Peptide for promoting proliferation of lactobacillus gasseri as well as preparation method and application of peptide

Abstract

The invention discloses a peptide for promoting proliferation of lactobacillus gasseri as well as a preparation method and application of the peptide. The peptide comprises any one or more amino acid sequences shown in SEQ ID NO.1-17. The molecular weight is 600-1400 Da, the glutamic acid content is 12.70%-48.91%, and the biological degree, the viable count and the nitrogen source utilization rate of the lactobacillus gasseri are remarkably improved. The strain proliferation promoting peptide is high in stability, simple in process and low in production cost, and the precise design theory of a high-density culture nitrogen source of lactobacillus gasseri is further improved; and scientific theoretical support is provided for design of an optimal culture medium and acquisition of high biomass through a fermentation engineering technology.

Description

Technical Field

[0001] The invention relates to a peptide for promoting the proliferation of Lactobacillus gasseri, in particular to a peptide for promoting the proliferation of Lactobacillus gasseri, a preparation method and application thereof. Background Art

[0002] Lactobacillus gasseri ( Lactobacillus gasseri ) is an important probiotic, widely distributed in the human intestine and fermented dairy products. It plays a positive role in maintaining intestinal and vaginal health, regulating human immunity and producing antibacterial bacteriocins.

[0003] Specific peptides can have a positive effect on the growth and metabolism of lactic acid bacteria. These peptides can not only serve as a nutrient source, but also promote bacterial proliferation by regulating bacterial gene expression and other mechanisms. Lactobacillus gasseri is very demanding in the use of nitrogen sources, and its growth is poor when casein and free amino acids are used as nitrogen sources. Although casein is the main source of essential amino acids and growth-stimulating amino acids for most lactic acid bacteria, due to the lack of cell wall proteases that hydrolyze casein and the underdeveloped amino acid transport system, Lactobacillus gasseri can only use peptides that enter the cell passively through active transport through specific transporters (Opp, Pep, Dpp, etc.) or paracellular pathways as nitrogen sources. Adding casein hydrolyzed as an additive to the culture medium is an effective measure for casein to be utilized by Lactobacillus gasseri. Nitrogen source is one of the key factors that restrict the effective proliferation of Lactobacillus gasseri, which increases its difficulty in cultivation. Although some peptides from natural sources have been discovered and applied to the proliferation of probiotics, specific peptides for Lactobacillus gasseri and their synthesis methods are still relatively rare. Therefore, the development of a simple and efficient peptide to promote the proliferation of Lactobacillus gasseri is of great significance to its development and application in the probiotics industry. Summary of the invention

[0004] In order to solve the problem that there are still relatively few specific peptides for Lactobacillus gasseri and their synthesis methods, the present invention provides a peptide for promoting the proliferation of Lactobacillus gasseri, a preparation method and application thereof, which can significantly increase the biomass, viable count and nitrogen source utilization rate of Lactobacillus gasseri.

[0005] In order to achieve the above object, the present invention provides, on one hand, a use of a peptide in promoting the proliferation of Lactobacillus gasseri, characterized in that the peptide comprises any one or more amino acid sequences shown in SEQ ID NOs. 1-17.

[0006] Preferably, the molecular weight of the peptide is 600-1400 Da.

[0007] Specifically, the glutamic acid content of the peptide is 12.70% to 48.91%.

[0008] Specifically, the peptide can significantly increase the biomass, viable bacterial count and nitrogen source utilization rate of Lactobacillus gasseri.

[0009] The second aspect of the present invention provides a method for preparing the above-mentioned peptide, which comprises the following steps: (1) Add papain to the casein solution for enzymatic hydrolysis, the enzyme dosage is 4000U / g pro, the enzymatic reaction temperature is 55℃, the pH is between 6.5±0.05, the reaction is carried out for 7h, and immediately after the reaction, the enzyme is inactivated by boiling water bath. After cooling, the solution pH is adjusted to 4.6 to precipitate the unhydrolyzed casein, and the supernatant is collected after centrifugation. After freeze-drying, the dry powder is collected for later use; Proteolysis is the process of breaking down casein into peptide chains. The length and sequence structure of these peptide chains directly affect their activity in promoting microbial growth. By selecting suitable enzymes (such as papain, etc.) for enzymatic hydrolysis of casein, small molecule peptides can be generated. These peptides are easier to be transported into the cell for use by bacteria, thus promoting the growth of Lactobacillus gasseri.

[0010] The present invention can specifically obtain peptide segments with high activity in promoting bacterial proliferation by controlling the reaction time and reaction pH of the enzymatic hydrolysis. The enzymatic hydrolysis step is the core of the whole process because it directly affects the type and activity of the final target peptide.

[0011] (2) using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa for separation, and collecting the lyophilized powder after ultrafiltration; Ultrafiltration technology uses a semipermeable membrane to separate small molecules and macromolecules, usually by selective filtration based on the pore size of the membrane. The present invention uses ultrafiltration to remove impurities after enzymatic hydrolysis (such as undecomposed proteins, macromolecular polymers, etc.) using a 2kDa ultrafiltration membrane, and concentrates and purifies the target small molecule peptides. Ultrafiltration can remove inactive impurities with larger molecular weight and unhydrolyzed casein, thereby increasing the concentration of small molecule peptides in the solution. This step effectively removes impurities, ensuring that subsequent separation and purification processes can more accurately screen and retain bioactive peptides with pro-microbial properties. It also provides high-concentration peptide samples for subsequent technologies (such as gel separation chromatography and nanofiltration), enhancing the separation effect.

[0012] (3) Further enriching the ultrafiltration fraction using gel filtration chromatography; Gel separation chromatography uses the molecular sieving effect of gel to separate substances based on molecular size. By selecting the appropriate gel medium and mobile phase, the enzymatic hydrolysis products can be further purified and the target peptides can be separated. By using different types of gel media (such as Sephadex, etc.) and adjusting conditions such as flow rate, peptides can be separated according to their molecular weight.

[0013] Gel separation chromatography technology helps to further improve the purity of the target peptide in the peptide mixture after ultrafiltration. This process not only improves the purity of the target peptide, but also provides a clear sample for subsequent peptide identification analysis.

[0014] (4) Use nanofiltration membrane to further nanofilter peptides with a molecular weight of 600 to 1400 Da.

[0015] Nanofiltration technology further separates small molecules and macromolecules in the sample through a semipermeable membrane. Its main function is to further concentrate and purify the target peptides through selective filtration of pore size. This process can remove some residual low-molecular impurities and peptides of other molecular weights, improve the purity of the peptides, and ensure the concentration and stability of the active peptides in the final product.

[0016] The role of nanofiltration technology in this process is to optimize the concentration of peptides and remove low molecular weight impurities that have not been completely removed in other steps. It can improve the stability and biological activity of the final product, ensuring that the final product has sufficient effect to promote the proliferation of Lactobacillus gasseri.

[0017] According to the molecular weight and structural characteristics of peptides utilized by Lactobacillus gasseri, the present invention adopts enzymatic hydrolysis, ultrafiltration, nanofiltration and gel chromatography separation technology to sequentially differentiate and concentrate peptide components, thereby obtaining peptides suitable for efficient proliferation of Lactobacillus gasseri. When Lactobacillus gasseri is cultured in a culture medium using the peptides as nitrogen sources, the nitrogen source utilization rate reaches more than 30%, thereby effectively improving the utilization efficiency of the nitrogen source and reducing the waste of the nitrogen source.

[0018] Before the nanofiltration step, the molecular weight and structure of the peptide can be determined by NanoLC-MS / MS technology. The specific steps are as follows: (1) Prepare 40 mL of Buffer A solution (39.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid) and 40 mL of Buffer B solution (7.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid, 32 mL of acetonitrile) for later use. Use 200 μL of methanol, 200 μL of Buffer B solution, and 200 μL of Buffer A to activate the C18 desalting column by centrifugation. Add 200 μL of sample solution to the C18 desalting column and centrifuge (2000 g / min, 12 min) and pour in the filtrate. Add 200 μL of Buffer A to the C18 desalting column and centrifuge (6000 g / min, 4 min) and pour in the filtrate. Repeat the operation 3 times and replace the collection tube. Add 180 μL of Buffer B to the C18 desalting column and centrifuge (2000 g / min, 4 min). Save the effluent. Repeat the operation 2 times. Dry the effluent in a centrifugal concentrator at room temperature and refrigerate for later use.

[0019] (2) Chromatographic conditions reference: Chromatographic column: C18 Chromatographic column: Acclaim PepMap Rplc C18 (1.9μm×150μm×150mm); Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid and 80% acetonitrile; Injection volume: 4 μL, flow rate: 800nL / min. Elution gradient is as follows: 0-8 min, 6%-9% B; 8-24 min, 9%-14% B; 24-60 min, 14%-30% B; 60-75 min, 30%-40% B; 75-80 min, 40%-90% B; 80-90 min, 90%-0% B. MS conditions: primary MS resolution: 70000 (at m / z 200); parent ion scan range: 350-1800 m / z; AGC target 3e6; maximum IT: 0 ms; secondary MS resolution: 17500 (at m / z 200); AGC target 1e5; maximum IT: 6 ms.

[0020] To determine the origin of the peptides and their positions in the protein sequences, the peptide sequences were analyzed using Thermo Proteome Discoverer2.2 (GPMAW, Denmark) software.

[0021] The analysis showed that the components that significantly promoted the proliferation of the strain were mainly hydrophilic peptides with a molecular weight between 600 and 1400 Da; the amino acid composition analysis of the transport peptide showed that it mainly contained glutamic acid, and the calculation of the proportion of glutamic acid in the peptide segment showed that the glutamic acid content was between 12.7% and 48.91%. The active peptide significantly increased the biomass, viable count and nitrogen source utilization rate of Lactobacillus gasseri.

[0022] NanoLC-MS / MS combines liquid chromatography (LC) and mass spectrometry (MS) techniques for high-resolution analysis of the chemical composition and molecular structure of peptides. Liquid chromatography first separates peptides based on their polarity differences, and mass spectrometry further analyzes the separated peptides at the molecular level, providing detailed molecular weight and amino acid sequence information. Through nano-liquid chromatography technology, the complex components of the sample are efficiently separated by a micro-chromatographic column and then sent to a mass spectrometer for tandem mass spectrometry analysis. Mass spectrometry data provides information about the quality, amino acid sequence, and function of peptides, which helps to screen out peptides with specific functions.

[0023] This technology identifies the structure and sequence of peptides that have the activity of promoting the growth of Lactobacillus gasseri through an efficient separation and analysis process. Compared with traditional mass spectrometry analysis, NanoLC-MS / MS can provide higher resolution and sensitivity, effectively avoiding potential sample contamination or loss of low-abundance active peptides.

[0024] Traditional protein hydrolysis and separation methods use simple gel chromatography or dialysis to purify peptides, but it is often difficult to efficiently separate all biologically active peptides. The introduction of NanoLC-MS / MS technology makes the analysis and identification of peptides more precise, and can analyze and identify those peptides that have a significant promoting effect on Lactobacillus gasseri with high resolution.

[0025] The introduction of nanofiltration technology solves the problem of interference from other peptide molecules that may occur during the purification process. It can not only improve the purity of the peptide and its activity in promoting the growth of Lactobacillus gasseri, but also optimize the stability of the product. Compared with previous methods, it has the following advantages: 1) Efficient bioactive peptide screening: Traditional casein hydrolysis and separation methods usually do not have a high enough resolution to screen out specific bioactive peptides. NanoLC-MS / MS technology can conduct in-depth analysis of peptides and identify structural sequences with significant microbial growth-promoting peptide activity, thereby providing a standard for the selection of nanofiltration molecular weight.

[0026] 2) Higher purity and activity: Nanofiltration technology provides more sophisticated screening when purifying peptides, removing peptides of other molecular weights that may affect biological activity, so that the final peptides have higher activity in promoting the growth of Lactobacillus gasseri and can more effectively promote the proliferation of Lactobacillus gasseri.

[0027] 3) Production process optimization: Compared with traditional methods, the multi-step separation and analysis technology in the present invention greatly improves the controllability and efficiency of the peptide production process, so that the purity of the Lactobacillus gasseri growth-promoting active peptide is the highest, the quality is stable, and the consistency can be maintained between different batches.

[0028] Through this innovative combination of technologies, the Lactobacillus gasseri growth-promoting peptides obtained have high purity and activity, can significantly promote the proliferation of Lactobacillus gasseri, and show better results than traditional methods. The high efficiency and stability of this process make it more valuable for application in industrial production and can better meet the needs of production of different scales.

[0029] Preferably, in step (1), the concentration of the casein solution is 5%.

[0030] Preferably, in step (1), the boiling water bath time is 10 to 15 min.

[0031] Preferably, in step (1), the centrifugation condition is 7000-8000 r / min for 15 min.

[0032] The third aspect of the present invention provides a peptide obtained by the above preparation method.

[0033] The fourth aspect of the present invention provides the use of the above peptide in promoting the proliferation of Lactobacillus gasseri.

[0034] Through the above technical solution, the present invention achieves the following beneficial effects: 1. The molecular weight of the peptide of the present invention is 600-1400Da, and the glutamic acid content is between 12.70% and 48.91%, which can significantly improve the biomass, viable count and nitrogen source utilization rate of Lactobacillus gasseri. The peptide promoting the proliferation of the strain has high stability, simple process and low production cost, and further improves the precise design theory of nitrogen source for high-density culture of Lactobacillus gasseri.

[0035] 2. The present invention uses enzymatic hydrolysis, ultrafiltration, nanofiltration and gel chromatography separation technology to sequentially differentiate and concentrate the peptide components based on the molecular weight and structural characteristics of the peptide utilized by Lactobacillus gasseri, thereby obtaining a peptide suitable for efficient proliferation of Lactobacillus gasseri. When Lactobacillus gasseri is cultured in a culture medium using the peptide as a nitrogen source, the nitrogen source utilization rate reaches more than 30%, which is significantly higher than the utilization rate of other nitrogen sources, thereby effectively improving the utilization efficiency of the nitrogen source and effectively reducing the waste of the nitrogen source. DETAILED DESCRIPTION

[0036] The specific implementation of the present invention is described in detail below in conjunction with the examples. It should be understood that the specific implementation described herein is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0037] The main raw materials, reagents and experimental equipment used in the following examples are as follows: The raw materials involved in the following examples are all commercially available unless otherwise specified. Lactobacillus gasseri ATCC 33323 involved in the following examples was purchased from the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a collection number of CGMCC 1.3396; Lactobacillus gasseri Lactobacillus gasseri LBV150N was purchased from Royal DSM of the Netherlands, and Lactobacillus gasseri OLL2716 strain was purchased from Meiji Dairy; Lactobacillus gasseri LG36 was purchased from DuPont of the United States; Lactobacillus gasseri BNR17 was purchased from Chr. Hansen of Denmark.

[0038] The culture medium involved in the following examples is as follows: Preparation of MRS liquid culture medium (1000 mL): Accurately weigh 54 g of MRS broth culture medium, dissolve it in 1000 mL of distilled water, divide it into 50 mL conical flasks, autoclave at 121°C for 15 min, and set aside.

[0039] Preparation of MRS solid culture medium (1000 mL): Accurately weigh 54 g of MRS broth medium and 15 g of agar powder, dissolve in 1000 mL of distilled water, stir to mix, autoclave at 121°C for 15 min, pour into plates and set aside.

[0040] Basic chemical synthesis medium (1000mL): 10 g glucose, 0.01 g guanine, 0.01 g adenine, 0.01 g xanthine, 0.001 g niacin, 0.01 g thiamine, 0.02 g cobalamin, 0.001 g pantothenic acid, 0.005 g inositol, 0.002 g vitamin B6, 1 g Tween 80, 1 g ammonium citrate, 6 g potassium dihydrogen phosphate, 5 g sodium acetate, 6 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.02 g ferrous sulfate, 0.025 g manganese sulfate.

[0041] Experimental equipment: clean bench; UV spectrophotometer; oscillator; high performance liquid chromatograph; anaerobic workstation; high performance liquid chromatograph; freeze dryer; electric constant temperature water bath; NanoLC-MS / MS; desktop high-speed refrigerated centrifuge; full-wavelength automatic microplate reader; multifunctional roll membrane test equipment, membrane PES2-1812.

[0042] Example 1 This embodiment provides a method for directional preparation of a peptide that can improve the proliferation efficiency of Lactobacillus gasseri, and the technical solution adopted is as follows: Step 1: Take out the bacteria preservation tube stored at -80℃, wait for it to thaw, use an inoculation loop to take a small amount of bacterial liquid and streak it on the MRS solid culture medium, and culture it at 37℃ in an anaerobic workstation for 48 hours. After the colonies grow, pick a single colony and inoculate it into 5mL MRS liquid tube culture medium, and culture it anaerobically for 18h~24h. Repeat the above operation 2-3 times to obtain the activated bacterial liquid.

[0043] Step 2: Prepare a casein solution of a certain concentration, add papain, and perform enzymatic hydrolysis. Adjust the temperature and pH to the optimal reaction conditions for the protease. After adding the protease, use the pH-STAT method to maintain the system at the optimal pH of 6.5±0.05. React for 7 hours. After the reaction is completed, immediately boil in water for 10 minutes to inactivate the enzyme. After cooling, set the solution to pH 4.6 to precipitate the unhydrolyzed casein. Centrifuge at 8000 r / min for 15 minutes, take the supernatant, and collect the dry powder after freeze-drying for later use.

[0044] Step 3: The casein hydrolysates produced by the hydrolysis of papain that promotes the growth of the five strains of Lactobacillus gasseri are separated by ultrafiltration membranes with a molecular weight cutoff of 2 kDa. The total reflux working mode is used, and the pressure is controlled by a constant flow pump throughout the process. The inlet working pressure is 0.3 MPa. The effluent is collected, concentrated by a rotary evaporator, and freeze-dried to collect the dry powder for later use.

[0045] Step 4: Based on the above analysis, peptides that improve the proliferation efficiency of Lactobacillus gasseri are further prepared, and component A collected in step 3 is selected for subsequent preparation. The main operations are as follows: (1) Filling of Sephadex G-15 gel chromatography column Weigh 16 g of Sephadex G-15 dextran gel, add appropriate deionized water and let it stand for a certain period of time before filling the chromatography column. Transfer the fully swollen gel filler to the glass chromatography column evenly through glass rod drainage, and use ultrapure water as the mobile phase for cleaning. After filling, use ultrapure water at a flow rate of 0.5 mL / min to rinse the column until the baseline is stable for use. (2) Gel filtration chromatography separation The lyophilized powder of component A obtained by ultrafiltration was prepared into a sample solution with a concentration of 60 mg / mL. After filtering through a 0.45 μm filter membrane, the sample was loaded in a volume of 2 mL. The mobile phase was ultrapure water with a flow rate of 0.5 mL / min. 3 mL of effluent was collected from each tube and the absorbance was detected. The detection wavelength of the ultraviolet detector was 220 nm. The collected components were freeze-dried and set aside. Component A was separated by Sephadex G-15 gel chromatography to obtain three components, namely, components B1, B2, and B3. Component B2 with significant growth-promoting activity was collected.

[0046] Step 5: Determine the molecular weight and structure of the peptide using NanoLC-MS / MS technology. The specific steps are as follows: (1) Prepare 40 mL of Buffer A solution (39.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid) and 40 mL of Buffer B solution (7.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid, 32 mL of acetonitrile) for later use. Use 200 μL of methanol, 200 μL of Buffer B solution, and 200 μL of Buffer A to activate the C18 desalting column by centrifugation. Add 200 μL of sample solution to the C18 desalting column and centrifuge (2000 g / min, 12 min) and pour in the filtrate. Add 200 μL of Buffer A to the C18 desalting column and centrifuge (6000 g / min, 4 min) and pour in the filtrate. Repeat the operation 3 times and replace the collection tube. Add 180 μL of Buffer B to the C18 desalting column and centrifuge (2000 g / min, 4 min). Save the effluent. Repeat the operation 2 times. Dry the effluent in a centrifugal concentrator at room temperature and refrigerate for later use.

[0047] (2) Chromatographic conditions reference: Chromatographic column: C18 Chromatographic column: Acclaim PepMap Rplc C18 (1.9μm×150μm×150mm); Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid and 80% acetonitrile; Injection volume: 4 μL, flow rate: 800nL / min. Elution gradient is as follows: 0-8 min, 6%-9% B; 8-24 min, 9%-14% B; 24-60 min, 14%-30% B; 60-75 min, 30%-40% B; 75-80 min, 40%-90% B; 80-90 min, 90%-0% B. MS conditions: primary MS resolution: 70000 (at m / z 200); parent ion scan range: 350-1800 m / z; AGC target 3e6; maximum IT: 0 ms; secondary MS resolution: 17500 (at m / z 200); AGC target 1e5; maximum IT: 6 ms.

[0048] To determine the origin of the peptides and their positions in the protein sequences, the peptide sequences were analyzed using Thermo Proteome Discoverer2.2 (GPMAW, Denmark) software.

[0049] Analysis showed that the B2 component that significantly promoted the proliferation of the strain was mainly a hydrophilic peptide with a molecular weight between 600 and 1400 Da; analysis of the amino acid composition of the transit peptide found that it mainly contained glutamic acid, and calculation of the proportion of glutamic acid in the peptide segment showed that the glutamic acid content was between 12.7% and 48.91%.

[0050] Table 1 Analysis of peptide characteristics of component B

[0051] Step 6: The collected B2 component was separated by a nanofiltration membrane with a cut-off of 600Da and 1400Da, and component C (600-1400Da) was collected and the dry powder was collected by vacuum freeze drying. The structure of the peptide segment in component C was determined by liquid chromatography-mass spectrometry technology, and the results showed that the glutamic acid content in the peptide segment of component C accounted for 35.83%.

[0052] Step 7: Select Lactobacillus gasseri ATCC 33323, Lactobacillus gasseri LBV150N, Lactobacillus gasseri OLL2716, Lactobacillus gasseri LG36, and Lactobacillus gasseri BNR17. Use the peptide component C prepared in step 6 to culture five strains of Lactobacillus gasseri, and determine the maximum biomass, viable count and nitrogen source utilization rate of the strains under this component.

[0053] (1) The prepared peptide component C was added to the basal culture medium at a concentration of 8 g / L. Five strains of Lactobacillus gasseri were cultured separately. The activated bacterial solution was inoculated at a 2% inoculation rate. The OD was measured every 1 h. 600 , the maximum biomass and viable counts of the five bacterial strains under the culture conditions were determined; (2) The nitrogen content before and after fermentation was determined by the Kjeldahl method, and the nitrogen source utilization rate before and after fermentation was calculated.

[0054] Example 2 This embodiment provides a method for directional preparation of a peptide, and the technical solution adopted is as follows: Step 1: Take out the bacteria preservation tube stored at -80℃, wait for it to thaw, use an inoculation loop to take a small amount of bacterial liquid and streak it on the MRS-L solid medium, and culture it at 37℃ in an anaerobic workstation for 48h. After the colony grows, pick a single colony and inoculate it in a 5mL MRS-L liquid tube, culture it anaerobically for 18h~24h, and repeat the above operation 2-3 times to obtain the activated bacterial liquid.

[0055] Step 2: Prepare a casein solution of a certain concentration, add papain, and perform enzymatic hydrolysis. Adjust the temperature and pH to the optimal reaction conditions for the protease. After adding the protease, use the pH-STAT method to maintain the system at pH 5.5±0.05. React for 5 hours. After the reaction, immediately boil in water for 10 minutes to inactivate the enzyme. After cooling, set the solution to pH 4.6 to precipitate the unhydrolyzed casein. Centrifuge at 8000 r / min for 15 minutes, take the supernatant, and collect the dry powder after freeze-drying for later use.

[0056] Step 3: The casein hydrolysates produced by the hydrolysis of papain that promotes the growth of the five strains of Lactobacillus gasseri are separated by ultrafiltration membranes with a molecular weight cutoff of 2 kDa. The total reflux working mode is used, and the pressure is controlled by a constant flow pump throughout the process. The inlet working pressure is 0.3 MPa. The effluent is collected, concentrated by a rotary evaporator, and freeze-dried to collect the dry powder for later use.

[0057] Step 4: Based on the above analysis, peptides that improve the proliferation efficiency of Lactobacillus gasseri are further prepared, and component A collected in step 3 is selected for subsequent preparation. The main operations are as follows: (1) Filling of Sephadex G-15 gel chromatography column Weigh 16 g of Sephadex G-15 dextran gel, add appropriate deionized water and let it stand for a certain period of time before filling the chromatography column. Transfer the fully swollen gel filler to the glass chromatography column evenly through glass rod drainage, and use ultrapure water as the mobile phase for cleaning. After filling, use ultrapure water at a flow rate of 0.5 mL / min to rinse the column until the baseline is stable for use. (2) Gel filtration chromatography separation The lyophilized powder of component A obtained by ultrafiltration was prepared into a sample solution with a concentration of 60 mg / mL. After filtering through a 0.45 μm filter membrane, the sample was loaded in a volume of 2 mL. The mobile phase was ultrapure water with a flow rate of 0.5 mL / min. 3 mL of effluent was collected from each tube and the absorbance was detected. The detection wavelength of the ultraviolet detector was 220 nm. The collected components were freeze-dried and set aside. Component A was separated by Sephadex G-15 gel chromatography to obtain three components, namely, components B1, B2, and B3. Component B2 with significant growth-promoting activity was collected.

[0058] Step 5: Determine the molecular weight and structure of the peptide using NanoLC-MS / MS technology. The specific steps are as follows: (1) Prepare 40 mL of Buffer A solution (39.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid) and 40 mL of Buffer B solution (7.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid, 32 mL of acetonitrile) for later use. Use 200 μL of methanol, 200 μL of Buffer B solution, and 200 μL of Buffer A to activate the C18 desalting column by centrifugation. Add 200 μL of sample solution to the C18 desalting column and centrifuge (2000 g / min, 12 min) and pour in the filtrate. Add 200 μL of Buffer A to the C18 desalting column and centrifuge (6000 g / min, 4 min) and pour in the filtrate. Repeat the operation 3 times and replace the collection tube. Add 180 μL of Buffer B to the C18 desalting column and centrifuge (2000 g / min, 4 min). Save the effluent. Repeat the operation 2 times. Dry the effluent in a centrifugal concentrator at room temperature and refrigerate for later use.

[0059] (2) Chromatographic conditions reference: Chromatographic column: C18 Chromatographic column: Acclaim PepMap Rplc C18 (1.9μm×150μm×150mm); Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid and 80% acetonitrile; Injection volume: 4 μL, flow rate: 800nL / min. Elution gradient is as follows: 0-8 min, 6%-9% B; 8-24 min, 9%-14% B; 24-60 min, 14%-30% B; 60-75 min, 30%-40% B; 75-80 min, 40%-90% B; 80-90 min, 90%-0% B. MS conditions: primary MS resolution: 70000 (at m / z 200); parent ion scan range: 350-1800 m / z; AGC target 3e6; maximum IT: 0 ms; secondary MS resolution: 17500 (at m / z 200); AGC target 1e5; maximum IT: 6 ms.

[0060] To determine the origin of the peptides and their positions in the protein sequences, the peptide sequences were analyzed using Thermo Proteome Discoverer2.2 (GPMAW, Denmark) software.

[0061] Analysis showed that the B2 component that significantly promoted the proliferation of the strain was mainly a hydrophilic peptide with a molecular weight between 1000 and 2000 Da; analysis of the amino acid composition of the transit peptide found that it mainly contained leucine, and calculation of the proportion of leucine in the peptide segment showed that the leucine content was between 9.00% and 31.69%.

[0062] Table 2 Analysis of peptide characteristics of B2 component

[0063] Step 6: The collected B2 component was separated by a nanofiltration membrane with a cut-off of 1000Da and 2000Da, and component C (1000-2000Da) was collected and freeze-dried to collect the dry powder. Liquid chromatography-mass spectrometry was used to determine the structure of the peptide segment in component C. The results showed that the leucine content in the peptide segment of component C reached 23.28%.

[0064] Step 7: Select Lactobacillus gasseri ATCC 33323, Lactobacillus gasseri LBV150N, Lactobacillus gasseri OLL2716, Lactobacillus gasseri LG36, and Lactobacillus gasseri BNR17. Use the peptide component C prepared in step 6 to culture five strains of Lactobacillus gasseri, and determine the maximum biomass, viable count and nitrogen source utilization rate of the strains under this component.

[0065] (1) The prepared peptide component C was added to the basal culture medium at a concentration of 8 g / L. Five strains of Lactobacillus gasseri were cultured separately. The activated bacterial solution was inoculated at a 2% inoculation rate. The OD was measured every 1 h. 600 , the maximum biomass and viable counts of the five bacterial strains under the culture conditions were determined; (2) The nitrogen content before and after fermentation was determined by the Kjeldahl method, and the nitrogen source utilization rate before and after fermentation was calculated.

[0066] Example 3 This embodiment provides a method for directional preparation of a peptide, and the technical solution adopted is as follows: Step 1: Take out the bacteria preservation tube stored at -80℃, wait for it to thaw, use an inoculation loop to take a small amount of bacterial liquid and streak it on the MRS-L solid medium, and culture it at 37℃ in an anaerobic workstation for 48h. After the colony grows, pick a single colony and inoculate it in a 5mL MRS-L liquid tube, culture it anaerobically for 18h~24h, and repeat the above operation 2-3 times to obtain the activated bacterial liquid.

[0067] Step 2: Prepare a casein solution of a certain concentration, add papain, and perform enzymatic hydrolysis. Adjust the temperature and pH to the optimal reaction conditions for the protease. After adding the protease, use the pH-STAT method to maintain the system at pH 6.0±0.05. React for 6 hours. After the reaction is completed, immediately boil in water for 10 minutes to inactivate the enzyme. After cooling, precipitate the unhydrolyzed casein at pH 4.6. Centrifuge at 8000 r / min for 15 minutes, take the supernatant, and collect the dry powder after freeze-drying for later use.

[0068] Step 3: The casein hydrolysates produced by the hydrolysis of papain that promotes the growth of the five strains of Lactobacillus gasseri are separated by ultrafiltration membranes with a molecular weight cutoff of 2 kDa. The total reflux working mode is used, and the pressure is controlled by a constant flow pump throughout the process. The inlet working pressure is 0.3 MPa. The effluent is collected, concentrated by a rotary evaporator, and freeze-dried to collect the dry powder for later use.

[0069] Step 4: Based on the above analysis, peptides that improve the proliferation efficiency of Lactobacillus gasseri are further prepared, and component A collected in step 3 is selected for subsequent preparation. The main operations are as follows: (1) Filling of Sephadex G-15 gel chromatography column Weigh 16 g of Sephadex G-15 dextran gel, add appropriate deionized water and let it stand for a certain period of time before filling the chromatography column. Transfer the fully swollen gel filler to the glass chromatography column evenly through glass rod drainage, and use ultrapure water as the mobile phase for cleaning. After filling, use ultrapure water at a flow rate of 0.5 mL / min to rinse the column until the baseline is stable for use. (2) Gel filtration chromatography separation The lyophilized powder of component A obtained by ultrafiltration was prepared into a sample solution with a concentration of 60 mg / mL. After filtering through a 0.45 μm filter membrane, the sample was loaded in a volume of 2 mL. The mobile phase was ultrapure water with a flow rate of 0.5 mL / min. 3 mL of effluent was collected from each tube and the absorbance was detected. The detection wavelength of the ultraviolet detector was 220 nm. The collected components were freeze-dried and set aside. Component A was separated by Sephadex G-15 gel chromatography to obtain three components, namely, components B1, B2, and B3. Component B2 with significant growth-promoting activity was collected.

[0070] Step 5: Determine the molecular weight and structure of the peptide using NanoLC-MS / MS technology. The specific steps are as follows: (1) Prepare 40 mL of Buffer A solution (39.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid) and 40 mL of Buffer B solution (7.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid, 32 mL of acetonitrile) for later use. Use 200 μL of methanol, 200 μL of Buffer B solution, and 200 μL of Buffer A to activate the C18 desalting column by centrifugation. Add 200 μL of sample solution to the C18 desalting column and centrifuge (2000 g / min, 12 min) and pour in the filtrate. Add 200 μL of Buffer A to the C18 desalting column and centrifuge (6000 g / min, 4 min) and pour in the filtrate. Repeat the operation 3 times and replace the collection tube. Add 180 μL of Buffer B to the C18 desalting column and centrifuge (2000 g / min, 4 min). Save the effluent. Repeat the operation 2 times. Dry the effluent in a centrifugal concentrator at room temperature and refrigerate for later use.

[0071] (2) Chromatographic conditions reference: Chromatographic column: C18 Chromatographic column: Acclaim PepMap Rplc C18 (1.9μm×150μm×150mm); Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid and 80% acetonitrile; Injection volume: 4 μL, flow rate: 800nL / min. Elution gradient is as follows: 0-8 min, 6%-9% B; 8-24 min, 9%-14% B; 24-60 min, 14%-30% B; 60-75 min, 30%-40% B; 75-80 min, 40%-90% B; 80-90 min, 90%-0% B. MS conditions: primary MS resolution: 70000 (at m / z 200); parent ion scan range: 350-1800 m / z; AGC target 3e6; maximum IT: 0 ms; secondary MS resolution: 17500 (at m / z 200); AGC target 1e5; maximum IT: 6 ms.

[0072] To determine the origin of the peptides and their positions in the protein sequences, the peptide sequences were analyzed using Thermo Proteome Discoverer2.2 (GPMAW, Denmark) software.

[0073] Analysis showed that the B3 component that significantly promoted the proliferation of the strain was mainly a hydrophilic peptide with a molecular weight between 900 and 1500 Da; analysis of the amino acid composition of the transit peptide found that it mainly contained serine, and calculation of the proportion of serine in the peptide segment showed that the serine content was between 7.31% and 27.59%.

[0074] Table 3 Analysis of peptide characteristics of component B

[0075] Step 6: The collected B3 component was separated by a nanofiltration membrane with a cut-off of 900Da and 1500Da, and component C (900-1500Da) was collected and freeze-dried to collect the dry powder. Liquid chromatography-mass spectrometry was used to determine the structure of the peptide segment in component C. The results showed that the serine content in the peptide segment of component C accounted for 26.38%.

[0076] Step 7: Select Lactobacillus gasseri ATCC 33323, Lactobacillus gasseri LBV150N, Lactobacillus gasseri OLL2716, Lactobacillus gasseri LG36, and Lactobacillus gasseri BNR17. Use the peptide component C prepared in step 6 to culture five strains of Lactobacillus gasseri, and determine the maximum biomass, viable count and nitrogen source utilization rate of the strains under this component.

[0077] (1) The prepared peptide component C was added to the basal culture medium at a concentration of 8 g / L. Five strains of Lactobacillus gasseri were cultured separately. The activated bacterial solution was inoculated at a 2% inoculation rate. The OD was measured every 1 h. 600 , the maximum biomass and viable counts of the five bacterial strains under the culture conditions were determined; (2) The nitrogen content before and after fermentation was determined by the Kjeldahl method, and the nitrogen source utilization rate before and after fermentation was calculated.

[0078] Example 4 This embodiment provides a method for directional preparation of a peptide, and the technical solution adopted is as follows: Step 1: Take out the bacteria preservation tube stored at -80℃, wait for it to thaw, use an inoculation loop to take a small amount of bacterial liquid and streak it on the MRS-L solid medium, and culture it at 37℃ in an anaerobic workstation for 48h. After the colony grows, pick a single colony and inoculate it in a 5mL MRS-L liquid tube, culture it anaerobically for 18h~24h, and repeat the above operation 2-3 times to obtain the activated bacterial liquid.

[0079] Step 2: Prepare a casein solution of a certain concentration, add papain, and perform enzymatic hydrolysis. Adjust the temperature and pH to the optimal reaction conditions for the protease. After adding the protease, use the pH-STAT method to maintain the system at pH 8.0±0.05. React for 10 hours. After the reaction, immediately boil in water for 10 minutes to inactivate the enzyme. After cooling, precipitate the unhydrolyzed casein at pH 4.6. Centrifuge at 8000 r / min for 15 minutes, take the supernatant, and collect the dry powder after freeze-drying for later use.

[0080] Step 3: The casein hydrolysates produced by the hydrolysis of papain that promotes the growth of the five strains of Lactobacillus gasseri are separated by ultrafiltration membranes with a molecular weight cutoff of 2 kDa. The total reflux working mode is used, and the pressure is controlled by a constant flow pump throughout the process. The inlet working pressure is 0.3 MPa. The effluent is collected, concentrated by a rotary evaporator, and freeze-dried to collect the dry powder for later use.

[0081] Step 4: Based on the above analysis, peptides that improve the proliferation efficiency of Lactobacillus gasseri are further prepared, and component A collected in step 3 is selected for subsequent preparation. The main operations are as follows: (1) Filling of Sephadex G-15 gel chromatography column Weigh 16 g of Sephadex G-15 dextran gel, add appropriate deionized water and let it stand for a certain period of time before filling the chromatography column. Transfer the fully swollen gel filler to the glass chromatography column evenly through glass rod drainage, and use ultrapure water as the mobile phase for cleaning. After filling, use ultrapure water at a flow rate of 0.5 mL / min to rinse the column until the baseline is stable for use. (2) Gel filtration chromatography separation The lyophilized powder of component A obtained by ultrafiltration was prepared into a sample solution with a concentration of 60 mg / mL. After filtering through a 0.45 μm filter membrane, the sample was loaded in a volume of 2 mL. The mobile phase was ultrapure water with a flow rate of 0.5 mL / min. 3 mL of effluent was collected from each tube and the absorbance was detected. The detection wavelength of the ultraviolet detector was 220 nm. The collected components were freeze-dried and set aside. Component A was separated by Sephadex G-15 gel chromatography to obtain three components, namely, components B1, B2, and B3. Component B3 with significant growth-promoting activity was collected.

[0082] Step 5: Determine the molecular weight and structure of the peptide using NanoLC-MS / MS technology. The specific steps are as follows: (1) Prepare 40 mL of Buffer A solution (39.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid) and 40 mL of Buffer B solution (7.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid, 32 mL of acetonitrile) for later use. Use 200 μL of methanol, 200 μL of Buffer B solution, and 200 μL of Buffer A to activate the C18 desalting column by centrifugation. Add 200 μL of sample solution to the C18 desalting column and centrifuge (2000 g / min, 12 min) and pour in the filtrate. Add 200 μL of Buffer A to the C18 desalting column and centrifuge (6000 g / min, 4 min) and pour in the filtrate. Repeat the operation 3 times and replace the collection tube. Add 180 μL of Buffer B to the C18 desalting column and centrifuge (2000 g / min, 4 min). Save the effluent. Repeat the operation 2 times. Dry the effluent in a centrifugal concentrator at room temperature and refrigerate for later use.

[0083] (2) Chromatographic conditions reference: Chromatographic column: C18 Chromatographic column: Acclaim PepMap Rplc C18 (1.9μm×150μm×150mm); Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid and 80% acetonitrile; Injection volume: 4 μL, flow rate: 800nL / min. Elution gradient is as follows: 0-8 min, 6%-9% B; 8-24 min, 9%-14% B; 24-60 min, 14%-30% B; 60-75 min, 30%-40% B; 75-80 min, 40%-90% B; 80-90 min, 90%-0% B. MS conditions: primary MS resolution: 70000 (at m / z 200); parent ion scan range: 350-1800 m / z; AGC target 3e6; maximum IT: 0 ms; secondary MS resolution: 17500 (at m / z 200); AGC target 1e5; maximum IT: 6 ms.

[0084] To determine the origin of the peptides and their positions in the protein sequences, the peptide sequences were analyzed using Thermo ProteomeDiscoverer 2.2 (GPMAW, Denmark) software.

[0085] Analysis showed that the B3 component that significantly promoted the proliferation of the strain was mainly a hydrophilic peptide with a molecular weight between 600 and 1100 Da; analysis of the amino acid composition of the transit peptide found that it mainly contained leucine, and calculation of the proportion of leucine in the peptide segment showed that the leucine content was between 12.74% and 40.59%.

[0086] Table 4B3 component peptide characteristics analysis

[0087] Step 6: The collected B3 component was separated by a nanofiltration membrane with a cut-off of 600Da and 1100Da, and component C (600-1100Da) was collected and freeze-dried to collect the dry powder. Liquid chromatography-mass spectrometry was used to determine the structure of the peptide segment in component C. The results showed that the leucine content in the peptide segment of component C reached 32.57%.

[0088] Step 7: Select Lactobacillus gasseri ATCC 33323, Lactobacillus gasseri LBV150N, Lactobacillus gasseri OLL2716, Lactobacillus gasseri LG36, and Lactobacillus gasseri BNR17. Use the peptide component C prepared in step 6 to culture five strains of Lactobacillus gasseri, and determine the maximum biomass, viable count and nitrogen source utilization rate of the strains under this component.

[0089] (1) The prepared peptide component C was added to the basal culture medium at a concentration of 8 g / L. Five strains of Lactobacillus gasseri were cultured separately. The activated bacterial solution was inoculated at a 2% inoculation rate. The OD was measured every 1 h. 600 , the maximum biomass and viable counts of the five bacterial strains under the culture conditions were determined; (2) The nitrogen content before and after fermentation was determined by the Kjeldahl method, and the nitrogen source utilization rate before and after fermentation was calculated.

[0090] Example 5 This embodiment provides a method for directional preparation of a peptide, and the technical solution adopted is as follows: Step 1: Take out the bacteria preservation tube stored at -80℃, wait for it to thaw, use an inoculation loop to take a small amount of bacterial liquid and streak it on the MRS-L solid medium, and culture it at 37℃ in an anaerobic workstation for 48h. After the colony grows, pick a single colony and inoculate it in a 5mL MRS-L liquid tube, culture it anaerobically for 18h~24h, and repeat the above operation 2-3 times to obtain the activated bacterial liquid.

[0091] Step 2: Prepare a casein solution of a certain concentration, add papain, and perform enzymolysis. Adjust the temperature and pH to the optimal reaction conditions for the protease. After adding the protease, use the pH-STAT method to maintain the system at the optimal pH of 7.5±0.05. React for 8 hours. After the reaction is completed, immediately boil in water for 10 minutes to inactivate the enzyme. After cooling, set the solution to pH 4.6 to precipitate the unhydrolyzed casein. Centrifuge at 8000 r / min for 15 minutes, take the supernatant, and collect the dry powder after freeze-drying for later use.

[0092] Step 3: The casein hydrolysates produced by the hydrolysis of papain that promotes the growth of the five strains of Lactobacillus gasseri are separated by ultrafiltration membranes with a molecular weight cutoff of 2 kDa. The total reflux working mode is used, and the pressure is controlled by a constant flow pump throughout the process. The inlet working pressure is 0.3 MPa. The effluent is collected, concentrated by a rotary evaporator, and freeze-dried to collect the dry powder for later use.

[0093] Step 4: Based on the above analysis, peptides that improve the proliferation efficiency of Lactobacillus gasseri are further prepared, and component A collected in step 3 is selected for subsequent preparation. The main operations are as follows: (1) Filling of Sephadex G-15 gel chromatography column Weigh 16 g of Sephadex G-15 dextran gel, add appropriate deionized water and let it stand for a certain period of time before filling the chromatography column. Transfer the fully swollen gel filler to the glass chromatography column evenly through glass rod drainage, and use ultrapure water as the mobile phase for cleaning. After filling, use ultrapure water at a flow rate of 0.5 mL / min to rinse the column until the baseline is stable for use. (2) Gel filtration chromatography separation The lyophilized powder of component A obtained by ultrafiltration was prepared into a sample solution with a concentration of 60 mg / mL. After filtering through a 0.45 μm filter membrane, the sample was loaded in a volume of 2 mL. The mobile phase was ultrapure water with a flow rate of 0.5 mL / min. 3 mL of effluent was collected from each tube and the absorbance was detected. The detection wavelength of the ultraviolet detector was 220 nm. The collected components were freeze-dried and set aside. Component A was separated by Sephadex G-15 gel chromatography to obtain three components, namely, components B1, B2, and B3. Component B2 with significant growth-promoting activity was collected.

[0094] Step 5: Determine the molecular weight and structure of the peptide using NanoLC-MS / MS technology. The specific steps are as follows: (1) Prepare 40 mL of Buffer A solution (39.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid) and 40 mL of Buffer B solution (7.2 mL of ultrapure water, 0.8 mL of 10% trifluoroacetic acid, 32 mL of acetonitrile) for later use. Use 200 μL of methanol, 200 μL of Buffer B solution, and 200 μL of Buffer A to activate the C18 desalting column by centrifugation. Add 200 μL of sample solution to the C18 desalting column and centrifuge (2000 g / min, 12 min) and pour in the filtrate. Add 200 μL of Buffer A to the C18 desalting column and centrifuge (6000 g / min, 4 min) and pour in the filtrate. Repeat the operation 3 times and replace the collection tube. Add 180 μL of Buffer B to the C18 desalting column and centrifuge (2000 g / min, 4 min). Save the effluent. Repeat the operation 2 times. Dry the effluent in a centrifugal concentrator at room temperature and refrigerate for later use.

[0095] (2) Chromatographic conditions reference: Chromatographic column: C18 Chromatographic column: Acclaim PepMap Rplc C18 (1.9μm×150μm×150mm); Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid and 80% acetonitrile; Injection volume: 4 μL, flow rate: 800nL / min. Elution gradient is as follows: 0-8 min, 6%-9% B; 8-24 min, 9%-14% B; 24-60 min, 14%-30% B; 60-75 min, 30%-40% B; 75-80 min, 40%-90% B; 80-90 min, 90%-0% B. MS conditions: primary MS resolution: 70000 (at m / z 200); parent ion scan range: 350-1800 m / z; AGC target 3e6; maximum IT: 0 ms; secondary MS resolution: 17500 (at m / z 200); AGC target 1e5; maximum IT: 6 ms.

[0096] To determine the origin of the peptides and their positions in the protein sequences, the peptide sequences were analyzed using Thermo Proteome Discoverer2.2 (GPMAW, Denmark) software.

[0097] Analysis showed that the B2 component that significantly promoted the proliferation of the strain was mainly a hydrophilic peptide with a molecular weight between 500 and 1300 Da; analysis of the amino acid composition of the transit peptide found that it mainly contained proline, and calculation of the proportion of proline in the peptide segment showed that the proline content was between 16.97% and 62.65%.

[0098] Table 5B2 Component Peptide Characteristic Analysis

[0099] Step 6: The collected B2 component was separated by a nanofiltration membrane with a cut-off of 500Da and 1300Da, and component C (500-1300Da) was collected and freeze-dried to collect the dry powder. Liquid chromatography-mass spectrometry was used to determine the structure of the peptide segment in component C. The results showed that the proline content in the peptide segment of component C reached 49.27%.

[0100] Step 7: Select Lactobacillus gasseri ATCC 33323, Lactobacillus gasseri LBV150N, Lactobacillus gasseri OLL2716, Lactobacillus gasseri LG36, and Lactobacillus gasseri BNR17. Use the peptide component C prepared in step 6 to culture five strains of Lactobacillus gasseri, and determine the maximum biomass, viable count and nitrogen source utilization rate of the strains under this component.

[0101] (1) The prepared peptide component C was added to the basal culture medium at a concentration of 8 g / L. Five strains of Lactobacillus gasseri were cultured separately. The activated bacterial solution was inoculated at a 2% inoculation rate. The OD was measured every 1 h. 600 , the maximum biomass and viable counts of the five bacterial strains under the culture conditions were determined; (2) The nitrogen content before and after fermentation was determined by the Kjeldahl method, and the nitrogen source utilization rate before and after fermentation was calculated.

[0102] Table 6 Comparison of the effects of various examples of Lactobacillus gasseri ATCC 33323

[0103] Table 7 Comparison of the effects of various embodiments of Lactobacillus gasseri LBV

[0104] Table 8 Comparison of the effects of various examples of Lactobacillus gasseri OLL2716

[0105] Table 9 Comparison of the effects of various embodiments of Lactobacillus gasseri LG36

[0106] Table 10 Comparison of the effects of various examples of Lactobacillus gasseri BNR17

[0107] The test results are shown in Tables 6-10. It can be seen from the results that the proliferation effect of Lactobacillus gasseri cultured with component C prepared in Example 1 is significantly higher than that of other case conditions. The final fermentation liquid OD 600All of them were higher than 1.3, and the nitrogen source utilization rates were higher than 38%. It can be seen that when component C was used for proliferation, the growth effect was good and the nitrogen source utilization rate was high.

[0108] In summary, the present invention uses enzymatic hydrolysis, ultrafiltration, nanofiltration and gel chromatography separation technology based on the molecular weight and structural characteristics of peptides used by Lactobacillus gasseri, and sequentially distinguishes and concentrates the peptide components to obtain peptides suitable for efficient proliferation of Lactobacillus gasseri. The peptide is used as a nitrogen source culture medium to culture Lactobacillus gasseri, and the nitrogen source utilization rate reaches more than 30%, which is significantly higher than the utilization rate of other nitrogen sources, effectively improving the utilization efficiency of nitrogen sources and reducing the waste of nitrogen sources. The present invention is to further improve the precise design theory of nitrogen sources for high-density cultivation of Lactobacillus gasseri; and to provide scientific theoretical support for the design of optimal culture medium and the acquisition of high biomass through fermentation engineering and metabolic engineering technology.

Claims

1. Use of a peptide in promoting the proliferation of Lactobacillus gasseri, characterized in that: The peptide comprises any one or more of the amino acid sequences shown in SEQ ID NOs. 1-17.

2. The use according to claim 1, characterized in that: The molecular weight of the peptide is 600-1400 Da.

3. The use according to claim 1, characterized in that: The glutamic acid content of the peptide is 12.70% to 48.91%.

4. The use according to claim 1, characterized in that: The peptide can significantly increase the biomass, viable bacterial count and nitrogen source utilization rate of Lactobacillus gasseri.

5. The method for preparing the peptide according to claim 1, characterized in that: The steps include: (1) Add papain to the casein solution for enzymatic hydrolysis, the enzyme dosage is 4000U / g pro, the enzymatic reaction temperature is 55℃, the pH is between 6.5±0.05, the reaction is carried out for 7h, and immediately after the reaction, the enzyme is inactivated by boiling water bath. After cooling, the solution pH is adjusted to 4.6 to precipitate the unhydrolyzed casein, and the supernatant is collected after centrifugation. After freeze-drying, the dry powder is collected for later use; (2) using an ultrafiltration membrane with a molecular weight cutoff of 2 kDa for separation, and collecting the lyophilized powder after ultrafiltration; (3) Further enriching the ultrafiltration fraction using gel filtration chromatography; (4) Use nanofiltration membrane to further nanofilter peptides with a molecular weight of 600 to 1400 Da.

6. The preparation method according to claim, characterized in that: In step (1), the concentration of the casein solution is 5%.

7. The preparation method according to claim 1, characterized in that: In step (1), the boiling water bath time is 10 to 15 min.

8. The preparation method according to claim, characterized in that: The following step is also included between steps (3) and (4): using NanoLC-MS / MS technology to analyze the peptide fragments and identify the structural sequences of peptides with significant microbial growth-promoting activity.

9. The peptide obtained by the method according to any one of claims 5 to 8.

10. Use of the peptide according to claim 9 in promoting the proliferation of Lactobacillus gasseri.

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