Saccharomyces cerevisiae producing gamma-aminobutyric acid and method for promoting production thereof

Abstract

The present application provides a kind of lactobacillus sakei of gamma-aminobutyric acid and the method for promoting its production, comprising: make lactobacillus sakei under the condition containing branched chain amino acid fermentation culture, to obtain the fermentation product containing gamma aminobutyric acid.The method provided by the present application can break through the limiting factor of the limited intake of L-glutamic acid sodium by cell membrane during the growth of fermentation strain, realize efficient metabolism substrate to produce gamma aminobutyric acid.Compared with not using branched chain amino acid, the method provided by the present application can make the yield of gamma aminobutyric acid increase by 10 times, and the method of the present application is simple, cheap, green, meet the health food requirements.

Description

Technical Field

[0001] This invention belongs to the field of food microbiology technology, specifically relating to a sake-producing Lactobacillus and a method for promoting its yield. Background Technology

[0002] γ Gamma-aminobutyric acid (GABA) Gamma-aminobutyric acid (GABA) is a key inhibitory neurotransmitter in the human nervous system, possessing multiple physiological regulatory functions such as lowering blood pressure, improving sleep, relieving anxiety, enhancing memory, and delaying nerve aging. It is currently widely marketed as a nutritional supplement. Currently, human GABA supplementation is achieved through two main pathways: dietary intake and exogenous supplementation. However, the background content of GABA in natural foods such as tomatoes, grapes, and potatoes is extremely low, insufficient to meet the body's daily adequate needs. Therefore, large-scale artificial production of GABA has become a research hotspot in the industry. Existing industrial production methods for GABA mainly fall into two categories: chemical synthesis and microbial fermentation. Chemical synthesis methods suffer from drawbacks such as raw material residues, low purity, and insufficient food safety, making them unsuitable for food processing. Microbial fermentation, on the other hand, offers advantages such as low production costs, high product safety, and the ability to be directly used as a natural functional food ingredient, making it the mainstream technology for large-scale production of food-grade GABA.

[0003] Glutamate decarboxylase (GAD) is the core functional enzyme that catalyzes the decarboxylation of monosodium glutamate (MSG) to γ-aminobutyric acid (GABA). The activity of this enzyme within microbial cells directly determines the efficiency of GABA synthesis. Microorganisms capable of metabolizing and synthesizing GABA include Escherichia coli, yeast, and lactic acid bacteria. Compared to other strains, lactic acid bacteria have a higher food safety rating, and the GABA produced during fermentation has better bioavailability and is more easily absorbed by the human body. Simultaneously, the fermentation process generates beneficial lactic acid, and the strains themselves possess antibacterial, anti-inflammatory, and immune-regulating probiotic effects, making them a core strain for the research and development of GABA-rich fermented foods. *Lactobacillus thuringiensis* is widely distributed in sake (…). Latilactobacillus sakei Lactobacillus globulus is a type of functional lactic acid bacteria that is resistant to low temperatures, high salt levels, and has extremely strong environmental adaptability. It has become a crucial fermentation strain in fermented meat products, pickled fruits and vegetables, fermented aquatic products, dairy products, and alcoholic beverage processing, possessing multiple application values ​​including preservation and pest control, flavor optimization, texture improvement, enhanced food safety, and the addition of probiotic properties to food. Increasing the yield of γ-aminobutyric acid (GABA) in sake-producing Lactobacillus globulus can enhance the bacteria's tolerance to fermentation stresses such as acid and high salt, maintain the stability of the fermentation system, and enable in-situ synthesis of natural GABA by microorganisms. This is beneficial for imparting more health benefits to fermented foods, achieving clean food labeling, and optimizing fermentation flavor.

[0004] Currently, existing technologies mainly improve the efficiency of substrate transmembrane transport, thereby increasing γ-aminobutyric acid (GABA) production, by regulating fermentation culture conditions and adding exogenous coenzymes and nutrients. For example, existing technology CN 116790687A optimizes the fermentation process of *Lactobacillus plantarum* by regulating the concentration of substrate and pyridoxal phosphate, as well as the culture time; existing technology CN202210402135.5 increases the permeability of *Lactobacillus brevis* cell membrane by adding nicotinic acid and arginine, resulting in a 40% increase in GABA production. However, these existing technologies only focus on enhancing the efficiency of substrate transmembrane transport without changing the physiological characteristics of the bacteria themselves. Without damaging the activity of the bacteria, the substrate transport capacity of the cell membrane has an inherent upper limit, which cannot fundamentally improve the intracellular substrate conversion efficiency and makes it difficult to achieve a breakthrough increase in GABA production. Summary of the Invention

[0005] To solve all or part of the above-mentioned technical problems, the present invention provides the following technical solutions: The first aspect of the present invention provides a method for promoting the production of γ-rays by *Lactobacillus globulus* in sake. Methods for obtaining GABA include: fermenting *Lactobacillus globulus* in sake under conditions containing branched-chain amino acids to obtain GABA-containing bacteria. Fermentation products of GABA.

[0006] The method provided by this invention adds branched-chain amino acids during the fermentation process of *Lactobacillus cereus* in sake to promote the metabolism of *Lactobacillus cereus*, thereby overcoming the limitation of the strain's low ability to metabolize L-glutamate. In some embodiments, it can also metabolize γ-glutamate. The yield of GABA was increased more than 10 times. The branched-chain amino acids used have the advantages of being green, safe, and inexpensive, and the method allows the fermentation strains to grow fully to provide high doses of widely distributed lactobacilli in sake and beneficial byproducts.

[0007] In some embodiments, the branched-chain amino acids include one or more combinations of L-leucine, L-isoleucine, and L-phenylalanine.

[0008] In some embodiments, the *Lactobacillus saccharidus* species included those carrying the glutamate decarboxylase gene (…). gad Lactobacillus is widely distributed in sake containing the gene.

[0009] In some embodiments, the *Lactobacillus sacchariformis* is *Lactobacillus sacchariformis* KLMM605, with the Latin name [missing information]. Latilactobacillus sakei It was isolated from Guizhou fermented meat and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 50040 and deposit date of August 12, 2026.

[0010] In some embodiments, the method includes: adding the branched-chain amino acids to a culture medium and fermenting Lactobacillus sakebi in the culture medium, wherein the concentration of the branched-chain amino acids in the culture medium is 400 μg / mL to 1200 μg / mL.

[0011] In some embodiments, the method specifically includes: The activated bacterial solution containing Lactobacillus sacchariformis was inoculated into culture medium A for the first stage of fermentation. The culture medium A contained monosodium glutamate (MSG). The culture was cultured to the logarithmic growth phase to obtain the first stage fermentation broth. The first-stage fermentation broth was mixed with culture medium B, which contains L-glutamate sodium and the branched-chain amino acids, to carry out a second-stage fermentation to obtain γ-glutamic acid. Fermentation products of GABA.

[0012] The second stage of fermentation involves culturing the stable-phase fermentation strain in a medium containing branched-chain amino acids. The intracellular L-glutamate content is increased by acylphosphatase transamination, followed by increased substrate γ-glutamate content by glutamate decarboxylase. Gamma-aminobutyric acid synthesis to achieve γ Efficient production of GABA.

[0013] In some embodiments, the activated bacterial solution is inoculated into the culture medium A at a volume ratio of 1-3%.

[0014] In some embodiments, the culture medium A is an MRS basal medium containing 1-2% (w / v) L-glutamate sodium.

[0015] In some embodiments, the first stage of fermentation includes: culturing at 28-32°C and 90-110 rpm until the logarithmic growth phase.

[0016] In some embodiments, the fermentation time in the first stage is 10-13 hours to reach the logarithmic growth phase, thereby giving the strain high viability.

[0017] In some embodiments, the amount of culture medium B is 8-12% (v / v) of the first-stage fermentation broth, and culture medium B is an MRS basal medium containing 400-1200 μg / mL branched-chain amino acids. For example, the preparation method of culture medium B may include: adding branched-chain amino acids to MRS basal culture medium containing 1-2% (w / v) L-glutamate, vortexing to mix, and filtering to remove bacteria.

[0018] In some embodiments, the second stage of fermentation includes: continuing to culture at 28~32℃ and 90~110 rpm for 10~24 h.

[0019] In some embodiments, the method specifically includes: First stage fermentation: Inoculate the activated bacterial solution at 1-3% (v / v) into medium A containing 1-2% (w / v) L-glutamate, and culture at 28-32℃ for 90-110 rpm until the logarithmic growth phase; Second stage fermentation: Add 8-12% volume of culture medium B containing branched-chain amino acids to the fermentation broth produced in the first stage fermentation, and continue culturing at 28-32℃ and 90-110 rpm for 10-24 h to obtain γ-amino acids. Fermentation products of GABA.

[0020] In some embodiments, the method for preparing the activated bacterial solution containing Lactobacillus sakebifolia includes: inoculating Lactobacillus sakebifolia strain into MRS basal medium at a ratio of 1-3% (v / v), culturing at 30-40°C for 12-24 hours, and repeating for two generations to obtain the activated bacterial solution.

[0021] The second aspect of the present invention provides a *Lactobacillus sacchariformis* species that produces γ-aminobutyric acid (GABA), namely *Lactobacillus sacchariformis* KLMM605, with the Latin name [missing information]. Latilactobacillus sakei It was isolated from Guizhou fermented meat and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 50040 and deposit date of August 12, 2026.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The method provided by the present invention adds branched-chain amino acids during the fermentation process of *Lactobacillus saccharina* to promote the metabolism of *Lactobacillus saccharina*, thereby overcoming the limitation that extracellular L-glutamate cannot fully penetrate the cell membrane to enter the cell. Through transamination, it promotes the generation of intracellular L-glutamate in the fermentation strain, overcoming the limitation of the strain's low ability to metabolize L-glutamate sodium, and promoting the full conversion of the substrate to γ-glutamate. Gamma-aminobutyric acid (GABA) is used to increase production. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The Lactobacillus sacchariformis KLMM605 used in this invention is... gad Agarose gel electrophoresis image of gene PCR products; Figure 2This is a graph showing the γ-aminobutyric acid (GABA) production of Lactobacillus sakebi strains KLMM605, KLMM606, and KLMM611 used in Comparative Examples 1-3 of this invention in medium A, which does not contain branched-chain amino acids. Figure 3 This is a graph showing the effect of adding different concentrations of L-leucine during fermentation in Examples 1-3 and Comparative Example 1 on the yield of γ-aminobutyric acid from Lactobacillus spp. KLMM605 in sake brewing. Figure 4 This is a graph showing the effect of adding different concentrations of L-isoleucine during fermentation in Examples 4-6 and Comparative Example 1 on the yield of γ-aminobutyric acid from Lactobacillus spp. KLMM605 in sake brewing. Figure 5 The graphs show the effects of adding different concentrations of L-phenylalanine during fermentation in Examples 7-9 on the yield of γ-aminobutyric acid from Lactobacillus thuringiensis KLMM605 in sake brewing. Figure 6 It is Lactobacillus thuringiensis KLMM605 in sake. AcP Agarose gel electrophoresis image of gene PCR products; Figure 7 It is Lactobacillus thuringiensis KLMM605 in sake. AcP q-PCR diagram of mRNA response to L-isoleucine. Detailed Implementation

[0025] The invention will be more fully understood through the following detailed description of its embodiments. The detailed embodiments disclosed herein are merely illustrative, and the invention may be embodied in various forms. Therefore, the specific functional details disclosed herein should not be construed as limiting, but rather as the basis for the claims.

[0026] In addition, unless otherwise specified, all raw materials used in the following embodiments can be purchased from the market or other sources, and all production and testing equipment used are known in the art, as are the testing methods used.

[0027] The sources and preparation methods of some of the raw materials used in the following examples and comparative examples are as follows: (1) Experimental culture medium: MRS (deMan, Rogosa and Sharpe) basal medium: purchased from Guangdong Huankai Microbial Technology Co., Ltd., used for the activation, subculture, and fermentation of Lactobacillus thuringiensis in sake.

[0028] (2) Experimental bacterial strains: Lactobacillus spp. KLMM605, Latin name: Latilactobacillus sakeiIt was isolated from Guizhou fermented meat and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 50040 and deposit date of August 12, 2026.

[0029] (3) Biochemical reagents: γ-Aminobutyric acid (GABA) standard (analytical grade) was purchased from Yuanye Biotechnology; monosodium glutamate (MSG) and phenol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; L-phenylalanine (food grade), L-isoleucine (food grade), and L-leucine (food grade) were purchased from Hebei Huayang Biotechnology Co., Ltd.; sodium hypochlorite solution (available chlorine 7.5%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. and stored at 4℃; sodium tetraborate and ethanol (analytical grade) were purchased from Shanghai Titan Technology Co., Ltd.; agarose, 50×TAE, nucleic acid dyes, nucleic acid D2000 Marker, bacterial genomic DNA extraction kit, and bacterial total RNA extraction kit were purchased from Sangon Biotech (Shanghai) Co., Ltd.; cDNA synthesis kit and Hieff Universal Blue qPCR SYBR Green Master Mix were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; primers gad- F ,gad- R AcP- F AcP- R, 16S -F、 16S -R was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0030] Table 1 Primer sequences involved in this study

[0031] (4) Preparation of main reagents: 6% (w / v) phenol: Take 6 g of phenol, add 100 mL of ultrapure water, and store in a refrigerator at 4℃ protected from light; 0.1 mol / L sodium tetraborate solution: Dissolve in a water bath at 45℃, and store in a sealed container at room temperature.

[0032] Example 1 This embodiment provides a method for promoting the production of γ-rays by *Lactobacillus globulus* in sake. The method for processing GABA includes the following steps: (1) The Lactobacillus sacchariformis KLMM605 strain was inoculated into MRS basal medium at a ratio of 2% (v / v) and cultured at 37℃ for 18 h. Activated bacterial solution was obtained after two generations. (2) First stage fermentation: The activated bacterial solution obtained in step (1) was inoculated into medium A containing 1% (w / v) L-glutamate at 2% (v / v) and cultured at 100 rpm in an environment of 30℃ until the logarithmic growth phase; (3) Second stage fermentation: Take 4.5 mL of the fermentation broth prepared in step (2), add 0.5 mL of medium B containing L-leucine, so that the final concentration of L-leucine is 400 μg / mL, and continue to culture at 100 rpm in a 30℃ environment for 18 h.

[0033] Examples 2-9 Examples 2-9 are basically the same as Example 1, except that the types of Lactobacillus thuringiensis, branched-chain amino acids, and the concentration of branched-chain amino acids in culture medium B for the second stage of fermentation are varied according to Table 1.

[0034] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that the culture medium B of Comparative Example 1 does not contain branched-chain amino acids.

[0035] Comparative Examples 2-3 Comparative Examples 2-3 are basically the same as Comparative Example 1, except that the changes in the *Lactobacillus spp.* strains in sake, as described in Table 1, are different.

[0036] Table 1. Types, concentrations, and γ-aminobutyric acid yields of branched-chain amino acids used in Examples 1-9 and Comparative Examples 1-3

[0037] The relevant testing methods of this invention are as follows: 1. Glutamate decarboxylase has the ability to convert the substrate L-glutamate into γ-aminobutyric acid (GABA). To test whether *Lactobacillus saccharidus* isolated from fermented meat has the ability to produce GABA, samples from *Lactobacillus saccharidus* KLMM 605 were analyzed. gad The gene was amplified by PCR and detected by agarose gel electrophoresis. The expected size of the PCR product was 600 bp.

[0038] Extraction of bacterial genomic DNA: Perform the procedure according to the bacterial genomic DNA extraction kit.

[0039] The specific steps for PCR amplification are as follows: (1) Mix 5.4 μL dd H2O, 0.8 μL gad Upstream primer, 0.8 μL gad The downstream primer and 10.0 μL of 2×PCRMix were added sequentially to a clean small centrifuge tube, and finally 3 μL of cDNA template was added. (2) Mix the liquid in the system thoroughly and centrifuge briefly; (3) Amplification was performed using a PCR instrument. The PCR amplification conditions were as follows: Phase 1: 95℃, 10 min; Phase 2: (95℃, 30 s; 58℃, 30 s; 72℃, 1 min), 30 cycles.

[0040] After the reaction, 6 μL of PCR product was taken and electrophoresed in a 1.5% (w / v) agarose gel at 110 V for 30 min, and the bands were detected under UV light.

[0041] 2. Quantitative detection of γ in fermentation products using colorimetric method Gamma-aminobutyric acid (GABA). The specific steps include: (1) Weigh 100 mg of analytical grade γ-aminobutyric acid and add it to a 50 mL volumetric flask. Prepare a 2 mg / mL γ-aminobutyric acid standard solution by diluting it with MRS medium containing 1% sodium glutamate (MSG). Use this as the stock solution and dilute it in a 50 mL volumetric flask to prepare 1, 0.5, 0.25, 0.125 and 0 mg / mL γ-aminobutyric acid standard solutions. (2) Take 4 mL of untreated Lactobacillus cereus culture, L-phenylalanine-treated Lactobacillus cereus culture, L-leucine-treated Lactobacillus cereus culture, and L-isoleucine-treated Lactobacillus cereus culture and centrifuge at 13000×g for 15 min at 4℃ and collect the fermentation supernatant. (3) Take 60 μL of the above γ-aminobutyric acid standard solution (γ-aminobutyric acid dissolved in MRS containing 1% MSG) or fermentation supernatant, add 200 μL of 0.1 mol / L sodium tetraborate buffer and 80 μL of 6% phenol solution, mix well, then mix with 90 μL of sodium hypochlorite solution (available chlorine is 7.5%), heat in a boiling water bath for 10 minutes, and then immediately place the solution in ice water for 20 minutes.

[0042] (4) When the solution turns blue-green, add 400 μL of 60% ethanol solution and shake until well mixed; (5) In OD 645 The absorbance was measured at nm. A standard curve and regression equation were established with absorbance as the ordinate and the concentration of each standard solution (mg / mL) as the abscissa. The content of γ-aminobutyric acid in the sample was then determined based on this.

[0043] 3. Acyl phosphatases (AcPs) are intermediary enzymes that regulate the synthesis of γ-aminobutyric acid (GABA) from branched-chain amino acids. To further investigate their promoting mechanism, [further research is needed]. AcP The DNA amplification of *Lactobacillus saccharidus* using genetically designed primer pairs specifically includes the following steps: (1) Add 4.4 μL dd H2O, 0.8 μL upstream primer, 0.8 μL downstream primer (see Table 1), and 10.0 μL 2×PCR master Mix to a clean small centrifuge tube in sequence, and finally add 4 μL Lactobacillus sake cDNA template. (2) Vortex the liquid in the system to mix it and then briefly centrifuge it; (3) Amplification was performed using a PCR instrument. The PCR amplification conditions were as follows: First stage, pre-denaturation: 94℃, 5 min; Second stage, DNA amplification: 94℃, 30 s; 61℃, 1 min; 72℃, 2 min, 30 cycles; Third stage, post-extension: 72℃, 10 min.

[0044] After the reaction, 6 μL of PCR product was taken and electrophoresed in a 2% agarose gel at 110 V for 30 min, and the bands were detected under UV light.

[0045] 4. Investigating the presence of widespread Lactobacillus in sake AcP The response of mRNA to L-isoleucine specifically includes the following steps: 1) Extraction of total RNA Following the steps of the column-based bacterial total RNA extraction kit, lysozyme at a concentration of 40 mg / mL was used to enzymatically digest the bacteria at 37°C for 50 min, vortexing every 5 min to mix thoroughly.

[0046] 2) cDNA synthesis RNA samples were subjected to cDNA synthesis according to the instructions of the cDNA synthesis kit. The system included 5 μL of 4×Hifair AdvanceFast One-step RT superMix, 1 μL of gDNA Remover Mix, RNase-free Water, and Total RNA, for a total volume of 20 μL. The reaction program was as follows: 37℃, 5 min; 85℃, 30 s.

[0047] 3) Real-time quantitative PCR (RT-PCR) The reaction mixture was prepared according to the qPCR Mix instructions. The mixture included 10 μL Hieff Universal Blue qPCR SYBR Green Master Mix, 2 μL cDNA, 0.2 μL (10 μM forward and reverse primers), and 8.4 μL sterilized ddH2O. The qPCR program was as follows: Stage 1, pre-denaturation: 95℃, 5 min; Stage 2, DNA amplification: 95℃, 20 s; 61℃, 30 s; 72℃, 1 min, 40 cycles.16S The procedure was as follows: First stage, pre-denaturation: 95℃, 5 min; Second stage, DNA amplification: 95℃, 20 s; 58℃, 30 s; 72℃, 1 min, 40 cycles.

[0048] Figure 1 The Lactobacillus sacchariformis KLMM605 used in this invention is... gad Agarose gel electrophoresis image of gene PCR products, as shown below. Figure 1 As shown, after 30 cycles of amplification, a band appeared at a size of approximately 600 bp, indicating the presence of *Lactobacillus sacchariformis* isolated from fermented meat in the previous stage. gad gene, It has the potential to produce γ-aminobutyric acid.

[0049] Figure 2 This is a graph showing the γ-aminobutyric acid (GABA) production of *Lactobacillus sakebi* strains KLMM605, KLMM606, and KLMM611 used in Comparative Examples 1, 2, and 3 of this invention in a culture medium without branched-chain amino acids. Figure 2 As shown, all three strains can produce γ-aminobutyric acid (GABA), and there is no significant difference in yield.

[0050] Figure 3 This is a graph showing the effect of adding different concentrations of L-leucine during fermentation in Examples 1-3 and Comparative Example 1 on the yield of γ-aminobutyric acid from Lactobacillus spp. KLMM605 in sake brewing. Figure 4 This is a graph showing the effect of adding different concentrations of L-isoleucine during fermentation in Examples 4-6 and Comparative Example 1 on the yield of γ-aminobutyric acid from Lactobacillus spp. KLMM605 in sake brewing. Figure 5 This is a graph showing the effect of adding different concentrations of L-phenylalanine during fermentation in Examples 7-9 on the yield of γ-aminobutyric acid (GABA) from *Lactobacillus globosum* KLMM605 in sake brewing. Combined with... Figure 3-5 It can be seen that, compared with Comparative Example 1, L-leucine, L-isoleucine and L-phenylalanine can all increase the yield of γ-aminobutyric acid, and the effect is better when the addition amount is 400 μg / mL-800 μg / mL, with the best effect when the addition amount is 800 μg / mL.

[0051] Figure 6 It is Lactobacillus thuringiensis KLMM605 in sake. AcP Agarose gel electrophoresis image of gene PCR products. Figure 6 As shown, the expected band appears at approximately 177 bp, indicating that *Lactobacillus sacchariformis* KLMM605 possesses... AcP Genes. L-Isoleucine has the lowest promoting ability. If it can promote the production of γ-aminobutyric acid (GABA) by affecting AcP mRNA expression, it indicates that branched-chain amino acids promote GABA production by affecting AcP mRNA expression.

[0052] Figure 7 It is Lactobacillus thuringiensis KLMM605 in sake. AcP mRNA response to L-isoleucine q-PCR diagram. (See diagram below.) Figure 7 As shown, compared with Example 5 and Comparative Example 1, it was found that the expression level of AcP mRNA in Lactobacillus sakebiophilus increased by 1.45 times after L-isoleucine treatment, indicating that branched-chain amino acids... AcP Gene expression ， The increased intracellular AcP enzyme then transaminates L-glutamate, which is decarboxylated by intracellular glutamate decarboxylase, ultimately promoting the large-scale production of γ-aminobutyric acid (GABA). AcP enzyme indirectly enhances the GABA synthesis capacity of *Lactobacillus sacchariformis* by regulating intracellular phosphate, energy, and acid-base homeostasis. This enzyme hydrolyzes high-energy acyl phosphate intermediates such as acetyl phosphate and 1,3-diphosphoglycerate accumulated during glycolysis. On one hand, it releases inorganic phosphate, promoting the phosphorylation of vitamin B6 to generate the key GAD coenzyme PLP, maintaining the catalytic activity of glutamate decarboxylase; on the other hand, it generates ATP through substrate-level phosphorylation, providing energy for intracellular glutamate transport, GAD and GadC protein expression, and extracellular secretion of GABA. Simultaneously, AcP enzyme stabilizes intracellular proton homeostasis, enhances the survival ability of *Lactobacillus sacchariformis* in acidic environments, prolongs the efficient expression cycle of the gad operon, and continuously drives the conversion of glutamate to GABA. In addition, this enzyme can relieve the feedback inhibition of glycolysis by acyl phosphate, ensure the supply of endogenous glutamate precursors, and ultimately achieve a significant increase in γ-aminobutyric acid (GABA) production.

[0053] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0054] The method provided by this invention can promote the production of γ-aminobutyric acid (GABA) by *Lactobacillus globosum* in sake brewing, increasing the yield several times compared to when branched-chain amino acids are not used, thus meeting the requirements for health food products. This method is simple, inexpensive, and environmentally friendly, overcoming the limitation of limited L-glutamate uptake by cell membranes during the growth of fermentation strains, and achieving the goal of efficiently metabolizing substrates to produce GABA.

[0055] All aspects, embodiments, features, and examples of this invention should be considered illustrative, used to explain and illustrate the invention, but not to limit it. The scope of the invention is defined only by the claims.

[0056] Although the invention has been described with reference to the above embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that substantially equivalents can be substituted for elements in the described embodiments. Therefore, the invention is not intended to be limited to the specific embodiments disclosed, but rather to encompass all embodiments falling within the scope defined by the claims.

Claims

1. A method to promote the production of γ-lactamases by widely distributed lactobacilli in sake. The method for aminobutyric acid is characterized by, include: The sake is fermented with *Lactobacillus globulus* in conditions containing branched-chain amino acids to obtain bacteria containing γ-amino acids. Fermentation products of GABA.

2. The method for promoting the production of γ-lactamase by *Lactobacillus globulus* in sake according to claim 1. The method for aminobutyric acid is characterized by, The branched-chain amino acids include one or more combinations of L-leucine, L-isoleucine, and L-phenylalanine.

3. The method for promoting the production of γ-lactamase by widely distributed Lactobacillus in sake according to claim 1. The method for aminobutyric acid is characterized by, The *Lactobacillus saccharidus* mentioned includes *Lactobacillus saccharidus* carrying the glutamate decarboxylase gene.

4. The method for promoting the production of γ-lactamase by widely distributed Lactobacillus in sake according to claim 3. The method for producing aminobutyric acid is characterized by: The *Lactobacillus sacchariformis* mentioned is *Lactobacillus sacchariformis* KLMM605, with the Latin name [missing information]. Latilactobacillus sakei It was isolated from Guizhou fermented meat and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 50040 and deposit date of August 12, 2026.

5. The method for promoting the production of γ-lactamase by *Lactobacillus globulus* in sake according to any one of claims 1-4 The method for aminobutyric acid is characterized by, include: The branched-chain amino acids were added to the culture medium and fermented with Lactobacillus sacchariformis. The concentration of the branched-chain amino acids in the culture medium was 400~1200 μg / mL.

6. The method for promoting the production of γ-rays by *Lactobacillus globulus* in sake according to claim 5. The method for aminobutyric acid is characterized by, Specifically, it includes: The activated bacterial solution containing Lactobacillus sacchariformis was inoculated into culture medium A for the first stage of fermentation. The culture medium A contained monosodium glutamate (MSG). The culture was cultured to the logarithmic growth phase to obtain the first stage fermentation broth. The first-stage fermentation broth was mixed with culture medium B, which contains L-glutamate sodium and the branched-chain amino acids, to carry out a second-stage fermentation to obtain γ-glutamic acid. Fermentation products of GABA.

7. The method for promoting the production of γ-rays by *Lactobacillus globulus* in sake according to claim 6. The method for aminobutyric acid is characterized by, The activated bacterial solution was inoculated into culture medium A at a volume ratio of 1-3%. And / or, the culture medium A contains 1-2 w / v% L-glutamate MRS basal medium; And / or, the first stage of fermentation includes: culturing at 28~32℃ and 90~110 rpm until the logarithmic growth phase; And / or, the fermentation time in the first stage is 10-13 hours to reach the logarithmic growth phase.

8. The method for promoting the production of γ-rays by *Lactobacillus globulus* in sake according to claim 6. The method for aminobutyric acid is characterized by, The amount of culture medium B is 8-12% of the fermentation broth in the first stage. Culture medium B is an MRS basic culture medium containing branched-chain amino acids at a concentration of 400-1200 μg / mL. And / or, the second stage of fermentation includes: continuing to culture at 28~32℃ and 90~110 rpm for 10~24 h.

9. The method for promoting the production of γ-rays by *Lactobacillus globulus* in sake according to claim 6. The method for aminobutyric acid is characterized by, The method for preparing the activated bacterial solution containing Lactobacillus sacchariformis includes: inoculating Lactobacillus sacchariformis strain into MRS basal medium at a volume ratio of 1-3%, culturing at 30-40℃ for 12-24 hours, and repeating for two generations to obtain the activated bacterial solution.

10. A sake-producing *Lactobacillus* species, characterized in that, It is Lactobacillus sacchariformis KLMM605, with the Latin name [missing information]. Latilactobacillus sakei It was isolated from Guizhou fermented meat and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 50040 and deposit date of August 12, 2026.

Images