Composite fermentation inoculant and method for producing gamma-aminobutyric acid by fermentation of composite fermentation inoculant

Abstract

The invention relates to the technical field of microorganisms, in particular to a compound fermentation inoculant and a method for producing gamma-aminobutyric acid by fermentation of the compound fermentation inoculant. The compound fermentation bacterium agent comprises lactobacillus fermentum ASAG and bacillus amyloliquefaciens ASAG. The invention develops a compound fermentation inoculant suitable for solid state fermentation of GABA by using rice bran as a single substrate, the use value of rice bran can be improved by fermenting and producing GABA by using the compound fermentation inoculant, the efficient value-added utilization of rice bran as a rice processing byproduct is realized, the production cost of GABA can be reduced, and the method is simple and convenient to operate and has a wide application prospect. The method is high in production efficiency, free of pollution and high in safety, fermentation products can be used for food ingredients or food additives, and the method has great economic benefits and wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a composite fermentation bacterial agent and a method for producing gamma-aminobutyric acid by fermenting the composite fermentation bacterial agent. Background Art

[0002] Rice bran is one of the main by-products of rice processing, accounting for about 8% of the mass of rice, with an annual output of 14 million tons. Rice bran is rich in nutrients, concentrating 64% of the nutrients in rice, including protein, fat, carbohydrates, dietary fiber and other ingredients, as well as active substances such as γ-aminobutyric acid, γ-oryzanol, tocopherol, gallic acid, squalene, etc.

[0003] γ-aminobutyric acid (GABA) is a non-protein amino acid that is widely found in vertebrates, plants and microorganisms, and has good water solubility and thermal stability. As an important inhibitory neurotransmitter in the central nervous system, GABA has a variety of regulatory effects on the body, such as lowering blood pressure, improving sleep, improving brain function, preventing obesity, and promoting growth hormone secretion. It is gradually being used in the fields of medicine and food.

[0004] There are three main methods for producing GABA, namely chemical synthesis, plant enrichment and microbial fermentation. Although the chemical synthesis method has a fast synthesis speed, it has disadvantages such as high cost, violent reaction and low safety, and is not suitable for the production of food-grade GABA. The plant enrichment method is simple to operate and highly safe, but its production efficiency is low and separation is difficult, so it is also difficult to be widely used. The microbial fermentation method produces GABA by selecting excellent, stable and non-toxic strains during the growth and reproduction process. Studies have found that lactic acid bacteria, molds, yeasts and Bacillus can all be used for the production of GABA, and mixed bacterial fermentation is usually better than single bacterial fermentation because mixed bacterial fermentation can produce complex proteases, which are more conducive to protein degradation.

[0005] Microbial fermentation can be divided into two forms: liquid fermentation and solid fermentation according to the physical properties of the culture medium. Solid fermentation has relatively loose requirements on fermentation conditions. In solid fermentation, microorganisms can maintain their original natural growth state, which is conducive to the accumulation and diversity of fermentation metabolites. Compared with liquid fermentation, it has the advantages of high yield, low investment, simple process, and easy drying and recovery.

[0006] At present, the commonly used GABA production methods mostly use single strains for liquid fermentation, and there are few reports on composite strain fermentation and solid fermentation, and there are relatively few reports on rice bran as a single substrate. Therefore, how to develop a composite fermentation agent suitable for solid-state fermentation of GABA using rice bran as a single substrate has become a technical problem that needs to be solved in this field. Summary of the invention

[0007] In order to solve the above technical problems, the present invention obtains a composite fermentation agent suitable for solid-state fermentation of GABA with rice bran as a single substrate by complex screening of a large number of strains, and proposes the following technical solution based on this.

[0008] Firstly, the present invention provides a strain of Lactobacillus fermentum ASAG and a strain of Bacillus amyloliquefaciens ASAG.

[0009] Lactobacillus fermentum ASAG was isolated from soil samples from a Sichuan pickle processing factory. The strain was deposited in the China Center for Type Culture Collection on December 16, 2024. Address of the depository: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Postal Code: 430072, Classification Name: Lactobacillus fermentum Lactobacillus fermentum , the deposit number is CCTCC No: M20242832.

[0010] Bacillus amyloliquefaciens ASAG was isolated from a soil sample of a corn field in Hubei Province. The strain was deposited in the China Center for Type Culture Collection on December 16, 2024. Address of the depository: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Postal Code: 430072, Classification Name: Bacillus amyloliquefaciens Bacillus amyloliquefaciens , the deposit number is CCTCCNo: M 20242831.

[0011] Furthermore, the present invention provides a composite fermentation bacterial agent, comprising the fermentation Lactobacillus ASAG and the amyloliquefaciens Bacillus ASAG.

[0012] Preferably, the composite fermentation bacterial agent consists of the fermentation Lactobacillus ASAG and the amyloliquefaciens Bacillus ASAG.

[0013] Preferably, the weight ratio of Lactobacillus fermentum ASAG to Bacillus amyloliquefaciens ASAG is 1:(0.1~9).

[0014] More preferably, the weight ratio of Lactobacillus fermentum ASAG to Bacillus amyloliquefaciens ASAG is 1:(2-9) or 1:(0.1-0.5).

[0015] More preferably, the weight ratio of Lactobacillus fermentum ASAG to Bacillus amyloliquefaciens ASAG is 1:(8-9).

[0016] Furthermore, the present invention provides the use of the composite fermentation agent in the fermentation production of γ-aminobutyric acid.

[0017] Furthermore, the present invention provides a method for producing γ-aminobutyric acid by fermentation, comprising fermenting rice bran with the composite fermentation agent.

[0018] Preferably, the rice bran is defatted rice bran.

[0019] Preferably, the fermentation is a solid state fermentation.

[0020] Preferably, the fermentation temperature is 28°C to 32°C; and / or the fermentation time is more than 7 days; and / or the mass volume ratio of rice bran to the composite fermentation agent is (1 to 3) g: 1 mL.

[0021] Preferably, in the composite fermentation bacterial agent, the number of viable bacteria of the fermentation lactobacillus ASAG or the amyloliquefaciens ASAG is 1×10 8 ~1×10 11 CFU / mL.

[0022] Preferably, the fermented Lactobacillus ASAG is used after being activated with MRS culture medium.

[0023] Preferably, the Bacillus amyloliquefaciens ASAG is used after being activated in LB medium.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The invention develops a composite fermentation agent suitable for solid-state fermentation of GABA using rice bran as a single substrate. By using the composite fermentation agent to ferment and produce GABA, not only can the use value of rice bran be improved, efficient value-added utilization of rice bran, a byproduct of rice processing, can be achieved, but also the production cost of GABA can be reduced. The method is simple to operate, has high production efficiency, is pollution-free, and has high safety. The fermentation product can be used for food ingredients or food additives, has great economic benefits, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram showing the results of GABA production by fermentation of different strains in Example 1.

[0026] Figure 2 This is a graph showing the results of GABA production by composite fermentation agents with different ratios in Example 2. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0029] The culture medium formula used in the following examples is as follows: (1) YPD medium: 10.0 g / L yeast extract, 20.0 g / L tryptone, and 20.0 g / L anhydrous glucose. Used for the activation and cultivation of Saccharomyces cerevisiae.

[0030] (2) PDA medium: 10.0 g / L potato extract powder, 20.0 g / L anhydrous glucose. Used for the activation and cultivation of Rhizopus oryzae.

[0031] (3) MRS medium: glucose 20.0 g / L, peptone 10.0 g / L, beef powder 5.0 g / L, sodium acetate 5.0 g / L, yeast powder 4.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 mL / L, used for the activation and cultivation of fermentation lactobacillus.

[0032] (4) LB medium: 5.0 g / L yeast extract, 10.0 g / L tryptone, and 10.0 g / L NaCl. Used for the activation and cultivation of Bacillus amyloliquefaciens.

[0033] (5) Fermentation medium: 50 g defatted rice bran.

[0034] Example 1 In this embodiment, different single strains and composite fermentation agents composed of different strains are used to ferment and produce GABA. The strains used are Lactobacillus fermentum ASAG with a preservation number of CCTCC No: M 20242832, Bacillus amyloliquefaciens ASAG with a preservation number of CCTCC No: M 20242831, Saccharomyces cerevisiae isolated from soil samples of Xinjiang grape plantations, and Rhizopus oryzae isolated from agricultural waste compost in Heilongjiang, which are respectively marked as: FJ-Lactobacillus fermentum ASAG; NJ-Saccharomyces cerevisiae; JDF-Bacillus amyloliquefaciens ASAG; MG-Rhizopus oryzae.

[0035] The fermentation steps are as follows: Saccharomyces cerevisiae, Rhizopus oryzae, and Bacillus amyloliquefaciens ASAG were activated in YPD, PDA, and LB medium for 24 hours, respectively, and then inoculated into the corresponding seed medium at a volume percentage of 3%, and cultured at 200 rpm and 30°C for 10 hours; Lactobacillus fermentum ASAG was activated in MRS medium for 24 hours and then inoculated into MRS seed medium at a volume percentage of 3%, and cultured at 37°C for 10 hours. All strains were cultured until the viable count was 1×10 10 CFU / mL, single strain fermentation test, four single strains were inoculated in 50g defatted rice bran at 25mL, and fermented at 30℃ for 7 days. Composite fermentation agent fermentation test, two different strains were inoculated in 50g defatted rice bran at 12.5mL each, and fermented at 30℃ for 7 days. Defatted rice bran with 25mL sterile water was used as the control group (CK).

[0036] The colorimetric analysis showed that Figure 1 As shown, groups with one identical letter are not significantly different, groups without identical letters are significantly different, p<0.05. In the single strain fermentation test, the fermented rice bran obtained by fermentation with Lactobacillus fermentum ASAG had the highest GABA content of 2.642 g / L. In the composite fermentation test, the fermented rice bran obtained by fermentation with a composite fermentation agent composed of Lactobacillus fermentum ASAG and Bacillus amyloliquefaciens ASAG had the highest GABA content of 2.791 g / L.

[0037] Example 2 This example further explores the effect of fermenting GABA by using a composite fermentation agent prepared by using Lactobacillus fermentum ASAG and Bacillus amyloliquefaciens ASAG in different ratios. The steps are different from those in Example 1 only in that: The inoculation volume of the composite fermentation agent was kept unchanged at 25 mL, and the volume ratios of Lactobacillus fermentum ASAG and Bacillus amyloliquefaciens ASAG were adjusted to 1:9, 3:7, 5:5, 7:3, and 9:1, respectively, and inoculated into 50 g of defatted rice bran, and fermented at 30°C for 7 days.

[0038] The colorimetric analysis showed that Figure 2 As shown, groups with one identical letter are not significantly different, groups without identical letters are significantly different, p<0.05. When the weight ratio of Lactobacillus fermentum ASAG to Bacillus amyloliquefaciens ASAG was 1:9, the GABA content in fermented rice bran was the highest, at 2.438 g / L.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Lactobacillus fermentum ( Lactobacillus fermentum ), characterized in that, It is fermented Lactobacillus ASAG, and its preservation number is CCTCC No: M 20242832.

2. A strain of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), characterized in that, It is Bacillus amyloliquefaciens ASAG, and its preservation number is CCTCC No: M 20242831.

3. A composite fermentation agent, characterized in that: It comprises the fermented Lactobacillus ASAG described in claim 1 and the Bacillus amyloliquefaciens ASAG described in claim 2.

4. The composite fermentation agent according to claim 3, characterized in that: The weight ratio of the fermented lactobacillus ASAG to the amyloliquefaciens Bacillus ASAG is 1:(0.1-9).

5. The composite fermentation agent according to claim 4, characterized in that: The weight ratio of the fermented lactobacillus ASAG to the amyloliquefaciens ASAG is 1:(2-9) or 1:(0.1-0.5).

6. The composite fermentation agent according to claim 5, characterized in that: The weight ratio of the fermented lactobacillus ASAG to the amyloliquefaciens Bacillus ASAG is 1:(8-9).

7. Use of the composite fermentation agent according to any one of claims 3 to 6 in fermentation production of γ-aminobutyric acid.

8. A method for producing γ-aminobutyric acid by fermentation, characterized in that: The method comprises fermenting rice bran using the composite fermentation agent according to any one of claims 3 to 6.

9. The method according to claim 8, characterized in that The fermentation is solid state fermentation.

10. The method according to claim 8, characterized in that The fermentation temperature is 28°C to 32°C; and / or the fermentation time is more than 7 days; and / or the mass volume ratio of rice bran to the composite fermentation agent is (1 to 3) g: 1 mL.

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