CN1276087C - Process for biologically synthesizing gamma-amino butyric acid - Google Patents

Abstract

The present invention discloses a method for biologically synthesizing gamma-amino butyric acid. The present invention is characterized in that after lactobacillus brevis whose the preservation number is CGMCC NO. 1306 is activated by an agar slant culture medium, the actobacillus brevis is inoculated to a GYP seed culture medium or an MRS seed culture medium; after the lactobacillus brevis is cultured for 10 to 30 hours, the lactobacillus brevis whose the inoculum size is from 0.5% to 5% is inoculated to a GYP fermentation culture medium or an MRS fermentation culture medium; at the temperature of 25 DEG C to 35 DEG C, after the lactobacillus brevis is cultured for 48h to 120h without moving, fermentation liquor containing mycelia can be obtained; the mycelia are collected in a centrifugal separating mode; the centrifugated mycelia are washed by deionized water which is sterilized; 0.25 to 2g of wet mycelia are taken and are suspended in 15 to 50mL of a citric acid-disodium hydrogen phosphate buffering system, wherein the content of L-sodium glutamate is from 5mM to 60mM, and the reaction time is from 1 to 10 hours; after the reaction solution is centrifugated, solution containing the gamma-amino butyric acid is obtained. The method has the advantages of low production cost, moderate reaction condition, little environment pollution, simple method, easy operation, high productive efficiency, high product purity, and the lightening of subsequent separation and purification processes.

Description

A method for biosynthesizing gamma-aminobutyric acid

technical field

The invention relates to a method for biosynthesizing γ-aminobutyric acid.

Background technique

γ-Aminobutyric acid (GABA for short) is a non-protein amino acid with important physiological activity and an important neurotransmitter in the central nervous system. A large number of literature reports that GABA can lower blood pressure and cholesterol, reduce neuron activity, and prevent nerve cell overheating. It not only has the effect of calming the nerves and anti-depression, but also enhances brain function and long-term memory, increases growth hormone secretion, prevents obesity, and strengthens the liver. Kidney benefit, improve menopausal syndrome and other functions. With the continuous in-depth research on the physiological activity of GABA, more and more physiological functions of γ-aminobutyric acid have been discovered in the latest research. GABA has also received more and more attention. Although GABA exists widely in organisms, its content is very low in animals and plants, and its separation is very difficult. It needs to be synthesized. There are mainly chemical synthesis method and biosynthesis method for its synthetic preparation method. Compared with the chemical synthesis method, the biosynthesis of GABA utilizes the glutamic acid decarboxylase in the organism as a catalyst, and L-sodium glutamate (L-MSG) is used as a substrate to convert L-sodium glutamate (L- MSG) α-decarboxylation to produce γ-aminobutyric acid and CO ₂ , the main advantages are mild synthesis conditions, no need for expensive raw materials, less environmental pollution, and less energy consumption. One of the keys to biosynthesize GABA is to screen out excellent strains with high glutamic acid decarboxylase activity. It has been reported in the literature that Escherichia coli is usually used as the production strain for the biosynthesis of GABA, but there are hidden dangers in food safety and hygiene when using Escherichia coli as the production strain. Our laboratory screened out a lactic acid strain with high-efficiency biosynthesis of GABA. This paper reports a method for the biosynthesis of GABA.

Contents of the invention

The purpose of the present invention is to provide a method for biosynthesizing γ-aminobutyric acid.

The preservation number is: Lactobacillus brevis (Lactobacillus brevis) of CGMCC NO.1306, after being activated by agar slant medium, transferred to GYP seed medium or MRS seed medium, after culturing for 10-30 hours, with 0.5%-5 % of the inoculum was inoculated in GYP or MRS fermentation medium, and cultured at 25°C-35°C for 48h-120h to obtain the fermentation liquid containing bacteria, and the bacteria were collected by centrifugation; the centrifuged bacteria were then Wash with sterilized deionized water, take 0.25-2g of wet bacteria, suspend in 15-50mL citric acid-disodium hydrogen phosphate buffer system, the content of L-sodium glutamate is 5mM-60mM, and react for 1-10 hours, the reaction solution was centrifuged to obtain a solution containing γ-aminobutyric acid.

The preservation number is: Lactobacillus brevis (Lactobacillus brevis) of CGMCC NO.1306, which produces lactic acid during fermentation, appears in pairs or chains under the microscope, does not move, has no spores, small colonies, smooth surface; belongs to Gram-positive strain, facultatively anaerobic.

In the present invention, Lactobacillus brevis is used as the strain, and the growth of the thallus reaches a stable period, and the thallus is collected, catalyzed by whole cells, with L-sodium glutamate (L-MSG) as the substrate, and citric acid-disodium hydrogen phosphate As a buffer system, there is no other medium components such as other carbon and nitrogen sources, and the highly active glutamic acid decarboxylase in lactic acid bacteria is used to decarboxylate L-sodium glutamate (L-MSG) to produce γ-aminobutyric acid. GABA production reached 1 ~ 6g / L. The method has the advantages of low production cost, mild reaction conditions, little environmental pollution, simple method, easy operation, short period, high production efficiency, and high product purity, which reduces the subsequent separation and purification process.

Detailed ways

A. strain

Screened by this laboratory.

B. Medium

1000mL agar slant preservation medium: glucose 10g, yeast extract 10g, peptone 5g, sodium acetate 2g, MgSO ₄ 7H ₂ O 20mg, MnSO ₄ 4H ₂ O 1mg, NaCl 1mg, FeSO ₄ 7H ₂ O 1mg, agar 15g , pH 6.8.

1000mL GYP seed medium: glucose 10g, yeast extract 10g, peptone 5g, sodium acetate 2g, MgSO ₄ 7H ₂ O 20mg, MnSO ₄ 4H ₂ O 1mg, NaCl 1mg, FeSO ₄ 7H ₂ O 1mg, pH 6.6- 7.0.

1000mL GYP fermentation medium: glucose 10g, yeast extract 10g, peptone 5g, sodium acetate 2g, MgSO ₄ 7H ₂ O 20mg, MnSO ₄ 4H ₂ O 1mg, NaCl 1mg, FeSO ₄ 7H ₂ O 1mg, monohydrate L - Sodium Glutamate 10g, pH 6.6-7.0.

1000mL MRS seed medium: beef extract 10g, yeast extract 5g, peptone 10g, K ₂ HpO ₄ 2g, ammonium citrate 2g, glucose 20g, Tween-801mL, sodium acetate 5g, inorganic salt solution 5mL (MgSO ₄ 7H ₂ O, 11.5%; _MnSO4-2H2O , 2.4% ₎ , pH 6.6.

1000mL MRS fermentation medium: beef extract 10g, yeast extract 5g, peptone 10g, K ₂ HPO ₄ 2g, ammonium citrate 2g, glucose 20g, Tween-801mL, sodium acetate 5g, inorganic salt solution 5mL (MgSO ₄ 7H ₂ O, 11.5%; MnSO ₄ ·2H ₂ O, 2.4%), 10 g of sodium L-glutamate monohydrate, pH 6.6.

C. Preparation process

After the strain was activated on agar slant medium, it was transferred to GYP seed medium or MRS seed medium. After culturing for 10-30 hours, inoculate GYP or MRS fermentation medium with an inoculation amount of 0.5%-5%. Under static culture for 48h ~ 120h, the fermentation liquid containing bacteria is obtained, and the bacteria are collected by centrifugation (8000r/min, 5min). After centrifugation, the cells were washed with sterilized deionized water, and 0.25-2 g of wet cells were taken, suspended in 25 mL of citric acid-disodium hydrogen phosphate buffer system, and the content of L-sodium glutamate was 5 mM-100 mM , react for 1 to 10 hours, and centrifuge the reaction solution to obtain a solution containing γ-aminobutyric acid.

After the strain was activated on the agar slant medium, it was transferred to GYP seed medium or MRS seed medium. After culturing for 10-30 hours, it was inoculated with GYP or MRS fermentation medium with an inoculation amount of 0.5%-5%. Under static culture for 48h ~ 120h, the fermentation liquid containing bacteria is obtained, and the bacteria are collected by centrifugation (8000r/min, 5min). After centrifugation, the cells were washed with sterilized deionized water, and 0.25-2 g of wet cells were taken, suspended in 25 mL of citric acid-disodium hydrogen phosphate buffer system, and the content of L-sodium glutamate was 5 mM-100 mM , react for 1 to 10 hours, and centrifuge the reaction solution to obtain a solution containing γ-aminobutyric acid.

Example 1

After the strain was activated on the agar slant medium, it was transferred to the GYP seed medium for 24 hours. The seed culture liquid was inoculated in the GYP fermentation medium with 1% inoculation amount, and 1% of Sodium L-glutamate monohydrate, the liquid volume of the fermentation medium is 200mL/500mL, and it is left to culture for 60 hours to obtain the fermentation liquid containing bacteria, and the bacteria are collected by centrifugation (8000r/min, 5min). After centrifugation, the cells were washed with sterilized deionized water, and 0.5 g of wet cells were taken, suspended in 25 mL of citric acid-disodium hydrogen phosphate buffer system, and the content of L-sodium glutamate was 10 mM. Reaction 1 hours, the reaction solution was centrifuged and analyzed by high-performance liquid chromatography, and the content of γ-aminobutyric acid was about 1g/L.

Example 2

After the strain was activated on the agar slant medium, it was transferred to the GYP seed medium for 24 hours. The seed culture solution was inoculated into the GYP fermentation medium with 1% inoculation amount, and 1% of the fermented medium was added to the fermentation medium. Sodium L-glutamate monohydrate, the liquid volume of the fermentation medium is 200mL/500mL, and it is left to culture for 60 hours to obtain the fermentation liquid containing bacteria, and the bacteria are collected by centrifugation (8000r/min, 5min). After centrifugation, the cells were washed with sterilized deionized water, and 0.5 g of wet cells were taken, suspended in 25 mL of citric acid-disodium hydrogen phosphate buffer system, and the content of L-sodium glutamate was 20 mM. Reaction 2 hours, the reaction solution was centrifuged and analyzed by high performance liquid chromatography, and the content of γ-aminobutyric acid was about 2g/L.

Example 3

After the strain was activated on the agar slant medium, it was transferred to the GYP seed medium for 24 hours. The seed culture liquid was inoculated in the GYP fermentation medium with 1% inoculation amount, and 1% of Sodium L-glutamate monohydrate, the liquid volume of the fermentation medium is 200mL/500mL, and it is left to culture for 60 hours to obtain the fermentation liquid containing bacteria, and the bacteria are collected by centrifugation (8000r/min, 5min). After centrifugation, the cells were washed with sterilized deionized water, and 0.5 g of wet cells were taken, suspended in 25 mL of citric acid-disodium hydrogen phosphate buffer system, and the content of L-sodium glutamate was 40 mM. Reaction 3 hours, the reaction solution was centrifuged and analyzed by high performance liquid chromatography, and the content of γ-aminobutyric acid was about 4g/L.

Example 4

After the strain was activated on the agar slant medium, it was transferred to the GYP seed medium for 24 hours. The seed culture liquid was inoculated in the GYP fermentation medium with 1% inoculation amount, and 1% of Sodium L-glutamate monohydrate, the liquid volume of the fermentation medium is 200mL/500mL, and it is left to culture for 60 hours to obtain the fermentation liquid containing bacteria, and the bacteria are collected by centrifugation (8000r/min, 5min). After centrifugation, the bacteria were washed with sterilized deionized water, and 0.5 g of wet bacteria was taken, suspended in 25 mL of citric acid-disodium hydrogen phosphate buffer system, and the content of L-sodium glutamate was 60 mM. hours, the reaction solution was centrifuged and analyzed by high performance liquid chromatography, and the content of γ-aminobutyric acid was about 6g/L.

Claims (1)

1. the method for a biosynthesizing γ-An Jidingsuan, it is characterized in that, deposit number is: the short lactobacillus of CGMCCNO.1306 (Lactobacillus brevis), after the agar slant culture-medium activation, transfer in GYP seed culture medium or MRS seed culture medium, cultivate after 10～30 hours, inoculum size with 0.5%～5% is inoculated in GYP or the MRS fermention medium, leave standstill under 25 ℃～35 ℃ and cultivate 48h～120h, promptly get the fermented liquid of mycetome, the thalline centrifugation is collected; Thalline after centrifugal is again with the deionized water wash of the bacterium of going out, get 0.25～2g wet thallus, be suspended in citric acid-Sodium phosphate dibasic buffer system of 15～50mL, L-Sodium Glutamate content is 5mM～60mM, reacted the centrifugal solution that contains γ-An Jidingsuan that promptly gets of reaction solution 1～10 hour.