A process for the whole cell catalyzed production of gamma-aminobutyric acid

Abstract

The application discloses a method for preparing gamma-aminobutyric acid by whole cell catalysis, which comprises the following steps: adding a substrate into a bacterial suspension of paralactobacillus, and converting the substrate into gamma-aminobutyric acid by whole cell catalysis under negative pressure. Further, the application preferably adds a surfactant in the process of conversion, and adopts six-pulley turbine straight-blade stirring. Through the improvements, the conversion efficiency of gamma-aminobutyric acid can be significantly improved. The conversion time of the application is only 4h-6h, the yield of gamma-aminobutyric acid can be up to more than 500g / L in a short time, and the conversion rate can be up to 99%, so that the time for synthesizing gamma-aminobutyric acid is greatly reduced, the production efficiency is significantly improved, and the application has far-reaching significance for industrial production.

Description

Technical Field

[0001] This invention relates to a method for preparing γ-aminobutyric acid (GABA), specifically a safe, efficient, high-conversion-efficiency, and high-yield whole-cell catalytic method for preparing GABA, belonging to the field of microbial fermentation engineering technology. Background Technology

[0002] Gamma-aminobutyric acid (GABA) is a naturally occurring functional non-protein amino acid with physiological functions such as improving brain function, lowering blood pressure, reducing anxiety, regulating hormone secretion, treating epilepsy, and protecting the liver and kidneys. It has broad application prospects and market demand in the fields of food, medicine and health care, beverage processing, and cosmetics.

[0003] The main methods for preparing γ-aminobutyric acid (GABA) include chemical synthesis, plant enrichment, microbial fermentation, and biotransformation. Chemical synthesis is subject to harsh conditions, high energy consumption, high cost, low yield, and poor safety, making it unsuitable for food and pharmaceutical applications. Plant enrichment results in low GABA content, making it unsuitable for large-scale production. Microbial fermentation has a long production cycle, low yield, and difficult subsequent separation and extraction, limiting its industrial application. Biotransformation, also known as whole-cell catalytic transformation, is increasingly favored due to its advantages of simple operation, mild conditions, high raw material utilization, high conversion rate, and low separation and purification costs.

[0004] Currently, microbial cell transformation is commonly used to produce GABA. For example, patent CN102174449B discloses a method for high-yield production of γ-aminobutyric acid (GABA), which involves strain isolation and screening, acridine orange-UV mutagenesis, and N... +A high-yield γ-aminobutyric acid (GABA) strain, *Lactobacillus brevis* TCCC (CGMCC NO. 3414), was obtained through mutagenesis. GABA production was then achieved through optimization of the fermentation medium and conditions, coupled with fermentation of growing cells and biotransformation of resting cells, yielding approximately 100 g / L. This process uses wild-type bacteria, but the yield is low. Patent CN111635898A discloses a glutamate decarboxylation mutant with significantly enhanced enzyme activity and its applications. The strain used is *Bacillus megaterium*. Based on this mutant, a recombinant engineered strain was constructed, and GABA was prepared using L-glutamate as a substrate via whole-cell catalysis. The molar conversion rate in the catalytic system was close to 100%, with no byproducts produced. The catalytic time was 6–12 h, and the highest GABA yield reached 625.6 g / L. However, the strains used in this process are engineered strains, which require the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) for induction during the cultivation stage, as well as the addition of antibiotics as screening markers. IPTG and antibiotics are toxic to humans, and there is a risk of IPTG and antibiotic residues in the products obtained through this process. Since γ-aminobutyric acid is mainly used in the food industry, any residues could harm consumers.

[0005] Patent CN109722402 A discloses a method for producing γ-aminobutyric acid (GABA) via whole-cell transformation. This method uses *Corynebacterium glutamicum* as the producing strain, heterologously expressing *Bacillus megaterium* glutamate decarboxylase, and obtaining protein-overexpressing *Corynebacterium glutamicum* somatic cells through high-density fermentation. Using L-glutamate or L-glutamate as a substrate, GABA is produced via whole-cell catalysis by *Corynebacterium glutamicum*, yielding 420–600 g / L of GABA after 5–24 hours of catalysis. This process also uses engineered bacteria, adding chloramphenicol as a selection marker for recombinant bacteria to the seed culture medium, and requires IPTG induction. All of these substances may remain in the final product, posing a risk to healthy individuals with long-term use.

[0006] Patent CN107475151B has screened a high-yield strain of *Lactobacillus parabrevis* HX12-19, which produces γ-aminobutyric acid (GABA). This strain was isolated and screened from food materials, exhibiting high safety and applicability in the food and other fields. It employs a segmented control method combining traditional fermentation and biotransformation, fully utilizing enzymes to obtain the GABA product, significantly improving production efficiency and reducing costs, achieving a fermentation yield of up to 235 g / L. While this patent represents a high level of achievement in the current industry for cell transformation using wild-type lactic acid bacteria, there is still potential for further improvement. Summary of the Invention

[0007] To address the problem of low conversion efficiency in the current whole-cell conversion method for producing γ-aminobutyric acid (GABA), this invention provides a whole-cell catalytic method for preparing GABA. This method has the advantages of whole-cell conversion, such as simple operation, mild conditions, high raw material utilization, high conversion rate, and low separation and purification cost. Furthermore, the method uses negative pressure during the conversion process, which improves the yield and conversion rate of GABA.

[0008] The specific technical solution of this invention is as follows: A method for whole-cell catalytic preparation of γ-aminobutyric acid (GABA) includes the following steps: adding a substrate to a suspension of Lactobacillus parabrevis, and converting the substrate into GABA under negative pressure via whole-cell catalysis.

[0009] Furthermore, in the above method, the whole-cell catalysis method refers to the biotransformation method mentioned in the background art, also known as the microbial cell transformation method or the whole-cell transformation method, which uses enzymes in microbial cells as catalysts to carry out chemical transformation to convert the substrate into γ-aminobutyric acid.

[0010] Furthermore, in the above method, the substrate can be monosodium glutamate (MSG) or glutamic acid. Using MSG as a substrate leads to excessively high sodium ion concentration and osmotic pressure in the conversion system, causing cell dehydration and rupture, resulting in the release of intracellular enzymes and a decrease in enzyme activity and conversion rate. Compared to MSG, glutamic acid as a substrate does not cause excessively high osmotic pressure in the conversion system, resulting in a higher conversion rate. Simultaneously, glutamic acid as a substrate does not produce inorganic salt byproducts, eliminating the need for desalination processes and improving production efficiency and process simplicity. Therefore, glutamic acid is preferred as the substrate.

[0011] Furthermore, a substrate is added to the bacterial suspension at a concentration of 500–900 g / L, more preferably 700–800 g / L.

[0012] Furthermore, in the method of this invention, whole-cell catalysis is carried out under negative pressure, that is, at a pressure below atmospheric pressure. Throughout the entire conversion process, the system is maintained under negative pressure, ranging from -0.1 MPa to -0.01 MPa. This negative pressure can be achieved through vacuuming. Vacuuming allows for more efficient conversion of the carbon dioxide byproduct generated in the decarboxylation reaction from the aqueous phase to the gas phase, which is then removed with the negative pressure. This eliminates product inhibition and facilitates a shift in the reaction equilibrium towards the synthesis of γ-aminobutyric acid (GABA). The separation of carbon dioxide also improves the solubility of the substrate, avoids the influence of carbon dioxide on the bacterial cells, and increases the conversion efficiency.

[0013] Furthermore, the *Lactobacillus parabrevis* suspension is obtained by resuspending *Lactobacillus parabrevis* cells obtained through fermentation in water. First, a *Lactobacillus parabrevis* fermentation broth is obtained by fermentation, then the wet *Lactobacillus parabrevis* cells are obtained by centrifugation, and then the cells are resuspended in water to obtain the suspension. The *Lactobacillus parabrevis* can be any *Lactobacillus parabrevis* strain reported in the prior art capable of catalyzing the conversion of the substrate monosodium glutamate or glutamate to γ-aminobutyric acid (GABA). The fermentation method for obtaining the *Lactobacillus parabrevis* cells can follow existing methods, generally involving culturing a seed culture to obtain a seed culture, which is then added to a fermentation medium for fermentation to obtain the fermentation broth.

[0014] Furthermore, the concentration of bacterial cells in the bacterial suspension is 5~10 g / L.

[0015] Preferably, the *Lactobacillus parabrevis* is *Lactobacillus parabrevis* HX12-19, with accession number CCTCCM2017307. This *Lactobacillus parabrevis* HX12-19 has been described in detail in patent CN107475151B, and its fermentation culture method can also be performed with reference to the method described in that patent. In practical use, the fermentation broth can be obtained using the method described in CN107475151B, and then the fermentation broth can be centrifuged to obtain the bacterial cells.

[0016] In a specific embodiment of the present invention, when obtaining *Lactobacillus parabrittlesis* cells through fermentation, fermentation is carried out until the cell density reaches OD0. 600 Fermentation was terminated when the pH was >2.5, yielding the fermentation broth. The fermentation broth was then centrifuged to obtain *Lactobacillus parabrittlesis* cells. During fermentation, the pH was adjusted to 4-6 with phosphate, and the culture temperature was 30 ± 2℃.

[0017] Furthermore, in whole-cell catalytic transformation, in addition to being carried out under negative pressure, there are also certain requirements for the transformation temperature and system pH. Preferably, the transformation temperature is 20~35℃. Preferably, the pH of the system is maintained at 4.7-5.0 during transformation. The pH can be adjusted by the dissolution of glutamate. The substrate glutamate is acidic, and its dissolution in water can maintain a certain pH in the system. After the dissolved glutamate is consumed, more glutamate continues to dissolve in, achieving a dynamic optimal pH balance.

[0018] Furthermore, the method of this invention has high conversion efficiency and requires little time; the entire conversion time is generally 4-6 hours, ending when the substrate L-glutamic acid or its sodium salt is completely consumed. Furthermore, the system is continuously stirred during the conversion process. The stirring can be performed using commonly used stirring methods in the prior art, such as paddle stirring, turbine stirring, etc., preferably turbine stirring, and more preferably a six-paddle turbine straight-blade stirring. The six-paddle turbine straight-blade stirring can effectively increase the radial and axial movement of the conversion liquid, thereby allowing the carbon dioxide contained in the conversion liquid to be released into the air in gaseous form under negative pressure with high efficiency, further improving the solubility of the substrate glutamic acid and the conversion efficiency.

[0019] Furthermore, during the conversion process, it is preferable to add a surfactant to the bacterial suspension to improve the conversion efficiency. The surfactant may be soybean oil, corn oil, polydimethylsiloxane, polyoxyethylene, sodium dodecylbenzenesulfonate, or a fatty acid salt (such as sodium palmitate).

[0020] Furthermore, the surfactant content in the bacterial suspension is 0.5~1g / L.

[0021] Furthermore, in one specific embodiment, a substrate is added to the bacterial suspension, stirred, and the conversion is carried out under a certain temperature and negative pressure. In another specific embodiment, a substrate is added to the bacterial suspension, and the mixture is stirred with a turbine while maintaining a certain temperature and negative pressure. In yet another specific embodiment, a substrate is added to the bacterial suspension, and the mixture is stirred with a six-bladed turbine while maintaining a certain temperature and negative pressure. In yet another specific embodiment, a substrate and a surfactant are added to the bacterial suspension, stirred, and the conversion is carried out under a certain temperature and negative pressure. In yet another specific embodiment, a substrate and a surfactant are added to the bacterial suspension, and the mixture is stirred with a turbine while maintaining a certain temperature and negative pressure. In yet another specific embodiment, a substrate and a surfactant are added to the bacterial suspension, and the mixture is stirred with a six-bladed turbine while maintaining a certain temperature and negative pressure.

[0022] The present invention has the following advantages: 1. This invention employs a negative pressure vacuum method during the conversion process, which more efficiently converts the carbon dioxide byproduct generated in the decarboxylation reaction from the aqueous phase to the gas phase and removes it with the negative pressure. This eliminates product inhibition and facilitates a shift in the reaction equilibrium towards the synthesis of γ-aminobutyric acid. Furthermore, excessively high carbon dioxide concentrations lead to high carbonic acid concentrations in the conversion system, and high concentrations of HCO3-... -This can cause an excessively high charge density in the cell membrane potential, which in turn obstructs the transmembrane transport pathway of the substrate, affecting the transformation efficiency. Ultimately, the bacterial cells will undergo morphological changes, metabolism will be inhibited, leading to cell fragmentation. The release of intracellular glutamate dehydrogenase will reduce enzyme activity. Therefore, negative pressure transformation can effectively solve this adverse effect.

[0023] 2. The substrate glutamic acid in the conversion system has low solubility in water. As carbon dioxide is removed during the negative pressure conversion process, its content decreases, thereby reducing the carbonic acid content in the solution and increasing the pH. The higher pH value can increase the solubility of the substrate glutamic acid. The increased substrate content causes the decarboxylation reaction to shift in the forward direction, improving the conversion efficiency of γ-aminobutyric acid and effectively solving the problem of glutamic acid solubility.

[0024] 3. In this invention, glutamic acid is preferably used as a substrate for conversion. Glutamic acid can effectively avoid the problem of high sodium ion concentration when using high concentrations of monosodium glutamate as a substrate, thus avoiding excessively high osmotic pressure in the conversion system, cell dehydration and rupture, and preventing the release of intracellular enzymes that would cause a significant decrease in enzyme activity and conversion rate. This effectively improves the conversion efficiency. At the same time, glutamic acid does not require an additional desalination process in industrial production, which can greatly improve production efficiency.

[0025] 4. The conversion system of the present invention preferably adopts a six-blade turbine straight-blade agitator, which can effectively increase the radial and axial movement of the conversion liquid, thereby enabling the carbon dioxide contained in the conversion liquid to be released into the air in gaseous form under negative pressure with high efficiency, further improving the solubility of the substrate glutamic acid and the conversion efficiency.

[0026] 5. In the transformation system of the present invention, surfactants are preferably added, which can improve cell membrane permeability, reduce cell surface tension, and efficiently realize the transmembrane transport of substrates to achieve intracellular transformation and extracellular secretion.

[0027] 6. The conversion time of this invention is only 4h~6h, and the yield of γ-aminobutyric acid can accumulate to more than 500g / L in a short time. The conversion rate can be nearly 100%, which greatly reduces the time for synthesizing γ-aminobutyric acid and significantly improves production efficiency. It has profound significance for industrial production. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments, but the present invention is not limited to the following embodiments. In the following embodiments, unless otherwise specified, the terms used are those defined in the prior art, and the operations described are all reported in the prior art.

[0029] Unless otherwise specified, all concentrations mentioned in the following embodiments are mass percentage concentrations.

[0030] In the following examples, Lactobacillus parabrevis HX12-19, reported in CN107475151B, is used to verify the advantages of the preparation method of the present invention, but this does not mean that the method of the present invention is only applicable to this one strain.

[0031] The preparation method of wet cells of *Lactobacillus parabrevis* HX12-19 is as follows: *Lactobacillus parabrevis* HX12-19 from test tube slant agar is inoculated into 100 ml of liquid seed culture medium. The culture is then incubated in a shake flask at 30°C for 15-20 h to obtain the seed culture. The seed culture is then inoculated into 3 L of fermentation medium at a 1% inoculation rate. The aeration rate is 0.1 vvm, the rotation speed is 20 rpm, and the culture is carried out at 30°C for 20 h. During this period, the pH is maintained between 4 and 6 by supplementing with phosphate. The bacterial OD... 600 Fermentation was stopped at 2.8 rpm to obtain fermentation broth. The fermentation broth was centrifuged at 8000 rpm to collect the cells. The supernatant was discarded to obtain 40g of wet cells.

[0032] The liquid seed culture medium was MRS medium; the fermentation medium formula was as follows: carbon source glucose 10g / L, nitrogen source yeast powder 10g / L, tryptone 10g / L, sodium acetate 5g / L, dipotassium hydrogen phosphate 2g / L, and trace elements magnesium sulfate 0.5g / L and manganese sulfate 0.3g / L.

[0033] Example 1 10g of wet Lactobacillus parabriquets HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 0.8g of sodium palmitate and 0.8kg of glutamic acid were added. The entire process was carried out using a six-blade turbine stirrer with a vacuum pump to maintain a pressure of -0.1MPa, a pH of 4.7-5.0, and a temperature of 30℃. The catalytic conversion was carried out for 5 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was determined by liquid chromatography, and the conversion rate was calculated.

[0034] Example 2 10g of wet Lactobacillus paragenesis HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 1.0g of sodium dodecylbenzenesulfonate and 0.7kg of glutamic acid were added. The entire process was carried out using a six-blade turbine stirrer with a vacuum pump to maintain a pressure of -0.01MPa, a pH of 4.7-5.0, and a temperature of 20℃. The catalytic conversion was carried out for 4 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was determined by liquid chromatography, and the conversion rate was calculated.

[0035] Example 3 10g of wet Lactobacillus parabrevis HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 0.8g of soybean oil and 0.8kg of glutamic acid were added. The entire process was carried out using a six-blade turbine straight-blade agitator. A vacuum pump was used to maintain a pressure of -0.01MPa in the reaction system, a pH of 4.7-5.0 during the conversion process, and a temperature of 30℃. The catalytic conversion was carried out for 5 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was detected by liquid chromatography, and the conversion rate was calculated.

[0036] Example 4 10g of wet Lactobacillus paragenesis HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 1.0g of sodium palmitate and 0.5kg of glutamic acid were added. The entire reaction was carried out using a six-blade turbine mixer with a vacuum pump to maintain a pressure of -0.1MPa, a pH of 4.7-5.0, and a temperature of 30℃. The catalytic conversion was carried out for 4 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was determined by liquid chromatography, and the conversion rate was calculated.

[0037] Example 5 10g of wet Lactobacillus paragenesis HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 1.0g of sodium palmitate and 0.9kg of glutamic acid were added. The entire reaction was carried out using a six-blade turbine mixer with a vacuum pump to maintain a pressure of -0.1MPa, a pH of 4.7-5.0, and a temperature of 30℃. The catalytic conversion was carried out for 6 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was determined by liquid chromatography, and the conversion rate was calculated.

[0038] Example 6 10g of wet Lactobacillus parabrevis HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 1.0g of sodium palmitate and 0.8kg of glutamic acid were added. The entire process was carried out using a six-blade turbine stirrer with a vacuum pump to maintain a pressure of -0.1MPa, a pH of 4.7-5.0, and a temperature of 30℃ in the reaction system. The catalytic conversion was carried out for 5 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was determined by liquid chromatography, and the conversion rate was calculated.

[0039] Example 7 10g of wet Lactobacillus parabriquets HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 0.5g of sodium palmitate and 0.8kg of glutamic acid were added. The entire reaction was carried out using a six-blade turbine mixer with a vacuum pump to maintain a pressure of -0.1MPa, a pH of 4.7-5.0, and a temperature of 30℃. The catalytic conversion was carried out for 5 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was determined by liquid chromatography, and the conversion rate was calculated.

[0040] Example 8 Take 5g of wet Lactobacillus paragenesis HX12-19 cells, place them in a 3L tank, and suspend them in 1L of purified water. Add 0.8g of sodium palmitate and 0.8kg of glutamic acid. The entire process is carried out using a six-blade turbine stirrer. A vacuum pump is used to maintain the pressure in the reaction system at -0.1MPa, the pH at 4.7-5.0, and the temperature at 30℃. Catalytic conversion is carried out for 5 hours. Then, the supernatant is collected by centrifugation. The supernatant is filtered through a 0.22μm filter, and the γ-aminobutyric acid content is detected by liquid chromatography. The conversion rate is calculated.

[0041] Example 9 10g of wet Lactobacillus paragenesis HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 0.8kg of glutamic acid was added. The entire reaction was carried out using a six-blade turbine with a straight blade. A vacuum pump was used to maintain the pressure in the reaction system at -0.1MPa, the pH at 4.7-5.0, and the temperature at 30℃. The catalytic conversion was carried out for 5 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was determined by liquid chromatography, and the conversion rate was calculated.

[0042] Example 10 10g of wet Lactobacillus parabriquets HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 0.8g of cocoyl diethanolamide and 0.8kg of glutamic acid were added. The entire reaction was carried out using a six-blade turbine mixer with a vacuum pump to maintain a pressure of -0.1MPa, a pH of 4.7-5.0, and a temperature of 30℃. The catalytic conversion was carried out for 5 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was determined by liquid chromatography, and the conversion rate was calculated.

[0043] Example 11 Take 10g of wet Lactobacillus parabrevis HX12-19 cells and place them in a 3L tank. Suspend them in 1L of purified water, add 0.8g of sodium palmitate and 0.8kg of glutamic acid. Use a four-blade propeller stirrer throughout the process. After stirring evenly, use a vacuum pump to evacuate the system and maintain the pressure at -0.1MPa, pH at 4.7-5.0, and temperature at 30℃. Catalytic conversion is carried out for 6 hours. Then, centrifuge to collect the supernatant. Filter the supernatant through a 0.22μm filter, detect the γ-aminobutyric acid content in the liquid phase, and calculate the conversion rate.

[0044] Example 12 10g of wet Lactobacillus paragenesis HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 0.4kg of sodium L-glutamate was added. The reaction was carried out using a six-blade turbine mixer. During the reaction, as the pH value increased, hydrochloric acid and 0.4kg of sodium L-glutamate were added intermittently. The pH value was controlled between 4.7 and 5.0 by adding hydrochloric acid. A vacuum pump was used to maintain the pressure in the reaction system at -0.1 MPa and the temperature at 30℃. The catalytic conversion was carried out for 5 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was detected by liquid chromatography, and the conversion rate was calculated.

[0045] Example 13 Take 10g of wet Lactobacillus paragenesis HX12-19 cells, place them in a 3L container, and suspend them in 1L of purified water. Add 0.8kg of glutamic acid. Stir the entire process using a four-blade propeller. After stirring evenly, use a vacuum pump to evacuate the system and maintain the pressure at -0.1MPa, pH at 4.7-5.0, and temperature at 30℃. Catalytic conversion is carried out for 5 hours. Then, centrifuge to collect the supernatant. Filter the supernatant through a 0.22μm filter, detect the γ-aminobutyric acid content using liquid chromatography, and calculate the conversion rate.

[0046] Example 14 10g of wet Lactobacillus paragenesis HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 0.4kg of monosodium glutamate was added. The reaction was carried out using a four-bladed propeller stirrer. During the reaction, as the pH value increased, hydrochloric acid and 0.4kg of L-glutamate were added intermittently. The pH value was controlled between 4.7 and 5.0 by adding hydrochloric acid. A vacuum pump was used to maintain the pressure in the reaction system at -0.1 MPa and the temperature at 30℃. The catalytic conversion was carried out for 5 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was detected by liquid chromatography, and the conversion rate was calculated.

[0047] Comparative Example 1 Take 10g of wet Lactobacillus paragenesis HX12-19 cells and place them in a 3L tank. Suspend them in 1L of purified water and add 0.8kg of glutamic acid. Stir the entire process using a four-blade rotary mixer. After stirring evenly, maintain normal pressure, pH 4.7-5.0, and temperature 30℃ for 6 hours of catalytic conversion. Then, centrifuge to collect the supernatant. Filter the supernatant through a 0.22μm filter cartridge, detect the γ-aminobutyric acid content in the liquid phase, and calculate the conversion rate.

[0048] Comparative Example 2 10g of wet Lactobacillus paragenesis HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 0.4kg of monosodium glutamate was added. The reaction was carried out using a four-bladed propeller agitator at atmospheric pressure. During the reaction, as the pH value increased, hydrochloric acid and 0.4kg of L-glutamate were added intermittently. The pH value was controlled between 4.7 and 5.0 by adding hydrochloric acid. The temperature was 30℃, and the catalytic conversion was carried out for 5 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was detected by liquid chromatography, and the conversion rate was calculated.

[0049] Comparative Example 3 10g of wet Lactobacillus paragenesis HX12-19 cells were placed in a 3L tank and suspended in 1L of purified water. 0.8kg of sodium glutamate was added. The entire reaction was carried out using a four-blade vortex mixer and a vacuum pump was used to maintain the pressure in the reaction system at -0.1MPa. The pH value was controlled between 4.7 and 5.0 by adding hydrochloric acid, and the temperature was 30℃. The catalytic conversion was carried out for 5 hours. The supernatant was then collected by centrifugation and filtered through a 0.22μm filter. The γ-aminobutyric acid content was detected by liquid chromatography, and the conversion rate was calculated.

[0050] Validation example: Detection of γ-aminobutyric acid and sodium glutamate 1. Instruments: A high-performance liquid chromatograph equipped with an ultraviolet detector, an autosampler, and a data processing system; Column: Hypersil ODS C18, 5μm, 4.6×250mm (column with equivalent or better separation performance); Analytical balance: accuracy 0.1 mg; Ultrasonic dissolving device; Microporous filter; Volumetric flask: 100mL; Needle filter (0.22μm).

[0051] 2. Reagents: Unless otherwise specified, all reagents used in the analysis were of analytical grade.

[0052] Methanol: chromatographic grade; Acetonitrile: chromatographic grade; o-phenylenedialdehyde (OPA); crystalline sodium acetate; glacial acetic acid; γ-aminobutyric acid standard: purity ≥99.0%; glutamic acid standard: purity ≥99.0%; boric acid; sodium hydroxide.

[0053] 3. Analysis Steps (1) Preparation of standard solutions Accurately weigh 0.5g of γ-aminobutyric acid or sodium glutamate standard, dissolve in water and bring to a final volume of 100mL. After mixing thoroughly, take 10mL and bring to a final volume of 100mL. Filter through a 0.22μm filter membrane and collect the filtrate as the standard solution.

[0054] (2) Preparation of sample solution Accurately measure 0.5 ml of the sample from the examples and comparative examples, dilute with water to 100 mL, mix well, take 10 mL and dilute to 100 mL, filter through a 0.22 μm filter membrane, collect the filtrate as the sample solution to be tested.

[0055] (3) Preparation of 0.4 mol / L borate buffer Accurately weigh 2.47g of boric acid, add about 80mL of water, adjust the pH to 10.2 with sodium hydroxide, and dilute to 100mL with water.

[0056] (4) Preparation of derivatizing reagents Weigh 0.1 g of o-phthalaldehyde (OPA), dissolve it in 1 mL of acetonitrile, add 130 μL of mercaptoethanol, and bring the volume to 10 mL with 0.4 mol / L borate buffer.

[0057] (5) Pre-column derivation Pre-column derivatization is performed using an autosampler. The pre-column derivatization steps are as follows: (6) Chromatographic analysis conditions mobile phase Phase A: Weigh 7.5g of crystalline sodium acetate, dissolve it in water and bring the volume to 1000 mL, add 5% acetic acid to adjust the pH to 7.20±0.02, filter, and set aside.

[0058] Phase B: Filtered with chromatographic grade acetonitrile, ready for use.

[0059] The mobile phase ratio during operation is: 75% phase A + 25% phase B (volume ratio).

[0060] Flow rate: 1.0 mL / min.

[0061] Detection wavelength: 338 nm.

[0062] Column temperature: 40℃.

[0063] (7) Sample determination Record the retention time and peak area of ​​the chromatographic peaks. The retention times of the sample and the standard solution should be consistent. Calculate the corresponding concentration of γ-aminobutyric acid or sodium glutamate using the external standard method.

[0064] (8) Calculation of results: The content of γ-aminobutyric acid or sodium glutamate in the sample is calculated using the following formula: In the formula: X1 — The content of γ-aminobutyric acid or sodium glutamate in the sample, in g / L; Ai — the peak area of ​​γ-aminobutyric acid or sodium glutamate in the sample; AS – Peak area of ​​γ-aminobutyric acid or sodium glutamate standards; CS – Concentration of γ-aminobutyric acid or sodium glutamate standard dilution, in g / L; n — the dilution factor of the sample; C – Purity of the standard; Conversion rate = (Amount of added sodium glutamate - Amount of remaining sodium glutamate) / Amount of added sodium glutamate * 100% (9) The experimental results are shown in Table 1 below: The results above show that adding a six-blade turbine straight-blade agitator and different surfactants to the negative pressure can further increase the yield, with a conversion rate of up to 99% and a cumulative concentration of γ-aminobutyric acid up to 604 g / L. This indicates that the present invention has a good effect on improving conversion efficiency.

Claims

1. A method for whole-cell catalytic preparation of γ-aminobutyric acid, characterized in that: The substrate was added to a suspension of Lactobacillus parabrevis, and the substrate was converted into γ-aminobutyric acid by whole-cell catalysis under negative pressure. The bacterial suspension also contains a surfactant, which is soybean oil, sodium dodecylbenzene sulfonate, or sodium palmitate. The entire conversion process is carried out under stirring, and the stirring method is a six-blade turbine straight-blade stirring. The substrate is glutamic acid.

2. The method according to claim 1, characterized in that: The initial concentration of the substrate in the bacterial suspension is 500–900 g / L.

3. The method according to claim 1, characterized in that: The initial concentration of the substrate in the bacterial suspension is 700–800 g / L.

4. The method according to claim 1, characterized in that: Throughout the entire conversion process, the system pressure was maintained at -0.1 MPa to -0.01 MPa.

5. The method according to claim 1, characterized in that: The bacterial suspension of Lactobacillus parabrevis is obtained by resuspending the fermented Lactobacillus parabrevis cells in water.

6. The method according to claim 5, characterized in that: The concentration of bacteria in the bacterial suspension is 5~10 g / L.

7. The method according to claim 1, characterized in that: The conversion temperature is 20~35℃; the conversion pH is 4.7-5.0; and the conversion time is 4-6h.

8. The method according to claim 1, characterized in that: The surfactant content in the bacterial suspension is 0.5~1g / L.

9. The method according to claim 1, characterized in that: The Lactobacillus parabrevis mentioned is Lactobacillus parabrevis HX12-19, with accession number CCTCC NO: M 2017307.

10. The method according to claim 1, characterized in that: When preparing Lactobacillus parabrevis cells by fermentation, fermentation is carried out until the cell density reaches OD. 600 Fermentation was stopped when the temperature reached >2.5℃, and the fermentation broth was obtained. The fermentation broth was then centrifuged to obtain Lactobacillus parabrevis cells.

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