A method for preparing lactobacillus hilgardii by fermentation to produce high yield of gamma-aminobutyric acid

Abstract

The application discloses a fermentation preparation method of Lactobacillus hilakii with high GABA yield, and the method comprises the following steps: two-stage fermentation is carried out by using Lactobacillus hilakii, acid stress and intermittent substrate feeding are used to induce and maintain high activity of GAD enzyme in the GABA synthesis stage; when GABA accumulates to a certain concentration, a semi-permeable membrane filtration system connected with a fermentation tank is started, the product is removed on line, and the circulating bacteria are intercepted, so that the product inhibition is released, and the substrate concentration is controlled; the permeate is collected, and high-purity food-grade gamma-aminobutyric acid is obtained through the steps of ultrafiltration, decolorization, ion purification, concentration, crystallization and drying. The application innovatively couples fermentation with on-line membrane separation, breaks through the bottleneck of the traditional process, realizes super-high yield (>360 g / L) and super-high efficiency production of GABA accumulation, significantly shortens the production cycle, reduces the cost, and the obtained product meets the new resource food standard.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and new resource food preparation technology, specifically involving a method for efficiently producing γ-aminobutyric acid (GABA) through two-stage precise fermentation coupled with online membrane separation technology, and its application in the preparation of food-grade GABA raw materials. Background Technology

[0002] Gamma-aminobutyric acid (GABA) is a natural non-protein amino acid. As an important inhibitory neurotransmitter in the central nervous system, it has physiological functions such as sedation, blood pressure regulation, and sleep improvement. In 2009, my country's Ministry of Health approved GABA as a new resource food, which can be widely used in functional foods, health products, and other fields.

[0003] Currently, industrial production of GABA primarily relies on microbial fermentation. Compared to chemical synthesis, fermentation offers key advantages such as mild reaction conditions, high food-grade safety, no harmful solvent residues, and environmental friendliness, making it the only compliant production method for new resource-based food-grade GABA. Among these strains, *Lactobacillus hilgardii* (formerly *Lactobacillus hilgardii*) is a lactic acid bacteria strain explicitly approved by the National Health Commission for use in the production of food-grade GABA. It possesses advantages such as naturally high glutamate decarboxylase (GAD) activity, high food-grade safety, and non-pathogenicity, making it a core potential strain for the industrial production of GABA.

[0004] However, existing technologies for producing GABA through fermentation of Lactobacillus schlegelii still face insurmountable industry bottlenecks, with the following core shortcomings:

[0005] 1) Production ceiling is difficult to break through and the level of industrialization is low: In the existing industrial mass production process, the conventional output of GABA fermented by Lactobacillus hessler is only 30-80 g / L; the existing technology patent CN116814468B discloses 151.7 g / L, which cannot break through the industry barrier of 200 g / L and is difficult to meet the cost control requirements of large-scale production.

[0006] 2) Dual inhibition of substrate and product leads to continuous decline in catalytic efficiency: During the process of GAD enzyme catalyzing the decarboxylation of L-glutamate to generate GABA, high concentrations of substrate L-glutamate directly inhibit the spatial conformation and catalytic activity of GAD enzyme; while when GABA accumulates in the fermentation broth to a certain concentration, it will produce strong product feedback inhibition, resulting in a rapid decline in GAD enzyme activity and a significant shortening of the catalytic half-life, making it impossible to achieve continuous and efficient synthesis.

[0007] 3) Long fermentation cycle and extremely low production efficiency: The fermentation cycle of existing high-yield processes is generally as long as 72-96 hours, with low equipment utilization, high energy consumption, slow batch turnover, and high industrial production costs; and there are no effective means to shorten the catalytic cycle, so it is impossible to achieve both high yield and high efficiency.

[0008] 4) Insufficient GAD enzyme expression: Current processes mostly use a single substrate induction method, and do not achieve efficient upregulation of GAD enzyme expression through environmental stress and substrate induction. Insufficient intracellular GAD enzyme content has become the core internal factor limiting high GABA production.

[0009] 5) Low process integration and disconnect between fermentation and separation: In the existing technology, membrane separation is only used for cell removal or purification after fermentation. It does not achieve in-situ coupling between the fermentation process and online separation, and cannot relieve product inhibition in real time during the catalytic process.

[0010] In summary, current technologies lack a solution that organically combines the four core technologies of two-stage precise fermentation, acid stress-substrate synergistic induction of GAD enzyme high expression, intermittent fed-batch controlled substrate, and in-situ coupling of fermentation and online membrane separation. This makes it impossible to simultaneously address the four major industry pain points: yield, cycle time, inhibition, and enzyme activity. Therefore, developing a *Lactobacillus schlegelii* fermentation process that can overcome yield bottlenecks, completely eliminate dual inhibition, significantly shorten the production cycle, and meet food-grade requirements throughout the entire process is of decisive significance for the industrial upgrading of GABA-based new resource foods. Summary of the Invention

[0011] This invention aims to significantly increase γ-aminobutyric acid (GABA) yield, shorten the production cycle, greatly improve production efficiency, completely eliminate substrate and product dual inhibition, and achieve continuous and efficient catalysis of GAD enzymes. To solve the above problems, this invention adopts the following solution:

[0012] A method for preparing high-yield γ-aminobutyric acid (GABA) by fermentation of Lactobacillus hilgardii is characterized by the following steps: Fermentation stage: Lactobacillus hilgardii is used to ferment and produce GABA in a fermenter; Online product removal stage: When GABA accumulates to a predetermined concentration in the fermentation broth, a membrane separation system connected to the fermenter is activated to separate GABA from the bacterial cells in the fermentation broth online, removing the permeate containing GABA from the fermentation system, while simultaneously recycling the retained bacterial cells back to the fermenter for continued fermentation.

[0013] The fermentation stage and the online product removal stage are carried out in synergy. By removing the product in situ, the feedback inhibition of γ-aminobutyric acid on glutamate decarboxylase (GAD enzyme) is relieved, thereby achieving continuous and efficient synthesis of γ-aminobutyric acid.

[0014] A further improvement of the present invention is that the Lactobacillus lt02 was deposited at the China Center for Type Culture Collection (CCTCC) on March 30, 2026, at Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2026547.

[0015] A further improvement of the present invention is that the fermentation stage includes a first fermentation stage - cell accumulation period and a second fermentation stage - GABA high-efficiency synthesis period;

[0016] First fermentation stage - cell accumulation period: Inoculate the seed culture medium with 8-12% (v / v) of the seed culture solution. Maintain a facultative anaerobic environment at 30-35℃, pH 5.0-5.5, and a rotation speed of 50-100 rpm, with nitrogen purging to maintain the facultative anaerobic environment. Continuously culture *Lactobacillus hessei* for 20-24 hours to obtain high-density cells with an OD500 concentration. 600 >6.5, proceed to the second fermentation stage;

[0017] Second fermentation stage - GABA high-efficiency synthesis period: Adjust the pH to 4.0-5.0 for acid stress induction, and add 0.05% (w / v) of pyridoxal phosphate (PLP, an essential coenzyme for GAD enzyme) to the fermentation broth. Intermittently add L-glutamate sodium and glucose, and control the concentration of L-glutamate sodium and glucose within the predetermined range.

[0018] A further improvement of the present invention is that, in the second fermentation stage, the intermittent feeding is performed by feeding an 800 g / L sodium L-glutamate solution and a 500 g / L glucose solution every 1.5 h, thereby controlling the concentration of sodium L-glutamate in the fermentation broth to be maintained at 35-45 g / L and the concentration of glucose to be maintained at 4-6 g / L.

[0019] A further improvement of the present invention is that the predetermined concentration is such that the membrane separation system is started when the γ-aminobutyric acid in the fermentation broth is ≥80 g / L.

[0020] A further improvement of the present invention is that the membrane separation system is a 0.22 μm food-grade sterile hollow fiber membrane separation system, in which the trapped bacteria are recycled back to the fermenter for continued fermentation and the permeate is collected.

[0021] A further improvement of this invention is that filtration is stopped when the permeate volume reaches 80% of the original fermentation broth volume, and sterile fermentation supplement medium is added simultaneously to maintain the stability of the fermenter volume (the supplement volume is consistent with the permeate volume removed by membrane separation), and 0.05% (w / v) of pyridoxal phosphate is added to the fermentation broth.

[0022] A further improvement of this invention is that, during the continued fermentation process, L-glutamate sodium and glucose are intermittently added, and the concentration of L-glutamate sodium in the fermentation broth is maintained at 35-45 g / L and the concentration of glucose is maintained at 4-6 g / L. Fermentation is stopped when the cumulative yield of γ-aminobutyric acid reaches more than 360 g / L.

[0023] A further improvement of the present invention is that it further includes purifying the permeate and the fermentation broth in the tank, the purification step comprising:

[0024] Ultrafiltration: Combine the permeate with the fermentation broth in the tank, and pass the liquid through a food-grade spiral wound ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The operating pressure is 0.2-0.3 MPa and the temperature is 25-35℃. Cross-flow filtration removes impurities such as large molecular proteins, polysaccharides, bacterial fragments, and colloids, and the ultrafiltration permeate is collected.

[0025] Decolorization: Add 0.5%-2% (w / v) activated carbon to the ultrafiltration permeate, stir at 50-60℃ for 30-60 min to decolorize, first filter through plate and frame filter to coarsely remove activated carbon, and then filter through a 0.45μm filter cartridge to obtain a decolorized and clear solution containing GABA.

[0026] Ion exchange: After adjusting the pH of the decolorized and clarified solution to 5.5-6.5, it is passed through a 001×7 type strong acid cation exchange resin column at a flow rate of 1-2 BV / h. GABA, as a basic amino acid, is specifically adsorbed by the resin, while impurities such as residual sugar, inorganic salts, and small molecule organic acids are discharged with the effluent. The GABA adsorbed on the resin is eluted with a 0.5 mol / L ammonia solution at a flow rate of 1 BV / h to obtain the GABA eluent.

[0027] Concentration and crystallization: After concentrating the eluent, add anhydrous ethanol for low-temperature crystallization;

[0028] Drying: Collect the crystals and dry them to obtain high-purity (purity greater than 99%) γ-aminobutyric acid product.

[0029] A further improvement of the present invention is that the crystallization conditions are as follows: the eluent containing γ-aminobutyric acid is concentrated under reduced pressure at 50-60°C to 300-500 g / L, cooled to 10-15°C, and anhydrous ethanol is added under stirring to 30%-50% by volume. The mixture is then allowed to stand at 4°C for 8-24 h to crystallize.

[0030] A further improvement of the present invention is that the seed culture medium is obtained by inoculating Lactobacillus hesslerii LT02 (CCTCC NO: M 2026547) into the seed culture medium and culturing it on a shaker at a temperature of 30-35°C for 16-24 h.

[0031] A further improvement of this invention is that the seed culture medium, fermentation culture medium, and fermentation supplement culture medium are all food-grade culture media. The seed culture medium comprises: glucose 18-22 g / L, peptone 8-12 g / L, beef extract powder 8-12 g / L, yeast powder 4-6 g / L, potassium dihydrogen phosphate 1-3 g / L, triammonium citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.2 g / L, manganese sulfate 0.04-0.06 g / L, and Tween-80 1-2 mL / L.

[0032] The fermentation medium consists of: glucose 18-22 g / L, L-glutamate sodium 9-11 g / L, peptone 9-11 g / L, beef extract powder 7-10 g / L, yeast powder 3-5 g / L, corn steep liquor powder 13-16 g / L, dipotassium hydrogen phosphate 2-3 g / L, diammonium hydrogen citrate 2-3 g / L, sodium acetate 4-5 g / L, magnesium sulfate 0.2-0.3 g / L, manganese sulfate 0.03-0.05 g / L, and Tween 80 1-2 mL / L.

[0033] The fermentation supplement medium consists of: 4-6 g / L peptone, 2-3 g / L yeast extract, 1-3 g / L potassium dihydrogen phosphate, 1-3 g / L triammonium citrate, 4-6 g / L sodium acetate, 0.1-0.2 g / L magnesium sulfate, and 0.04-0.06 g / L manganese sulfate.

[0034] Beneficial effects

[0035] 1) Breakthrough improvement in GABA production: The two-stage regulation and membrane separation coupling of this invention achieve a cumulative total GABA production of ≥360 g / L within a short period of 44-48 h, which is 5-10 times the current industrial level and more than 2 times the highest level of existing publicly available technology, completely breaking through the production bottleneck of GABA fermentation by Lactobacillus schrei.

[0036] 2) The production cycle is significantly shortened and the production efficiency is greatly improved: The cumulative output of this invention in 46 hours is 372.5 g / L, with a production efficiency of 8.1 g / L / h; the output of the existing technology in 72 hours is 151.7 g / L, with a production efficiency of 2.11 g / L / h. The production efficiency is improved by 284%, which doubles the equipment utilization rate and greatly reduces energy consumption and labor costs, giving it a strong industrial competitiveness.

[0037] 3) Complete elimination of dual inhibition by substrate and product, significantly improving GAD enzyme catalytic efficiency: By intermittent feeding and precise control of substrate concentration within the low inhibition range, the inhibition of GAD enzyme by high concentrations of monosodium glutamate is eliminated; by continuously removing the product in situ through online membrane separation, the GABA concentration in the tank is stably controlled within the non-inhibition range, completely solving the industry pain point of product feedback inhibition; in addition, the dual induction synergistic effect of acid stress and substrate enhances GAD enzyme expression, achieving continuous and efficient catalysis.

[0038] 4) The strains, reagents, and consumables used in this invention are all food-grade, without genetic engineering modification or the introduction of toxic or harmful solvents. The purity of the resulting GABA product is ≥99%, which meets the relevant requirements for new resource foods and can be directly applied in the food and health food fields.

[0039] 5) The process is highly stable and easy to scale up industrially; the online membrane separation system can realize the recycling of bacterial cells, has high batch stability, can be directly adapted to existing fermentation industrial equipment, does not require large-scale modification, and has extremely strong industrial application value. Attached Figure Description

[0040] Figure 1 This is a process flow diagram of the present invention;

[0041] Figure 2 Photograph of Lactobacillus hesitant LT02 colony morphology;

[0042] Figure 3 This is a comparison chart of the cumulative GABA production of Example 3 and Comparative Examples 1-3;

[0043] Figure 4 Figure showing the effect of different membrane separation start-up times on fermentation parameters;

[0044] Figure 5 The image shows the fermentation results of different batches of Lactobacillus hesitant LT02. Detailed Implementation

[0045] The following embodiments are included to illustrate preferred embodiments of the present invention. Those skilled in the art should recognize that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that are effective in practicing the present invention. Based on this disclosure, those skilled in the art should recognize that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention; that is, any equivalent results or equivalent procedural transformations made using the content of this specification, or direct or indirect applications in other related technical fields, should be included within the scope of protection of this patent.

[0046] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0047] To achieve the above-mentioned objectives, the present invention discloses the following technical solution.

[0048] This invention discloses a screened strain of Lactobacillus hilgardii, named LT02, which was deposited on March 30, 2026, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 2026547.

[0049] The culture media used in the examples and comparative examples are as follows:

[0050] The seed culture medium consisted of: 20.0 g / L glucose, 10.0 g / L peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 2.0 g / L potassium dihydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1.0 mL / L Tween-80. The culture medium was autoclaved at 121°C for 20 min, and glucose was autoclaved separately at 115°C for 20 min.

[0051] Fermentation medium composition: glucose 20.0 g / L, L-glutamate sodium 10.0 g / L, peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast powder 4.0 g / L, corn steep liquor powder 15.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 mL / L; the medium was autoclaved at 121℃ for 20 min; glucose was autoclaved separately at 115℃ for 20 min.

[0052] The fermentation supplement medium consisted of: 5.0 g / L peptone, 2.5 g / L yeast extract, 2.0 g / L potassium dihydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, and 0.05 g / L manganese sulfate. The medium was autoclaved at 121°C for 20 min.

[0053] Example 1: Obtaining the strain

[0054] The bacterial strain was isolated from a kimchi sample purchased from a vegetable market in Nanjing, Jiangsu Province. The specific strain screening steps are as follows:

[0055] After chopping the purchased kimchi sample, wash it with sterile water and collect the washing solution. Then, use sterile water to perform a 10-fold serial dilution of the washing solution to a final concentration of 10. -10Spread each gradient of bacterial suspension evenly onto bromocresol purple-MRS medium plates and incubate upside down in a 30°C incubator for 2-3 days. Once colonies have grown, select those that turn the plate yellow and continue to isolate and purify them until single colonies are obtained.

[0056] Six candidate strains (#1~#6) were inoculated into shake flasks containing seed culture medium and cultured at 30°C for 24 h to obtain a seed pre-culture solution. This seed pre-culture solution was then inoculated into seed culture medium at a rate of 10% (v / v) and cultured at 30°C for 24 h to obtain the seed culture solution. This seed culture solution was then inoculated into 6 L of fermentation medium at a rate of 10% (v / v) for further cultivation. During fermentation, the pH of the fermentation broth was controlled at 5.5, the temperature at 30°C, and the dissolved oxygen at 5-10%. After fermentation, the cells were collected by centrifugation. The cells were resuspended in an acetic acid / sodium acetate buffer solution at pH 4.8, and the OD of the bacterial count was measured. 600 The concentration was controlled at 10. 100 g / L of sodium L-glutamate was added to the bacterial suspension, followed by 0.5 g / L of pyridoxal phosphate to initiate the transformation. After 24 h of transformation, the supernatant of the transformation solution was collected, and the concentration of GABA was determined using HPLC (AccQ-tag amino acid analyzer, Waters Corporation; mobile phase A was ultrapure water, and mobile phase B was 60% acetonitrile). The concentration of GABA in the supernatant of the transformation solution after 24 h of transformation for different strains is shown in Table 1. The results showed that strain #3 produced a GABA concentration of 60.3 g / L, and the highest substrate molar conversion rate (theoretical conversion rate) was 99%. The colony morphology of strain #3 is shown in the image below. Figure 2 As shown.

[0057] Table 1. GABA concentration in the transformation solution after 24 h of catalytic transformation by different strains

[0058]

[0059] Example 2: Strain Identification

[0060] Molecular biological identification of strain #3: Strain #3 was inoculated into MRS slant medium for expansion, and the 16S rDNA of the expanded strain was sequenced using universal 16S rDNA primers SEQ ID NO: 2 and SEQ ID NO: 3. Homology comparison was performed using the Blast search program, and the sequencing result is shown in SEQ ID No: 1. Combining morphological and physiological observations, as well as physiological and biochemical identification, this strain was finally identified as a novel *Lentilactobacillus hilgardii*, named *Lentilactobacillus hilgardii* LT02. This strain was deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, China on March 30, 2026, with accession number CCTCC NO: M 2026547. Under laboratory conditions, *Lentilactobacillus hilgardii* LT02 was activated with MRS liquid medium, mixed 1:1 with cryopreservation medium, and aliquoted into glycerol tubes for cryopreservation at -78°C.

[0061] Example 3

[0062] (1) Seed culture preparation: Lactobacillus LT02 was inoculated into seed culture medium and cultured on a shaker at 30℃ for 24 h to obtain seed culture medium.

[0063] (2) First stage fermentation (cell accumulation period): The seed culture was inoculated into the fermentation medium at an inoculation rate of 10% (v / v). The culture conditions were: temperature 30℃, pH 5.5, rotation speed 80 rpm, nitrogen gas was purged to maintain a facultative anaerobic environment, and the culture was carried out continuously for 22 h. The cell concentration OD 600 It reached 7.2.

[0064] (3) Second stage fermentation (GABA high-efficiency synthesis period): Adjust the pH to 4.5 for acid stress induction, and add 0.05% (w / v) pyridoxal phosphate to the fermentation broth. At the same time, intermittently add 800 g / L L-glutamate sodium solution and 500 g / L glucose solution every 1.5 h, and control the concentration of L-glutamate sodium in the fermentation broth to maintain at 35-45 g / L and the concentration of glucose to maintain at 4-6 g / L each time.

[0065] (4) Fermentation-membrane separation coupled catalysis: After fermentation for 28 h, the concentration of GABA in the fermentation broth was measured to be ≥80 g / L. An online 0.22 μm food-grade sterile hollow fiber membrane separation system was started, and all the retained cells were recycled back to the fermenter. The permeate was collected. Filtration was stopped when the volume of the permeate was 80% of the original fermentation broth. Sterile fermentation supplement medium was added to maintain volume stability (the supplement volume was consistent with the volume of permeate removed by membrane separation), and 0.05% (w / v) pyridoxal phosphate was added to the fermentation broth. Fermentation continued, with L-glutamate sodium solution and glucose solution added every 1.5 h to control the concentration of L-glutamate sodium in the fermentation broth at 35-45 g / L and the concentration of glucose at 4-6 g / L.

[0066] (5) End of fermentation: Continue fermentation until 46 h, when the cumulative GABA yield is >360 g / L, and stop fermentation.

[0067] (6) Purification and refining: The permeate and fermentation broth in the tank were combined and treated with a 3000 Da food-grade spiral wound ultrafiltration membrane at an operating pressure of 0.25 MPa and a temperature of 30℃. The permeate was collected to remove macromolecular impurities. 1% (w / v) food-grade activated carbon was added to the ultrafiltration permeate, and the mixture was stirred at 55℃ for 45 min to decolorize. After coarse removal of activated carbon by plate and frame filtration, the mixture was then finely filtered through a 0.45 μm filter cartridge to obtain a decolorized and clarified solution containing γ-aminobutyric acid. The pH of the decolorized and clarified solution was adjusted to 5.5 and then passed through a 001×7 type strong acid cation exchange resin column at a flow rate of 2 BV / h. GABA was adsorbed by the resin as a basic amino acid, and impurities (residual sugar, inorganic salts, etc.) were discharged with the effluent. The GABA adsorbed by the resin was eluted with a 0.5 mol / L ammonia solution at a flow rate of 1 BV / h to obtain the GABA eluent. The eluent containing GABA was concentrated under reduced pressure at 60°C to a GABA concentration of 400 g / L. The concentration was then lowered to 15°C, and anhydrous ethanol was slowly added with stirring until the ethanol volume fraction reached 40%. Crystallization was carried out at 4°C for 12 h, and the wet crystals were collected by centrifugation. The wet crystals were then dried under vacuum at 55°C for 10 h to obtain a GABA product with a purity greater than 99%.

[0068] Test results:

[0069] Fermentation endpoint: 46 h, cumulative GABA yield 372.5 g / L, L-glutamate molar conversion rate 98.6%;

[0070] Finished product specifications: GABA purity 99.3%.

[0071] Comparative Example 1: Acid-Stress-Free Process

[0072] The only difference between this comparative example and Example 3 is that the pH is not adjusted in the second stage, and the pH is maintained at 5.5 throughout the process. There is no acid stress induction, and the remaining process parameters are completely consistent with those of Example 3. Fermentation is stopped after 46 hours.

[0073] Test results:

[0074] Fermentation for 46 h: cumulative GABA yield 279.3 g / L, L-sodium glutamate molar conversion rate 86.4%;

[0075] Results analysis: Acid stress is the core factor inducing efficient expression of GAD enzyme. Without acid stress, enzyme expression is insufficient and GABA production decreases significantly.

[0076] Comparative Example 2: Batch process for single-use high-concentration substrate addition

[0077] The only difference between this comparative example and Example 3 is that: in the second stage, L-glutamate sodium was added at a final concentration of 300 g / L all at once, without intermittent feeding; the other process parameters were completely consistent with Example 3, and fermentation was stopped after 46 h.

[0078] Test results:

[0079] Fermentation for 46 h: cumulative GABA yield 132.6 g / L, L-glutamate molar conversion rate 72.5%;

[0080] Results analysis: Adding a high concentration of substrate at once will produce strong substrate inhibition, significantly reduce GAD enzyme activity, and greatly reduce GABA yield and conversion rate.

[0081] Comparative Example 3: Conventional feed process without online membrane separation coupling

[0082] The only difference between this comparative example and Example 3 is that the online membrane separation coupling system was not started. All other process parameters, strains, culture media, and culture conditions were completely consistent with Example 3. Fermentation parameters were measured at 48 h and 72 h, respectively. Detection results:

[0083] Fermentation for 48 h: cumulative GABA yield 125.3 g / L, L-glutamate molar conversion rate 82.1%;

[0084] Fermentation for 72 h: cumulative GABA yield 138.5 g / L, L-sodium glutamate molar conversion rate 85.3%;

[0085] Results analysis: Without online membrane separation, the product feedback inhibition could not be eliminated, resulting in a significant decrease in GABA yield and conversion rate, a significant extension of the fermentation cycle, and an inability to achieve high yield and high efficiency.

[0086] Comparison of membrane separation start-up time in Example 4

[0087] The membrane separation system was started at concentrations of γ-aminobutyric acid (GABA) of 80 g / L, 90 g / L, 100 g / L, and 110 g / L, respectively. The remaining operating steps were the same as in Example 3. Key fermentation indicators and finished product indicators were tested, and the results are shown in Table 2.

[0088] Table 2. Effects of different membrane separation start-up times on fermentation parameters

[0089]

[0090] Results analysis: When starting at 80 g / L too early, GAD enzyme expression was not fully induced, resulting in low product accumulation despite the same fermentation cycle; when starting at 90 g / L, the product had just reached the inhibition threshold, and timely removal could completely eliminate feedback inhibition, achieving optimal yield and conversion rate; when starting at 100 g / L and 110 g / L too late, significant product inhibition was observed in the fermentation broth, GAD enzyme activity partially decreased, and the inhibitory effect intensified with delayed start time, leading to a continuous decline in yield and conversion rate, and a slight extension of the fermentation cycle; different start times had no significant impact on the purity of the finished product, which remained ≥99.0%.

[0091] Example 4: Batch Stability Verification

[0092] To demonstrate the feasibility of industrial-scale production of the process, three consecutive pilot-scale tests were conducted using a 10 L fermenter. The process parameters of Example 3 were strictly followed, and key fermentation and finished product indicators were tested. The results are shown in Table 3.

[0093] Table 3. Batch stability verification results of GABA production by Lactobacillus hilus LT02 fermentation.

[0094]

[0095] Results analysis: The relative standard deviation (RSD) of the fermentation cycle, GABA yield, substrate conversion rate, and finished product purity of the three pilot batches were all ≤1.1%, which is far lower than the requirement of RSD≤5% for industrial production. This proves that the process parameters of the present invention are highly controllable and have excellent batch stability, and can be directly scaled up to industrial-grade fermenters for large-scale production.

[0096] The above description is merely an embodiment of the present invention and does not limit the scope of the patent. Any equivalent results or equivalent process transformations made using the content of this specification, or direct or indirect applications in other related technical fields, should be included within the scope of protection of this patent.

Claims

1. A method for preparing high-yield γ-aminobutyric acid (GABA) Lactobacillus schlegelii by fermentation, characterized in that, Includes the following stages: Fermentation stage: γ-aminobutyric acid (GABA) was produced by two-stage fermentation in a fermenter using Lactobacillus hilgardii LT02, which was selected independently. Lactobacillus hilgardii LT02 was deposited at the China Center for Type Culture Collection (CCTCC) on March 30, 2026, at Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2026547. The fermentation process includes a first fermentation stage and a second fermentation stage; First fermentation stage: Inoculate the seed culture medium with 8-12% of the seed culture solution. Maintain a facultative anaerobic environment at 30-35℃, pH 5.0-5.5, and a rotation speed of 50-100 rpm, with nitrogen purging to ensure proper anaerobic conditions. Continuously culture *Lactobacillus hessei* for 20-24 hours, observing the OD500 concentration. 600 >6.5; Second fermentation stage: Adjust the pH to 4.2-4.8 to induce acid stress, and add 0.05% pyridoxal phosphate to the fermentation broth. Intermittently add sodium L-glutamate and glucose, and control the concentration of sodium L-glutamate to 35-45 g / L and the concentration of glucose to 4-6 g / L. Online product removal stage: When the γ-aminobutyric acid in the fermentation broth is ≥80 g / L, the membrane separation system is started. The membrane separation system connected to the fermenter is started to separate the γ-aminobutyric acid and the bacterial cells in the fermentation broth online. The permeate containing γ-aminobutyric acid is removed from the fermentation system, while the retained bacterial cells are recycled back to the fermenter to continue fermentation. The fermentation stage and the online product removal stage are carried out in synergy. By removing the product in situ, the feedback inhibition of γ-aminobutyric acid on glutamate decarboxylase is relieved, thereby achieving the continuous and efficient synthesis of γ-aminobutyric acid.

2. The method for preparing high-yield γ-aminobutyric acid (GABA) Lactobacillus schlegelii by fermentation as described in claim 1, characterized in that, In the second fermentation stage, the intermittent feeding consisted of adding 800 g / L L-glutamate sodium solution and 500 g / L glucose solution every 1.5 h.

3. The method for preparing high-yield γ-aminobutyric acid (GABA) Lactobacillus schlegelii by fermentation as described in claim 1, characterized in that, The membrane separation system is a 0.22 μm food-grade sterile hollow fiber membrane separation system. When in use, the trapped bacteria are recycled back to the fermenter for continued fermentation, and the permeate is collected.

4. The method for preparing high-yield γ-aminobutyric acid (GABA) Lactobacillus schlegelii by fermentation as described in claim 3, characterized in that, When the permeate volume reaches 80% of the original fermentation broth volume, filtration is stopped, and sterile fermentation supplement medium is added simultaneously to maintain the stability of the fermenter volume. 0.05% pyridoxal phosphate is also added to the fermentation broth.

5. The method for preparing high-yield γ-aminobutyric acid (GABA) Lactobacillus schlegelii by fermentation as described in claim 4, characterized in that, Fermentation was continued with intermittent addition of monosodium glutamate (MSG) and glucose. The concentration of MSG in the fermentation broth was maintained at 35-45 g / L and the concentration of glucose was maintained at 4-6 g / L. Fermentation was stopped when the cumulative yield of γ-aminobutyric acid (GABA) reached more than 360 g / L.

6. The method for preparing high-yield γ-aminobutyric acid (GABA) Lactobacillus schlegelii by fermentation as described in claim 5, characterized in that, It also includes purifying the permeate and the fermentation broth in the tank, the purification steps of which include: Ultrafiltration: Combine the permeate with the fermentation broth in the tank, and pass the liquid through a food-grade spiral wound ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The operating pressure is 0.2-0.3 MPa and the temperature is 25-35℃. Collect the ultrafiltration permeate. Decolorization: Add 0.5%-2% activated carbon to the ultrafiltration permeate, stir at 50-60℃ for 30-60 min to decolorize, first filter through plate and frame filter to coarsely remove activated carbon, and then filter through a 0.45 μm filter cartridge to obtain a decolorized and clear solution containing GABA. Ion exchange: After adjusting the pH of the decolorized clarified solution to 5.5-6.5, it is passed through a 001×7 type strong acid cation exchange resin column at a flow rate of 1-2 BV / h; the GABA adsorbed on the resin is eluted with 0.5 mol / L ammonia solution at a flow rate of 1 BV / h to obtain the GABA eluent. Concentration and crystallization: The eluent is concentrated and then anhydrous ethanol is added for low-temperature crystallization; Drying: Collect the crystals and dry them to obtain γ-aminobutyric acid product with a purity greater than 99%.

7. The method for preparing high-yield γ-aminobutyric acid (GABA) Lactobacillus schlegelii by fermentation as described in claim 6, characterized in that, The crystallization conditions are as follows: the eluent containing γ-aminobutyric acid is concentrated under reduced pressure at 50-60℃ to 300-500 g / L, cooled to 10-15℃, and anhydrous ethanol is added with stirring until the ethanol volume fraction is 30%-50%. The mixture is then allowed to stand at 4℃ for 8-24 hours to crystallize.

8. The method for preparing high-yield γ-aminobutyric acid (GABA) Lactobacillus schlegelii by fermentation as described in claim 1, characterized in that, The seed culture medium was obtained by inoculating Lactobacillus hessei LT02 into the seed culture medium and culturing it on a shaker at a temperature of 30-35°C for 16-24 hours.

9. The method for preparing high-yield γ-aminobutyric acid (GABA) Lactobacillus schlegelii by fermentation as described in claim 8, characterized in that, The seed culture medium, fermentation culture medium, and fermentation supplement culture medium are all food-grade culture media. The seed culture medium includes: glucose 18-22 g / L, peptone 8-12 g / L, beef extract powder 8-12 g / L, yeast powder 4-6 g / L, potassium dihydrogen phosphate 1-3 g / L, triammonium citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.2 g / L, manganese sulfate 0.04-0.06 g / L, and Tween 80 1-2 mL / L. The fermentation medium consists of: glucose 18-22 g / L, L-glutamate sodium 9-11 g / L, peptone 9-11 g / L, beef extract powder 7-10 g / L, yeast powder 3-5 g / L, corn steep liquor powder 13-16 g / L, dipotassium hydrogen phosphate 2-3 g / L, diammonium hydrogen citrate 2-3 g / L, sodium acetate 4-5 g / L, magnesium sulfate 0.2-0.3 g / L, manganese sulfate 0.03-0.05 g / L, and Tween 80 1-2 mL / L. The fermentation supplement medium comprises: peptone 4-6 g / L, yeast extract 2-3 g / L, potassium dihydrogen phosphate 1-3 g / L, triammonium citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.2 g / L, and manganese sulfate 0.04-0.06 g / L.

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