Composite fermentation agent and method for producing gamma-aminobutyric acid by fermentation using the same

Abstract

The present invention relates to the field of microbial technology, and in particular to a composite fermentation agent and a method for producing gamma-aminobutyric acid using the composite fermentation agent. The composite fermentation agent includes Lactobacillus fermentum ASAG and Bacillus amyloliquefaciens ASAG. The present invention develops a composite fermentation agent suitable for solid-state fermentation of GABA using rice bran as a single substrate. The use of the composite fermentation agent to produce GABA by fermentation not only increases the use value of rice bran, achieving efficient value-added utilization of rice bran, a byproduct of rice processing, but also reduces the production cost of GABA. Furthermore, the method is simple to operate, has high production efficiency, is pollution-free, and is highly safe. The fermentation product can be used as a food ingredient or food additive, is highly economical, and has broad application prospects.

Description

Technical Field

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

[0002] Rice bran is a major byproduct of rice processing, accounting for approximately 8% of the rice grain's mass, with an annual production of 14 million tons. Rice bran is highly nutritious, containing 64% of the nutrients in rice, including protein, fat, carbohydrates, dietary fiber, and active ingredients such as gamma-aminobutyric acid, gamma-oryzanol, tocopherol, gallic acid, and squalene.

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

[0004] There are three main methods for producing GABA: chemical synthesis, plant enrichment, and microbial fermentation. While chemical synthesis offers rapid synthesis, it suffers from high costs, intense reactions, and low safety, making it unsuitable for producing food-grade GABA. Plant enrichment offers ease of operation and high safety, but its low production efficiency and difficulty in separation make widespread application difficult. Microbial fermentation involves selecting high-quality, stable, and non-toxic strains to produce GABA during their growth and reproduction. Studies have found that lactic acid bacteria, molds, yeasts, and Bacillus can all be used for GABA production, and mixed fermentation is generally superior to single-bacteria fermentation because it produces complex proteases that are more conducive to protein degradation.

[0005] Microbial fermentation can be categorized into liquid fermentation and solid-state fermentation, depending on the physical properties of the culture medium. Solid-state fermentation has relatively loose requirements for fermentation conditions, allowing microorganisms to maintain their original, natural growth state, which is conducive to the accumulation and diversity of fermentation metabolites. Compared to liquid fermentation, it offers advantages such as higher yields, lower investment, simpler processes, and easier drying and recovery.

[0006] Currently, most commonly used GABA production methods utilize liquid fermentation with a single bacterial strain. Few reports exist on fermentation using composite bacterial strains or solid-state fermentation, and relatively few reports on using rice bran as a single substrate. Therefore, developing a composite fermentation agent suitable for solid-state fermentation of GABA using rice bran as a single substrate has become a technical challenge that urgently needs to be addressed 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 using rice bran as a single substrate through a large number of composite screening of bacterial strains, and proposes the following technical solution based on this.

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

[0009] Lactobacillus fermentum ASAG was isolated from a soil sample from a Sichuan pickle processing factory. The strain was deposited in the China Center for Type Culture Collection on December 16, 2024. The depository address is 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 from a corn field in Hubei Province. The strain was deposited in the China Center for Type Culture Collection on December 16, 2024. The depository address is: 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 agent, comprising the Lactobacillus fermentum ASAG and the Bacillus amyloliquefaciens ASAG.

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

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

[0019] Preferably, the fermentation is 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 agent, the viable cell count of the Lactobacillus fermentum ASAG or the Bacillus amyloliquefaciens ASAG is 1×10 8 ~1×10 11 CFU / mL.

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

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

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present 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, and efficient value-added utilization of rice bran, a byproduct of rice processing, 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 is highly safe. The fermentation product can be used as a food ingredient or food additive, thus having great economic benefits and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram showing the results of GABA production by fermentation of different strains in Example 1.

[0027] Figure 2 This is a graph showing the results of GABA production by composite fermentation agents with different ratios in Example 2. DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0029] Where specific techniques or conditions are not specified in the examples, all methods were performed according to conventional methods, techniques or conditions described in literature in the field, or according to product specifications. Reagents and instruments used, for which the manufacturers are not specified, are conventional products that can be purchased through regular channels.

[0030] The culture medium formula used in the following examples is as follows:

[0031] (1) YPD medium: yeast extract 10.0 g / L, tryptone 20.0 g / L, anhydrous glucose 20.0 g / L. Used for the activation and cultivation of Saccharomyces cerevisiae.

[0032] (2) PDA medium: potato extract powder 10.0 g / L, anhydrous glucose 20.0 g / L. Used for the activation and cultivation of Rhizopus oryzae.

[0033] (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 Lactobacillus fermentum.

[0034] (4) LB medium: 5.0 g / L yeast extract, 10.0 g / L tryptone, 10.0 g / L NaCl. Used for activation and cultivation of Bacillus amyloliquefaciens.

[0035] (5) Fermentation medium: 50 g defatted rice bran.

[0036] Example 1

[0037] In this example, GABA was fermented using different single strains and composite fermentation agents composed of different strains. The strains used were 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 Province, and were labeled as: FJ-Lactobacillus fermentum ASAG; NJ-Saccharomyces cerevisiae; JDF-Bacillus amyloliquefaciens ASAG; MG-Rhizopus oryzae.

[0038] The fermentation steps were as follows: Saccharomyces cerevisiae, Rhizopus oryzae, and Bacillus amyloliquefaciens ASAG were activated in YPD, PDA, and LB medium for 24 h, 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 h; Lactobacillus fermentum ASAG was activated in MRS medium for 24 h, then inoculated into MRS seed medium at a volume percentage of 3% and cultured at 37°C for 10 h. All strains were cultured until the viable count was 1×10 10 CFU / mL. For single-strain fermentation tests, 25 mL of each of the four strains was inoculated into 50 g of defatted rice bran and the fermentation was allowed to proceed at 30°C for 7 days. For combined fermentation tests, 12.5 mL of each of the two strains was inoculated into 50 g of defatted rice bran and the fermentation was allowed to proceed at 30°C for 7 days. Defatted rice bran supplemented with 25 mL of sterile water served as the control group (CK).

[0039] 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 experiments, the rice bran fermented with Lactobacillus fermentum ASAG had the highest GABA content, at 2.642 g / L. In the composite fermentation experiments, the rice bran fermented with a composite fermentation inoculum composed of Lactobacillus fermentum ASAG and Bacillus amyloliquefaciens ASAG had the highest GABA content, at 2.791 g / L.

[0040] Example 2

[0041] This example further explores the effect of fermentation on GABA production by composite fermentation bacteria prepared by combining Lactobacillus fermentum ASAG and Bacillus amyloliquefaciens ASAG in different ratios. The steps differ from those in Example 1 only in that:

[0042] 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 a constant temperature of 30°C for 7 days.

[0043] The colorimetric analysis showed that Figure 2 The results are shown in Table 1. Groups with one identical letter are not significantly different, while groups without identical letters are significantly different (p < 0.05). The highest GABA content in fermented rice bran was 2.438 g / L when the weight ratio of Lactobacillus fermentum ASAG to Bacillus amyloliquefaciens ASAG was 1:9.

[0044] 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 various embodiments of the present invention.

Claims

1. A composite fermentation agent, characterized in that: These include Lactobacillus fermentum ASAG and Bacillus amyloliquefaciens ASAG; Among them, the preservation number of Lactobacillus fermentum ASAG is CCTCC No: M 20242832, and the preservation number of Bacillus amyloliquefaciens ASAG is CCTCC No: M 20242831.

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

3. The composite fermentation agent according to claim 2, 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).

4. The composite fermentation agent according to claim 3, characterized in that The weight ratio of the fermented lactobacillus ASAG to the amyloliquefaciens ASAG is 1:(8-9).

5. Use of the composite fermentation agent according to any one of claims 1 to 4 in the fermentation production of γ-aminobutyric acid.

6. A method for producing γ-aminobutyric acid by fermentation, characterized in that: The method comprises fermenting rice bran by using the composite fermentation agent according to any one of claims 1 to 4.

7. The method according to claim 6, characterized in that The fermentation is solid-state fermentation.

8. The method according to claim 6, 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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