Screening and application of a high-yield γ-aminobutyric acid lactic acid bacteria

Abstract

The present invention belongs to the field of microbial fermentation technology, and particularly relates to the screening and application of a high-yield γ-aminobutyric acid lactic acid bacteria. The present invention utilizes the newly screened fermentation mucus lactobacillus SJM8 to produce a high content of γ-aminobutyric acid. By optimizing the fermentation process, the content of γ-aminobutyric acid produced by SJM8 is compared with that before optimization, and the γ-aminobutyric acid production is increased by 2.5 times, while the fermentation cycle is reduced, and the energy consumption is reduced while the γ-aminobutyric acid production is increased, thereby reducing the production cost.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to the screening and application of high-yield gamma-aminobutyric acid lactic acid bacteria. Background Art

[0002] γ-aminobutyric acid (GABA) is an amino acid that is widely found in vertebrates, plants, and microorganisms. It is a natural inhibitory neurotransmitter that plays an important role in the brain and central nervous system. It has anti-anxiety, anti-blood pressure, sleep-promoting, anti-epileptic, and anti-depressant effects. Today, there are three main pathways for the synthesis of GABA: one is the chemical synthesis method using chemical raw materials and certain reaction conditions; the second is the plant enrichment method using plants to enrich GABA; and the third is the use of microbial fermentation to produce metabolites of GABA. Among them, microbial fermentation produces GABA mainly through the process of glutamate decarboxylase (GAD) converting glutamate into GABA. Different microorganisms (especially lactic acid bacteria, yeast, and some Gram-negative bacteria) have been shown to have the ability to produce GABA, including lactic acid bacteria, yeast, and Clostridium cells. At present, the research on the production of GABA by microbial fermentation mainly focuses on the screening of superior strains, the optimization of fermentation processes, and their anti-inflammatory, sleep-inducing, and anti-fatigue effects. Some research progress has been made, but the following problems and challenges still exist: (1) Although many strains can produce GABA under experimental conditions, the GABA yield in large-scale industrial production is still low and has not yet met the requirements of commercial production. (2) In the actual fermentation process, the growth, metabolic pathways, and GABA yield of the strains are often affected by factors such as fermentation conditions and culture medium components, resulting in instability in the production process. (3) Strains with high GABA yields usually require higher culture costs, and the current fermentation process also requires higher investment, resulting in a high production cost of GABA, which restricts its large-scale application.

[0003] For example, the prior art CN117721033B discloses a strain of fermented mucus lactobacillus KS6, and the accumulation of GABA in the fermentation supernatant after 24h of stable period culture is 85.80pg / mL; the prior art CN117402768B discloses a strain of reuteri mucus lactobacillus KA1, and the accumulation of GABA is 32.24pg / mL; the prior art CN117695266A discloses that reuteri lactobacillus produces GABA through glutamate decarboxylation, which can induce cardioprotection; the prior art CN117603947A discloses bridge mucus lactobacillus JT3, and the accumulation of GABA in the fermentation supernatant is 43.61pg / mL; the prior art CN116814464B discloses fermented mucus lactobacillus JF5, and the accumulation of GABA after fermentation is 18.16pg / mL. Its GABA yield still needs to be improved to achieve industrial production.

[0004] In response to the above problems, with the ultimate goal of increasing GABA production, a strain of high-yield GABA was obtained through strain identification, screening and separation, the production process was optimized, the best process flow for high-yield GABA was determined, and GABA powder products were developed to provide technical support for achieving GABA production at an industrial level and broadening its application in the food field. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to propose the screening and application of a fermentative Lactobacillus mucus with high γ-aminobutyric acid production, and solves the problems of long fermentation cycle and low GABA industrialization level by screening and directional addition of conversion substrates and cofactors in the fermentation medium.

[0006] In order to achieve the above purpose, the following technical solutions are proposed:

[0007] In one aspect, the present invention provides a fermented mucus lactobacillus, wherein the fermented mucus lactobacillus is fermented mucus lactobacillus SJM8, which is classified as fermented mucus lactobacillus Limosilactobacillus fermentum , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 8, 2025, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 33359.

[0008] In a second aspect, the present invention provides an application of the microorganism in producing GABA.

[0009] Furthermore, the application is at least one of the following:

[0010] (1) Application in increasing the content of glutamate decarboxylase

[0011] (2) Application in increasing the content of γ-aminobutyric acid

[0012] In a third aspect, the present invention provides a method for preparing high-content γ-aminobutyric acid, characterized in that it comprises the following steps:

[0013] S1. Prepare fermentation medium using MRS medium, sodium L-glutamate and vitamin B6 as raw materials

[0014] S2. The fermented mucus lactobacillus was cultured in MRS medium until the viable count reached 1×10 8 CFU / mL or more, get activated strains

[0015] S3. Adding the activated strain in step S1 to the fermentation medium prepared in step S1 to ferment and produce high-content γ-aminobutyric acid

[0016] Furthermore, the fermentation medium in step S1 includes 15 g / L peptone, 6 g / L yeast extract, 25 g / L glucose, 5 g / L sodium succinate, 3 g / L sodium L-glutamate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 2 mmol / L vitamin B6, and pH 6.2.

[0017] Furthermore, according to the step of producing high-content GABA of the present invention, the content of γ-aminobutyric acid is higher than 10 g / L.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) The present invention screened and obtained a probiotic strain with the ability to increase GABA production, which was identified as Lactobacillus mucilaginosus (named SJM8).

[0020] (2) The present invention provides a method for producing high-content GABA using SJM8. The microorganism has excellent fermentation performance and can shorten the fermentation cycle while increasing the GABA yield.

[0021] (3) Compared with the existing methods for producing GABA, the method used in the present invention has a higher GABA yield (increased by 2.5 times). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 To screen 15 dominant lactic acid bacteria strains;

[0023] Figure 2 This is a plate image of the colony morphology of fermenting Lactobacillus mucilaginosus SJM8;

[0024] Figure 3 This is the molecular evolutionary tree of Lactobacillus mucilaginosus SJM8;

[0025] Figure 4 It is a single factor optimization diagram;

[0026] Figure 5 is the response surface test diagram;

[0027] Figure 6 Comparison of GABA content before and after optimization;

[0028] Figure 7 The antioxidant activity and cholesterol degradation ability of GABA. DETAILED DESCRIPTION

[0029] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0030] In the following examples, fermented mucus lactobacillus was derived from naturally fermented sauerkraut samples, and the isolated strains were deposited in the General Microbiological Center of the China National Microbiological Culture Collection Administration. The chemical reagents used were purchased from regular chemical reagent suppliers and were analytically pure.

[0031] Example 1 Isolation and purification of bacterial strains

[0032] The collected naturally fermented products such as soursop and sauerkraut were diluted 10 times in a clean bench with sterile saline, and 0.1 mL of the bacterial solution with an appropriate dilution multiple was evenly coated on the MRS solid medium and placed in a 30°C incubator for constant temperature culture for 12-48 h. Then, the culture base was taken out to pick a single colony with a diameter of 1-2 mm and a smooth surface under a sterile environment, and inoculated onto a new MRS solid medium by streaking, and repeated many times until a pure colony was isolated and purified. The screened microorganisms were diluted in a gradient with sterile saline and cultured at 30°C for 24 hours using an MRS solid culture base. Then, according to the size, color, gloss, and transparency of the colony, a single colony with a smooth surface and milky white color was picked, and the strain was purified by streaking on the MRS solid plate for 2-3 times, and then stored in a refrigerator at 4°C.

[0033] Example 2 Screening of dominant strains producing high levels of GABA

[0034] Prepare the fermentation medium according to 15 g / L peptone, 6 g / L yeast extract, 25 g / L glucose, 5 g / L sodium succinate, 3 g / L sodium L-glutamate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and pH 6.2. Place the medium in a 250 mL conical flask at a 20% inoculum amount, sterilize at 121°C for 15 min, and cool for use.

[0035] The activated 15 lactic acid bacteria were inoculated into the sterilized MRS liquid medium in the clean bench for 3-4 subcultures. When the number of viable bacteria reached 1×10 8 When the CFU / mL is above, prepare seed solution for later use.

[0036] The seed liquid of 15 lactic acid bacteria strains was inoculated into the fermentation medium at a rate of 5% for fermentation. The fermentation conditions were as follows: constant temperature and static culture at 30°C for 120 h, and the pH and OD were measured every 24 h. 600 The values ​​of iodine, GABA content, and 6 sample time points were measured, namely 0, 24, 48, 72, 96, and 120 h. The suitable fermentation strain was selected by comparing the results of multiple indexes.

[0037] Basic measurement indicators include:

[0038] pH value determination: using pH meter;

[0039] OD 600 : The absorbance value was measured at 600 nm using an enzyme-labeled instrument.

[0040] GABA content: GABA content was measured using a GABA content detection kit

[0041] The data were processed and analyzed using GraphPad Prism 8 software.

[0042] Analysis of measurement results: Figure 1 As shown, by measuring OD 600 , pH, and GABA content were used to screen the superior strains. With the extension of fermentation time, OD600 and GABA content showed a trend of first increasing and then maintaining stability, and pH showed a trend of first decreasing and then maintaining stability. However, the amount of GABA produced by different strains was different. The GABA content of M4, SJM8, L6 and M9 reached the highest at 72 h. As the fermentation time continued to extend, the GABA content decreased. Therefore, SJM8 was selected as the superior strain with high GABA production based on the comprehensive growth of microorganisms and GABA content.

[0043] Therefore, according to the above experimental results, it can be seen that fermentative mucus lactobacillus SJM8 has strong adaptability in the fermentation medium system and produces higher GABA content than other strains. The above strains were selected and deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on January 8, 2025, and subsequent research was carried out.

[0044] The strain deposit information is as follows:

[0045] Lactobacillus fermentans SJM8 was classified as Lactobacillus fermentans Limosilactobacillus fermentum , and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 10, 2025, with the deposit number CGMCC NO. 33359.

[0046] Example 3 Bacterial species identification

[0047] The isolated strains were systematically identified by phenotypic characteristics, 16SrDNA and ITS.

[0048] (1) The colony morphology of the isolated strains is as follows Figure 3 As shown, the colonies of strain SJM8 on MRS plates were milky white, with regular round edges and a smooth surface ( Figure 2 ).

[0049] (2) The isolated SJM8 was inoculated into MRS liquid medium for activation for 24 h, and 1 mL of bacterial liquid was taken into a 1.5 mL sterile centrifuge tube. The genome was extracted using Plant Zol (TransGen kit). The extracted genomic DNA was amplified using bacterial universal primers 27F (SEQ ID NO: 1 5′-AGAGT TTGAT CCTGG CTCAG-3′) and 1492R (SEQ ID NO: 2 5′-CTACG GCTAC CTTGT TACGA -3′). The PCR conditions were as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 57°C annealing for 30 s, 72°C extension for 1.5 min, 25 cycles; 72°C extension for 7 min; 3% agarose gel electrophoresis detection, and then the amplified sample was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing.

[0050] Log in to the NCBI website, perform BLAST homology analysis and comparison between the SJM8 16S (SEQ ID NO: 3) rDNA sequencing results and the relevant information in the website database, identify the strain and construct a phylogenetic tree. The results are as follows: Figure 3 shown by Figure 3 It can be seen that SJM8 is a fermentative Lactobacillus mucus.

[0051] SEQ ID NO:3:

[0052]

[0053] Example 4 Optimization of high-yield GABA fermentation process

[0054] SJM8 was used as the bacterial agent, and the fermentation medium was prepared using the above fermentation medium (15 g / L peptone, 6 g / L yeast extract, 25 g / L glucose, 5 g / L sodium succinate, 3 g / L sodium L-glutamate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, pH 6.2). The fermentation medium was placed in a 250 mL conical flask at a 20% inoculum, sterilized at 121°C for 15 min, and cooled for use), and cultured at 30°C for 48 h to produce GABA. On this basis, the single factor ( Figure 4 ) and response surface experiments to optimize the fermentation process for GABA production ( Figure 5 ), using MRS as the basic culture medium, by adjusting the inoculum amount (5%, 10%, 15%, 20%, 25%), fermentation temperature (20℃, 25℃, 30℃, 35℃, 40℃), sodium glutamate concentration (40 g / L, 60 g / L, 80 g / L, 100 g / L, 120 g / L), vitamin B6 content (0.5mmol / L, 1 mmol / L, 1.5 mmol / L, 2 mmol / L, 2.5 mmol / L) and other parameters, the effects of different parameters on GABA production were analyzed to determine the optimal fermentation process.

[0055] Result analysis: With the increase of inoculation amount, fermentation temperature, sodium glutamate concentration and cofactor content, the content of GABA showed a trend of first increasing and then decreasing. According to the results of single factor test, taking GABA content as the response value, the Box-Behnken test was used to obtain the optimal process parameters: inoculation amount of 17.5%, temperature of 38.32℃, sodium glutamate addition of 63.90 g / L, vitamin B6 content of 2.00 mmol / L. After verification, the optimal process parameters were inoculation amount of 17.5%, temperature of 38℃, sodium glutamate addition of 63.90 g / L, cofactor content of 2.00 mmol / L, and the obtained GABA content was 9.3 mg / mL, which was 2.5 times higher than the GABA content produced by the process before optimization (3.72 mg / mL). Figure 6 ).

[0056] Example 5 Antioxidant activity and cholesterol degradation ability of GABA

[0057] The antioxidant capacity of the produced GABA was investigated by evaluating its ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals. DPPH was dissolved in a methanol solution, usually at a concentration of 0.1 mM. The sample solution to be tested was added to the reaction solution containing DPPH and reacted for 30 min. The absorbance value was measured at 570 nm, and the DPPH free radical scavenging capacity (%) was calculated according to the following formula:

[0058]

[0059] A0 is the initial absorbance of the DPPH free radical solution, and A1 is the absorbance after the reaction

[0060] Prepare a 7 mM ABTS free radical solution, mix ABTS salt with hydrogen peroxide solution, and react to generate ABTS• + Free radicals. ABTS• + After the solution reacted with the sample for 15 min, the absorbance was measured at 734 nm, and the ABTS free radical scavenging capacity (%) was calculated according to the following formula:

[0061]

[0062] A0 is the initial absorbance of the ABTS free radical solution, and A1 is the absorbance after the reaction.

[0063] Sterilized cholesterol concentrate (cholesterol concentrate: 10mL N, N-dimethylformamide, 0.5mL Tween 80, 0.5g cholesterol, fully mixed and sterilized at 121℃ for 20min.) was added to MRS medium to make the cholesterol concentration in the medium 100 μg / mL. SJM8 culture was inoculated at 17.5% inoculum. Then the bacterial solution was centrifuged at 4℃, 5200×g for 10min. 1mL of supernatant was taken, 2ml of o-phthalaldehyde reagent was added, and it was fully mixed. After 10min, 1mL of concentrated sulfuric acid was added, and the concentrated sulfuric acid flowed along the inside of the tube, and then mixed on a tube shaker. After 10min, the absorbance OD (550nm) was read at 550nm, and the degradation rate of GABA to cholesterol was calculated using the standard curve method.

[0064] GABA produced at different fermentation times was used as the evaluation sample. The DPPH and ABTS free radical scavenging abilities and cholesterol content were measured to clarify the functional characteristics of GABA. The results showed that GABA obtained by SJM8 fermentation for 72 h had the highest free radical scavenging ability, with a DPPH scavenging ability of 47% and an ABTS free radical scavenging ability of 58%, and the lowest cholesterol content of 0.22 mg / mL. Previous studies have confirmed that GABA obtained at 72 h had the highest yield, so it had the strongest functional characteristics ( Figure 7 ).

[0065] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus mucilaginosus, characterized in that: The fermented mucus lactobacillus is fermented mucus lactobacillus SJM8, which is classified and named as fermented mucus lactobacillus Limosilactobacillus fermentum , and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 10, 2025, with the deposit number CGMCC NO. 33359.

2. Use of the fermented mucus lactobacillus according to claim 1 in producing gamma-aminobutyric acid.

3. A bacterial agent, characterized in that: The bacterial agent comprises the fermented mucus lactobacillus according to claim 1 (Limosilactobacillus fermentum SJM8 strain.

4. A method for preparing γ-aminobutyric acid, characterized in that: The method comprises the following steps: culturing the fermented mucus lactobacillus as claimed in claim 1, and harvesting and purifying to extract gamma-aminobutyric acid.

5. The method according to claim 4, characterized in that The following steps are also included: S1. Using MRS medium, sodium L-glutamate and vitamin B6 as raw materials, preparing a fermentation medium; S2. The fermented mucus lactobacillus according to claim 1 is cultured in MRS medium until the viable count reaches 1×10 8 CFU / mL or more, the activated strain was obtained; S3. Adding the strain activated in step S1 to the fermentation medium prepared in step S1 to carry out fermentation to produce high-content γ-aminobutyric acid.

6. The method according to claim 5, characterized in that Step S1: the fermentation medium includes 15 g / L peptone, 6 g / L yeast extract, 25 g / L glucose, 5 g / L sodium succinate, 3 g / L sodium L-glutamate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 2 mmol / L vitamin B6, and pH 6.

2.

7. The method according to claim 5 or 6, characterized in that: The inoculation amount is 15-25%, the temperature is 35-40℃, the amount of sodium glutamate added is 55-110g / L, and the content of vitamin B6 is 1-2.5 mmol / L.

8. The method according to claim 7, characterized in that The inoculum size is 16-18%, the temperature is 37-38.5℃, the amount of sodium glutamate added is 60-65 g / L, and the content of cofactors is 1.5-2.3 mmol / L.

9. The fermented mucus lactobacillus according to claim 1 (Limosilactobacillus fermentum) The use of the SJM8 strain in the preparation of an in vitro antioxidant is characterized in that: The fermented Lactobacillus mucilaginosus (Limosilactobacillus fermentum) The SJM8 strain exerts its antioxidant effect by scavenging DPPH free radicals and ABTS free radical activity.

10. The fermented mucus lactobacillus according to claim 1 (Limosilactobacillus fermentum) Application of SJM8 strain in preparing preparations for degrading or reducing cholesterol in vitro.

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