Method for efficiently preparing high-F-value oligopeptide by utilizing bacterium-enzyme synergism

Abstract

The invention belongs to the technical field of bioengineering, and discloses a method for efficiently preparing high-F-value oligopeptide from soybean meal. The method comprises the following steps: 1) treating soybean meal with cellulase to release protein in the soybean meal; (2) hydrolyzing soybean meal protein by adopting alkaline protease to obtain a culture medium with high protein content; 3) sterilizing the culture medium and then inoculating bacillus subtilis which can utilize soybean meal protein as a nitrogen source, and meanwhile, autonomously secreting protease and specifically cutting aromatic amino acid in the soybean meal protein in the fermentation process by the bacillus subtilis; 4) selectively removing aromatic amino acid after protein hydrolysis through activated carbon dearomatization treatment, retaining branched chain amino acid, and finally obtaining an F value gt; 29 is an oligopeptide. According to the method, the utilization rate of protein is remarkably increased through the synergistic effect of multi-stage enzymolysis and bacterial strain self-produced enzyme, the production cost of preparing high-F-value oligopeptide from soybeans is reduced by combining a directional dearomatization process, and the method can be widely applied to the field of functional feed and food.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a method for efficiently preparing oligopeptides with high F values ​​by utilizing bacterial enzymes. Background Art

[0002] Soybean meal, also known as "soybean meal," is a byproduct of soybean oil extraction. Its primary component is protein, reaching 40%-50%, along with a rich source of amino acids, vitamins, and minerals. It is a high-quality source of plant protein. However, in industry, approximately 85% of soybean meal is used for poultry and pig farming, with little development of its protein at high value, resulting in a waste of protein resources. With the development of bioactive peptides, their proteins should be considered as part of research and application.

[0003] High-F value oligopeptides are composed of specialized amino acids with high content of branched-chain amino acids (BCAAs: valine, isoleucine, and leucine) and low content of aromatic amino acids (AAAs: tryptophan, tyrosine, and phenylalanine). The Fischer ratio (F value) is the molar ratio of branched-chain amino acids to aromatic amino acids. High-F value oligopeptides should have an F value greater than 20. Their primary biological activities include anti-fatigue and alcohol detoxification. They can also aid in the treatment of hepatic encephalopathy and phenylketonuria, and improve protein nutritional status. Soybean meal, with its high content of leucine and isoleucine, offers significant advantages for the production of high-F value oligopeptides. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for efficiently preparing oligopeptides with high F values ​​by utilizing bacterial enzymes. In the second enzymatic hydrolysis process, a fermentation method is adopted to utilize the hydrolyzate of soybean meal as a culture medium, and the fermentation product simultaneously performs the second enzymatic hydrolysis on the enzymatic hydrolyzate, thereby achieving high-value utilization, saving costs, and simplifying the process.

[0005] In order to achieve the above purpose, the present invention has taken the following steps: S1: drying and grinding soybean meal, sieving to obtain low-temperature soybean meal powder, adding distilled water to prepare soybean meal suspension A; S2 adjusts the pH value of the suspension to acidic, then adds cellulase, and inactivates it in a boiling water bath after the reaction to obtain enzymatic solution B1; S3 adjusts the pH of the enzymatic hydrolysate B1 to alkaline, adds alkaline protease, and sterilizes at high temperature after the reaction to obtain the enzymatic hydrolysate B2; S4 uses the enzymatic hydrolysate B2 as the culture medium, inoculates Bacillus subtilis, and obtains the fermentation liquid C1 after cultivation; S5: After centrifuging the fermentation liquid C1, activated carbon is added for dearomatization, and then centrifugation is performed to obtain a high F value oligopeptide solution D. In S1, the soybean meal is passed through a 100-200 mesh sieve and prepared into a 5%-15% suspension A with distilled water.

[0006] In the S2, the pH is adjusted to 4-6, the amount of cellulase added is 0.5%-3%, the reaction temperature is 40-60°C, the reaction time is 1-2h, and the reaction speed is 100-200rpm.

[0007] In the S3, the pH is adjusted to 9-11, the amount of alkaline protease added is 2%-5% of the solid content, the reaction temperature is 40-60°C, the reaction time is 3-5h, the reaction speed is 100-200rpm, and the high temperature sterilization conditions are 121°C for 20min.

[0008] In the S4, the seed culture medium is LB culture medium, the inoculation amount is 0.3%-1%, and the fermentation culture medium culture time is 48-72 hours.

[0009] In S4, the Bacillus subtilis is introduced with genes encoding carboxypeptidase and / or subtilisin C (Subtilisin Carlsberg), preferably the Bacillus subtilis is Bacillus subtilis 168, and specifically the two genes are introduced into different strains or simultaneously into the same strain.

[0010] In S5, the fermentation liquid is centrifuged at 8000-10000 rpm for 15-20 min, the amount of activated carbon added is 5-10% of the reaction system, the reaction temperature is 25-35° C., the reaction time is 1-3 h, and the reaction speed is 100-200 rpm.

[0011] The exoprotease used in the present invention is alkaline protease, and the endoprotease is carboxypeptidase YpwA produced by fermentation. In the process of combined enzymatic and fermentation preparation, Bacillus subtilis can utilize the carbon and nitrogen sources after enzymatic hydrolysis of cellulase and alkaline protease, and the carboxypeptidase YpwA produced by Bacillus subtilis can also specifically cleave aromatic amino acids in the hydrolyzate.

[0012] The method for preparing oligopeptides with high F value by using bacterial enzymes in collaboration is further preferably: S1, soybean meal was passed through a 200-mesh sieve and prepared into a 15% suspension A with distilled water.

[0013] S2, pH was adjusted to 5, the amount of cellulase added was 0.5%, the reaction temperature was 45°C, the reaction time was 2 h, and the reaction speed was 150 rpm.

[0014] S3, pH is adjusted to 10.0, the amount of alkaline protease added is 5% of the solid content, the reaction temperature is 40-60°C, the reaction time is 4h, the reaction speed is 100-200rpm, and the high temperature sterilization conditions are 121°C for 20min.

[0015] S4, the seed culture medium is LB medium, the inoculation amount is 0.3%-0.5%, and the fermentation medium culture time is 48h.

[0016] S5, the fermentation broth was centrifuged at 8000-10000 rpm for 15-20 min, the amount of activated carbon added was 10% of the reaction system, the reaction temperature was 30°C, the reaction time was 2 h, and the reaction speed was 100-200 rpm.

[0017] The F value of the soybean meal high F value oligopeptide prepared by the above method is between 22 and 34, and can reach a maximum of 33.06.

[0018] The beneficial effects of the present invention are as follows: Bacillus subtilis utilizes the carbon and nitrogen sources obtained from enzymatic hydrolysis of soybean meal to culture the bacteria, while the endoprotease produced by the bacteria continues to enzymatically hydrolyze the protein in the system, thereby combining fermentation enzyme production and enzymatic hydrolysis into one step, which not only fully utilizes the protein in the soybean meal, but also simplifies the process and saves costs. DETAILED DESCRIPTION

[0019] The following examples are used to further illustrate the present invention, but are not intended to limit the present invention in any form. Methods not described in detail in the examples can be achieved by conventional techniques in the art. Reagents not specified in the examples are all commercially available.

[0020] The cellulase in the present invention was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd. with an enzyme activity of 10,000-20,000 U / g; the alkaline protease was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with an enzyme activity of 200 U / mg.

[0021] The bacteria in the present invention are Bacillus subtilis 168 / pMA5-apr and 168 / pMA5-ypwA, which are derived from patent CN112592950B. The pma5 plasmid containing the ypwa gene is introduced into Bacillus subtilis 168 to obtain Bacillus subtilis 168 / pMA5-apr, and the pma5 plasmid containing the apr gene is introduced into Bacillus subtilis 168 to obtain Bacillus subtilis 168 / pMA5-apr. Because the ypwa and apr genes are from Bacillus subtilis, they encode carboxypeptidase (Carboxypeptidase) and subtilisin C (Subtilisin Carlsberg), respectively.

[0022] The method for culturing Bacillus subtilis seed solution is as follows: In a clean bench, 200 μl of bacteria were pipetted from the glycerol tube into LB medium, cultured at 37°C in a constant temperature shaker for 18 h, and then inoculated into the fermentation medium.

[0023] LB medium (g / L): peptone 10.0, yeast powder 5.0, sodium chloride 10.0, distilled water 1 L, pH 7.0±0.2.

[0024] Example 1 A method for preparing oligopeptides with high F values ​​by using bacterial enzymes in collaboration comprises the following steps: S1, soybean meal was passed through a 200-mesh sieve and prepared into a 15% suspension A with distilled water.

[0025] S2, pH was adjusted to 5, the amount of cellulase added was 1%, the reaction temperature was 45°C, the reaction time was 2 h, and the reaction speed was 150 rpm.

[0026] S3, pH is adjusted to 10.0, the amount of alkaline protease added is 5% of the solid content, the reaction temperature is 40-60°C, the reaction time is 4h, the reaction speed is 100-200rpm, and the high temperature sterilization conditions are 121°C for 20min.

[0027] S4, the seed culture medium is LB medium, and Bacillus subtilis 168 / pMA5-apr and 168 / pMA5-ypwA are inoculated simultaneously, with an inoculation amount of 0.5% each, and the fermentation medium culture time is 48 h.

[0028] S5, the fermentation broth was centrifuged at 8000-10000 rpm for 15-20 min, the amount of activated carbon added was 10% of the reaction system, the reaction temperature was 30°C, the reaction time was 2 h, and the reaction speed was 100-200 rpm. After the reaction was completed, centrifugation was performed at 8000-10000 rpm for 15-20 min. A small amount of activated carbon remained in the solution, which was filtered with filter paper and the supernatant was retained. The hydrolyzed amino acid content was determined using an amino acid analyzer and the F value was calculated.

[0029] Example 2 A method for preparing high F value oligopeptides by combining enzyme method and fermentation method using soybean meal, comprising the following steps: S1, soybean meal was passed through a 200-mesh sieve and prepared into a 5% suspension A with distilled water.

[0030] S2, pH was adjusted to 5, the amount of cellulase added was 0.5%, the reaction temperature was 45°C, the reaction time was 2 h, and the reaction speed was 150 rpm.

[0031] S3, pH is adjusted to 10.0, the amount of alkaline protease added is 2% of the solid content, the reaction temperature is 40-60°C, the reaction time is 4h, the reaction speed is 100-200rpm, and the high temperature sterilization conditions are 121°C for 20min.

[0032] S4, the seed culture medium is LB medium, and Bacillus subtilis 168 / pMA5-apr and 168 / pMA5-ypwA are inoculated simultaneously, with an inoculation amount of 0.3% each, and the fermentation medium culture time is 48 h.

[0033] S5, the fermentation broth was centrifuged at 8000-10000 rpm for 15-20 min, the amount of activated carbon added was 10% of the reaction system, the reaction temperature was 30°C, the reaction time was 2 h, and the reaction speed was 100-200 rpm. After the reaction was completed, centrifugation was performed at 8000-10000 rpm for 15-20 min. A small amount of activated carbon remained in the solution, which was filtered with filter paper and the supernatant was retained. The hydrolyzed amino acid content was determined using an amino acid analyzer and the F value was calculated.

[0034] Example 3: A method for preparing high F value oligopeptides by combining enzyme method and fermentation method using soybean meal, comprising the following steps: S1, soybean meal was passed through a 200-mesh sieve and prepared into a 25% suspension A with distilled water.

[0035] S2, pH was adjusted to 5, the amount of cellulase added was 3%, the reaction temperature was 45°C, the reaction time was 2 h, and the reaction speed was 150 rpm.

[0036] S3, pH is adjusted to 10.0, the amount of alkaline protease added is 5% of the solid content, the reaction temperature is 40-60°C, the reaction time is 4h, the reaction speed is 100-200rpm, and the high temperature sterilization conditions are 121°C for 20min.

[0037] S4, the seed culture medium is LB medium, and Bacillus subtilis 168 / pMA5-apr and 168 / pMA5-ypwA are inoculated simultaneously, with an inoculation amount of 0.5% each, and the fermentation medium culture time is 48 h.

[0038] S5, the fermentation broth was centrifuged at 8000-10000 rpm for 15-20 min, the amount of activated carbon added was 10% of the reaction system, the reaction temperature was 30°C, the reaction time was 2 h, and the reaction speed was 100-200 rpm. After the reaction was completed, centrifugation was performed at 8000-10000 rpm for 15-20 min. A small amount of activated carbon remained in the solution, which was filtered with filter paper and the supernatant was retained. The hydrolyzed amino acid content was determined using an amino acid analyzer and the F value was calculated.

[0039] Comparative Example 1 The difference between Comparative Example 1 and Example 2 is that in S4, after Bacillus subtilis 168 / pMA5-apr and 168 / pMA5-ypwA are cultured in LB medium, the fermentation medium is another medium with a formula of 12 g / L peptone, 24 g / L yeast powder, 4 g / L glycerol, 2.31 g / L potassium dihydrogen phosphate, and 2.54 g / L potassium hydrogen phosphate. The pH is adjusted to 7.0 and sterilized at 121 ° C for 20 min before use. The seed culture medium inoculation amount is 0.3%. The two bacteria are cultured separately, and the supernatant is obtained by centrifugation after 48 h of culture. The supernatants of the two bacteria are mixed and added to the soybean meal hydrolyzate after high temperature sterilization in S3 for further enzymatic hydrolysis. The enzymatic hydrolysis conditions are pH = 6.0, temperature of 40 ° C, and time of 3-5 h. After the reaction is completed, continue to operate as in S5.

[0040] The F values ​​of the products finally obtained in Examples 1-3 and Comparative Example 1 are shown in Table 1.

[0041] F value = moles of branched-chain amino acids / moles of aromatic amino acids Table 1 F value comparison table

[0042] It can be seen from Table 1 that the maximum F value of Example 2 among Examples 1-3 is 29.2, which is greater than 20, meeting the requirements of high F value oligopeptides. Comparative Example 1 is an experiment in which fermentation enzyme production and enzymatic hydrolysis are carried out separately, and Example 2 is an experiment in which the two are combined in one step. The results are not much different, but a lot of culture medium materials and fermentation and reaction time are saved, which reduces costs and simplifies the process.

Claims

1. A method for efficiently preparing high F value oligopeptides by using bacterial enzymes, characterized in that: It includes the following steps: S1: drying and grinding soybean meal, sieving to obtain soybean meal powder, adding distilled water to prepare soybean meal suspension A; S2 adjusts the pH value of suspension A to acidic, then adds cellulase and inactivates it in a boiling water bath to obtain enzymatic solution B1; S3 adjusts the pH of the enzymatic hydrolysate B1 to alkaline, adds alkaline protease, and sterilizes at high temperature to obtain the enzymatic hydrolysate B2; S4 uses the enzymatic hydrolysate B2 as the culture medium, inoculates Bacillus subtilis, and obtains the fermentation liquid C1 after cultivation; S5 centrifuges the fermentation broth C1, adds activated carbon to the obtained supernatant for dearomatization, and then centrifuges and filters to obtain a high F value oligopeptide solution D.

2. The method according to claim 1, characterized in that In S1, soybean meal is passed through a 100-200 mesh sieve and prepared into a 5%-15% suspension A with distilled water.

3. The method according to claim 1, characterized in that In the S2, the pH is adjusted to 4-6, the amount of cellulase added is 0.5%-3%, the reaction temperature is 40-60°C, and the reaction time is 1-2 hours.

4. The method according to claim 1, wherein In the S3, the pH is adjusted to 9-11, the amount of alkaline protease added is 2%-5% of the solid content, the reaction temperature is 40-60°C, the reaction time is 3-5h, and the high temperature sterilization condition is 121°C for 20min.

5. The method according to claim 1, wherein In the S4, the seed culture medium is LB culture medium, the inoculation amount is 0.3%-1%, and the fermentation culture medium culture time is 48-72 hours.

6. The method according to claim 1, characterized in that In S4, the Bacillus subtilis is introduced with genes encoding carboxypeptidase and / or subtilisin C (Subtilisin Carlsberg), preferably the Bacillus subtilis is Bacillus subtilis 168, and specifically the two genes are introduced into different strains or simultaneously into the same strain.

7. The method according to claim 1, characterized in that In S5, the fermentation liquid is centrifuged at 8000-10000 rpm for 15-20 min, the amount of activated carbon added is 5-10% of the reaction system, the reaction temperature is 25-35° C., and the reaction time is 1-3 h.

8. The method according to claim 1, characterized in that The high F value oligopeptide refers to an oligopeptide with an F value greater than 22, specifically an F value between 22-34.

9. A high F value oligopeptide obtained by the method according to any one of claims 1 to 8.

10. Use of the high F value oligopeptide according to claim 9 in preparing functional feed or food.

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