A method for producing food-grade gamma-aminobutyric acid using lactobacillus brevis

Abstract

The application discloses a method for efficiently producing food-grade gamma-aminobutyric acid (GABA) by Lactobacillus brevis. The method comprehensively considers fermentation and whole-cell transformation two sections, and adopts a global optimization method to improve the total yield of GABA. Under the optimal conditions, the yield of GABA is greater than 180 g / L after 36 h of fermentation, the yield of GABA is greater than 300 g / L after 6 h of whole-cell transformation, and the substrate conversion rates of the two sections are both greater than 99%, namely, the production cycle of GABA by single fermentation and transformation is 42 h, and the yield is greater than 480 g / L. The application can greatly improve the efficiency of Lactobacillus brevis in producing GABA, and has the advantages of simplicity, easy operation, easy separation of products, and good integration with the existing fermentation process.

Description

Technical Field

[0001] This invention relates to a method for the efficient production of "food-grade" γ-aminobutyric acid (GABA) by Lactobacillus brevis, belonging to the field of bioengineering technology. Background Technology

[0002] GABA is a four-carbon non-protein amino acid widely found in nature, distributed in animals, plants, and microorganisms. It has effects such as lowering blood pressure, improving liver and kidney function, regulating hormone secretion, and delaying aging. It has wide applications in the medical, cosmetic, and animal feed industries.

[0003] The main methods for producing GABA include chemical synthesis, plant enrichment, microbial fermentation, and whole-cell (or enzymatic) conversion. Chemical synthesis yields relatively high output, but the process is energy-intensive, has poor safety, high cost, and pollutes the environment. The resulting GABA is not "food grade" and cannot be used in the food and pharmaceutical industries. Plant enrichment is under mild conditions, but the yield of GABA is low, making it unsuitable for extraction and purification. GABA-rich plants are often used as functional raw materials, such as "Kilmi" developed by the China National Rice Research Institute. Microbial fermentation produces high-yield GABA, but it has disadvantages such as a long fermentation cycle, complex culture medium, and difficulties in separation and purification. Whole-cell (or enzymatic) conversion offers advantages such as high yield, mild reaction conditions, short cycle, and convenient separation and purification. In particular, whole-cell conversion avoids complex enzyme purification processes, reducing costs and improving conversion stability, and allows for multiple conversion cycles, making its advantages even more pronounced.

[0004] Currently, the main microorganisms used in microbial fermentation and whole-cell transformation are *Escherichia coli*, *Corynebacterium glutamicum*, and lactic acid bacteria. *E. coli* itself has very low GAD activity; therefore, the *E. coli* strains used for production are genetically engineered strains with enhanced GAD activity. Furthermore, because *E. coli* produces endotoxins, the GABA produced poses certain safety risks. Therefore, engineered *E. coli* strains are often used to produce feed-grade GABA. *Corynebacterium glutamicum* lacks a GAD system; therefore, producing GABA using *Corynebacterium glutamicum* fermentation requires genetic engineering to integrate an exogenous GAD system to modify the strain. The advantage of this type of genetically engineered strain is that it does not require the addition of the substrate glutamate or glutamate salt when fermenting GABA from glucose. Of course, GABA produced using genetically engineered strains has potential risks and cannot be used as a "food-grade" product. Lactic acid bacteria are recognized as food-grade safe strains, but the National Health and Family Planning Commission's new food ingredient catalog stipulates that only GABA produced by *Lactobacillus brevis* and *Lactobacillus hessei* can be used as a new food ingredient. Other lactic acid bacteria fermenting to produce GABA are only suitable for developing lactic acid bacteria fermented products, such as yogurt containing GABA, and cannot isolate and extract GABA for use as a new food ingredient.

[0005] Our research group previously screened a GABA-producing *Lactobacillus brevis* strain. This strain was not genetically engineered; rather, its GABA production capacity was enhanced through combined mutagenesis and process optimization, achieving a competitive level of "food-grade" GABA production. Previous studies on GABA production from *Lactobacillus brevis* often optimized either the microbial fermentation process or the whole-cell transformation process separately. However, in reality, the fermentation process not only produces a GABA-rich fermentation broth but also generates a large number of bacterial cells, which can be collected and used for whole-cell transformation to produce GABA. Therefore, comprehensively considering the fermentation and whole-cell transformation processes of *Lactobacillus brevis* for global optimization and efficient GABA production is of great significance for promoting the industrial-scale production of food and pharmaceutical-grade GABA. Summary of the Invention

[0006] The purpose of this invention is to provide a new method for the efficient production of "food-grade" GABA using Lactobacillus brevis.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution: A novel method for the efficient production of "food-grade" GABA using Lactobacillus brevis includes seed culture, fermentation culture, wet cell preparation, and whole-cell transformation using Lactobacillus brevis as the production strain.

[0008] The *Lactobacillus brevis* used is described in: A method for preparing γ-aminobutyric acid by biotransformation. Application No.: 201510626277.X, Authorization Announcement Date: February 23, 2018.

[0009] The fermentation culture conditions are as follows: static culture at 37 °C for 12–48 h.

[0010] The fermentation culture medium consisted of: glucose 10–100 g / L, yeast extract 10–50 g / L, Tween 80 2 mL / L, and a total of 2.04 mol / L of monosodium glutamate (MSG) and L-glutamic acid. Specifically, MSG was 0–50 g / L and L-glutamic acid was 300–257 g / L. A typical composition was: glucose 50 g / L, yeast extract 30 g / L, Tween 80 2 mL / L, MSG 20 g / L, and L-glutamic acid 283 g / L.

[0011] The conditions for whole-cell transformation are 40–50 °C and 180 r / min for 4–12 h.

[0012] The whole-cell transformation system consists of: 30-80 g / L wet bacterial cells, 250-650 g / L L-glutamic acid, 0.1 mmol / L pyridoxal 5'-phosphate, and 1 mL / L Tween 80.

[0013] The wet bacterial cells referred to are obtained by centrifuging the fermentation broth at 4°C and 6000 r / min for 10 min after the fermentation culture is completed, washing it twice with physiological saline, and then centrifuging it under the same conditions to obtain wet bacterial cells.

[0014] The method for producing food-grade γ-aminobutyric acid using Lactobacillus brevis disclosed in this invention has the following advantages compared to existing technologies: This invention establishes a method for the efficient production of "food-grade" GABA from *Lactobacillus brevis*. When optimizing the carbon and nitrogen source concentrations, substrate concentrations, and fermentation time during fermentation, not only is the GABA yield considered, but also the cell mass and GAD activity required for subsequent conversion. When optimizing the substrate concentration and conversion time required for whole-cell conversion, the focus is on GABA conversion yield and conversion rate. Under optimal conditions, fermentation for 36 hours yields more than 180 g / L of GABA, and whole-cell conversion for 6 hours yields more than 300 g / L of GABA, with substrate conversion rates exceeding 99% in both stages. This means a single fermentation and conversion cycle for GABA production is 42 hours, with a yield exceeding 480 g / L. This invention significantly improves the efficiency of GABA production from *Lactobacillus brevis*, and has the advantages of being simple, easy to operate, and allowing for easy product separation, and it can be well integrated with existing fermentation processes.

[0015] This invention primarily addresses the problem of separately optimizing the fermentation and conversion stages in microbial GABA production, neglecting the inherent connection between the two stages. It focuses on examining the GABA yield, cell mass, and GAD enzyme activity after fermentation, both of which are closely related to the subsequent conversion stage and thus affect the GABA yield. The main challenge lies in considering the fermentation and conversion stages holistically in the design, which necessitates changing all optimization conditions previously applied to individual fermentation or conversion processes. Attached Figure Description

[0016] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0017] Figure 1 The effect of glucose concentration on GABA yield during fermentation; Figure 2 The effect of glucose concentration on cell growth during fermentation; Figure 3 GAD activity of cells after 36 h of fermentation at different glucose concentrations; Figure 4 The effect of yeast concentration on GABA yield during fermentation; Figure 5 The effect of yeast concentration on cell growth during fermentation; Figure 6GAD activity of cells after 36 h of fermentation under different yeast powder concentrations; Figure 7 The effect of the ratio of L-glutamate monosodium glutamate to glutamate on cell growth; Figure 8 The effect of the ratio of L-glutamate monosodium glutamate to glutamate on GABA production; Figure 9 GAD activity of cells after 36 h of fermentation under different ratios of L-glutamate monosodium glutamate and glutamate; Figure 10 The effect of L-glutamate concentration on GABA production during whole-cell transformation; Figure 11 The effect of L-glutamate concentration on conversion rate during whole-cell transformation. Detailed Implementation

[0018] The embodiments of the present invention are described in detail below. The solutions described in these embodiments are not limited to the present invention. Those skilled in the art can make improvements and variations based on the spirit of the present invention, and all such improvements and variations should be considered within the scope of the present invention. The scope and essence of the present invention are defined by the claims. Unless otherwise specified, the reagents used in this invention are commercially available products, and DNFB refers to 2,4-dinitrofluorobenzene.

[0019] General notes: Slant culture medium: peptone 5 g / L, yeast extract 20 g / L, glucose 5 g / L, ammonium sulfate 0.05 g / L, pH 4.5, agar 20 g / L.

[0020] Seed culture medium: 5 g / L peptone, 20 g / L yeast extract, 5 g / L glucose, 0.05 g / L ammonium sulfate, pH 4.5.

[0021] The above culture medium was sterilized at 115–121°C for 15–20 min (glucose needs to be sterilized separately and then mixed with other components in the culture medium).

[0022] Preparation of seed culture: Use an inoculation loop to scrape one loop of bacterial culture from the bacteria on the slant culture medium into the seed culture medium, and incubate at 37℃ for 12 h to activate two generations.

[0023] Determination of growth: Take 1 mL of bacterial culture after a certain period of time, then dilute the bacterial culture 10 to 20 times, and measure it at a wavelength of 600 nm using a UV-Vis spectrophotometer.

[0024] GAD relative enzyme activity assay: A certain amount of wet bacterial cells as crude enzyme was reacted in a mixed reaction solution of 0.2 mol / L sodium acetate solution, 50.0 g / L L-glutamate sodium, and pH 4.8. The reaction was carried out at 180 r / min for 30 min at 40℃, followed by boiling for 5 min to terminate the reaction. The amount of γ-aminobutyric acid generated after the reaction was determined by high performance liquid chromatography. GAD enzyme activity (U) is defined as the number of cells required to produce 1 μg of GABA in 1 min. The relative enzyme activity of GAD was calculated by defining the experimental group with the highest GAD activity within the same group as 100%, and calculating the relative GAD activity percentage of the others based on the highest GAD activity.

[0025] Determination of GABA content Sample pretreatment: The fermentation or conversion broth was centrifuged at 8000 r / min for 5 min, and the supernatant was collected. After diluting 70 times with distilled water, 10 μL was transferred to a 1.5 mL centrifuge tube. 100 μL of derivatization buffer (4.2 g sodium carbonate diluted to 100 mL with ultrapure water), 0.1 mL of derivatizing agent DNFB (2,4-dinitrofluorobenzene) diluted to 50 mL with acetonitrile, and 790 μL of volume buffer (3.4 g dipotassium hydrogen phosphate dissolved in 145.5 mL of 0.1 mol / L sodium hydroxide solution, diluted to 1000 mL with ultrapure water) were added sequentially. The mixture was shaken to mix and incubated at 60 ℃ for 1 h. After the reaction, the mixture was filtered through a 0.22 μm cellulose acetate membrane, and the GABA yield was determined by high-performance liquid chromatography (HPLC).

[0026] High-performance liquid chromatography (HPLC) conditions: A C18 column was used; mobile phase A was water:acetonitrile = 1:1; mobile phase B was sodium acetate solution (4.1 g sodium acetate diluted to 1 L of ultrapure water, pH adjusted to 6.2 with glacial acetic acid) with gradient elution; flow rate: 0.8 mL / min; injection volume: 10 μL; UV detection wavelength: 360 nm; column temperature: 30℃. The gradient elution program was: 0–2 min, 16%–30% A; 2–4 min, 30%–34% A; 4–9 min, 34%–43% A; 9–19 min, 43%–55% A; 19–21 min, 55%–16% A; 21–23 min, 16% A.

[0027] Example 1 The specific steps are as follows: Fermentation culture bases with different glucose concentrations were prepared in 250 mL Erlenmeyer flasks, and seed culture was inoculated at a rate of 10%. The flasks were then incubated at 37 °C for 48 h (all experiments were repeated 3 times, and the results are the average of the 3 times).

[0028] Fermentation medium: glucose 10-100 g / L, yeast extract 20 g / L, monosodium glutamate 20 g / L, glutamic acid 283 g / L, Tween-80 2 mL / L.

[0029] The GABA production and cell biomass during fermentation, as well as the GAD activity in the cells after 36 h of fermentation, were measured. The results are shown in [Table missing]. Figures 1-3 As shown in the figure, regardless of the glucose concentration, the cell biomass reached its maximum at 36 h of fermentation, while the GABA yield showed no significant difference between 36 h and 48 h. Therefore, 36 h is the optimal fermentation time. At this time, the GAD activity in the cells decreased with increasing glucose concentration. When the glucose concentration was 50 g / L, the relative activity reached 97%. When the glucose concentration was 10 or 30 g / L, although the relative GAD activity was higher, the cell biomass and GABA yield were significantly lower than when the glucose concentration was 50 g / L. Therefore, a glucose addition of 50 g / L was determined to be the optimal condition, at which the fermentation GABA yield was 171 g / L, and the OD... 600 It reached 14, with a relative GAD activity of 97%.

[0030] Example 2 The specific steps are as follows: Fermentation cultures with different yeast powder concentrations were prepared in 250 mL Erlenmeyer flasks. Seed culture was inoculated at a rate of 10% and cultured statically at 37 °C for 48 h (all experiments were repeated 3 times, and the results are the average of the 3 times).

[0031] Fermentation medium: glucose 50 g / L, yeast powder 10-50 g / L, monosodium glutamate 20 g / L, glutamic acid 283 g / L, Tween-80 2 mL / L.

[0032] The GABA production and cell biomass during fermentation, as well as the GAD activity in the cells after 36 h of fermentation, were measured. The results are shown in [Table missing]. Figures 4-6 As shown in the figure, the yeast powder addition of 30–50 g / L had little effect on GABA production during fermentation. After 36 h of fermentation, GABA production tended to stabilize and no longer increased significantly. Lower yeast powder concentrations required a longer time to reach maximum biomass, and there was no significant difference in OD values ​​after 36 h of fermentation with yeast powder additions of 20–50 g / L. Therefore, 36 h of fermentation was deemed optimal. At this time, GAD activity in the cells exhibited a saddle-shaped curve with increasing yeast powder concentration, reaching a peak at a yeast powder concentration of 30 g / L. Considering that cell biomass and GABA production were also high at a yeast powder concentration of 30 g / L, a yeast powder addition of 30 g / L was determined to be the optimal condition, at which point the fermentation GABA production was 184 g / L, and the OD value was [not specified in the original text]. 600It reached 13.58, with a relative GAD activity of 100%.

[0033] Example 3 The specific steps are as follows: Fermentation cultures with different combinations of L-glutamic acid and L-glutamate sodium concentrations (Table 1) were prepared in 250 mL Erlenmeyer flasks. Seed culture was inoculated at a rate of 10% and cultured statically at 30 °C for 48 h (all experiments were repeated 3 times, and the results are the average of the 3 times).

[0034] Table 1 Different combinations of L-glutamate monosodium glutamate and L-glutamic acid The GABA production and cell biomass during fermentation, as well as the GAD activity in the cells after 36 h of fermentation, were measured. The results are shown in [Table missing]. Figures 7-9 As shown in the figure, combination 2 can significantly improve the cell growth rate and GABA production in the early stage of fermentation. When the L-glutamate sodium concentration exceeds 20 g / L, it does not accelerate the growth of the strain or increase the GABA concentration. Moreover, the relative activity of GAD in the cells after 36 h of fermentation decreases. Therefore, combination 2 is the optimal combination.

[0035] Example 4 The specific steps are as follows: The seed culture was inoculated at a rate of 10% into a 250 mL Erlenmeyer flask containing 100 mL of fermentation medium. After static incubation at 37 °C for 36 h, the precipitate was obtained by centrifugation at 6000 r / min for 10 min at 4 °C. The precipitate was washed twice with physiological saline and centrifuged under the same conditions to obtain wet cells for whole-cell transformation experiments.

[0036] The whole-cell transformation system consisted of: 30 g / L wet bacterial cells, 250–650 g / L L-glutamic acid, 0.1 mmol / L pyridoxal 5'-phosphate, and 1 mL / L Tween-80. Transformation was performed at 40 °C and 180 rpm for 12 h. (All experiments were repeated three times, and the results are the average of the three trials.) The yield and conversion rate of GABA in the conversion solution were detected, and the results are shown in [the table below]. Figures 10-11 As the concentration of L-glutamic acid increases, the yield of GABA after conversion also increases. However, when the L-glutamic acid concentration is between 450 and 650 g / L, there is no significant difference in GABA yield within 8 hours of conversion. Furthermore, when the L-glutamic acid concentration is between 250 and 450 g / L, no L-glutamic acid remains after conversion in just 6 hours. Therefore, the optimal concentration of L-glutamic acid in the conversion system is 450 g / L, at which point the GABA yield can reach 302 g / L after 6 hours of conversion, achieving a conversion rate of 100%.

[0037] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for synergistically coupling fermentation and conversion in a two-stage process to produce food-grade γ-aminobutyric acid, characterized in that, This method uses *Lactobacillus brevis* as the production strain for seed culture, fermentation culture, wet cell preparation, and whole-cell transformation. The fermentation medium consists of: glucose 10–100 g / L, yeast extract 10–50 g / L, Tween 80 2 mL / L, and a total of 2.04 mol / L of monosodium glutamate (MSG) and L-glutamic acid (L-glutamic acid); wherein MSG is 0–50 g / L and L-glutamic acid is 300–257 g / L. Wet cell preparation involves centrifuging the fermentation broth at 4°C and 6000 r / min for 10 min after fermentation to obtain a precipitate, washing it twice with physiological saline, and then centrifuging again under the same conditions. The whole-cell transformation system consists of: wet cells 30–80 g / L, L-glutamic acid 250–650 g / L, pyridoxal 5'-phosphate 0.1 mmol / L, and Tween 80 1 mL / L. The whole-cell transformation process is carried out at 40–50°C and 180 r / min for 4–12 minutes. h; The fermentation culture conditions are static culture at 37℃ for 12-48 hours.

2. The method for producing food-grade γ-aminobutyric acid by synergistic coupling of fermentation and conversion in two stages as described in claim 1, wherein the fermentation medium consists of 50 g / L glucose, 30 g / L yeast extract, 2 mL / L Tween 80, 20 g / L sodium L-glutamate, and 283 g / L L-glutamic acid.

Images