Method for producing gamma-aminobutyric acid with high yield through mixed fermentation

Abstract

The invention belongs to the technical field of fermentation engineering and enzyme engineering, and particularly relates to a method for producing gamma-aminobutyric acid at high yield through mixed fermentation. Specifically, a mixed fermentation mode is adopted, the growth of lactobacillus hilgardii is promoted through the growth of bifidobacteria, and the synergistic interaction of the two strains is realized, so that the yield of GABA is greatly improved. Experimental results show that compared with traditional single-bacterium fermentation, the mixed fermentation method has the advantages that the yield of the GABA can be increased by 2-3 times, the fermentation period is shortened by 50% or above, and remarkable technical advantages and economic benefits are achieved. Therefore, the mixed fermentation method provided by the technical scheme provides an efficient and environment-friendly new way for the production of high-yield GABA, and has important application value and market prospect.

Description

Technical Field

[0001] The invention belongs to the technical fields of fermentation engineering and enzyme engineering, and particularly relates to a method for high-yield gamma-aminobutyric acid by mixed fermentation. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] γ-Aminobutyric acid (GABA) is an important non-protein amino acid widely found in animals, plants, and microorganisms, exhibiting diverse biological activities. As a major inhibitory neurotransmitter in the central nervous system, GABA regulates neuronal excitability and is crucial for maintaining normal nervous system function. Furthermore, GABA exhibits physiological effects such as anti-anxiety, lowering blood pressure, improving sleep, and promoting brain recovery. Therefore, it holds broad application prospects in medicine, food, and functional beverages. Currently, GABA production primarily involves chemical synthesis and biofermentation. While chemical synthesis is a mature process, it suffers from high costs, significant environmental pollution, and low product purity. In contrast, biofermentation, due to its environmental advantages, low cost, and high product purity, has gradually become the mainstream method for GABA production.

[0004] In the bio-fermentation method, Lactobacillus hilgridis ( Lactobacillus hilgardii ) is one of the most commonly used strains. Lactobacillus hilgensis possesses a unique metabolic pathway, enabling it to directly convert glutamate into gamma-aminobutyric acid (GABA) through transamination. This bioconversion process is not only highly efficient but also requires no complex chemical reagents, making it widely used in GABA production. However, despite the significant advantages of Lactobacillus hilgensis in GABA production, traditional single-strain fermentation methods still have some significant limitations. First, single-strain fermentation often results in low GABA yields. This is primarily because the growth of a single strain is significantly affected by environmental factors such as pH, temperature, and nutrient concentrations. Changes in these factors can significantly affect strain growth and GABA production. Second, single-strain fermentation methods require a long fermentation cycle, typically requiring a significant time to achieve the desired GABA yield. This not only increases production costs but also limits improvements in production efficiency. Summary of the Invention

[0005] To address the shortcomings of the aforementioned prior art, the present invention provides a method for high-yield γ-aminobutyric acid (GABA) through mixed fermentation. The present invention screens and obtains a strain of Bifidobacterium lactis. Mixing this strain with the GABA-producing Lactobacillus hilgeni for fermentation significantly increases GABA production in Lactobacillus hilgeni. Experiments have demonstrated that Bifidobacterium lactis can grow in large quantities during fermentation and produce metabolites such as lactic acid. These metabolites not only lower the pH of the fermentation broth, inhibiting the growth of harmful bacteria, but also promote Lactobacillus hilgeni's growth and GABA synthesis through competitive inhibition and metabolic regulation. Furthermore, Bifidobacterium lactis can further enhance GABA production by producing enzymes such as glutamate decarboxylase (GAD). Based on these research findings, the present invention was completed.

[0006] In order to achieve the above technical objectives, the present invention relates to the following technical solutions: The first aspect of the present invention provides a strain of Bifidobacterium lactis ( Bifidobacterium lactis )ga-2, deposited in the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), the deposit date is May 26, 2025, and its biological deposit number is CCTCC NO: M 20251181.

[0007] A second aspect of the present invention provides the use of the above-mentioned Bifidobacterium lactis ga-2 in any one or more of the following: (a) promoting the growth of Lactobacillus hilgridis; (b) Promote the production of γ-aminobutyric acid by Lactobacillus hilarii.

[0008] The third aspect of the present invention provides a microbial composition comprising the above-mentioned Bifidobacterium lactis ga-2 and Lactobacillus hilgeni.

[0009] The fourth aspect of the present invention provides the use of the above-mentioned microbial composition in the production of γ-aminobutyric acid.

[0010] A fifth aspect of the present invention provides a method for producing γ-aminobutyric acid by fermentation, the method comprising: The activated Bifidobacterium lactis ga-2 and Lactobacillus hilgensis are mixed and inoculated into a culture medium, and a substrate sodium glutamate is added to carry out fermentation culture to produce gamma-aminobutyric acid.

[0011] Furthermore, the method includes: S1, picking the single colonies of Bifidobacterium lactis ga-2 and Lactobacillus hilgeni respectively to the first culture medium for primary activation culture, and then picking the activated Bifidobacterium lactis ga-2 and Lactobacillus hilgeni single colonies to the second culture medium for secondary activation culture; S2. The Lactobacillus hilgensis activated for the second time is inoculated into the third culture medium for fermentation and culture for a period of time, and then Bifidobacterium lactis is inoculated. Sodium glutamate is added in a fed-batch manner to continue fermentation and culture to produce γ-aminobutyric acid.

[0012] Beneficial technical effects of one or more of the above technical solutions: The above technical solution uses a mixed fermentation method to promote the growth of Lactobacillus hilarii by the growth of Bifidobacterium, achieving synergistic synergy between the two strains, thereby significantly increasing GABA production. Experimental results show that compared with traditional single-bacteria fermentation, the mixed fermentation method can increase GABA production by 2-3 times and shorten the fermentation cycle by more than 50%, with significant technical advantages and economic benefits. Therefore, the mixed fermentation method proposed in the above technical solution provides a new, efficient and environmentally friendly way to produce high-yield GABA, with important application value and market prospects. DETAILED DESCRIPTION

[0013] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0014] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0015] In a typical embodiment of the present invention, a strain of Bifidobacterium lactis ( Bifidobacterium lactis )ga-2, deposited in the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), the deposit date is May 26, 2025, and its biological deposit number is CCTCC NO: M 20251181.

[0016] A second aspect of the present invention provides the use of the above-mentioned Bifidobacterium lactis ga-2 in any one or more of the following: (a) promoting the growth of Lactobacillus hilgridis; (b) Promote the production of γ-aminobutyric acid by Lactobacillus hilarii.

[0017] The third aspect of the present invention provides a microbial composition comprising the above-mentioned Bifidobacterium lactis ga-2 and Lactobacillus hilgeni.

[0018] The fourth aspect of the present invention provides the use of the above-mentioned microbial composition in the production of γ-aminobutyric acid.

[0019] A fifth aspect of the present invention provides a method for producing γ-aminobutyric acid by fermentation, the method comprising: The activated Bifidobacterium lactis ga-2 and Lactobacillus hilgensis are mixed and inoculated into a culture medium, and a substrate sodium glutamate is added to carry out fermentation culture to produce gamma-aminobutyric acid.

[0020] Furthermore, the method includes: S1, picking the single colonies of Bifidobacterium lactis ga-2 and Lactobacillus hilgeni respectively to the first culture medium for primary activation culture, and then picking the activated Bifidobacterium lactis ga-2 and Lactobacillus hilgeni single colonies to the second culture medium for secondary activation culture; S2. The Lactobacillus hilgensis activated for the second time is inoculated into the third culture medium for fermentation and culture for a period of time, and then Bifidobacterium lactis is inoculated. Sodium glutamate is added in a fed-batch manner to continue fermentation and culture to produce γ-aminobutyric acid.

[0021] Wherein, in step S1, the composition of the first culture medium is: Each 1000 mL contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and 15-20 g agar.

[0022] The second culture solution is composed of: Each 1000 mL contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 2 g fructooligosaccharides, 1 mL Tween 80, 2 g sodium glutamate, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate.

[0023] In step S2, the third culture solution is composed of: Each 1000 mL contains 10 g peptone, 5 g beef powder, 5 g soybean peptide, 5 g wheat peptide, 4 g yeast powder, 2 g glucose, 2 g oligofructose, 1 mL Tween 80, 2 g sodium glutamate, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate.

[0024] The specific conditions of the fermentation culture for a period of time are: 30-37° C., 80-120 r / min, culture for 2-4 hours, and OD600 value reaching 1.0-1.5.

[0025] In step S2, the inoculation amounts of Lactobacillus hilgensis and Bifidobacterium lactis are both 2%-3% (v / v).

[0026] The specific conditions for adding sodium glutamate in the fed-batch method are: the fed-batch amount of sodium glutamate is 5-20 g / L; The conditions for continued fermentation culture are: 30-37°C, 20-80 r / min, and culture for 10-30 h.

[0027] Furthermore, the method includes: Separately, select the preserved Lactobacillus hilgensis and Bifidobacterium lactis strains and perform streak activation culture on solid culture medium (prepared by adding 1.5-2% agar to the first culture medium). Culture Lactobacillus hilgensis at 30°C for 18-24 hours, and Bifidobacterium lactis at 30°C for 18-24 hours. After the culture is completed, select a single colony and inoculate it into the second culture medium for liquid static culture. Culture Lactobacillus hilgensis at 30°C for 18-24 hours, and Bifidobacterium lactis at 30°C for 18-24 hours to restore the strains to maximum growth activity. v / v ) inoculated into the third culture medium, cultured at 37 ℃, 100 r / min for about 2-4 h, measured the OD600 value of Lactobacillus hilgensis in the culture medium, and when the OD600 value reached 1.0-1.5, added 3%-5% ( v / v ) was inoculated into the third culture medium, and sodium glutamate was added by feeding at a rate of 10 g / L. The culture was cultured at 37 °C and 50 r / min for 24 h.

[0028] Wherein, the composition of the first culture solution is: Each 1000 mL contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate.

[0029] The second culture solution is composed of: Each 1000 mL contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 2 g fructooligosaccharides, 1 mL Tween 80, 2 g sodium glutamate, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate.

[0030] The composition of the third culture solution is: Each 1000 mL contains 10 g peptone, 5 g beef powder, 5 g soybean peptide, 5 g wheat peptide, 4 g yeast powder, 2 g glucose, 2 g oligofructose, 1 mL Tween 80, 2 g sodium glutamate, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate.

[0031] Below by embodiment, the present invention is further explained, but does not constitute limitation of the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the invention.In embodiment, lactobacillus hilgeni CGMCC NO.26755, lactobacillus hilgeni CGMCC NO.26138 are disclosed in Chinese patent CN116814468A and CN116286513A respectively, and plant lactobacillus GDMCC NO.64769 is disclosed in Chinese patent CN118497086A.

[0032] The solid culture medium containing the first culture solution is composed of: Each 1000 mL contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and 20 g agar.

[0033] The composition of the second culture medium is: Each 1000 mL contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 2 g fructooligosaccharides, 1 mL Tween 80, 2 g sodium glutamate, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate.

[0034] The composition of the third culture medium is: Each 1000 mL contains 10 g peptone, 5 g beef powder, 5 g soybean peptide, 5 g wheat peptide, 4 g yeast powder, 2 g glucose, 2 g oligofructose, 1 mL Tween 80, 2 g sodium glutamate, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate.

[0035] Example 1 Mixed fermentation of Lactobacillus hilgeni CGMCC No. 26755 and Bifidobacterium lactis Preserved strains of Lactobacillus hilgensis CGMCC No. 26755 and Bifidobacterium lactis strain ga-2 were inoculated onto solid culture medium containing the first culture medium for streak activation (incubation at 30°C for approximately 24 hours). Single colonies of each strain were then plated and incubated in liquid culture medium containing the second culture medium (incubation at 30°C for approximately 24 hours) to restore maximum growth activity. In the experimental group, Lactobacillus hilgensis was inoculated into the third culture medium at a 2% inoculum size and cultured at 37°C, 100 rpm, for approximately 3 hours. After that, sodium glutamate was fed at a rate of 10 g / L, and fermentation continued at 37°C, 50 rpm, for 24 hours. In the control group, Bifidobacterium lactis was inoculated into the third culture medium at a 2% inoculum size and cultured at 37°C, 100 rpm, for approximately 3 hours. After that, sodium glutamate was fed at a rate of 10 g / L, and fermentation continued at 37°C, 50 rpm, for 24 hours. In control group 2, Lactobacillus hilgensis was inoculated into the third culture medium at a 2% inoculum size and cultured at 37°C, 100 r / min for about 3 h. The OD of Lactobacillus hilgensis in the culture medium was measured. 600 When the OD600 value reached 1.0, Bifidobacterium lactis was inoculated into the third culture medium at a 3% inoculum size. Sodium glutamate was then added via a fed-batch method at a rate of 10 g / L. Fermentation was continued at 37°C and 50 rpm for 24 hours. Samples were collected every 12 hours to measure GABA production and the OD600 value of the culture medium.

[0036] The results showed that when Bifidobacterium lactis was cultured alone, no GABA was produced, while when Lactobacillus hilgris was cultured alone, the GABA production was low. After 24 hours of mixed fermentation, the GABA production reached 16.3 g / L, which was 1.4 times higher than that of single bacteria fermentation (6.8 g / L); the bacterial growth (OD600) increased by 88%, and the fermentation cycle was shortened to 24 hours (single bacteria required 48 hours to achieve similar production).

[0037] Table 1 GABA production and OD value determination of different groups

[0038] Example 2 Mixed fermentation of Lactobacillus hilgeni CGMCC NO.26138 and Bifidobacterium lactis Preserved strains of Lactobacillus hilgensis CGMCC No. 26138 and Bifidobacterium lactis strain ga-2 were inoculated onto solid culture medium containing the first culture medium for streak activation (incubation at 30°C for approximately 24 hours). Single colonies of each strain were then plated and placed in liquid culture medium containing the second culture medium (incubation at 30°C for approximately 24 hours) to restore maximum growth activity. In the experimental group, Lactobacillus hilgensis was inoculated into the third culture medium at a 2% inoculum size and cultured at 37°C, 100 rpm, for approximately 3 hours. After that, sodium glutamate was fed at a rate of 10 g / L, and fermentation continued at 37°C, 50 rpm, for 24 hours. In the control group, Bifidobacterium lactis was inoculated into the third culture medium at a 2% inoculum size and cultured at 37°C, 100 rpm, for approximately 3 hours. After that, sodium glutamate was fed at a rate of 10 g / L, and fermentation continued at 37°C, 50 rpm, for 24 hours. In control group 2, Lactobacillus plantarum was inoculated into the third culture medium at a 2% inoculum size and cultured at 37°C, 100 r / min for about 3 h. The OD of Lactobacillus plantarum in the culture medium was measured. 600 When the OD600 value reached 1.0, Bifidobacterium lactis was inoculated into the third culture medium at a 3% inoculum size. Sodium glutamate was then added via a fed-batch method at a rate of 10 g / L. Fermentation was continued at 37°C and 50 rpm for 24 hours. Samples were collected every 12 hours to measure GABA production and the OD600 value of the culture medium.

[0039] The results showed that when Bifidobacterium lactis was cultured alone, no GABA was produced, while when Lactobacillus hilgris was cultured alone, the GABA production was low. After 24 hours of mixed fermentation, the GABA production reached 36.3 g / L, which was 93% higher than that of single bacteria fermentation (18.8 g / L); the bacterial growth (OD600) increased by 125%, and the fermentation cycle was shortened to 24 hours (single bacteria required 48 hours to achieve similar production).

[0040] Table 2 GABA production and OD value determination of different groups

[0041] Example 3 Mixed fermentation of Lactobacillus plantarum GDMCC No. 64769 and Bifidobacterium lactis Preserved strains of Lactobacillus plantarum GDMCC No. 64769 and Bifidobacterium lactis strain ga-2 were inoculated onto solid culture medium containing the first culture medium for streak activation (incubation at 30°C for approximately 24 hours). Single colonies of each strain were then plated and placed in liquid culture medium containing the second culture medium (incubation at 30°C for approximately 24 hours) to restore maximum growth activity. In the experimental group, Lactobacillus plantarum was inoculated into the third culture medium at a 2% inoculum size and cultured at 37°C at 100 rpm for approximately 3 hours. After that, sodium glutamate was fed at a rate of 10 g / L, and fermentation continued at 37°C at 50 rpm for 24 hours. In the control group, Bifidobacterium lactis was inoculated into the third culture medium at a 2% inoculum size and cultured at 37°C at 100 rpm for approximately 3 hours. After that, sodium glutamate was fed at a rate of 10 g / L, and fermentation continued at 37°C at 50 rpm for 24 hours. In control group 2, Lactobacillus plantarum was inoculated into the third culture medium at a 2% inoculum size and cultured at 37°C, 100 r / min for about 3 h. The OD of Lactobacillus plantarum in the culture medium was measured. 600 When the OD600 value reached 1.0, Bifidobacterium lactis was inoculated into the third culture medium at a 3% inoculum size. Sodium glutamate was then added via a fed-batch method at a rate of 10 g / L. Fermentation was continued at 37°C and 50 rpm for 24 hours. Samples were collected every 12 hours to measure GABA production and the OD600 value of the culture medium.

[0042] Table 3 GABA production and OD value determination of different groups

[0043] The results showed that when Bifidobacterium lactis was cultured alone, it did not produce GABA. Another GABA-producing Lactobacillus plantarum had no promoting effect (including growth promotion and GABA production promotion) when used in combination with the Bifidobacterium lactis in this application.

[0044] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A strain of Bifidobacterium lactis ( Bifidobacterium lactis )ga-2, deposited in the China Center for Type Culture Collection, the deposit date is May 26, 2025, and its biological deposit number is CCTCC NO: M 20251181.

2. Use of the Bifidobacterium lactis ga-2 according to claim 1 in any one or more of the following: (a) promoting the growth of Lactobacillus hilgridis; (b) Promote the production of γ-aminobutyric acid by Lactobacillus hilarii.

3. A microbial composition, characterized in that The microbial composition comprises the Bifidobacterium lactis ga-2 and Lactobacillus hilgeni according to claim 1.

4. Use of the microbial composition according to claim 3 in the production of γ-aminobutyric acid.

5. A method for producing γ-aminobutyric acid by fermentation, characterized in that: The method comprises: The activated Bifidobacterium lactis ga-2 according to claim 1 is mixed with Lactobacillus hilgensis and inoculated into a culture medium, and a substrate sodium glutamate is added to carry out fermentation culture to produce gamma-aminobutyric acid.

6. The method according to claim 5, wherein The method comprises: S1, picking the single colonies of Bifidobacterium lactis ga-2 and Lactobacillus hilgeni respectively to the first culture medium for primary activation culture, and then picking the activated Bifidobacterium lactis ga-2 and Lactobacillus hilgeni single colonies to the second culture medium for secondary activation culture; S2. The Lactobacillus hilgensis activated for the second time is inoculated into the third culture medium for fermentation and culture for a period of time, and then Bifidobacterium lactis is inoculated. Sodium glutamate is added in a fed-batch manner to continue fermentation and culture to produce γ-aminobutyric acid.

7. The method according to claim 6, wherein In the step S1, The composition of the first culture medium is: Each 1000 mL contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and 15-20 g agar; The composition of the second culture solution is: Each 1000 mL contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 2 g fructooligosaccharides, 1 mL Tween 80, 2 g sodium glutamate, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate.

8. The method according to claim 6, wherein In step S2, the third culture solution is composed of: Each 1000 mL contains 10 g peptone, 5 g beef powder, 5 g soybean peptide, 5 g wheat peptide, 4 g yeast powder, 2 g glucose, 2 g oligofructose, 1 mL Tween 80, 2 g sodium glutamate, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate.

9. The method according to claim 6, wherein In step S2, the fermentation culture for a period of time is carried out under the following conditions: 30-37°C, 80-120 r / min, culture for 2-4 hours, and OD600 value reaches 1.0-1.

5.

10. The method according to claim 6, wherein In step S2, the inoculation amounts of Lactobacillus hilgeni and Bifidobacterium lactis are both 2%-3% (v / v); The specific conditions for adding sodium glutamate in the fed-batch method are: the fed-batch amount of sodium glutamate is 5-20 g / L; The conditions for continued fermentation culture are: 30-37°C, 20-80 r / min, and culture for 10-30 h.

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