A strain of Lactobacillus hilarii GZ2 and its application in efficient synthesis of γ-aminobutyric acid

Abstract

The present invention discloses a strain of Lactobacillus hilgeni GZ2 and its application in the efficient synthesis of γ-aminobutyric acid. Lactobacillus hilgeni GZ2 is isolated from Baijiu Daqu and has a preservation number of CGMCC NO.26755. The present invention adopts the strain of Lactobacillus hilgeni GZ2, takes lactose as the only carbon source, does not add sodium glutamate, and can produce 230 mg / L γ-aminobutyric acid. Taking sodium glutamate as a substrate and adopting a multiple fermentation control strategy of controlling acid-flow and adding glucose carbon source, the ability of high production of γ-aminobutyric acid can be achieved, and the cumulative concentration of γ-aminobutyric acid is 151.7 g / L. Lactic acid products obtained by using Lactobacillus hilgeni GZ2, such as dairy products, sourdough and fermented juice, are all internationally recognized safe and healthy foods. The γ-aminobutyric acid produced by biological methods does not have safety risks and can reach food safety level.

Description

Technical Field

[0001] The invention belongs to the technical field of food microorganisms, and particularly relates to a strain of Lactobacillus hilgris GZ2 and application thereof in efficiently synthesizing gamma-aminobutyric acid. Background Art

[0002] The applications of γ-aminobutyric acid include: (1) Medical field: lowering blood pressure, treating epilepsy, anti-tumor, treating alcohol dependence, improving brain function, lipid metabolism, promoting mental stability, promoting memory, secretion of growth hormone, activation of kidney function, activation of liver function, etc.; (2) Food field: increasing food flavor, additives, etc.; (3) Cosmetic field: preventing skin aging, removing body odor, anti-fatigue, anti-oxidation; (4) Chemical field: 2-pyrrolidone, biodegradable bioplastic nylon 4 synthetic precursor, etc. At the same time, the strategy of developing and producing γ-aminobutyric acid is a very important issue for products to meet different fields, including not only high value-added food and medical fields, but also the chemical industry of bioplastics and biochemicals. Due to the wide range of applications of γ-aminobutyric acid, the market demand has greatly increased.

[0003] It is stipulated that monosodium glutamate is used as raw material and Lactobacillus hilgardii ( Lactobacillus hilgardii ) GABA produced by the steps of fermentation, heat sterilization, cooling, activated carbon treatment, filtration, addition of auxiliary materials, spray drying, etc. can be added to beverages, chocolate and chocolate products, candy, baked goods, puffed foods, etc. This means that chemically synthesized GABA as food or food raw material does not comply with food regulations and is a food additive of non-food substances. As people pay more attention to food safety, more and more researchers are focusing on the production of GABA using lactic acid bacteria. Therefore, it is very important to obtain safe and industrially valuable GABA-producing bacteria from both the regulatory and safety perspectives.

[0004] The biological method uses strains to use sodium glutamate as a substrate to prepare γ-aminobutyric acid, including biological fermentation and biotransformation. Although the yield of γ-aminobutyric acid can be significantly increased by adjusting the culture medium and optimizing the fermentation process, in the actual production process, the efficient production process on a laboratory scale cannot meet the needs of low cost and high profit for industrial production. Only when the production strain itself has a high yield can it have more practical production significance. Therefore, screening high-yield γ-aminobutyric acid strains that are efficient and then optimized by different means is still the basis for promoting the industrial application of γ-aminobutyric acid. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a strain of Lactobacillus hilgeni GZ2 and its application in the efficient synthesis of γ-aminobutyric acid. The present invention separates and screens lactic acid bacteria with high γ-aminobutyric acid production from white wine Daqu to obtain a strain of Lactobacillus hilgeni GZ2, which has a simple preparation method for producing γ-aminobutyric acid and broad industrial application prospects.

[0006] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:

[0007] A strain of Lactobacillus hilgeni GZ2, wherein the Lactobacillus hilgeni GZ2 is isolated from Baijiu Daqu, and is deposited in the China General Microbiological Collection Center (CGMCC) on March 6, 2023, with a deposit number of CGMCC NO.26755, and a taxonomic name of Lactobacillus hilgeni Lactobacillus hilgardii.

[0008] The Lactobacillus hilarii GZ2 is a milky white or light yellow colony with a colony diameter of 0.05±0.02 cm, a neat edge, Gram-positive, no spores, no movement, and oxygen-resistant.

[0009] As an improvement, the microbial agent comprises one or more combinations of living cells of Lactobacillus hilgeni GZ2, dried bacteria of Lactobacillus hilgeni GZ2, immobilized cells of Lactobacillus hilgeni GZ2, liquid agent of Lactobacillus hilgeni GZ2, or solid agent of Lactobacillus hilgeni GZ2.

[0010] The above-mentioned Lactobacillus hilarii GZ2 is derived from Baijiu Daqu and is separated by gradient dilution method, and the specific steps are as follows:

[0011] Step 1, medium preparation

[0012] MRS liquid medium, GYP liquid medium, MRS and GYP liquid medium supplemented with sodium glutamate;

[0013] Step 2: Place the liquor koji powder in sterile saline, absorb the bacterial suspension, dilute it with sterile saline, and then spread it on an MRS medium plate containing 1% (W / V) CaCO3 and 0.5% (W / V) sodium glutamate. Culture it anaerobically at 37°C for 48 hours. When milky white or light yellow colonies appear, they are preliminarily identified as lactic acid bacteria based on the hydrolysis circles that appear on the culture medium around the colonies.

[0014] Step 3, pick a single colony, further separate and purify it, and then transfer it to an MRS solid plate and culture it at 37°C for 48 hours;

[0015] Step 4, inoculate the seeds prepared from the colonies on the MRS plate in step 3 into a GYP liquid culture medium containing 50 g / L sodium glutamate at a 10% inoculation rate, and detect the content of γ-aminobutyric acid by high performance liquid chromatography to obtain Lactobacillus hirsutigensis GZ2 with high γ-aminobutyric acid production.

[0016] Application of the above-mentioned Lactobacillus hilgri GZ2 or microbial agent in fermentation to produce γ-aminobutyric acid.

[0017] As an improvement, sodium glutamate is not added to the reaction system of the application, and glucose or lactose is used as the carbon source to produce γ-aminobutyric acid.

[0018] As an improvement, sodium glutamate is added to the reaction system of the application, and glucose, xylose, lactose, fructose, sucrose, cellobiose, starch, xylan, and β-cyclodextrin are used as carbon sources. The strain can produce high levels of γ-aminobutyric acid, wherein the concentration of sodium glutamate is 50 g / L.

[0019] The application of the gamma-aminobutyric acid produced by the above fermentation in food or medicine. Beneficial Effects

[0020] Compared with the prior art, the present invention discloses a strain of Lactobacillus hilgeni GZ2 and its application in efficiently synthesizing gamma-aminobutyric acid, separates and obtains Lactobacillus hilgeni GZ2 with high gamma-aminobutyric acid production from white wine Daqu, and simultaneously prepares a fermentation liquid containing a high gamma-aminobutyric acid product. Lactobacillus hilgeni GZ2 comes from traditional lactic acid bacteria fermentation products, and lactic acid bacteria products are internationally recognized as the safest health food, so the gamma-aminobutyric acid produced by this method does not have any safety hazards and can reach a food safety level. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the colony characteristic diagram of Lactobacillus hilgardii GZ2;

[0022] Figure 2 This is a cell morphology characteristic diagram of Lactobacillus hilgridis GZ2;

[0023] Figure 3 This is the electrophoresis diagram of 16S rDNA amplification of Lactobacillus hilgardii GZ2;

[0024] Figure 4 Lactobacillus hilarii GZ2 produces γ-aminobutyric acid using different carbon sources;

[0025] Figure 5 The effect of sodium glutamate concentration on the production of γ-aminobutyric acid by Lactobacillus hilgardii GZ2;

[0026] Figure 6 A continuous feeding strategy was developed for the efficient synthesis of GABA by Lactobacillus hilgri GZ2. DETAILED DESCRIPTION

[0027] MRS liquid medium: glucose 2%, yeast extract 0.5%, peptone 1%, beef extract 1%, dipotassium hydrogen phosphate 0.2%, triammonium citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.058%, manganese sulfate 0.025%, Tween 80 1 mL, pH 6.2, all are mass-volume fractions. MRS solid medium is MRS liquid medium with 1.5% (W / V) agar added.

[0028] GYP liquid medium: glucose 1%, yeast extract 1%, peptone 0.5%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01%, sodium chloride 0.01%, all in mass-volume fraction. GYP solid slant medium is GYP liquid medium with 1.5% (W / V) agar added. GYP fermentation medium is GYP liquid medium with sodium glutamate added.

[0029] Example 1 Screening of Lactobacillus hilgarnii with high γ-aminobutyric acid production

[0030] Using the gradient dilution separation method, 0.5 g of liquor koji from different wineries was weighed and placed in 10 mL of MRS liquid medium containing 50 g / L sodium glutamate. The culture was kept at 37°C for 72 h. 0.1 mL of the bacterial suspension was taken and gradient dilution (10 -1 ~10 -5 ), take 0.1 mL of the solution with a concentration of 10 -4 , 10 -5 The bacterial suspension was spread on the MRS solid culture medium plate and incubated at 37°C for 48 h. A single colony with a calcium dissolution zone on the MRS plate was picked for enrichment and purification. The colony enriched on the MRS solid culture medium plate was inoculated into the GYP liquid culture medium containing 50 g / L sodium glutamate and incubated at 37°C for 72 h. The content of γ-aminobutyric acid was detected by HPLC.

[0031] The high-yield γ-aminobutyric acid strain GZ2 was screened for 16S rDNA identification. The bacterial liquid was cultured in GYP liquid medium at 37℃ for 24 hours, and the genomic DNA was extracted using conventional bacterial genome extraction methods.

[0032] 16S rDNA electrophoresis and sequencing results are as follows Figure 3 As shown in the figure, it can be seen that its length is 1452 bp. After Blast comparative analysis, the homology of 16S rDNA of Lactobacillus hilgeni GZ2 and Lactobacillus hilgeni is 99%. Therefore, it is identified as Lactobacillus hilgeni and named Lactobacillus hilgeni. Lactobacillus hilgardii .

[0033] Example 2 Method for preparing γ-aminobutyric acid using Lactobacillus hilgardii GZ2

[0034] 1. Lactobacillus hilarii GZ2 uses glucose or lactose as the sole carbon source to produce γ-aminobutyric acid

[0035] Pick an appropriate amount of Lactobacillus hilgeni GZ2 from the activated GYP solid slant medium, inoculate it in GYP liquid medium, and culture it at 37℃, 200 rpm for 24 h; take the 24 h seed culture solution, inoculate it in GYP liquid medium at 10% inoculation, and culture it at 37℃, 200 rpm for 15 h as fermentation seeds; put 10 mL GYP liquid medium in the test tube, the carbon sources are glucose and lactose, take the fermentation seeds that have been cultured for 15 h, and inoculate them in GYP liquid medium with different carbon sources at 10% inoculation, and culture them at 37℃, 200 rpm for 48 h. The fermentation broth was detected by high performance liquid chromatography. With glucose as the carbon source and without the addition of sodium glutamate, the γ-aminobutyric acid production of Lactobacillus hilgeni GZ2 was 680 mg / L, and with lactose as the carbon source and without the addition of sodium glutamate, the γ-aminobutyric acid production of Lactobacillus hilgeni GZ2 was 260 mg / L.

[0036] 2. Preparation of γ-aminobutyric acid by Lactobacillus hilarii GZ2 using different carbon sources

[0037] An appropriate amount of Lactobacillus hilarii GZ2 was picked from the activated GYP solid slant culture medium, inoculated into the GYP liquid culture medium, and cultured at 37°C, 200 rpm for 24 h; the seed culture solution of 24 h was taken, inoculated into the GYP liquid culture medium at a 10% inoculation rate, and cultured at 37°C, 200 rpm for 15 h as fermentation seeds; and inoculated into GYP liquid culture media with glucose, xylose, lactose, fructose, sucrose, cellobiose, starch, xylan, and β-cyclodextrin as carbon sources at a 10% inoculation rate, respectively, and 50 g / L sodium glutamate was added, and cultured at 37°C, 200 rpm for 72 h.

[0038] The results are as follows Figure 4 As shown, Lactobacillus hilgri GZ2 can produce γ-aminobutyric acid with the above carbon sources. When glucose is used as the carbon source, the highest γ-aminobutyric acid yield is 7.19 g / L.

[0039] 3. Preparation of γ-aminobutyric acid by Lactobacillus hilaris GZ2 using different concentrations of sodium glutamate

[0040] Pick an appropriate amount of Lactobacillus hilarii GZ2 from the activated GYP solid slant culture medium, inoculate it into GYP liquid culture medium, and culture it at 37°C, 200 rpm for 24 h; take the 24-hour seed culture solution, inoculate it into GYP liquid culture medium at 10% of the inoculation amount, culture it at 37°C, 200 rpm for 15 h as fermentation seeds, and inoculate it into GYP liquid culture medium containing 10-50 g / L sodium glutamate concentration at 10% of the inoculation amount, and culture it at 37°C, 200 rpm for 72 h.

[0041] The results are as follows Figure 5 As shown in the figure, with the increase of sodium glutamate concentration, the production of γ-aminobutyric acid gradually increased. When the concentration of sodium glutamate was 50 g / L, the production of γ-aminobutyric acid was 11.0 g / L.

[0042] 4. Synthesis of GABA by acid-controlled fed-batch fermentation using Lactobacillus hilgardii GZ2

[0043] The activated Lactobacillus hilarii GZ2 on the slant was inoculated into GYP liquid medium and cultured at 37℃ for 24 h to make a first-level seed culture solution. The first-level seed culture solution was inoculated at 10% in GYP liquid medium at 37℃ and 200 rpm for 15 h to make a second-level seed solution. The second-level seed solution was inoculated at 10% in GYP liquid medium containing 50 g / L sodium glutamate, 50 g / L glucose, pH 5, and a 5 L bioreactor was used to control the temperature at 37℃ and stir at 200 rpm. The pH was controlled at 5.0 with 2.5 mol / L sulfuric acid. Sodium glutamate at a concentration of 0.2 g / mL was used. Continuous flow feeding was performed from 24 h to 60 h, and the feeding rate was 1 mL / min. The fermentation was 72 h. Samples were taken every 6 h, and the content of γ-aminobutyric acid was analyzed by HPLC.

[0044] The results are as follows Figure 6 As shown, the concentration of γ-aminobutyric acid of Lactobacillus hilgardii GZ2 was 151.7 g / L at 72 h of fermentation.

[0045] Example 3 Preparation of dairy products, fruit juice drinks and sourdough containing γ-aminobutyric acid by fermentation with Lactobacillus hilgarnii GZ2

[0046] Pick an appropriate amount of Lactobacillus hilarii GZ2 from the activated GYP solid slant medium, inoculate it into the GYP liquid medium, and culture it at 37℃ for 24 hours; take the 24-hour seed culture solution, centrifuge it, remove the culture medium, resuspend it with physiological saline, and inoculate it into pure milk or pure fruit juice at a 10% inoculation rate, and culture it at 37℃ for 12 hours as fermentation seeds; 5 mL of pure milk or pure fruit juice was filled into 15 mL test tubes, and the fermentation seeds cultured for 12 hours were taken and inoculated at a 4% inoculation rate, and cultured at 37℃ for 48 hours. The fermentation broth was detected by high-performance liquid chromatography, and the content of γ-aminobutyric acid in the fermented milk was 30 mg / L, and the content of γ-aminobutyric acid in the fermented fruit juice was 500 mg / L.

[0047] Prepare Lactobacillus hilarii GZ2 into bacterial powder, resuspend it in warm water (30℃), and press 10 7 CFU / g, mixed evenly with wheat flour to make dough, and cultured at 37℃ for 48 hours. The fermented dough was used to prepare sourdough bread. The γ-aminobutyric acid content in sourdough bread was analyzed by HPLC, which could reach 50 mg / 100 g bread.

[0048] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for synthesizing GABA by acid-controlled fed-batch fermentation using Lactobacillus hilgardii GZ2, It is characterized in that The specific steps of the method are as follows: inoculating the activated Lactobacillus hilgardii GZ2 on the slant into a GYP liquid culture medium, culturing at 37°C for 24 h to prepare a primary seed culture solution, inoculating the primary seed culture solution at 10% in a GYP liquid culture medium at 37°C and 200 rpm for 15 h to prepare a secondary seed solution, inoculating the secondary seed solution at 10% in a GYP liquid culture medium containing 50 g / L sodium glutamate, 50 g / L glucose, and a pH of 5, using a 5 L bioreactor, controlling the temperature at 37°C, stirring at 200 rpm, controlling the pH to 5.0 with 2.5 mol / L sulfuric acid, using 0.2 g / mL sodium glutamate, performing continuous flow feeding at a feeding rate of 1 mL / min from 24 h to 60 h, and fermenting for 72 h; h sampling, and the content of γ-aminobutyric acid was analyzed by HPLC; wherein the Lactobacillus hilgeni GZ2 was deposited in the China General Microorganism Collection Center (CGMCC) on March 6, 2023, with a deposit number of CGMCC NO.26755, and a taxonomic name of Lactobacillus hilgeni Lactobacillus hilgardii .

2. The method according to claim 1, It is characterized in that The Lactobacillus hilarii GZ2 is a milky white or light yellow colony with a colony diameter of 0.05±0.02 cm, a neat edge, Gram-positive, no spores, no movement, and oxygen-resistant.

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