medium (CN202310663637.8, CN202211495838.3) and developing active ingredients to improve lactic acid bacteria acid tolerance.

[0003] Proteins play an important role in providing amino acids, peptides, and other growth factors required for cell growth, improving cell resistance to adverse environments, and increasing the activity of amino peptides and proteases. Casein is the main protein in milk and is the main source of essential and growth-stimulating amino acids for lactic acid bacteria, and contains many oligopeptides containing growth-stimulating amino acids. Moreover, appropriate peptides can be more effectively and widely absorbed by lactic acid bacteria than free amino acids. However, casein cannot be directly utilized and needs to be broken down by proteases to obtain peptides and amino acids, which are then utilized by active transport through specific transport proteins (PepT1, Dpp, DtpT, etc.) or passive transport through the paracellular pathway. Adding casein hydrolysate as an additive to lactic acid bacteria culture medium is an effective measure for the utilization of casein by cells. Due to differences in cleavage sites of different enzymes and conditions such as enzyme hydrolysis time and temperature, casein can be hydrolyzed to produce casein hydrolysate containing different amino acids and peptides, thereby producing different effects on cell growth. However, there is currently a lack of research on the active polypeptides in casein hydrolysate that enhance lactic acid bacteria acid tolerance.

[0004] Therefore, it is an urgent problem for those skilled in the art to provide a polypeptide and casein hydrolyzed powder containing the polypeptide and application in promoting lactic acid bacteria proliferation. SUMMARY

[0005] Therefore, it is an urgent problem for those skilled in the art to provide a polypeptide and casein hydrolyzed powder containing the polypeptide and application in promoting lactic acid bacteria proliferation.

[0006] A polypeptide capable of improving acid tolerance of lactic acid bacteria during culture of Lactobacillus bulgaricus sp1.1, which is derived from casein hydrolysate.

[0007] In order to achieve the above object, the present application adopts the following technical scheme:

[0008] A polypeptide, the amino acid sequence of which is KEGIHAQ.

[0009] Further, the polypeptide is used for promoting proliferation of lactic acid bacteria.

[0010] Further, the polypeptide is used for improving acid tolerance and culture density of lactic acid bacteria.

[0011] Further, the polypeptide is added in an amount of 0.003-5 g / L at the beginning of the stable phase of lactic acid bacteria culture.

[0012] Further, a casein hydrolysate powder containing the polypeptide.

[0013] Further, a preparation method of the casein hydrolysate powder, comprising the following steps: dissolving casein in deionized water to prepare a 5% (w / v) casein solution, hydrolyzing the casein by papain at an enzyme-to-substrate ratio (E / S) of 2000 U / g casein for 2 h, wherein the hydrolysis is carried out at a pH of 7.0 and a temperature of 37 ℃; immediately inactivating all enzymes after the enzymolysis by heating the mixture in a boiling water bath for 3-20 min; then centrifuging at 6000-10000 x g for 10-30 min, and freeze-drying the obtained supernatant to prepare a casein hydrolysate powder or storing it at-80 ℃.

[0014] Further, the casein hydrolysate powder or the casein hydrolysate powder prepared by the preparation method is used for promoting proliferation of lactic acid bacteria.

[0015] Further, the casein hydrolysate powder or the casein hydrolysate powder prepared by the preparation method is used for improving acid tolerance and culture density of lactic acid bacteria.

[0016] Further, the casein is derived from cow milk.

[0017] Via the technical solution, compared with the prior art, the application provides a polypeptide, casein hydrolyzed powder containing the polypeptide and application of the polypeptide in promoting proliferation of lactic acid bacteria. The polypeptide can significantly improve acid resistance of the lactic acid bacteria. When 0.003-5 g / L of the polypeptide is added, the number of viable lactic acid bacteria can be increased by 1.2-3 times. The short peptide can be prepared by hydrolysis of casein powder. The casein hydrolysate containing the peptide segment obtained by hydrolysis and the freeze-dried powder also have the effect of promoting proliferation of lactic acid bacteria, and are suitable for actual industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and all other drawings obtained by those skilled in the art without creative labor based on the provided drawings also belong to the protection scope of the present application.

[0019] Figure 1 The figure is the molecular formula of the polypeptide of the present application;

[0020] Figure 2 The figure is the influence of different concentrations of active ingredients on the growth of L.bulgaricus sp1.1;

[0021] Figure 3 The figure is the influence of casein hydrolysates with different degrees of hydrolysis on the proliferation of L.bulgaricus sp1.1 in the stationary phase;

[0022] A: degree of hydrolysis of casein hydrolyzed by papain; B: influence of hydrolysates added with different hydrolysis times on the proliferation of L.bulgaricus sp1.1 in the stationary phase;

[0023] Figure 4 The figure is the influence of casein hydrolysates on the proliferation of L.bulgaricus sp1.1 at critical pH; Control: without casein and its hydrolysates; Casein: containing casein; hydrolysates: containing casein hydrolysates after 2h of hydrolysis. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor also belong to the protection scope of the present application.

[0025] Example 1 KEGIHAQ (molecular formula of polypeptide is shown below) Figure 1 ) to improve lactic acid culture density

[0026] Lactobacillus delbrueckii subsp. bulgaricus sp1.1 (L. bulgaricus sp1.1) with 4*10 8 CFU / mL was normally cultured in MRS medium to the early stationary phase (12 h, at which time the OD 600nm of the bacterial solution was 1.2), and synthetic KEGIHAQ powder (synthesized by Shengong Bioengineering Co., Ltd.) was added at a concentration of 0 (control group), 0.003, 0.03, 0.3, 0.5, 1, 3, 5 g / L. The culture was continued to be cultured at 37°C for 12 h, and then the growth of the bacterial cells was determined. The viable cell count was determined using the pour plate method. The bacterial solution diluted with a 0.9% NaCl gradient was poured into a petri dish with 20 mL of MRS solid medium, and after cooling and solidification, it was incubated in a 37°C incubator for 48 h before counting.

[0027] The results are shown in Table 1. Figure 2 As shown in Table 1, the addition of KEGIHAQ can significantly increase the viable cell count of lactic acid bacteria, and when the addition amount reaches 1 g / L, the viable cell count is increased by nearly 3 times.

[0028] Example 2 Casein hydrolysis to produce KEGIHAQ polypeptide and its effect on acid tolerance and culture density of lactic acid bacteria

[0029] Casein (C9260-250, Solarbio) was dissolved in deionized water to prepare a 5% (w / v) casein solution, and was hydrolyzed by papain at an enzyme to substrate ratio (E / S) of 2000 U / g protein for 0.5 h, 1 h, 2 h, 4 h, and 6 h. The hydrolysis conditions were pH 7.0 and temperature 37°C. After the enzyme hydrolysis was completed, all enzymes were inactivated immediately by heating the mixture in a boiling water bath for 5 min. Then it was centrifuged at 6000 x g for 15 min, and the supernatant obtained was freeze-dried for later use.

[0030] Nano LC Ultimate 3000 series system was used in combination with a Q-Enactive mass spectrometer (Thermos Scientific, USA) with an electrospray ionization source to identify whether the peptide spectrum of the hydrolysis product contained KEGIHAQ. The results showed that when the hydrolysis time was 2 hours, KEGIHAQ polypeptide could be obtained, and it was not found at other hydrolysis times.

[0031] Lactobacillus delbrueckii subsp. bulgaricus sp1.1 (L. bulgaricus sp1.1) was normally cultured to the early stationary phase (12 h, at which time the OD 600nm= 1.2), different hydrolysis time of casein hydrolysate lyophilized powder was added at a concentration of 1% (w / v). The culture was continued to be incubated at 37°C for 12 h, and then the growth of bacteria was determined. The viable cell count was determined by pour plate method. The bacteria solution diluted by 0.9% NaCl gradient was poured into 20 mL MRS solid medium in a petri dish, and then incubated at 37°C for 48 h in an incubator. The results are shown in Table 2. Figure 3 .

[0032] Figure 3 The results show that the casein hydrolysate containing KEGIHAQ with 2 hours of hydrolysis has the best effect on the proliferation of lactic acid bacteria.

[0033] The pH of MRS broth medium was adjusted to complete acid inhibition pH 4.3 by adding lactic acid. Casein hydrolysate lyophilized powder obtained after 2 hours of hydrolysis was added to the medium at an addition amount of 1% (w / v). The activated third generation L. bulgaricus sp1.1 (1*10 8 CFU / mL) was inoculated at 2% (v / v) into the above sterilized MRS liquid medium with or without casein and its hydrolysate, and then incubated at 37°C for 24 h in an incubator. The results are shown in Table 3. Figure 4 .

[0034] Figure 4 The results show that the casein hydrolysate containing KEGIHAQ with 2 hours of hydrolysis has the effect on the proliferation of lactic acid bacteria under acid inhibition conditions, indicating that it can improve the acid tolerance of lactic acid bacteria.

[0035] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a polypeptide in promoting proliferation of lactic acid bacteria, characterized in that, The amino acid sequence of the polypeptide is KEGIHAQ; the lactic acid bacteria are Lactobacillus delbrueckii subsp. bulgaricus sp1.

1.

2. Use of a polypeptide for improving acid tolerance and culture density of lactic acid bacteria, characterized in that, The amino acid sequence of the polypeptide is KEGIHAQ; the lactic acid bacteria are Lactobacillus delbrueckii subsp. bulgaricus sp1.

1.

3. Use according to claim 1 or 2, characterized in that, The polypeptide is added in an amount of 0.003-5 g / L at the initial stage of the stable phase of the lactic acid bacteria culture.

4. Use of a casein hydrolyzed powder for promoting proliferation of lactic acid bacteria, characterized in that, The casein hydrolysate contains polypeptides with an amino acid sequence of KEGIHAQ; the lactic acid bacteria are Lactobacillus delbrueckii subsp. bulgaricus sp1.

1. The casein hydrolysate is prepared by dissolving casein in deionized water to prepare a 5% (w / v) casein solution, hydrolyzing the casein by papain at an enzyme-to-substrate ratio of 2000 U / g casein for 2 h under the following hydrolysis conditions: pH 7.0 and temperature 37℃; after the enzymatic hydrolysis, all the enzymes are inactivated by heating the mixture in a boiling water bath for 3-20 min; then the mixture is centrifuged at 6000-10000 x g for 10-30 min, and the obtained supernatant is freeze-dried to prepare the casein hydrolysate or stored at -80℃.

5. Use of a casein hydrolyzed powder for improving acid tolerance and culture density of lactic acid bacteria, characterized in that, The casein hydrolysate contains polypeptides with an amino acid sequence of KEGIHAQ; the lactic acid bacteria are Lactobacillus delbrueckii subsp. bulgaricus sp1.

1. The casein hydrolysate is prepared by dissolving casein in deionized water to prepare a 5% (w / v) casein solution, hydrolyzing the casein by papain at an enzyme-to-substrate ratio of 2000 U / g casein for 2 h under the following hydrolysis conditions: pH 7.0 and temperature 37℃; after the enzymatic hydrolysis, all the enzymes are inactivated by heating the mixture in a boiling water bath for 3-20 min; then the mixture is centrifuged at 6000-10000 x g for 10-30 min, and the obtained supernatant is freeze-dried to prepare the casein hydrolysate or stored at -80℃.

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