Pediococcus pentosaceus NY-2-105 and application thereof

Abstract

The invention belongs to the field of microbial fermentation, and relates to pediococcus pentosaceus NY-2-105 and application thereof. The classification name of the strain is pediococcus pentosaceus, the strain is preserved in the China General Microbiological Culture Collection Center on November 11, 2025, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No. 36565. The strain provided by the invention has excellent environmental tolerance, can adapt to fermentation environments with high acid, high ethanol, high osmotic pressure and the like, and has the capacity of high yield of GABA and stable high yield heredity. The content of GABA (gamma-aminobutyric acid) in the fermented food prepared by utilizing the pediococcus pentosaceus NY-2-105 disclosed by the invention is 7.1-85 times that of a traditional process, and meanwhile, the sensory quality is improved. According to the invention, an excellent strain resource is provided for producing GABA-rich food, food additives and mental disease drugs.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and in particular relates to a strain of Pediococcus pentosaceus NY-2-105 and its applications. Background Technology

[0002] Gamma-aminobutyric acid (GABA), also known as aminobutyric acid or gamma-aminobutyric acid, is a four-carbon non-protein amino acid widely found in animals, plants, and microorganisms. It is an important inhibitory neurotransmitter in the mammalian nervous system. Numerous studies on mammals have shown that GABA can regulate circadian rhythms, treat insomnia, combat depression, enhance immunity, alleviate menopausal syndrome, lower blood pressure, and combat obesity. Due to its diverse biological activities, developing GABA production methods and GABA-rich foods has been a research hotspot both domestically and internationally.

[0003] Microbial fermentation is currently considered a safe, effective, rapid, and efficient method for GABA production, offering significant advantages as it is not limited by resources, environment, or space. However, existing microbial strains used for GABA production still face numerous challenges in practical applications. Although some natural lactic acid bacteria have been reported to possess the ability to produce GABA, their yields are generally low, and their adaptability to complex fermentation environments is limited. While genetically engineered bacteria can achieve high yields, they often suffer from genetic instability and performance degradation after several generations, limiting their long-term and large-scale application. Therefore, breeding a strain that combines high GABA production capacity, excellent environmental tolerance, and good genetic stability is of great significance for the preparation of GABA-rich fermented foods. Summary of the Invention

[0004] To address the above technical problems, this invention provides a strain of Pediococcus pentosaceus NY-2-105 and its applications. This strain has a high GABA production capacity and excellent tolerance to stress factors such as acetic acid, ethanol, and high osmotic pressure in the fermentation environment. At the same time, its high GABA production characteristic can be stably maintained after multiple generations, and it can be widely used in the industrial production of food, pharmaceuticals, etc.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of *Pediococcus pentosaceus* NY-2-105, whose taxonomic name is *Pediococcus pentosaceus* (… Pediococcus pentosaceus The sample was deposited on November 11, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0006] Its nucleotide sequence of 16S rRNA is shown in SEQ ID No. 1.

[0007] This invention first screened a wild-type Pediococcus pentosaceus NY-2 capable of producing GABA from vinegar mash, and then directionally bred Pediococcus pentosaceus NY-2-105 using ARTP mutagenesis. This mutant strain is superior to the original strain NY-2 in both GABA production and environmental tolerance. High-performance liquid chromatography (HPLC) analysis showed that after 48 h of cultivation under suitable conditions, the GABA yield in the fermentation supernatant of NY-2-105 reached 6.911 ± 0.11 mg / mL, an increase of approximately 3.69 times compared to the original strain (1.870 ± 0.02 mg / mL), with a conversion rate of up to 98.6% for the substrate L-glutamic acid. Furthermore, the high GABA production characteristic of this strain exhibits genetic stability; its GABA production capacity did not decline after 12 consecutive subcultures.

[0008] Application experiments show that, without changing the original brewing process of fermented foods, introducing strain NY-2-105 as a starter culture into the fermentation system can increase the GABA content in fermented foods by 7.1-85 times, and simultaneously improve the flavor and taste of fermented foods, thereby enhancing the overall quality.

[0009] Secondly, the present invention also provides a fermentation product of the above-mentioned Pediococcus pentosacchari NY-2-105, said fermentation product being obtained by fermentation of the Pediococcus pentosacchari NY-2-105 strain as described in claim 1 or a microbial combination containing the Pediococcus pentosacchari NY-2-105 strain as described in claim 1.

[0010] The fermentation products mentioned in this invention refer to all substances obtained by fermenting *Pediococcus pentosaceus* NY-2-105, including but not limited to live cells, inactivated cells, various primary and secondary metabolites, and fermentation broth. These products can be in the form of fermentation broth, concentrated broth, dried product, or various preparations derived therefrom.

[0011] Preferably, the fermentation product is a fermentation seed liquid; the fermentation seed liquid is prepared by a method comprising the following steps: activating and expanding *Pediococcus pentosaceus* NY-2-105 as described in claim 1 in a liquid culture medium, collecting the cells by centrifugation, and washing and resuspending with a sterile suspension medium to obtain a viable cell concentration of not less than 1 × 10⁻⁶. 8 A bacterial suspension of cfu / mL.

[0012] Thirdly, the present invention also provides a fermentation agent comprising the above-mentioned Pediococcus pentosaceus NY-2-105 strain or a combination of microorganisms containing the above-mentioned Pediococcus pentosaceus NY-2-105.

[0013] The microbial agent may also include biologically acceptable microbial carriers and excipients. The excipients include, but are not limited to, starch, glucose, maltodextrin, sucrose, lactose, skim milk powder, trehalose, and chitosan; the carriers include, but are not limited to, culture media, water, starch, cellulose, and gelatin. The *Pediococcus pentosaceus* NY-2-105 strain or microbial combinations containing the above-mentioned *Pediococcus pentosaceus* NY-2-105 provided by this invention can be prepared into microbial agents of different dosage forms by selecting suitable carriers and / or excipients, thereby improving the stability, activity, and efficacy of the microbial agent. The dosage forms of the microbial agent include, but are not limited to, liquids, powders, granules, tablets, and capsules.

[0014] Preferably, the fermenting agent is a food fermenting agent, composed of the above-mentioned Pediococcus pentosaceus NY-2-105 and at least one food fermentation microorganism selected from yeast, acetic acid bacteria, Aspergillus oryzae or Mucor.

[0015] More preferably, the yeast is Saccharomyces cerevisiae; the acetic acid bacteria is Acetobacter pasteurellii; and the Aspergillus is Aspergillus oryzae.

[0016] Fourthly, the present invention also provides the use of the above-mentioned Pediococcus pentosaceus NY-2-105 or the above-mentioned fermentation product or starter in any of the following: (1) Application in the preparation of fermented foods rich in γ-aminobutyric acid; (2) Application in the production of γ-aminobutyric acid; (3) Application in the preparation of functional foods; (4) Application in the preparation of food additives; (5) Use in the preparation of drugs for the prevention and / or treatment of mental illness.

[0017] The fermented foods described in this patent refer to a class of foods obtained by processing and modifying natural food raw materials using beneficial microorganisms (such as bacteria, yeasts, or molds) and their enzymes. GABA-rich fermented foods produced using Pediococcus pentosaceus NY-2-105 provided by this invention include, but are not limited to, fermented condiments (brewed vinegar, soy sauce, bean paste, etc.), fermented by-products (fermented bean curd, fermented black beans, pickled vegetables, sauerkraut, etc.), or fermented liquid beverages (fermented dairy products, fermented fruit and vegetable juices, etc.).

[0018] Preferably, its application is in the preparation of functional foods that help improve sleep.

[0019] Preferably, its use in the preparation of medicines for the prevention and / or treatment of insomnia, anxiety or depression.

[0020] Fifthly, the present invention also provides a method for producing fermented food rich in γ-aminobutyric acid, the method comprising the step of fermenting food raw materials or processed food raw materials using the above-mentioned Pediococcus pentosus NY-2-105 or the above-mentioned fermenting agent.

[0021] Preferably, the inoculum size of *Pediococcus pentosaceus* NY-2-105 is 10. 5 -10 7 cfu / g or 10 5 -10 7 cfu / mL.

[0022] Preferably, the fermented food is brewed vinegar; the method includes inoculating the mash and / or wine mash with the above-mentioned Pediococcus pentosaceus NY-2-105 for fermentation during the alcoholic fermentation stage and / or acetic acid fermentation stage.

[0023] The brewed vinegar described in this invention refers to a liquid condiment produced by microbial fermentation using various materials containing starch, sugar, or alcohol, either alone or in combination. This includes, but is not limited to, solid-state fermented vinegar, rice vinegar, or fruit vinegar.

[0024] More preferably, the brewed vinegar is solid-state fermented vinegar; the method includes inoculating the mash with the above-mentioned Pediococcus pentosaceus NY-2-105 during the alcoholic fermentation stage for fermentation.

[0025] Preferably, the fermented food is soy sauce; the method includes adding brine and the above-mentioned Pediococcus pentosaceus NY-2-105 to the koji for fermentation.

[0026] Preferably, the fermented food is soybean paste; the method includes adding brine and the above-mentioned Pediococcus pentosaceus NY-2-105 to the starter culture for fermentation.

[0027] The koji material mentioned herein is the material fermented by Aspergillus in the brewing of soy sauce or bean paste.

[0028] Preferably, the fermented food is fermented bean curd; the method includes adding the above-mentioned Pediococcus pentosaceus NY-2-105 to the fermented bean curd brine.

[0029] Preferably, the fermented food is chili sauce; the method includes adding the above-mentioned Pediococcus pentosaceus NY-2-105 to a fermentation substrate with chili as the main raw material for fermentation or adding the above-mentioned Pediococcus pentosaceus NY-2-105 to the chili sauce for secondary fermentation.

[0030] Preferably, the fermented food is a fermented jujube product; the method includes adding the above-mentioned Pediococcus pentosaceus NY-2-105 to a fermentation substrate with jujube as the main raw material for fermentation.

[0031] Preferably, the fermented food is kimchi; the method includes adding the above-mentioned Pediococcus pentosaceus NY-2-105 to dehydrated vegetables for fermentation.

[0032] The pickled vegetables described in this invention refer to an acidic fermented food made from fresh vegetables as the main raw material, which are pre-treated and then fermented in a salt solution or brine of a specific concentration by naturally or artificially inoculated lactic acid bacteria.

[0033] More preferably, the vegetable is Chinese cabbage.

[0034] Preferably, the fermented food is a fermented fruit and vegetable juice; the method includes adding the above-mentioned Pediococcus pentosaceus NY-2-105 to the fruit and vegetable juice for fermentation.

[0035] The fermented fruit and vegetable juice described in this invention refers to a liquid or pulpy beverage product obtained by using fruits and / or vegetables and their processed products (such as fruit and vegetable pulp or clear juice) as raw materials and fermenting them primarily with lactic acid bacteria.

[0036] More preferably, the fruit and vegetable juice is sea buckthorn juice.

[0037] Preferably, the fermented food is a fermented dairy product; the method includes adding the above-mentioned Pediococcus pentosaceus NY-2-105 to milk and / or milk protein products for fermentation.

[0038] In a sixth aspect, the present invention also provides fermented foods prepared according to any one of the preceding methods. Attached Figure Description

[0039] Figure 1 The colony morphology of Pediococcus pentosaceus strain NY-2 in Example 1; Figure 2 The microscopic morphology of Pediococcus pentosaceus NY-2 cells in Example 1; Figure 3 This is the phylogenetic tree of Pediococcus pentosaceus strain NY-2 in Example 1. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0041] The strains or fermenting agents used in the following embodiments of the present invention are from the following sources: Pediococcus pentosaceus NY-2-105 was deposited on November 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36565. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

[0042] Pediococcus pentosaceus NY-2 was obtained from the Systems Microbiology and Biomanufacturing Engineering Laboratory of Tianjin University of Science and Technology.

[0043] The starter culture (Daqu) comes from Shanxi Zilin Vinegar Industry Co., Ltd.; the fermented mash (Huomai) comes from Shanxi Zilin Co., Ltd.; the active dry yeast (Angel Yeast Co., Ltd.); the starter culture (Yiqu) comes from Shanxi Zilin Vinegar Industry Co., Ltd.; the Aspergillus oryzae comes from Jining Yuyuan Biotechnology Co., Ltd., product name: JY309 Aspergillus oryzae (starter culture, soy sauce starter); the conventional fermentation agent comes from Beijing Chuanxiu International Trade Co., Ltd., product name: Chuanxiu Classic Old Yogurt Fermentation Powder (30 strains); the Rhizopus comes from Beijing Chuanxiu International Trade Co., Ltd., product name: Chuanxiu Fermented Bean Curd Starter Culture.

[0044] Grape juice contains Hansenula polymorpha ( Hanseniaspora uvarum CGMCC No. 29509 originated from the Systems Microbiology and Biomanufacturing Engineering Laboratory of Tianjin University of Science and Technology. This strain has been disclosed in patent application number CN202410172246; Acetobacter pasteurellosis ( Acetobacter pasteurianus CGMCC No. 3089 originated from the Systems Microbiology and Biomanufacturing Engineering Laboratory of Tianjin University of Science and Technology and has been published in patent publication number CN119524021A.

[0045] The defatted soybeans, soybeans, wheat, sorghum, apples, rice, red dates, tofu, Chinese cabbage, sea buckthorn berries, raw milk, red yeast rice powder, star anise, cinnamon, etc. are all from ordinary commercially available sources, while the chili sauce is collected from the fermentation tanks of Tianjin Limin Condiment Brewing Group Co., Ltd.

[0046] The culture medium used in the following embodiments of the present invention is as follows: MRS medium: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5 g / L, glucose 20.0 g / L, sodium acetate 3.02 g / L, triammonium citrate 2.0 g / L, Tween 80 1.0 1 mL / L, dipotassium hydrogen phosphate 1.16 g / L, magnesium sulfate 0.05 g / L, manganese sulfate 0.03 g / L; when preparing solid medium, add an additional 20.0 g / L agar. The medium is autoclaved at 121°C for 15 min before use.

[0047] MRSS screening medium: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5 g / L, glucose 20.0 g / L, sodium acetate 3.02 g / L, triammonium citrate 2.0 g / L, Tween 80 1.0 1 mL / L, dipotassium hydrogen phosphate 1.16 g / L, magnesium sulfate 0.05 g / L, manganese sulfate 0.03 g / L, glyoxylic acid 2.0 g / L, succinic acid 2.0 g / L, bromocresol green 0.1 g / L, glutamic acid 10.0 g / L.

[0048] Seed culture medium: Soak apples for 24 hours, peel and core them, crush them, filter them, add white sugar to the apple juice to adjust the sugar content to 15 g / 100 g, and sterilize at 115℃ for 20 min to obtain the seed culture medium. Unless otherwise specified, all raw materials used in the following embodiments of the present invention are commercially available.

[0049] The detection method involved in the following embodiments of the present invention: γ-Aminobutyric acid: The content of γ-aminobutyric acid (GABA) was determined by high performance liquid chromatography (HPLC) according to Method 1 in QBT5633.7-2022 "Amino acids, amino acid salts and their analogues Part 7: γ-aminobutyric acid".

[0050] Lactic acid and acetic acid (quantitative): The content of lactic acid and acetic acid in food was determined by high performance liquid chromatography (HPLC) in accordance with GB 5009.157-2016 "National Food Safety Standard - Determination of Organic Acids in Food".

[0051] Volatile flavor compounds (ethyl lactate, phenylethanol, tetramethylpyrazine, etc.): The volatile flavor components in the samples were qualitatively and quantitatively analyzed by gas chromatography-mass spectrometry (GC-MS).

[0052] Total acidity (general and pickled vegetables): The total acid content was determined by acid-base titration or pH potentiometric titration, referring to GB 12456-2021 "National Food Safety Standard - Determination of Total Acidity in Food".

[0053] Total acidity (vinegar): The total acidity (calculated as acetic acid) content was determined by acid-base titration method according to GB / T 18187-2000 "Brewn Vinegar".

[0054] Amino acid nitrogen (soy sauce / vinegar): Determined according to GB 5009.235-2016 "National Food Safety Standard - Determination of Amino Acid Nitrogen in Food", using the formaldehyde value method or pH meter method.

[0055] Amino acid nitrogen (fermented bean curd): Determined by formaldehyde value method according to SB / T 10170-2007 Fermented Bean Curd.

[0056] Reducing sugar / total sugar: Determined by direct titration (Rhine-Enon method) in accordance with GB 5009.7-2016 National Food Safety Standard for the Determination of Reducing Sugar in Food.

[0057] Salt (chloride): Determined by silver nitrate titration (Mohr's method) in accordance with GB 5009.44-2016 "National Food Safety Standard - Determination of Chloride in Food".

[0058] Alcohol content (ethanol): Determined according to GB 5009.225-2023 "National Food Safety Standard - Determination of Ethanol Concentration in Wine", using the density bottle method or alcohol meter method.

[0059] Moisture content: Determined by direct drying method according to GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food".

[0060] Lactic acid bacteria viable count: The plate count method was used to determine the count, referring to GB 4789.35-2016 "National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria Examination".

[0061] Total bacterial count: The plate count method was used to determine the total bacterial count, referring to GB 4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Bacterial Count".

[0062] Example 1 This embodiment provides a strain of Pediococcus pentosaceus NY-2-105, which was obtained by screening Pediococcus pentosaceus from vinegar mash samples and then conducting high-throughput screening using ARTP mutagenesis.

[0063] 1. Screening and identification of Pediococcus pentosaceus NY-2 (1) Under aseptic conditions, accurately weigh 10 g of vinegar mash sample (from Shanxi Zilin Vinegar Industry Co., Ltd.), put it into an Erlenmeyer flask containing 90 mL of sterile physiological saline, gently shake for 30 min, and set aside for use.

[0064] (2) Isolation: Take 1 mL of the above bacterial suspension and slowly inject it into a test tube containing 9 mL of sterile water for serial dilution. Repeat this process. Select an appropriate dilution gradient solution and spread it on an MRS medium plate. Incubate at 37°C for 48 h. After colonies grow, pick a single colony and streak it onto an MRS medium plate for purification. Store at 4°C for later use.

[0065] (3) Screening: The isolated bacteria were inoculated into MRS liquid medium for 1-2 generations for activation, and then transferred into MRS liquid medium supplemented with 10.0 g / L glutamate (L-Glu) and cultured at 37℃ for 48 h. The supernatant was collected and the content of γ-aminobutyric acid (GABA) was determined by high performance liquid chromatography (HPLC). The strain with the highest GABA production was identified as NY-2, with a yield of 1.870±0.02 mg / mL. Therefore, this strain was selected as the starting strain for subsequent mutagenesis breeding.

[0066] 2. Identification of Pediococcus pentosaceus NY-2 2.1 Colony and cell morphology characteristics of NY-2 strain The NY-2 strain was inoculated onto MRS agar plates and incubated at 37°C for 48 h. Under aseptic conditions, NY-2 samples were picked from the plates and Gram-stained.

[0067] Colony morphology observation: Colonies are milky white, smooth, opaque, with regular edges. See details. Figure 1 Microscopic examination of bacterial cells: Gram-positive, arranged in pairs or tetrads, 0.8-1.0 μm in diameter. See details. Figure 2 Based on morphology and microscopic examination, it was initially identified as Pediococcus pentosaceus. Pediococcus pentosaceus ).

[0068] 2. Molecular biological identification of NY-2 strain The NY-2 strain was streaked onto MRS medium and cultured at 37°C for 48 h. The bacterial cells were collected and sent to Suzhou Genewiz Biotechnology Co., Ltd. for 16S rRNA sequencing. The sequencing results are shown in SEQ ID NO.1.

[0069] Based on the 16S rRNA gene results, a phylogenetic tree was constructed using MEGA software. Figure 3 It can be seen that strain NY-2 and Pediococcus pentosaceus ( Pediococcus pentosaceus They cluster on the same branch.

[0070] In summary, based on morphological characteristics and molecular biological analysis, strain NY-2 was identified as Pediococcus pentosaceus. Pediococcus pentosaceus It was named Pediococcus pentosaceus NY-2.

[0071] 3. Acid and ethanol resistance tests of Pediococcus pentosaceus NY-2 The NY-2 strain was inoculated into MRS medium with different concentrations of acetic acid (1%, 1.5%, 2%, 3%) and ethanol (1%, 3%, 5%, 7%), and cultured at 37°C for 48 h. OD200 was used to analyze the results. 600The values ​​reflect the growth status of the strain. The test results are shown in Table 1. The NY-2 strain exhibits strong ethanol tolerance but poor acetic acid tolerance. Growth of NY-2 is inhibited when the acetic acid concentration is 1.5%.

[0072] Table 1. Tolerance of strain NY-2 to different concentrations of acetic acid and ethanol

[0073] 4. Mutagenesis and screening process of Pediococcus pentosaceus NY-2-105 (1) Take 1 mL of bacterial suspension of strain NY-2 cultured to the logarithmic phase, centrifuge at 4000 rpm for 10 min, discard the supernatant, wash the bacterial sludge 2-3 times with sterile physiological saline, and finally resuspend in 1 mL of sterile physiological saline to obtain the bacterial suspension to be mutated. Under aseptic conditions, take 10 μL and place it on a sterile metal mutagenesis slide, and transfer it into the ARTP operating chamber. Set the time to 120 s. After the treatment is completed, place the slide into an EP tube pre-filled with 980 μL of sterile physiological saline, shake for 2 min, and then transfer it to an ice-water bath to avoid light for 2 h to obtain the mutagenic bacterial solution. Inoculate the mutagenic bacterial suspension into MRS liquid medium and culture for 2 h to allow the activity of the strain to recover and adapt to the culture environment.

[0074] (2) Add 3.5% acetic acid and 3% ethanol to the culture medium to create a certain pressure environment. This step simulates the environment during vinegar fermentation. Then, place the culture medium in a 37℃ constant temperature incubator and culture for 5-7 days to simulate the growth of the strain in the vinegar fermentation environment.

[0075] (3) After the culture was completed, the optical density (OD) of the culture medium was measured. 600 This is used to assess the growth of the strain. If the OD of the mutant strain... 600 If the value is greater than that of the control group (blank culture medium), it is considered that the strain has undergone an acetic acid-resistant mutation and has a strong acid resistance.

[0076] (4) Preliminary screening method: Dilute the bacterial suspension of the acetic acid-resistant mutant to 10. -4 Spread 100 μL onto MRS solid medium, pick a single colony and add it to MRSS selection medium. After incubation at 37°C for 72 h, measure the OD of the bacterial culture. 620 The absorbance value, compared with the original strain, when the OD of the mutant strain... 620 OD greater than that of the originating strain 620 At this point, it can be preliminarily concluded that the strain has undergone a high-GABA-producing mutation. The bacterial culture was diluted to 10⁻⁶ with sterile physiological saline. -6The bacteria were isolated by spreading on MRS solid medium and incubated at 37°C for 48 h. This process was repeated twice. Vigorous single colonies were picked and streaked on MRS slant medium and incubated again at 37°C for 48 h. After that, the culture was transferred to a 4°C refrigerator for storage.

[0077] (5) Secondary screening method: After the strains initially screened were activated by MRS liquid medium for 1-2 generations, the bacterial culture in the logarithmic phase was inoculated into MRS liquid medium containing precursor (glutamic acid), cultured at 37℃ for 48 h, the fermentation supernatant was collected and its GABA content was detected, and the strain with the highest GABA content was selected.

[0078] Table 2. GABA content in the culture supernatant of each mutant strain

[0079] In this stage of mutagenesis, six strains with enhanced acetic acid tolerance and increased GABA production were screened. The strain numbers and yields are shown in Table 2. Among them, strain NY-2-105 had the highest GABA production, at 6.911±0.11 mg / mL, which was 3.69 times higher than the original strain, and the conversion rate of glutamic acid was as high as 98.6%.

[0080] This strain was deposited on November 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36565; the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

[0081] Example 2 1. Assay of glutamate decarboxylase (GAD) activity (1) Strains culture and fermentation broth acquisition: The mutant strain NY-2-105 and the starting strain NY-2 were streaked onto MRS solid medium and incubated upside down at 37°C for 48 h. Colonies were picked and inoculated into MRS liquid medium and incubated at 37°C for 24 h to obtain activated bacterial culture. 2% (v / v) of the culture was transferred to 50 mL of MRS liquid medium and incubated at 37°C for 24 h to obtain seed culture for the experimental group and the control group, respectively.

[0082] The above seed culture was inoculated into a 500 mL Erlenmeyer flask (containing 250 mL of MRS liquid medium) at an inoculation rate of 10% (v / v) and incubated statically at 37°C for 48 h. Three biological replicates were set up for both the experimental and control groups, and the resulting cultures were used as fermentation broth for enzyme activity detection.

[0083] (2) Obtaining crude GAD enzyme solution: Take 1 mL of fermentation broth, centrifuge at 12000 r / min to collect the bacterial cells, and wash once with PBS buffer. Add 0.5 mL of Na2HPO4-citric acid buffer (pH 5.0, containing 0.15 mmol / L LLP) containing 50 mmol / L to rehydrate the bacterial cells, and add lysozyme (final concentration 0.6 mg·mL). -1 After reacting at 37℃ for 30 min, the sample was placed in ice water and ultrasonically broken up (power 25%, ultrasonic 3 s, interval 5 s, total time 10 min), followed by 12000 r·min -1 Centrifuge for 5 minutes and collect the supernatant, which is the crude GAD enzyme solution.

[0084] (3) Determination of GAD crude enzyme activity: The modified Berthelot colorimetric method was used to detect GAD enzyme activity.

[0085] Substrate solution preparation: Prepare a Na2HPO4-citric acid buffer (pH 4.8) containing 50 mmol / L L-MSG sodium glutamate (L-MSG) with 0.15 mmol / L pyridoxal phosphate (PLP) added as a coenzyme.

[0086] Enzymatic reaction: Mix 0.2 mL of crude GAD enzyme solution with 0.2 mL of substrate solution (total reaction volume 0.4 mL) and incubate in a 40℃ water bath for 60 min.

[0087] Colorimetric analysis and determination: After the reaction was complete, add 0.1 mL of Na₂CO₃ solution (1 mol / L), 0.5 mL of borate buffer (0.2 mol / L, pH 10.0), and 1 mL of 6% phenol to the above 0.4 mL reaction solution. Mix well, then add 1 mL of 0.1 mol / L NaClO solution and mix again. Let stand for 4 min, then immediately boil in a water bath for 10 min, followed by an ice bath for 20 min. After the solution turns blue-green, add 2 mL of 60% ethanol, mix well, and place in a 20℃ water bath for 40 min until the color stabilizes. Measure the absorbance at 640 nm. Use a blank control by boiling the crude enzyme solution in water for 10 min to inactivate it, and repeat the same steps as above. The enzyme activity detection results are shown in Table 3.

[0088] Enzyme activity definition: Under the above reaction conditions (pH 4.8, 40℃), the amount of enzyme required to catalyze the production of 1 μmol GABA per hour is defined as one enzyme activity unit (U).

[0089] Table 3 Enzyme activity assays of the starting strain and the mutant strain

[0090] As shown in Table 3, the enzyme activity of strain NY-2-105 reached 78.2 U / mL, significantly higher than that of the original strain NY-2 (11.5 U / mL), representing a 6.8-fold increase. This indicates that the enhanced GABA synthesis capacity of this strain is mainly due to the increased activity of its key synthase GAD.

[0091] 2. Passage stability of strain NY-2-105 The preserved Pediococcus pentosaceus NY-2-105 (referred to as generation 0) was inoculated into fresh MRS liquid medium (containing 10.0 g / L glutamate) at an inoculum size of 2% (v / v) and incubated statically at 37°C for 48 h. Thereafter, it was transferred to fresh medium every 48 h at the same inoculum size, and passaged continuously until the 12th generation.

[0092] Culture broths from generations 0, 2, 4, 6, 8, 10, and 12 of the strain were collected, centrifuged, and the supernatant was used to determine the GABA yield using high-performance liquid chromatography (HPLC). Three parallel experiments were performed for each sample, and the results are shown in Table 4.

[0093] Table 4. GABA content of the mutant strains after passage.

[0094] Large-scale fermentation production requires strain performance to remain stable over a long period. Genetic stability experiments showed that the high GABA production characteristic of strain NY-2-105 did not decline during the 12 passages tested. As shown in Table 4, its GABA production remained consistently high at 6.81-6.99 mg / mL, demonstrating the excellent genetic stability of this mutagenic strain.

[0095] 3. Tolerance test The preserved *Pediococcus pentosaceus* NY-2-105 strain was inoculated into MRS liquid medium with different concentrations of acetic acid (2%, 3%, 4%, 5%), ethanol (1%, 3%, 5%, 7%), lactic acid (3%, 4%, 5%, 6%), sodium chloride (3%, 5%, 7%, 9%), sodium lactate (5%, 7%, 9%, 11%), and glucose (200 g / L, 300 g / L, 400 g / L, 500 g / L). The cultures were incubated at 37°C for 48 h, and the growth of the strain was observed. Additionally, strain NY-2-105 was inoculated into MRS liquid medium and cultured at 30°C, 40°C, 45°C, and 50°C for 48 h, respectively, and the growth of the strain was observed. The results are shown in Table 5.

[0096] Table 5. Results of tolerance test for strain NY-2-105

[0097] Tolerance tests showed that *Pediococcus pentosaceus* NY-2-105, obtained through mutagenesis and selection, exhibited superior environmental tolerance compared to the original strain NY-2. The tolerance concentration of acetic acid for NY-2-105 increased significantly from less than 1.5% in NY-2 to 5%, while also demonstrating good growth capacity in 7% ethanol, 9% sodium chloride, 11% sodium lactate, 500 g / L glucose, and at high temperatures. This demonstrates the potential of this strain for application in various fermented foods.

[0098] Example 3 This embodiment provides a fermentation broth of *Pediococcus pentosaceus* NY-2-105, which is prepared by the following method: (1) Inoculate strain NY-2-105 into MRS liquid medium and culture at 37℃ for 24 h to obtain primary seed culture.

[0099] (2) The primary seed culture was transferred to MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37°C for 24 h to obtain the secondary seed culture.

[0100] (3) The secondary seed culture was transferred to MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37°C for 48 h to obtain the fermentation broth.

[0101] Example 4 This embodiment provides a fermentation seed culture of *Pediococcus pentosaceus* NY-2-105, which is prepared by the following method: The activated NY-2-105 fermentation broth was inoculated into MRS liquid medium at an inoculum rate of 4% (v / v) and incubated statically at 37°C until the cell concentration reached 10⁻⁶. 8 CFU / mL. Subsequently, the bacterial cells were collected by centrifugation at 8000 r / min for 10 min. The resulting bacterial sludge was washed twice with 0.85% physiological saline, resuspended, and its concentration adjusted to 10. 8 The cfu / mL concentration yields the fermentation seed culture of Pediococcus pentosaceus NY-2-105.

[0102] Application Example 1 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in the preparation of solid-state fermented vinegar. By inoculating it into the vinegar mash to participate in the acetic acid fermentation stage, the yield of GABA in the vinegar and the quality of the vinegar are effectively improved.

[0103] 1. Solid-state fermentation vinegar preparation (1) Raw material processing: Crush the dried sorghum to 30-40 mesh using a pulverizer, put it into a saccharification tank, add water at 88-90℃, and moisten for 4-8 hours.

[0104] (2) Liquefaction and saccharification: Heat-resistant α-amylase is added to sorghum at a ratio of 1000 U / kg, and liquefaction is carried out at 88-90℃ for 20-40 min. After liquefaction, the temperature of the mash is lowered to 58-62℃, 1500 U / kg of saccharifying enzyme is added, and the temperature is kept constant for 1 h.

[0105] (3) Mixing the starter culture: Reduce the temperature of the mash to 28-32℃, add Daqu (Chinese starter culture) at a ratio of sorghum:Daqu = 1:0.625, and then add water to make the material-to-water ratio 1:3.3. Add active dry yeast (rehydrated and activated at 35-37℃ for 30 min) to the mash at an inoculation rate of 2‰.

[0106] (4) Alcoholic fermentation: The mash is placed in a 50 L alcoholic fermentation tank. Fermentation is started open for the first three days, and then closed for the next three days until the 16th day. During fermentation, the temperature is maintained at 28-30℃. When the ethanol content in the mash is 7.0%-8.0% (w / w) and the total acid content is about 1 g / 100 g of mash, alcoholic fermentation is stopped, and mash is obtained.

[0107] (5) Acetic acid fermentation: The mash was placed in a mixing tank, and auxiliary materials were added according to the ratio of mash:wheat bran:rice husk of 5:1.1:0.6 (by weight). 2% of the mash was added by weight. The initial alcohol content of the vinegar mash was approximately 4.5%-5.0% (w / w), and the moisture content was 60%-65%. Simultaneously with mixing, 5% (v / v) of Pediococcus pentosaceus NY-2-105 fermentation seed liquid (provided in Example 4) was added, and fermentation lasted for 9 days.

[0108] (6) Smoking: After fermentation, the vinegar mash is transferred to a smoking tank for smoking.

[0109] (7) Vinegar rinsing: After the smoking process is completed, vinegar is rinsed to obtain new vinegar.

[0110] 2. Determination of GABA content during acetic acid fermentation During the 9-day acetic acid fermentation stage, samples were collected on days 1, 3, 5, 7, and 9. Representative vinegar mash samples were obtained immediately after each daily turning of the mash using a five-point sampling method, and their GABA content was measured. The results are shown in Table 6.

[0111] Table 6. Changes in GABA content during vinegar fermentation.

[0112] Dynamic monitoring of GABA content during acetic acid fermentation showed that after inoculation with strain NY-2-105, GABA in the vinegar mash began to accumulate rapidly from the early stage of fermentation, reaching the first peak at 5 days with a content of 1.032±0.065 g / 100g vinegar mash. After a slight decrease, it rose again, reaching 1.297±0.029 g / 100g vinegar mash at the end of fermentation (9 days).

[0113] 3. Determination of main physicochemical indicators The main physicochemical indicators of the newly fermented vinegar were determined according to the method in "Geographical Indication Product Shanxi Aged Vinegar" (GB / T19777-2013), and the new vinegar obtained by fermentation without the addition of Pediococcus pentosus NY-2-105 was used as a control. The results are shown in Table 7.

[0114] Table 7. Results of Physicochemical Indicators

[0115] As shown in Table 7, adding Pediococcus pentosaceus NY-2-105 during the acetic acid fermentation stage of solid-state fermented vinegar significantly increased the GABA content in the finished vinegar. Simultaneously, the non-volatile acid (calculated as lactic acid) content in the experimental group reached 1.86 g / 100 mL, an increase of 58.9% compared to the control group. This significant increase in non-volatile acid content complements the volatile acetic acid, effectively neutralizing the irritating acidity and resulting in a more mellow and lingering sour taste.

[0116] Application Example 2 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in solid-state fermented vinegar. By inoculating it into the mash to participate in the alcoholic fermentation stage, the yield of GABA in vinegar and the quality of vinegar are effectively improved.

[0117] 1. Solid-state fermentation vinegar preparation Steps (1)-(3) are the same as in Application Example 1.

[0118] (4) Alcoholic fermentation: The mash was placed in a 50 L alcoholic fermentation tank. Fermentation was started over for the first three days. After three days, Pediococcus pentosaceus NY-2-105 fermentation seed liquid (provided in Example 4) was inoculated at a rate of 10% (v / v). Then, fermentation was stopped and continued for a total of 16 days. During fermentation, the temperature was maintained at 28-30℃. When the ethanol content in the mash was 7.0%-8.0% (w / w) and the total acid content was about 1 g / 100 g of mash, alcoholic fermentation was stopped to obtain mash.

[0119] (5) Acetic acid fermentation: Same as application case 1, but without inoculating Pediococcus pentosus NY-2-105.

[0120] (6) Smoking: Same as application case 1.

[0121] (7) Drip vinegar: Same as application case 1.

[0122] 2. Determination of the content of various components in the fermented mash Referring to the measurement method in Application Example 1, the GABA content and physicochemical indicators of the prepared mash were determined, and the mash without the addition of NY-2-105 fermentation seed liquid was used as a control. The results are shown in Table 8.

[0123] Table 8. Determination of the content of various components in the mash

[0124] As shown in the table, the GABA content of the experimental group mash reached 2.15 mg / mL, which was 42 times higher than that of the control group.

[0125] The main physicochemical indicators of the newly fermented vinegar were determined according to the method in "Geographical Indication Product Shanxi Aged Vinegar" (GB / T19777-2013), and the new vinegar obtained by fermentation without the addition of Pediococcus pentosus NY-2-105 was used as a control. The results are shown in Table 9.

[0126] Table 9. Determination results of physicochemical indicators of finished vinegar (content)

[0127] As shown in Table 9, inoculating the vinegar with Pediococcus pentosaceus NY-2-105 during the alcoholic fermentation stage resulted in a GABA content of 0.62 g / 100 mL in the final product, an increase of 11.4 times compared to the control group. Furthermore, the non-volatile acid (calculated as lactic acid) content in the experimental group significantly increased to 1.98 g / 100 mL, a 69.2% increase compared to the control group. This indicates that the metabolites (GABA and lactic acid, etc.) of NY-2-105 during the alcoholic fermentation stage can be effectively retained and transferred to the final product.

[0128] Application Example 3 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in the brewing of fruit vinegar.

[0129] 1. Preparation of fruit vinegar (1) Preparation of seed culture medium: Activated grape juice Hansenula polymorpha CGMCC No.29509 was inoculated into seed culture medium and cultured until the stable phase to prepare Saccharomyces cerevisiae seed culture medium; activated Acetobacter pasteurellii CGMCC No.3089 was inoculated into seed culture medium and cultured until the stable phase, and cultured in a shaker at 30℃ and 180 r / min for 24 h to obtain Acetobacter pasteurellii seed culture medium.

[0130] (2) Raw material processing: After soaking the apples, peel and core them, crush them, juice them, add 0.35% (w / w) pectinase, and bathe them in a 50℃ water bath for 2 h; after enzymatic hydrolysis, centrifuge at 8000 r / min for 10 min and filter them with 4 layers of gauze to obtain clear juice. Adjust the sugar content to 15.0 g / 100 g with white sugar to obtain apple juice.

[0131] (3) Alcoholic fermentation: 3% (v / v) of grape juice seed culture of Hansenula polysaccharide CGMCCNo.29509 was added to the obtained apple juice and cultured under anaerobic conditions at 30℃ for 6 days to obtain apple wine fermentation liquid.

[0132] (4) Acetic acid fermentation: 10% (v / v) Acetobacter pasteurellium seed culture and 10% (v / v) Pediococcus pentosacchari NY-2-105 fermentation seed culture (provided in Example 4) were added to the apple wine fermentation broth and cultured at 30°C for 6 days to obtain apple cider vinegar.

[0133] 2. Determination of the content of various components in fruit vinegar The main physicochemical properties of apple cider vinegar were determined, and apple cider vinegar prepared by fermentation without the addition of Pediococcus pentosus NY-2-105 was used as a control. The test results are shown in Table 10.

[0134] Table 10. Determination of the content of various components in fruit vinegar

[0135] 3. Sensory evaluation To assess the quality of the fermented apple cider vinegar, a sensory evaluation team was formed. The team consisted of 10 experienced professional sensory evaluators (5 men and 5 women). The sensory evaluation form is shown in Table 11, and the sensory evaluation results are shown in Table 12.

[0136] Table 11 Sensory Evaluation Criteria for Apple Cider Vinegar

[0137] Table 12 Sensory rating results of apple cider vinegar

[0138] Experimental results showed that the introduction of Pediococcus pentosaceus NY-2-105 significantly increased the GABA content in apple cider vinegar by nearly 60 times, while the content of non-volatile acids also increased, effectively reducing the irritation of volatile acids and improving its quality and taste.

[0139] Application Example 4 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in the brewing of rice vinegar.

[0140] 1. Preparation of rice vinegar (1) Raw material pretreatment: Weigh 500 g of high-quality rice, wash it with clean water and soak it for 4-6 hours until the rice grains fully absorb water and expand; drain the rice and place it in a steamer to steam until the rice is not hard in the center and the grains are distinct. Then spread the rice out and let it cool naturally to 30-35℃ for later use.

[0141] (2) Saccharification and alcoholic fermentation: Transfer the cooled rice into a sterile fermentation tank, add 600 mL of sterile water and 1.5 g of yeast, and stir thoroughly. Carry out saccharification and alcoholic fermentation at 28-30℃ for 5-7 days until the mash is clear, the aroma is rich and there are no significant bubbles, and the mash is obtained.

[0142] (3) Acetic acid fermentation: The rice wine liquid obtained by filtering the mash was placed in a sterile fermentation tank and inoculated with 2% (v / v) Pediococcus pentosaceus NY-2-105 fermentation seed culture (prepared from Example 4) and 2% (v / v) Acetobacter pasteurellium seed culture (provided from Application Example 3). The fermentation tank was sealed with sterile gauze and placed in a constant temperature incubator at 30-35℃ for acetic acid fermentation. During fermentation, the tank was shaken 1-2 times daily to ensure oxygen supply. Fermentation continued for 10-15 days. When the sour taste became prominent and the alcoholic taste basically disappeared, the fermentation was considered to have ended, and rice vinegar was obtained.

[0143] 2. Determination of components in rice vinegar The main physicochemical properties of the prepared rice vinegar were determined, and rice vinegar prepared by fermentation with Pediococcus pentosus NY-2-105 seed culture medium without the addition of pentosus was used as a control. The test results are shown in Table 13.

[0144] Table 13 Determination of components in rice vinegar

[0145] 3. Sensory evaluation To assess the quality of the fermented rice vinegar, a sensory evaluation team conducted a sensory evaluation. The team consisted of 10 experienced professional sensory evaluators (5 men and 5 women), and the sensory evaluation form (Table 14) is shown below. Table 14 Sensory Evaluation Criteria for Rice Vinegar

[0146] Table 15 Sensory Evaluation Results of Rice Vinegar

[0147] Experimental results showed that the introduction of Pediococcus pentosaceus NY-2-105 significantly increased the GABA content in rice vinegar by nearly 21.5 times. At the same time, the content of lactic acid and key flavor substances such as ethyl lactate and tetramethylpyrazine also increased. This not only effectively reduced the irritation of volatile acids, but also enriched the aroma and flavor of rice vinegar, significantly improving its quality and taste.

[0148] Application Example 5 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in brewing soy sauce.

[0149] 1. Soy sauce preparation (1) Raw material pretreatment: Defatted soybeans were steamed and cooked. The steaming process was controlled at a pressure of 0.15 MPa and a temperature of 120℃ for 8-10 min to obtain steamed soybeans. Wheat was roasted at 0.05 MPa and 300℃, and then the roasted wheat was crushed to a size of about 80 mesh to obtain roasted wheat flour. The steamed soybeans and roasted wheat flour were mixed evenly in a weight ratio of 1:1 to obtain a mixture.

[0150] (2) Koji making: When the temperature of the mixed materials drops to 28°C, Aspergillus oryzae is added at a ratio of 0.1%; the material inoculated with Aspergillus oryzae is placed in an environment of 30°C for koji making, and the koji making time lasts for 38 hours to obtain koji material.

[0151] (3) Fermentation: The koji material is mixed evenly with 23°Bé brine at a ratio of 1:2 (w / w), and then 0.1% (v / v) of Pediococcus pentosaceus NY-2-105 fermentation seed liquid (provided in Example 4) is added. Finally, the mixture is sealed for fermentation. After being mixed evenly, the mixture is placed in a fermentation tank and sealed for fermentation. The fermentation temperature is controlled at around 30°C, and the fermentation cycle is 90-120 days. During the fermentation period, the mixture is stirred regularly to promote the growth and metabolism of microorganisms. After fermentation, the mash is pressed and filtered, and the filtrate is collected to obtain crude soy sauce.

[0152] 2. Determination of the content of various components in soy sauce The GABA content, amino acid nitrogen content, total acid and salt content in the fermented crude oil were detected, and the total nitrogen utilization rate was calculated. Soy sauce prepared by fermentation with Pediococcus pentosus NY-2-105 seed culture medium without the addition of pentosus was used as a control. The test results are shown in Table 16.

[0153] Table 16 Determination of the content of various components in soy sauce

[0154] The results showed that after fermentation with Pediococcus pentosaceus NY-2-105, the GABA content in soy sauce increased by nearly 33.8 times, while the amino acid nitrogen and total acid content also increased significantly. These changes not only enhanced the umami intensity and nutritional value of the soy sauce, but also made its flavor more harmonious and its taste more mellow.

[0155] Application Example 6 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in the brewing of soybean paste.

[0156] 1. Soybean paste preparation (1) Raw material pretreatment: Soak soybeans for 6-12 hours after washing, and steam them at 100℃ for 10 minutes to obtain steamed soybeans; steam wheat flour for 30 seconds, and mix the steamed soybeans and wheat flour evenly at a weight ratio of 1:1.5 to obtain a mixture.

[0157] (2) Koji making: When the temperature of the mixed materials drops to 28℃, Aspergillus oryzae is added at a ratio of 0.1% to make koji. The koji making temperature is 40℃ and the koji making time is 48 h to obtain koji material.

[0158] (3) Fermentation: Mix the starter culture with 23°Bé brine at a ratio of 1:2 (w / w), then inoculate with 0.1% (v / v) Pediococcus pentosaceus NY-2-105 fermentation seed liquid (provided in Example 4), mix well, and place in a fermentation container and seal. Ferment at a constant temperature of 30-35℃ for 60-90 days. Stir the sauce regularly during fermentation to ensure uniform fermentation. Stop fermentation when the sauce is reddish-brown and glossy with a rich aroma to obtain the finished soybean paste.

[0159] 2. Component determination in soybean paste The content of GABA, amino acid nitrogen, total acid, moisture, salt and reducing sugar in the fermented raw soy sauce was detected, and the total nitrogen utilization rate was calculated. Soy sauce prepared by fermentation with Pediococcus pentosus NY-2-105 seed culture solution without the addition of pentosus was used as a control. The test results are shown in Table 17.

[0160] Table 17 Content of various components in soybean paste

[0161] The results showed that the introduction of strain NY-2-105 significantly increased the GABA content in soybean paste by nearly 33.2 times. At the same time, the significant increase in amino acid nitrogen and total acid content made the soybean paste more umami and more mellow and smooth in taste.

[0162] Application Example 7 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in fermented chili sauce.

[0163] 1. Preparation of chili sauce Chili sauce that has been naturally fermented for one year was used as the fermentation substrate. Pediococcus pentosus NY-2-105 fermentation seed liquid (provided by Example 4) was inoculated at an inoculation rate of 3% (v / v) to start secondary fermentation. After inoculation, the container was sealed and allowed to ferment at 37°C for 15 days.

[0164] 2. Component determination in chili sauce The contents of GABA, acetic acid and lactic acid in chili sauce were detected, and chili sauce that underwent secondary fermentation without the addition of Pediococcus pentosus NY-2-105 seed culture was used as a control. The results are shown in Table 18.

[0165] Table 18 Determination of various components in chili sauce

[0166] 3. Sensory evaluation To evaluate the prepared chili sauce, a sensory evaluation team conducted a sensory assessment. The team consisted of 10 experienced professional sensory evaluators (5 men and 5 women), and the sensory rating scale is shown in Table 19. Table 19 Sensory Evaluation Criteria for Chili Sauce

[0167] Table 20 Sensory rating results for chili sauce

[0168] Experimental results showed that the introduction of Pediococcus pentosaceus NY-2-105 significantly increased the GABA content in chili sauce by nearly 7.1 times. At the same time, the contents of lactic acid and acetic acid were also significantly increased, effectively buffering the irritation of chili sauce, making the saltiness and spiciness more harmonious and the taste more mellow.

[0169] Application Example 8 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in fermented jujube powder.

[0170] 1. Preparation of fermented jujube powder (1) Raw material processing: After cleaning the red dates, slice them and soak them in water at a ratio of 1:9 (w / w) for 8 hours. Then, blend them into a paste, centrifuge at 1000 r / min for 1 min to remove the red date skin, and sterilize at 80℃ for 30 min to obtain date paste.

[0171] (2) Fermentation: The fermentation seed liquid of Pediococcus pentosus NY-2-105 (provided by Example 4) was inoculated into the jujube pulp at an inoculation rate of 3% (v / v) and fermented at 37°C for 24 h to obtain fermented jujube pulp.

[0172] (3) Spray drying: 2% skim milk powder and 0.5% pectinase were added to the fermented jujube pulp. After enzymatic hydrolysis at 50℃ for 1 h, spray drying was carried out. The spray drying conditions were: inlet temperature 150℃, outlet temperature 80℃, atomization flow rate 500 L / h, and peristaltic pump speed 20%, to obtain probiotic fermented jujube powder.

[0173] The fermented jujube powder containing probiotics had a GABA content of 1.21 mg / g, a powder yield of 32%, and a viable count of 1.12 × 10⁻⁶ cells / g. 10 cfu / g.

[0174] 2. Sensory evaluation Sensory evaluation was used to comprehensively assess the quality of the prepared probiotic fermented jujube powder. Based on the quantitative scoring criteria established in Table 21, a sensory panel consisting of 10 experienced professional evaluators (half male and half female) evaluated the samples. The evaluation results showed that the final sensory score of the fermented jujube powder was 72 points.

[0175] Table 21 Sensory Evaluation Standards for Probiotic Fermented Jujube Powder

[0176] Experimental results show that the jujube powder prepared by fermentation with Pediococcus pentosaceus NY-2-105 has a sweet and sour taste, fine powder texture, and a harmonious blend of jujube aroma and fermentation aroma. It also has an extremely high number of viable bacteria and is rich in GABA (1.21 mg / g), giving the jujube powder additional functional properties.

[0177] Application Example 9 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in the preparation of fermented bean curd.

[0178] 1. Preparation of fermented bean curd (1) Raw material pretreatment: Take firm tofu and cut it into cubes with a length, width and height of 3 cm. Drain at 4℃ for 2 h, sterilize at 105℃ for 15 min, and cool to room temperature for later use.

[0179] (2) Mucor inoculation and culture: Dissolve 3 g of Chuanxiu fermented bean curd starter in water, prepare a suspension and spray it evenly onto the tofu blocks. Place them in a sterile bamboo sieve and incubate them in an incubator at 25℃ and 80% humidity for 72 h until the tofu is covered with white fluff mycelium.

[0180] (3) Pickling: Transfer the tofu blocks to a sterile glass jar with the mycelium side facing up, then sprinkle in salt at a rate of 12g / 100g. Place a sterile stainless steel block (weighing 1 / 5 of the total weight of the tofu blocks) on top and pickle at 18℃ for 10 days.

[0181] (4) Preparation of fermented bean curd brine: Take 500 mL of sterile water, add 3% red yeast rice powder, 1 g star anise and 1 g cinnamon, heat in a 100℃ water bath for 30 min, cool to room temperature, add salt to adjust the salt content of the brine to 10%, stir to dissolve, filter twice with sterile gauze, and then inoculate the brine with 5% (v / v) Pediococcus pentosacchari NY-2-105 fermentation seed liquid (provided by Example 4) to obtain fermented bean curd brine.

[0182] (5) Secondary fermentation: Slowly pour the fermented bean curd brine into the glass jar containing tofu blocks, ensuring that it completely covers the tofu blocks by more than 1 cm. Seal the jar and transfer it to a 37℃ incubator for 25 days to obtain fermented bean curd.

[0183] 2. Determination of components in fermented bean curd The GABA content, amino acid nitrogen, and colony count in fermented bean curd were tested to detect key indicators, and fermented bean curd prepared without the addition of seed liquid of this bacterium was used as a control (traditional process). The test results are shown in Table 22.

[0184] Table 22 Determination of various components in fermented bean curd

[0185] 3. Sensory evaluation To assess the quality of the fermented bean curd, a sensory evaluation team conducted a sensory evaluation. The team consisted of 10 experienced professional sensory evaluators (5 men and 5 women), and the sensory evaluation form is shown in Table 23. Table 23 Sensory Evaluation Criteria for Fermented Tofu

[0186] Table 24 Sensory evaluation results of fermented bean curd

[0187] Experimental results show that using Pediococcus pentosaceus NY-2-105 to prepare fermented bean curd can increase the GABA content by nearly 85 times compared with the control group, and the amino acid nitrogen content is also significantly increased, making the fermented bean curd taste more delicious and mellow.

[0188] Application Example 10 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in the preparation of kimchi.

[0189] 1. Preparation of pickled vegetables (1) Raw material pretreatment: Take fresh Chinese cabbage, remove the outer old leaves and cut into small pieces of 4-5 cm square, wash with sterile water and drain; place the cabbage in layers in a sterile glass jar, sprinkle 2%-3% (w / w) salt evenly on each layer and press lightly, press the top with a sterile weight, and let stand at 25℃ for 4-6 hours; after the cabbage has fully released water and softened, take it out and rinse it with 0.5% (w / v) sterile saline 1-2 times to remove excess salt on the surface, and finally drain to obtain dehydrated Chinese cabbage.

[0190] (2) Canning: Thoroughly mix the dehydrated cabbage and auxiliary materials (ginger, garlic, chili paste) in a sterile glass container, and then transfer the mixed material into a sterile glass jar. When filling the jar, use a layered filling method. After each layer of mixed material, sprinkle a layer of 10% (w / v) glucose solution evenly, and gently press until there are no obvious gaps. Fill the jar to 80% of its total capacity.

[0191] (3) Inoculation and fermentation: Spray the fermentation seed liquid of Pediococcus pentosus NY-2-105 (provided by Example 4) evenly onto the surface of the cabbage in the tank at an inoculation amount of 10% (v / w), and then add 0.5% sterile light saline until the liquid completely covers the material.

[0192] (4) Fermentation: Cover the glass jar with the lid and wrap the mouth of the jar with sterile gauze. Move the fermentation jar to a 30℃ incubator and let it ferment for 7 days to obtain the finished kimchi.

[0193] 2. Component analysis of kimchi The GABA content, total acid, and colony count in the prepared kimchi were detected, with kimchi prepared without the addition of the bacterial seed liquid as a control (traditional process). The test results are shown in Table 25.

[0194] Table 25 Determination of various components in kimchi

[0195] 3. Sensory evaluation To assess the quality of the fermented kimchi, a sensory evaluation team conducted a sensory evaluation. The team consisted of 10 experienced professional sensory evaluators (5 men and 5 women), and the sensory evaluation form is shown in Table 26. Table 26 Sensory Evaluation Criteria for Kimchi

[0196] Table 27 Sensory Evaluation Results of Kimchi

[0197] In conclusion, adding strain NY-2-105 during the fermentation process of kimchi can increase the GABA and lactic acid content in kimchi, thus improving its quality and taste. This indicates that NY-2-105 can be used as a functional production strain for kimchi. Experimental results show that using *Pediococcus pentosaceus* NY-2-105 to prepare kimchi can increase the GABA content by nearly 7.2 times, while the moderate increase in total acid content gives the kimchi a crisper, more tangy, and flavorful fermented taste. Furthermore, the viable cell count in the finished fermented product remains at 10... 8 The high level of cfu / g or higher endows kimchi with excellent probiotic functional properties, enhancing the product's nutritional value and sensory quality.

[0198] Application Example 11 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in the preparation of fermented sea buckthorn juice.

[0199] 1. Preparation of fermented sea buckthorn juice (1) Preparation of sea buckthorn juice fermentation substrate: Wash fresh sea buckthorn fruits, drain them, and put them into a juicer to extract sea buckthorn juice. Filter the juice with sterile gauze to obtain clear sea buckthorn juice. Adjust the sugar content of the sea buckthorn juice with sucrose to 10% (10g / 100g), and then sterilize it at 105℃ for 20 min to obtain the sea buckthorn juice fermentation substrate. (2) Inoculation and fermentation: The fermentation seed liquid of Pediococcus pentosaceus NY-2-105 (provided in Example 4) was inoculated into the sea buckthorn juice fermentation substrate at an inoculation rate of 10% (v / v), mixed evenly, and then fermented statically in an incubator at 37°C for 48 h. Fermented sea buckthorn juice was obtained.

[0200] 2. Determination of components in fermented sea buckthorn juice The GABA content, sugar content, and acidity of the prepared fermented sea buckthorn juice were detected, with unfermented sea buckthorn juice as a control. The results are shown in Table 28.

[0201] Table 28 Determination of components in sea buckthorn juice

[0202] 3. Sensory evaluation To assess the quality of the fermented sea buckthorn juice, a sensory evaluation team conducted a sensory evaluation. The team consisted of 10 experienced professional sensory evaluators (5 men and 5 women), and the sensory evaluation form is shown in Table 29. Table 29 Sensory Evaluation Criteria for Seabuckthorn Juice

[0203] Table 30 Sensory Evaluation Results of Sea Buckthorn Juice

[0204] Experimental results show that fermenting sea buckthorn juice with Pediococcus pentosaceus NY-2-105 can increase the GABA content by about 31 times, while effectively improving the rough and sour taste of the original sea buckthorn juice, giving the product a unique fermented flavor and delicate taste.

[0205] Application Example 12 This embodiment provides the application of Pediococcus pentosaceus NY-2-105 in the preparation of yogurt.

[0206] 1. Yogurt preparation (1) Raw material pretreatment and sterilization: Take 1000 mL of fresh raw milk, add 50 g of white sugar, stir until completely dissolved, and then homogenize to break down the fat globules; then pasteurize at 85-90℃ for 15-20 min.

[0207] (2) Inoculation and fermentation: After the sterilized milk is cooled to below 43°C, add 1 g of Chuanxiu Classic Old Yogurt Fermentation Powder, and then inoculate with Pediococcus pentosus NY-2-105 fermentation seed liquid (provided by Example 4) at an inoculation amount of 1% (v / v). After mixing evenly, let it ferment in a constant temperature incubator at 42°C for 4-6 h until the milk coagulates and the pH value reaches about 4.5-4.6.

[0208] (3) Post-fermentation and refrigeration: After fermentation, immediately transfer the yogurt to a refrigerator at 2-6℃ for post-fermentation for at least 12 hours.

[0209] 2. Yogurt component determination After the post-ripening process, the GABA content, lactic acid content, and total number of live bacteria in the yogurt were tested, with conventionally fermented yogurt as a control (the starter was Chuanxiu Classic Old Yogurt Starter Powder). The test results are shown in Table 31.

[0210] Table 31 Determination of various components in yogurt

[0211] 3. Sensory evaluation To assess the quality of the prepared yogurt, a sensory evaluation panel was formed. The panel consisted of 10 experienced professional sensory evaluators (5 men and 5 women), and the sensory scoring sheet was based on Chinese Patent Application No. CN202411166753 (Table 1). The results are shown in the table below.

[0212] Table 32 Sensory evaluation results of yogurt

[0213] Experimental results show that adding strain NY-2-105 during the fermentation of yogurt can increase the GABA content, lactic acid content and viable bacteria count in yogurt, and the strain provided by this invention can be used to develop high-quality functional yogurt.

[0214] In summary, the *Pediococcus pentosaceus* NY-2-105 strain bred using ARTP mutagenesis technology in this invention significantly improves GABA production and environmental tolerance while retaining the safety of non-gene-edited foods. This strain is widely adaptable to various fermentation substrates, providing excellent germplasm resources for developing high-quality fermented foods rich in GABA, and has a very broad application prospect.

[0215] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Pediococcus pentosaceus NY-2-105, characterized in that, Its classification name is Pediococcus pentosaceus ( Pediococcus pentosaceus The sample was deposited on November 11, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The fermentation product of *Pediococcus pentosaceus* NY-2-105 according to claim 1, characterized in that, The fermentation product is obtained by fermentation of the Pediococcus pentosacchari NY-2-105 strain as described in claim 1 or a combination of microorganisms containing Pediococcus pentosacchari NY-2-105 as described in claim 1.

3. A fermenting agent, characterized in that, The fermentation agent comprises the Pediococcus pentosaceus NY-2-105 strain as described in claim 1 or a microbial combination containing the Pediococcus pentosaceus NY-2-105 strain as described in claim 1.

4. The fermenting agent as described in claim 3, characterized in that, The fermenting agent is a food fermenting agent, composed of Pediococcus pentosaceus NY-2-105 as described in claim 1 and at least one food fermentation microorganism selected from yeast, acetic acid bacteria, Aspergillus oryzae or Mucor.

5. The use of *Streptomyces coccidioides* HA04 according to claim 1, the fermentation product according to claim 2, or the starter culture according to claim 3 in any of the following: (1) Application in the preparation of fermented foods rich in γ-aminobutyric acid; (2) Application in the production of γ-aminobutyric acid; (3) Application in the preparation of functional foods; (4) Application in the preparation of food additives; (5) Use in the preparation of drugs for the prevention and / or treatment of mental illness.

6. A method for producing fermented food rich in γ-aminobutyric acid, characterized in that, The method includes the step of fermenting food raw materials or processed food raw materials using Pediococcus pentosus NY-2-105 as described in claim 1 or the fermenting agent as described in claims 3 and 4.

7. The method as described in claim 6, characterized in that, The fermented food is brewed vinegar; the method includes inoculating the Pediococcus pentosaceus NY-2-105 into the mash and / or wine mash during the alcoholic fermentation stage and / or acetic acid fermentation stage for fermentation.

8. The method as described in claim 6, characterized in that, The fermented food is soy sauce; the method includes adding brine and the Pentosacchariformis NY-2-105 to the koji material for fermentation.

9. The method as described in claim 6, characterized in that... The fermented food is fermented bean curd; the method includes adding Pediococcus pentosaccharis NY-2-105 to the fermented bean curd brine.

10. Fermented food prepared according to any one of claims 6-9.

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