Lactobacillus brevis JS231 with high yield of gamma-aminobutyric acid and application of lactobacillus brevis JS231

Abstract

The invention discloses lactobacillus brevis JS231 for high yield of gamma-aminobutyric acid and application of the lactobacillus brevis JS231, and belongs to the technical field of biology, the lactobacillus brevis JS2311 is preserved in Guangdong Microbial Culture Collection Center on August 30, 2024, the preservation address is building 59, No. 100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC 65079. The invention also provides any one of the following applications of the lactobacillus brevis JS231: (1) application in production of gamma-aminobutyric acid; (2) application in preparation of a fermentation product with antioxidant activity; (3) application in inhibiting the activity of pathogenic bacteria; (4) application in degradation of nitrite; and (5) application in development of lactic acid bacteria products rich in gamma-aminobutyric acid. The lactobacillus brevis JS231 provided by the invention provides an original material for the development of lactic acid bacteria resources.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a strain of Lactobacillus brevis JS231 with high gamma-aminobutyric acid production and application thereof. Background Art

[0002] Lactobacillus brevis is a type of lactic acid bacteria in a broad sense and is a generally recognized as safe (GRAS) food-grade microorganism. Lactic acid bacteria from food sources have higher safety. γ-Aminobutyric acid (GABA) is a natural amino acid widely found in animals, plants and microorganisms. It is converted from glutamic acid by decarboxylation of glutamate through glutamate decarboxylase (GAD) and has multiple active functions. In the field of functional foods and medicines, it has the effects of lowering blood pressure, promoting sleep, and relieving anxiety. In the field of cosmetics, it has the effect of smoothing the skin and enhancing skin elasticity. In the field of agriculture, it has the effect of regulating plant growth, development and stress response.

[0003] At present, there are two main methods for synthesizing GABA: chemical method and biological method. The biological method includes plant enrichment and microbial fermentation. Microorganisms have been widely used in the production of GABA in recent years due to their short growth cycle and fast reproduction rate. The production of GABA by fermentation is mostly based on lactic acid bacteria, which are recognized as safe and have multiple probiotic functions. Various studies have shown that lactic acid bacteria have glutamate decarboxylase activity, which can catalyze the decarboxylation of glutamate to produce GABA. Compared with the chemical method, GABA-rich foods prepared by microbial fermentation are produced by natural fermentation, without the need to add chemically purified GABA, are green and healthy, and have good application prospects.

[0004] Although there have been many reports on GABA-producing lactic acid bacteria, it is still very important for the food industry to conduct research on the isolation, identification, and application of high-yield GABA lactic acid bacteria in order to obtain stable and safe GABA for the development of functional foods rich in GABA. At present, there are few lactic acid bacteria strains that have the potential to industrially produce GABA and can be used for food fermentation, and the research on the probiotic functional characteristics of lactic acid bacteria with GABA production potential is far from enough, resulting in the inability to directly use the strains in food, limiting the expansion of the GABA product market. Therefore, screening out lactic acid bacteria with high GABA production from food sources is of great significance for the development of functional foods rich in γ-aminobutyric acid. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a strain of Lactobacillus brevis JS231 with high yield of γ-aminobutyric acid and its application. The Lactobacillus brevis JS231 provided by the present invention can efficiently convert glutamate into GABA.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: A strain of Lactobacillus brevis JS231, characterized in that the Lactobacillus brevis JS231 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 30, 2024, and the storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the storage number is GDMCC 65079.

[0008] Technical solution 2: An application of the Lactobacillus brevis JS231, the application comprising any of the following applications:

[0009] (1) Application in the production of γ-aminobutyric acid;

[0010] (2) Application in the preparation of fermentation products with antioxidant activity;

[0011] (3) Application in inhibiting the activity of pathogenic bacteria;

[0012] (4) Application in degradation of nitrite;

[0013] (5) Application in the development of lactic acid bacteria products rich in γ-aminobutyric acid.

[0014] Furthermore, the γ-aminobutyric acid is produced by efficiently converting glutamate.

[0015] Furthermore, the fermented product includes Gastrodia elata fermented extract.

[0016] Furthermore, the pathogenic bacteria include Staphylococcus aureus, Bacillus subtilis and Escherichia coli.

[0017] Technical solution three: A fermentation product rich in γ-aminobutyric acid, wherein the γ-aminobutyric acid in the fermentation product is obtained by fermentation of the Lactobacillus brevis JS231.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects:

[0019] The present invention provides a strain of Lactobacillus brevis JS231 that can efficiently convert glutamate to produce GABA. The strain has a slow growth rate in the early stage of fermentation, but has a high bacterial density after reaching the stable period, and has the potential for high-density culture; can use glucose to produce acid and gas at the same time; has the ability to metabolize a variety of carbohydrates; the fermentation liquid of the strain has good antibacterial properties and is inhibitory to Escherichia coli, Staphylococcus aureus, and Bacillus subtilis; at the same time, the strain has excellent nitrite degradation ability; can efficiently convert glutamate to produce GABA; has good artificial gastrointestinal fluid tolerance and non-hemolytic properties, and provides raw materials for the development of lactic acid bacteria resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The colony morphology (a) and microscopic examination image (b) of the JS231 strain;

[0022] Figure 2 The growth curve (a) and acid production curve (b) of JS231 strain;

[0023] Figure 3 is the inhibitory ability of JS231 strain against pathogenic bacteria;

[0024] Figure 4 The peak graphs of each standard product detected by high performance liquid chromatography; a is the blank group; b is the GABA standard product; c is the L-Glu standard product;

[0025] Figure 5 The peak diagram of the cell-free supernatant of lactic acid bacteria detected by high performance liquid chromatography, wherein a is the control group; b is the cell-free supernatant of JS231 strain;

[0026] Figure 6 The results of the hemolytic test of the JS231 strain; wherein (a) is the JS231 strain; (b) is Staphylococcus aureus;

[0027] Figure 7 To identify the antioxidant capacity of the cell-free supernatant of Gastrodia elata fermented by JS231 strain. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0034] MRS broth medium, MRS agar medium and MRS agar medium containing 1% CaCO3: MRS broth medium and MRS agar medium were purchased from Qingdao Haibo Biotechnology Co., Ltd. MRS agar medium was supplemented with 1g agar per 100ml of medium when used. MRS agar medium containing 1% CaCO3 is MRS agar medium supplemented with 1g agar and 1g CaCO3 per 100ml of medium when used.

[0035] MH broth (MHB) medium: purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0036] Gastrodia elata fermentation medium: 1g Gastrodia elata extract, 4g sucrose, 1g peptone, and 100ml water.

[0037] The above culture media were sterilized at 118°C for 15 min.

[0038] Preparation of simulated gastric fluid: Prepare simulated gastric fluid of pH 2.0 and pH 3.0 respectively. Take 10 g of pepsin, dissolve it in water and adjust the pH, make the volume to 1 L, so that the final mass concentration is 0.01 g / mL, sterilize it with a 0.22 μm microporous filter membrane, and store it at 4°C for use.

[0039] Preparation of simulated intestinal fluid: Take 6.8 g of KH2PO4, dissolve it in water, and adjust the pH to 6.8 with NaOH solution; take 10 g of trypsin, dissolve it in water, mix the two solutions to make up to 1 L, sterilize with 0.22 μm microporous filter membrane, and store at 4°C for use.

[0040] Method for strain activation and seed solution preparation: inoculate the stored glycerol tube of bacteria into MRS solid culture medium, activate at 35-37°C for 14-18h, pick 1 ring of single colony into a centrifuge tube of 1mL MRS culture medium, and culture at 35-37°C for 12-14h to obtain seed solution.

[0041] Example 1

[0042] 1. Screening, isolation and identification of strains

[0043] The strain JS231 was isolated and screened from the Hanzhong Jiangshui. The specific scheme is as follows: the Jiangshui sample was mixed and then diluted to 10 with physiological saline. -3 , 10 -4 , 10 -5 , 100 μL was spread on MRS agar medium containing 1% CaCO3, and cultured at 37°C for 24 hours. Single colonies that can produce CaCO3 dissolution circles were picked and streaked multiple times on MRS-CaCO3 medium for purification, and the purified single colonies were picked and cultured in MRS liquid medium at 37°C for 24 hours, and stored in a -80°C refrigerator with 15% glycerol.

[0044] Morphological identification: observe the color, size, and edge morphology of the purified single colony, perform Gram staining to observe the microscopic bacterial morphology and Gram identification results, and record the colony morphology and microscope images (see Figure 1 ).

[0045] 16S rRNA sequencing identification: Pick the preserved glycerol bacteria and streak them on MRS agar medium, culture at 37℃ for 24h, pick a single colony in MRS liquid medium at 37℃ and culture it statically for 24h. Use a bacterial genomic DNA purification kit to extract genomic DNA. Use 16S rRNA universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1) and 1429R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.2) to amplify the full-length sequence of strain 16S rRNA. The PCR amplification system is shown in Table 1. The PCR amplification program is: 95℃ pre-denaturation for 5min, 30 cycles (95℃ denaturation for 1min, 50℃ annealing for 30s, 72℃ extension for 90s), and 72℃ full extension for 10min. The amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0046] The sequencing results were compared with the NCBI database Blast to confirm the biological classification of the strain, and the identification showed that the bacteria was Lactobacillus brevis. Lactobacillus brevis JS231 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 30, 2024. The collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the collection number is GDMCC 65079.

[0047] Table 1 PCR amplification program

[0048] Reagents Volume (μL) 2×Mix 20 Genomic DNA 1 Primer 27F (10 μM) 2 Primer 1429R (10 μM) 2 <![CDATA[ddH2O]]> Make up to 40 μL

[0049] Example 2

[0050] The growth ability, acid production ability, physiological and biochemical identification, gastrointestinal fluid tolerance, cell surface characteristics, probiotic properties and safety of the Lactobacillus brevis JS231 identified above were evaluated. The specific scheme is as follows:

[0051] 1. Growth and acid production capacity

[0052] The activated Lactobacillus brevis JS231 was inoculated into MRS liquid medium at 1% inoculation amount and cultured at 37°C. The OD was measured within 0 to 28 hours. 600 and pH value, with culture time as the horizontal axis, OD 600 The growth curve is drawn on the ordinate, and the results are shown in Figure 2 a. The acid production curve was drawn with the strain culture time as the horizontal axis and the pH value as the vertical axis. The results are shown in Figure 2 b.

[0053] 2. Carbohydrate fermentation characteristics

[0054] The carbohydrate fermentation pattern of Lactobacillus brevis JS231 was determined by API 50CHL reagent (BioMérieux, Marcyl'Etoile, France). The results showed that Lactobacillus brevis JS231 utilized a wide range of carbon sources and had good carbohydrate fermentation performance, and was able to metabolize 14 carbon sources, namely L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, methyl-α-D-pyranoglucopyranoside, N-acetylglucosamine, D-maltose, D-sucrose, potassium gluconate, potassium 2-ketogluconate, and potassium 5-ketogluconate (Table 2).

[0055] Table 2 Carbohydrate metabolism identification results of Lactobacillus brevis JS231

[0056]

[0057]

[0058] Note: “+” indicates positive; “-” indicates negative.

[0059] 3. Artificial stomach and intestinal fluid tolerance

[0060] The activated Lactobacillus brevis JS231 seed liquid was inoculated into simulated gastric fluid at a 10% inoculation volume, mixed and placed at 37°C for static culture, and samples were taken at 0h and 3h for viable plate counts. After culturing in simulated gastric fluid for 3h, it was inoculated into simulated intestinal fluid at a 10% inoculation volume, mixed and placed at 37°C for static culture, and samples were taken at 0h, 3h, and 6h for viable plate counts. The experiment was repeated 3 times. The survival rates are shown in Table 3.

[0061] Table 3 Survival rate of JS231 in simulated gastric and intestinal fluids

[0062]

[0063] Usually, the pH of gastric juice is about 2.0 when people are fasting, and it can rise to 3.0 after eating in moderation, and the time for food to pass through the stomach is generally 0-3h. The present invention finds that the tolerance of Lactobacillus brevis JS231 to pH 2.0 gastric juice is all weaker, and the survival rate is 40.84%. 2-3 is stronger to the tolerance of pH 3.0 gastric juice, and the 3h survival rate is higher than 100%, indicating that the bacterial volume under the stress of pH 3.0 gastric juice is not only not lost, but also has proliferation, and the bacterial volume of Lactobacillus brevis JS231 increases to 2 times after 6h of simulated intestinal fluid stress. The result shows that Lactobacillus brevis JS231 has good stomach and intestinal fluid tolerance.

[0064] 4. Evaluation of the probiotic properties of Lactobacillus brevis JS231

[0065] 4.1 Detection of inhibitory ability against common pathogens

[0066] Escherichia coli (ATCC 25922), Bacillus subtilis (CMCC(B)63501), and Staphylococcus aureus (CMCC(B)26003) were selected as indicator bacteria, and the activated Lactobacillus brevis JS231 was cultured for 24 hours and centrifuged at 6000r / min for 15 minutes to obtain a cell-free supernatant (CFS). The indicator bacterial suspension cultured for 12 hours was diluted to an OD of 600 The culture medium was 0.5, and the culture medium was inoculated with JS231 CFS and MHB at a ratio of 1:2:17. The OD of the bacterial solution was detected at 0, 6, 12, and 24 hours. 600 The value is recorded as Ax, and the indicator bacteria OD without adding Lactobacillus brevis JS231CFS 600 The value is recorded as A0,

[0067]

[0068] The results showed that the cell-free supernatant of Lactobacillus brevis JS231 had good inhibitory ability against Staphylococcus aureus and Bacillus subtilis within 6 hours, and had a certain inhibitory ability against Escherichia coli. As the culture time increased, the inhibitory ability decreased to varying degrees (see Figure 3 ).

[0069] 4.2 Detection of the ability of Lactobacillus brevis JS231 to degrade sodium nitrite

[0070] Take one loop of JS231 bacteria from the glycerol storage tube with an inoculation loop and inoculate it into MRS liquid culture medium. Incubate at 37°C for 10 h and measure the OD value of the bacterial solution. 600 The value was adjusted to 0.6 as the seed solution. Then JS231 was inoculated with 1% inoculum in MRS liquid medium with a sodium nitrite mass concentration of 125 mg / L, and cultured at 37°C for 24 hours. 0.1 mL of bacterial solution was placed in a 10 mL centrifuge tube, and 0.25 mL of saturated borax solution (50 g / L), 0.1 mL of potassium ferrocyanide solution (106 g / L), 0.1 mL of zinc acetate solution (220 g / L) and 4 mL of ultrapure water were added in sequence. After mixing, the mixture was centrifuged at 8000 r / min for 5 minutes. Take 2.5 mL of supernatant and add it to a 10 mL centrifuge tube, add 0.50 mL of p-aminobenzenesulfonic acid solution (4 g / L), vortex mix and let stand in the dark for 5 min, then add 0.25 mL of naphthylethylenediamine hydrochloride solution (2 g / L) and 1.75 mL of ultrapure water, vortex mix and let stand in the dark for 10 min, measure the absorbance at a wavelength of 538 nm, and use water as the control group.

[0071] The present invention group was denoted as C1, and the control group was denoted as C0.

[0072] Excessive intake of nitrite can cause symptoms such as acute poisoning. Microorganisms such as lactic acid bacteria and a few Bacillus have been proven to have the function of degrading nitrite, among which lactic acid bacteria are currently the most common microorganisms for degrading nitrite. The present invention found that JS231 has a degradation rate of nearly 100% for 125 mg / L NaNO2 added to the culture medium within 24 hours.

[0073] 4.3 Detection of γ-aminobutyric acid content produced by Lactobacillus brevis JS231

[0074] The activated Lactobacillus brevis JS231 was inoculated into MRS liquid medium containing 10g / L L-Glu at 1%, cultured at 37°C for 48h, centrifuged at 8000r / min for 5min to remove the bacteria, and the fermentation supernatant was collected for determination of γ-aminobutyric acid (GABA) content. The GABA content detection method was carried out in accordance with QBT5633.7-2022. Sample derivatization reaction: Take 1mL of fermentation broth sample, add 24mL of ultrapure water, then add 5mL of derivatization buffer (42g / L sodium bicarbonate), 2.5mL of derivatization reagent solution (10g / L FDNB), 60°C water bath for 1h, cool and add constant volume solution (1.36g / L potassium dihydrogen phosphate) to 50mL. Filter with a 0.22μm filter membrane and collect the filtrate as the sample solution. Derivatization reaction of standard: Weigh 0.05g of GABA and L-Glu reference substances and 0.05g of each as mixed reference substances, dilute to 100mL with pure water, measure 25mL and place in a volumetric flask, add 5mL of derivatization buffer, 2.5mL of derivatization reagent solution, and place in a 60℃ water bath for 1h. After cooling, add constant volume solution to 50mL. Filter with a 0.22μm filter membrane and collect the filtrate as the standard solution. The elution procedure is shown in Table 4. HPLC conditions: C18 column (250mm×4.6mm×5μm), mobile phase A is 4.1g / L sodium acetate with 60μL glacial acetic acid, mobile phase B is acetonitrile, flow rate is 0.8mL / min, column temperature is 40℃, injection volume is 10μL, and detection wavelength is 360nm.

[0075] Table 4 Mobile phase gradient elution program

[0076] Time / min Mobile phase A / % Mobile phase B / % 0 90 10 10 70 30 15 70 30 16 90 10 30 90 10

[0077] The present invention establishes a high performance liquid chromatography detection method by derivatizing FDNB with GABA and L-Glu. The present invention finds that the utilization rate of L-Glu by Lactobacillus brevis JS231 reaches 97.7%, and the 48h GABA production reaches 4.7g / L, indicating that Lactobacillus brevis JS231 has good GABA production capacity (see Figure 4 and Figure 5 ).

[0078] 5. Safety identification of Lactobacillus brevis JS231

[0079] 5.1 Identification of hemolytic activity of Lactobacillus brevis JS231

[0080] The activated strain was streaked onto Columbia blood agar medium, cultured at 37°C for 24 hours, and observed for hemolysis. Staphylococcus aureus (CMCC(B)26003) was used as the positive control strain. When a grass-green hemolytic ring is produced around the colony, it is α-hemolysis, i.e., incomplete hemolysis; when a clearly defined, completely transparent hemolytic ring appears, it is β-hemolysis, i.e., complete hemolysis of red blood cells; when there is no hemolysis around the colony, it is γ-hemolysis, i.e., no hemolysis. Identification on Columbia blood agar medium revealed that the positive control Staphylococcus aureus was β-hemolytic, and JS2-3 was γ-hemolytic (see Figure 6 ).

[0081] 5.2 Antibiotic sensitivity test of Lactobacillus brevis JS231

[0082] The drug-sensitive paper agar diffusion method (KB method) was used to evenly spread the bacterial solution of Lactobacillus brevis JS231 on MRS agar medium, and the drug-sensitive paper was placed in a plum blossom shape, cultured at 37°C for 24h, and the diameter of the inhibition zone was measured and recorded. The present invention selected 7 types of antibiotics, a total of 14 species, and performed antibiotic sensitivity testing on Lactobacillus brevis JS231, where S, I and R represent sensitive, moderately sensitive and resistant, respectively. The present invention found that Lactobacillus brevis JS231 was resistant to enrofloxacin to kanamycin, norfloxacin, vancomycin and sulfamethoxazole, and was moderately sensitive to cefotaxime (Table 5).

[0083] Table 5 Antibiotic sensitivity of JS231

[0084]

[0085] Example 3

[0086] The antioxidant capacity of the cell-free supernatant of Gastrodia elata fermented by Lactobacillus brevis JS231 was detected. Lactobacillus brevis JS231 was inoculated into the Gastrodia elata fermentation medium for fermentation and the antioxidant capacity was detected. The specific scheme is as follows:

[0087] The glycerol preservation tube of Lactobacillus brevis JS231 was streaked on MRS solid medium for activation, cultured overnight at 37°C, single colonies were picked, inoculated into 1ml MRS liquid medium, and cultured at 37°C for 8-12h. 5% inoculum was inoculated into 1% Gastrodia elata fermentation medium, samples were taken at 0, 12, and 24h, centrifuged at 12000r / min for 5min, and the fermentation supernatant was taken to detect the DPPH free radical scavenging ability. The results showed that after the Gastrodia elata extract was fermented by JS231, the antioxidant capacity of the supernatant after 12h of fermentation was significantly enhanced compared with 0h (see Figure 7 ).

[0088] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Lactobacillus brevis JS231, characterized in that: The Lactobacillus brevis JS231 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 30, 2024, with the storage address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the storage number is GDMCC 65079.

2. An application of Lactobacillus brevis JS231 as claimed in claim 1, characterized in that: The application includes any of the following applications: (1) Application in the production of γ-aminobutyric acid; (2) Application in the preparation of fermentation products with antioxidant activity; (3) Application in inhibiting the activity of pathogenic bacteria; (4) Application in degradation of nitrite; (5) Application in the development of lactic acid bacteria products rich in γ-aminobutyric acid.

3. The use according to claim 2, characterized in that: The produced gamma-aminobutyric acid is obtained by efficiently converting glutamate.

4. The use according to claim 2, characterized in that: The fermented product includes Gastrodia elata fermented extract.

5. The use according to claim 2, characterized in that: The pathogenic bacteria include Staphylococcus aureus, Bacillus subtilis and Escherichia coli.

6. A fermented product rich in γ-aminobutyric acid, characterized in that: The gamma-aminobutyric acid in the fermented product is obtained by fermenting the Lactobacillus brevis JS231 described in claim 1.

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