CN117946946A - Lactobacillus brevis for producing gamma-aminobutyric acid and application thereof - Google Patents
Lactobacillus brevis for producing gamma-aminobutyric acid and application thereof Download PDFInfo
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- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title claims abstract description 114
- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 68
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 title claims abstract description 46
- 240000001929 Lactobacillus brevis Species 0.000 title claims abstract description 42
- 235000013957 Lactobacillus brevis Nutrition 0.000 title claims abstract description 38
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 238000000855 fermentation Methods 0.000 claims description 51
- 230000004151 fermentation Effects 0.000 claims description 51
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 35
- 229940073490 sodium glutamate Drugs 0.000 claims description 34
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 33
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 239000002609 medium Substances 0.000 claims description 18
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
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- 238000000034 method Methods 0.000 claims description 16
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- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 11
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biology, and provides lactobacillus brevis Lactobacillus brevis CE701 capable of efficiently producing gamma-aminobutyric acid, and the number is CGMCC 24975. The lactobacillus brevis disclosed by the invention can be used as a probiotic with high biological safety and can be used as a potential production strain of food-grade gamma-aminobutyric acid.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to lactobacillus brevis for producing gamma-aminobutyric acid by efficiently converting sodium glutamate and application thereof.
Background
Gamma-aminobutyric acid (gamma-aminobutyric acid, GABA) is a naturally occurring non-protein four-carbon amino acid that is widely found in plants, animals and microorganisms. Gamma-aminobutyric acid is an important inhibitory neurotransmitter in mammals and has the effects of lowering blood pressure, reducing blood ammonia, promoting urination, improving sleep, promoting mental tranquility and resisting anxiety. Recent studies have also found that gamma-aminobutyric acid can effectively promote insulin secretion while inhibiting the activities of alpha-amylase and alpha-glucosidase, thereby contributing to improvement of physiological conditions of diabetics. Meanwhile, the gamma-aminobutyric acid also has the functions of promoting ethanol metabolism, promoting wound healing, improving liver and kidney functions, promoting reproduction, inhibiting cancer cell growth and the like. In addition, the gamma-aminobutyric acid also has a higher application prospect in the adjuvant treatment of epilepsy, parkinson, schizophrenia, insomnia, depression, autonomic nerve disorder and other nervous system diseases. Therefore, the gamma-aminobutyric acid has wide application prospect in the fields of medicines and functional foods. This has led to an increased worldwide demand for food grade gamma-aminobutyric acid, especially consumer demand for food and beverage, which is a major factor in driving the growth of the gamma-aminobutyric acid market.
Although gamma-aminobutyric acid can be prepared by a chemical method, gamma-aminobutyric acid synthesized by the method has great health safety hazards such as generation of toxic byproducts, and the like, so the chemically synthesized gamma-aminobutyric acid is forbidden to be used as a food additive. The microbial fermentation method takes L-glutamic acid or MSG as raw material, and the biological preparation of gamma-aminobutyric acid is realized after alpha-decarboxylation is catalyzed by intracellular glutamate decarboxylase of the microorganism, thus having great attraction and industrial production prospect. The production of food grade gamma-aminobutyric acid by probiotics has become a consensus. The health committee of China approves gamma-aminobutyric acid as a new resource food in the No. 12 document of 2009, and meanwhile, food grade gamma-aminobutyric acid is also required to be prepared by fermenting L-sodium glutamate (MSG) serving as a raw material through lactobacillus shii or lactobacillus brevis.
The research shows that the synthesis capacity of gamma-aminobutyric acid of lactobacillus brevis strains from different sources is quite different. Lactobacillus brevis FPA 3709 isolated from soymilk by Ko et al had a gamma aminobutyric acid yield of only 5.42 g/L. Lactobacillus brevis NPS-QW-145 screened by Wu et al can produce 25.83 g/L gamma-aminobutyric acid in MRS medium. And the yield of gamma-aminobutyric acid of lactobacillus brevis RK03 is even as high as 41.25 g/L.
Therefore, the lactobacillus brevis with excellent gamma-aminobutyric acid production capacity is determined to be important for green and efficient preparation of food-grade gamma-aminobutyric acid.
Disclosure of Invention
The invention aims to provide a lactobacillus brevis from pickle and a method for producing gamma-aminobutyric acid by using the lactobacillus brevis for efficiently preparing food-grade gamma-aminobutyric acid.
In a first aspect, the invention provides a strain of lactobacillus brevis (Lactobacillus brevis) CE701 for producing gamma-aminobutyric acid, wherein the preservation number of the lactobacillus brevis is CGMCC No.24975.
The structure of the 16S rDNA sequence of the lactobacillus brevis is shown as SEQ ID NO. 1.
In a second aspect, the invention provides a method for producing gamma-aminobutyric acid, comprising converting sodium glutamate into gamma-aminobutyric acid using lactobacillus brevis CE701 of the invention.
Preferably, the method specifically comprises the following steps:
step a, performing primary activation culture on lactobacillus brevis CE701 until the OD600 reaches 2.0-3.0;
step b, performing secondary activation culture on the lactobacillus brevis CE701 until the OD600 reaches 2.0-3.0;
Step c, in the case of containing sodium glutamate, lactobacillus brevis CE701 is subjected to fermentation culture, and the synthesis of gamma-aminobutyric acid is completed in the process.
In a preferred embodiment, the seed medium used for the primary activation culture is MRS medium, and the culture temperature is 25-35 ℃, preferably 30 ℃, and the culture time is 16-20h.
In a preferred embodiment, the seed medium used in the secondary activation culture is an aqueous medium containing an absorbable carbon source, a nitrogen source and an inorganic salt containing sulfate. Optionally, the medium may also include suitable minerals, metals, and other nutritional components; the carbon source is selected from one or more of glucose, fructose, sucrose, galactose, dextrin, glycerol, starch, syrup and molasses; the nitrogen source is selected from one or more of yeast powder, peptone, beef extract, corn steep liquor and casein; the inorganic salt containing sulfate is selected from one or more of MgSO4、FeSO4、MnSO4、Na2SO4、ZnSO4、K2SO4、CaSO4; the suitable minerals, metals and other nutritional ingredients are selected from one or more of sodium acetate, sodium chloride, sodium glutamate. Preferably, the medium components include glucose, yeast powder, peptone, sodium acetate, sodium chloride, mgSO 4·7H2O、FeSO4·7H2 O and MnSO 4·4H2 O; further preferred the seed medium comprises: 20.0g/L glucose, 10.0g/L yeast powder, 5.0g/L peptone, 2.0g/L sodium acetate 、0.01g/L NaCl、0.02g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.01g/L MnSO4·4H2O; to facilitate the growth of bacteria in the seed stage and to increase the production of gamma-aminobutyric acid, sodium glutamate is added to the secondary activation medium, preferably at a concentration of 10-30g/L, for example 20g/L sodium glutamate.
The initial pH value of the step b) is 6.8, the culture temperature is 25-35 ℃, preferably 30 ℃, and the culture time is 20-24 hours; the primary activation culture is inoculated in a proportion of 0.1 to 0.5%, preferably 0.1% by volume for secondary activation.
In a preferred embodiment, in step c, the fermentation medium used in the fermentation culture is an aqueous medium comprising an absorbable carbon source, a nitrogen source and an inorganic salt comprising sulphur sulphate. Optionally, the medium may also include suitable minerals, metals, and other nutritional components; the carbon source is selected from one or more of glucose, fructose, sucrose, galactose, dextrin, glycerol, starch, syrup and molasses; the nitrogen source is selected from one or more of yeast powder, peptone, beef extract, corn steep liquor and casein; the sulfate-containing inorganic salt is selected from one or more of MgSO4、FeSO4、MnSO4、Na2SO4、ZnSO4、K2SO4、CaSO4; the minerals, metals and other nutritional components are selected from one or more of sodium acetate and sodium chloride. Preferably, the medium components include glucose, yeast powder, peptone, sodium acetate, sodium chloride, mgSO 4·7H2O、FeSO4·7H2 O and MnSO 4·4H2 O; further preferably, the fermentation medium comprises: 50.0g/L glucose, 15.0g/L yeast powder, 5.0g/L peptone, 3.0g/L sodium acetate 、0.01g/L NaCl、0.03g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.02g/L MnSO4·4H2O.
Further, the fermentation culture conditions in the step c are as follows: the pH value is 4.0-6.0, preferably 4.4-4.8; the temperature is 20-40 ℃, preferably 35-40 ℃; fermenting for 48-120h; in the fermentation culture, the concentration of sodium glutamate is 60-120g/L, preferably 110-120 g/L. Preferably, in step c, the secondary activator is inoculated at a ratio of 1% to 12%, preferably 8% to 12%, most preferably 10% by volume for fermentation culture.
In one embodiment, the fermentation conditions of step c are: the inoculation amount is 10%, the fermentation temperature is 40 ℃, the pH=4.4, the concentration of sodium glutamate is 120g/L, and the fermentation time is 96-108 h.
The fermentation culture of the invention activates the strain in dormant state step by step, and then restores the activity of the strain, finally the obtained seed liquid with good activity is fermented and cultured, and the conversion process from sodium glutamate as substrate to gamma-aminobutyric acid is synchronously carried out in the growth process of the strain.
In a third aspect, there is provided a composition comprising lactobacillus brevis of the invention. Preferably, the composition is a microbial inoculum and further comprises other auxiliary materials required for preparing the microbial inoculum.
In a fourth aspect, there is provided the use of a lactobacillus brevis or composition of the invention in the production of gamma-aminobutyric acid.
The Lactobacillus brevis (Lactobacillus brevis) CE701 is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is North Star Xilu No.1, 3 in the Korean region of Beijing, the preservation number is CGMCC No.24975, and the preservation time is 2022, 5, 26 days.
The beneficial effects of the invention are as follows: the lactobacillus brevis Lactobacillus brevis CE CGMCC 24975 provided by the invention has the capability of catalyzing sodium glutamate to synthesize gamma-aminobutyric acid. After the fermentation conditions are optimized, the gamma-aminobutyric acid yield of the strain is increased along with the increase of the sodium glutamate concentration (the sodium glutamate concentration is 60-120 g/L), the conversion efficiency is 98-99%, and the final yield is 72.46 g/L. The lactobacillus brevis for producing the gamma-aminobutyric acid and the application thereof provided by the invention have wide application prospects in the aspect of industrial production of food-grade gamma-aminobutyric acid.
Drawings
Fig. 1: shaking the bottle and re-screening the lactobacillus.
Fig. 2: single colony and cell morphology of strain CE 701.
Fig. 3: 16S rDNA phylogenetic tree of strain CE 701.
Fig. 4: the strain CE701 was fermented to prepare an optimum inoculum size result map of GABA.
Fig. 5: and (3) preparing an optimal fermentation pH value result graph of GABA by fermenting the strain CE 701.
Fig. 6: and (3) preparing a result diagram of the optimal fermentation temperature value of GABA by fermenting the strain CE 701.
Fig. 7: results for strain CE701 producing gamma-aminobutyric acid at different sodium glutamate concentrations.
Detailed Description
The present invention will be further illustrated by the following examples, but is not limited thereto.
Unless otherwise indicated, the reagents and apparatus used in the examples below are conventional in the art and are commercially available. The methods used are conventional, and the person skilled in the art will be able to know without any doubt how to carry out the described solutions and obtain the corresponding results, based on the description of the implementation.
Example 1: acquisition of lactobacillus brevis:
1.1 preparation for experiment
Sampling time: 2021 month 12.
Place of purchase of the pickle sample: the northeast Jintang vegetable market (117 DEG 17'N,39 DEG 2'E) at the intersection of the Nanshan road and Beijing street in the Nanshan region of Tianjin, city.
MRS medium: 10.0g/L casein peptone, 10.0g/L beef extract, 5.0g/L yeast extract, 5.0g/L glucose, 5.0g/L sodium acetate, and diamine citrate 2.0g/L,Tween 80 1.0 g/L,K2HPO4 2.0 g/L,MgSO4·7H2O 0.2 g/L,MnSO4·H2O 0.05 g/L,121℃ for sterilization 20 min.
Plate screening medium: caCO 3 (20.0 g/L) and agar (20 g/L) were added to MRS medium, and then sterilized at 121℃for 20 minutes.
Shake flask screening medium: after adding sodium glutamate (20.0 g/L) to MRS medium, it was sterilized at 121℃for 20min.
1.2 Screening of Lactobacillus brevis
1.2.1 Flat plate Primary Screen
After the solid-liquid coexisting pickled Chinese cabbage sample is crushed and homogenized, 200 mu L of the homogenized liquid is inoculated into 5mL of MRS medium, and the pickled Chinese cabbage sample is cultured for 24 hours at 30 ℃ and 200rpm to enrich microorganisms in the pickled Chinese cabbage sample. Then, the culture broth was diluted with a gradient such as sterile physiological saline (dilution gradient 10 -1 to 10 -7), the dilution (100. Mu.L) at a gradient 10 -5 to 1 0-7 was spread on the screening plate, and incubated at 30℃for 48-72 hours. The individual colonies of the hydrolytic circle on the plate were observed and recorded as lactic acid bacteria microorganisms.
1.2.2 Shaking flask re-screening verification
100 Strains of lactobacillus with the diameter ratio of the transparent ring to the colony diameter being more than 5 are selected and inoculated in a shake flask screening culture medium, and are subjected to standing fermentation for 48 hours at 30 ℃. Subsequently, the supernatant product of 8 strains of lactic acid bacteria having the best growth potential was measured by high performance liquid chromatography, and as shown in FIG. 1, the strain having the highest GABA yield was selected therefrom for the next study and designated as CE701.
1.3 Identification of strains
1.3.1 Morphological characteristics of strains
After the strain CE701 was activated, it was diluted and spread on a MRS solid plate, and cultured upside down at 30℃for 48 hours, and it was observed that the single colony on the surface of the medium had a diameter of about 1mm and was flat and round, convex upward, milky white, opaque, smooth in surface and regular in edge, as shown in FIG. 2 a. The single colony growing on the plate is picked up by an inoculating loop to prepare a water immersed tablet, and the bacterial forms are observed under an electron microscope to be in a short bar shape, mainly arranged in pairs or in a splayed shape, and the cells do not move, have no spores and no flagellum, as shown in figure 2 b.
1.3.2 Identification of the physiological and Biochemical Properties of the Strain
The strain CE701 was identified by reference to the physiological and biochemical identification experiments of lactic acid bacteria in the "classification and identification of lactic acid bacteria and the" Bojie's bacteria identification Manual "and the results are shown in Table 1, and the strain CE701 was primarily considered to be of Lactobacillus.
TABLE 1 identification of physiological and biochemical characteristics of Strain CE701
Note that: "+" represents positive reaction and "-" represents negative reaction
1.3.3 Homology analysis and construction of phylogenetic Tree
The strain CE701 was submitted to the Souzhou Jin Wei intelligent technology Co., ltd for genome extraction and sequencing of 16S rDNA (sequencing primer 27FC:5'-AGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2), 1490RC:5'-GTTACCTTGTTACGACTTC-3' (SEQ ID NO: 3)), and the obtained 16S rDNA sequence had the structure shown in SEQ ID NO: 1. The strain CE701 was identified as Lactobacillus brevis Lactobacillus brevis by BLAST comparison with strain CE701, which was submitted to NCBI and found to have the highest homology (98.26%) to the 16S rDNA of Lb.breviscrain HDE-9, in combination with a phylogenetic tree (as shown in FIG. 3).
Lactobacillus brevis Lactobacillus brevis CE701,26 days of 2022 is deposited in China general microbiological culture Collection center, with a deposit number: CGMCC No.24975, the preservation address is: the institute of microbiology, national academy of sciences, china, the area North Star, west way 1, 3, beijing, chaoyang.
Example 2 Strain activation
(1) Primary seed culture: the deposited strain CE701 was inoculated into MRS medium and shake-cultured at 30℃and 220rpm for 20 hours until the OD600 reached 2.0.
(2) Secondary seed culture: the primary seed solution was inoculated into a 250mL shaking flask containing 50mL of the secondary seed culture solution at an inoculation volume ratio of 0.1%, and was subjected to stationary culture at 30℃for 24 hours. The secondary seed culture medium comprises the following components: 20.0g/L glucose, 10.0g/L yeast powder, 5.0g/L peptone, 2.0g/L sodium acetate 、0.02g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.01g/L MnSO4·4H2O、0.01g/L NaCl、20.0g/L sodium glutamate, and an initial pH value of 6.8; until an OD600 of 3.0 is reached.
Example 3 fermentation Process optimization of GABA production
(1) Fermentation medium composition: 50.0g/L glucose, 15.0g/L yeast powder, 5.0g/L peptone, 3.0g/L sodium acetate 、0.03g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.02g/L MnSO4·4H2O、0.01g/LNaCl.
(2) Main technical parameters and optimized screening
Basic technical parameters: 1% of inoculation amount; the fermentation temperature is 30 ℃; initial ph=5.0; fermentation time was 48h.
Based on the basic technical parameters, shake flask tests are carried out, and optimized screening is carried out on factors such as inoculum size, fermentation temperature, pH value and the like. Shake flask test method: the secondary seed solution is inoculated into a 1L shaking flask containing 200mL of fermentation medium according to the inoculation volume ratio of 1%, fermentation culture is carried out, and 60g/L sodium glutamate is added into the fermentation medium. And precisely measuring the GABA content in the fermentation supernatant by using a high performance liquid chromatography.
3.1 Effect of inoculum size
The effect of different inoculum sizes on the catalytic synthesis of GABA by the strain CE701 was examined, and the inoculum sizes were 1%, 2%, 4%, 6%, 8%, 10% and 12% respectively. The bacterial OD600 was 3.0. Other technical parameter conditions: the fermentation temperature is 30 ℃; initial ph=5.0; fermentation time was 48h. The results are shown in FIG. 4.
As can be seen from FIG. 4, GABA had higher yields at 1% to 12% of the inoculum size, with 8% to 12% of the inoculum size being better and the highest yield obtained at 10% of the inoculum size.
3.2 Effect of fermentation broth pH
The effect of different pH values on the catalytic synthesis of GABA by the strain CE701 was examined, and the pH values were increased from 3.6 to 0.4 to 8.0 in sequence. Other technical parameter conditions: 1% of inoculation amount; the fermentation temperature is 30 ℃; fermentation time was 48h. The results are shown in FIG. 5.
As can be seen from fig. 5, the pH value has a great influence on the catalytic synthesis of GABA by sodium glutamate, and the lactobacillus brevis engineering strain of the invention is better at pH 4.0-6.0, wherein the GABA yield is more prominent at ph=4.4-4.8, and the GABA fermentation conversion effect is best at ph=4.4.
3.3 Effect of fermentation temperature
The influence of different fermentation temperatures on the catalytic synthesis of GABA by the strain CE701 is examined, and the temperature is increased from 20 ℃ to 60 ℃ sequentially. Other technical parameter conditions: 1% of inoculation amount; ph=5.0; fermentation time was 48h. The results are shown in FIG. 6.
As can be seen from FIG. 6, the fermentation temperature has a certain influence on the fermentation and conversion of GABA by sodium glutamate, the effect of 20-40 ℃ is obviously better than that of more than 40 ℃, and the range of 35-40 ℃ is more remarkable, so that the yield is higher. Fermentation at 40℃is optimal.
3.4 Effect of sodium glutamate concentration
Under the optimal conditions, the influence of different sodium glutamate concentrations on the catalytic synthesis of GABA by the strain CE701 is examined, and the sodium glutamate concentration is sequentially increased from 60g/L to 120g/L. Other technical parameter conditions: the inoculation amount is 10%; the fermentation temperature is 40 ℃; ph=4.4.
The results are shown in FIG. 7. Wherein, under the condition of 60-120 g/L sodium glutamate, the GABA has obvious output; wherein the GABA yield is highest when the sodium glutamate is between 110 and 120 g/L. In addition, as can be seen from fig. 7, as the concentration of sodium glutamate increases, the time for the transformation effect of the strain CE701 of the present invention to reach the peak is prolonged, which may be due to the fact that the high concentration of sodium glutamate brings about a larger osmotic pressure, which affects the normal fermentation performance of the strain CE 701. Even though the excessive concentration of sodium glutamate (100 g/L) in the fermentation liquor can influence the GABA yield in the initial stage of fermentation, the subsequent fermentation process shows that the strain CE701 can still convert high-concentration sodium glutamate (120 g/L) into GABA (72.46 g/L), the conversion rate is as high as 99%, and the lactobacillus brevis engineering strain has good GABA fermentation performance and has great potential to relieve the current situation of GABA supply and demand in the market.
SEQ ID NO:1
GATCATCTGTACATCTTAGACGGCTGACTCCCGAAGGTTATCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTAGCCTCACGACTTCGCAACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTGAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGTCTTATCTCTAAGATTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACTAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAACCCTTGAACAGTTACTCTTCAAAGTTGTCTCTTTAACACAGAGTTTACGAGCCGAAACCTTCTTCACTCCACGCGGCATGCTCATCAGACTTCGTCCATTGTGAAGATTCCTACTGCTGCCTCCCGTAGAGAGTTTGGGCCGTG
Claims (10)
1. A strain of lactobacillus brevis (Lactobacillus brevis) CE701 for producing gamma-aminobutyric acid, wherein the preservation number of the lactobacillus brevis is CGMCC No.24975.
2. A method of producing gamma-aminobutyric acid comprising converting sodium glutamate to gamma-aminobutyric acid using lactobacillus brevis CE701 as claimed in claim 1.
3. The method according to claim 2, wherein the method comprises in particular the steps of:
step a, performing primary activation culture on a Lactobacillus brevis CE701 strain until the OD600 reaches 2.0-3.0;
step b, performing secondary activation culture on the lactobacillus brevis CE701 strain until the OD600 reaches 2.0-3.0;
and c, fermenting and culturing the lactobacillus brevis CE701 under the condition of containing sodium glutamate, and completing the synthesis of gamma-aminobutyric acid.
4. The method according to claim 3, wherein in step a, the seed medium used for the primary activation culture is MRS medium at a temperature of 25-35 ℃, preferably 30 ℃, for a period of 16-20 hours.
5. The method of claim 3, wherein the seed medium used in the secondary activation culture in step b is an aqueous medium comprising an absorbable carbon source, a nitrogen source, and an inorganic sulfate-containing salt, and optionally, minerals, metals, and other nutrients; preferably, the carbon source is selected from one or more of glucose, fructose, sucrose, galactose, dextrin, glycerol, starch, syrup and molasses; the nitrogen source is selected from one or more of yeast powder, peptone, beef extract, corn steep liquor and casein; the inorganic salt containing sulfate is selected from one or more of MgSO4、FeSO4、MnSO4、Na2SO4、ZnSO4、K2SO4、CaSO4; the suitable minerals, metals and other nutritional ingredients are selected from one or more of sodium acetate, sodium chloride and sodium glutamate; preferably, the medium components include glucose, yeast powder, peptone, sodium acetate, sodium chloride, mgSO 4·7H2O、FeSO4·7H2 O and MnSO 4·4H2 O; further preferably, the seed medium used for the secondary activation culture includes: more preferably, the seed culture medium used for the secondary activation culture further comprises 10-30g/L sodium glutamate, wherein the seed culture medium comprises 20.0g/L glucose, 10.0g/L yeast powder, 5.0g/L peptone and 2.0g/L sodium acetate 、0.01g/L NaCl、0.02g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.01g/L MnSO4·4H2O;.
6. The method according to claim 5, wherein step b has an initial pH of 6.8, a cultivation temperature of 25-35 ℃, preferably 30 ℃, and a cultivation time of 20-24 hours; the primary activation culture is inoculated at a ratio of 0.1 to 0.5%, preferably 0.1% by volume for secondary activation.
7. A process according to claim 3, wherein in step c the fermentation medium used in the fermentation culture is an aqueous medium containing an absorbable carbon source, nitrogen source and inorganic salts of sulphur-containing sulphate, and optionally also minerals, metals and other nutrients;
The carbon source is selected from one or more of glucose, fructose, sucrose, galactose, dextrin, glycerol, starch, syrup and molasses; the nitrogen source is selected from one or more of yeast powder, peptone, beef extract, corn steep liquor and casein; the sulfate-containing inorganic salt is selected from one or more of MgSO4、FeSO4、MnSO4、Na2SO4、ZnSO4、K2SO4、CaSO4; the mineral, metal and other nutritional ingredients are selected from one or more of sodium acetate and sodium chloride; preferably, the medium components include glucose, yeast powder, peptone, sodium acetate, sodium chloride, mgSO 4·7H2O、FeSO4·7H2 O and MnSO 4·4H2 O; further preferably, the fermentation medium comprises: 50.0g/L glucose, 15.0g/L yeast powder, 5.0g/L peptone, 3.0g/L sodium acetate 、0.01g/L NaCl、0.03g/L MgSO4·7H2O、0.01g/LFeSO4·7H2O、0.02g/L MnSO4·4H2O.
8. The method of claim 7, wherein in step c, the fermentation culture conditions are: the pH value is 4.0-6.0, preferably 4.4-4.8; the temperature is 20-40 ℃, preferably 35-40 ℃; fermenting for 48-120h; the concentration of the sodium glutamate is 60-120g/L, preferably 110-120 g/L; inoculating the secondary activator in a volume ratio of 1-12%, preferably 8-12%, most preferably 10% for fermentation culture;
Preferably, in step c, the fermentation conditions are: the inoculation amount is 10%, the fermentation temperature is 40 ℃, the pH=4.4, the concentration of sodium glutamate is 120g/L, and the fermentation time is 96-108 h.
9. A composition comprising the lactobacillus brevis CE701 of claim 1; preferably, the composition is a microbial inoculum and further comprises other auxiliary materials required for preparing the microbial inoculum.
10. Use of lactobacillus brevis CE701 as claimed in claim 1 in the production of gamma-aminobutyric acid.
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