A marine-derived halophilic Bacillus Velezii strain producing branched-chain amino acid transaminase and its application

Abstract

The present invention relates to the field of microbial mutagenesis and fermentation technology, and discloses a marine-derived halophilic Bacillus Velezii strain that produces high-yield branched-chain amino acid transaminase and its application. The present invention selects a strain that produces high-yield branched-chain amino acid transaminase from traditional fermented shrimp paste as a starting strain, and obtains a mutant halophilic bacterium that produces high-yield branched-chain amino acid transaminase with high safety performance, good fermentation performance and genetic stability through ARTP mutagenesis, and the strain is named Bacillus Velezii ( Bacillus velezensis )T‑11, has been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20231091, and can be used for industrial fermentation of aquatic products.

Description

Technical Field

[0001] The invention relates to the technical field of microbial mutation breeding and fermentation, and in particular to a marine-derived halophilic Velezius bacillus with high branched-chain amino acid transaminase production and application thereof. Background Art

[0002] Traditional Chinese fermented aquatic products are popular among Asian consumers for their strong fermented aroma, unique seafood flavor and high nutritional value. Traditional fermented aquatic products are usually made by mixing aquatic raw materials such as shrimp, scallops or low-value fish with 10-30% salt, exposing them to the sun and dew at night, stirring them every day, and fermenting them naturally. During the fermentation process, macromolecules such as proteins or lipids in the raw materials are decomposed to produce flavor precursors such as polypeptides or fatty acids under the action of endogenous enzymes of the raw materials themselves and branched-chain amino acid transaminases produced by the metabolism of dominant halophilic bacteria in the fermentation system. Then, through secondary metabolic pathways such as lipid oxidation, Strecker degradation or Ehrlich pathway, numerous free amino acids, aldehydes, ketones, esters and sulfur-containing compounds are generated, giving shrimp paste a strong fermented aroma and characteristic seafood flavor.

[0003] Branched-chain amino acid transaminases are class IV folding enzymes that use 5'-pyridoxal phosphate as a coenzyme and remove the α-amino group in branched-chain amino acids such as leucine, valine and isoleucine through transamination reactions to produce the corresponding α-keto acids. Branched-chain amino acid transaminases are key enzymes that catalyze the Ehrlich pathway. They can degrade branched-chain amino acids into key flavor precursor substances, α-keto acids, which are then decarboxylated or reduced by α-keto acid decarboxylase to produce higher aldehydes such as isovaleraldehyde and isobutyraldehyde, and continue to oxidize to produce higher alcohols. In addition, the higher alcohols produced by the Ehrlich pathway can continue to react with acids to produce esters, which have an important impact on the flavor of fermented aquatic products. Branched-chain amino acid transaminases are widely distributed, but their content in most organisms is low. Considering the extraction cost and application potential, branched-chain amino acid transaminases present in certain specific microorganisms have gradually become a research hotspot. Although some strains capable of producing branched-chain amino acid transaminase have been screened out, the enzyme activity of the strains currently produced is only 30U / mL to 45U / mL. When in a high-salt environment, the enzyme activity is even lower than 10U / mL, making it difficult to use them for the industrial production of fermented aquatic foods.

[0004] Atmospheric room temperature plasmas (ARTP) mutagenesis technology is a new type of physical mutagenesis technology based on radio frequency atmospheric pressure discharge plasma. The operating principle of ARTP is to use radio frequency airflow discharge under normal pressure to form a plasma flow close to room temperature, which causes gene damage by releasing free radicals, nitrogen and oxygen atoms, and electromagnetic fields, resulting in significant changes in gene sequences. Compared with traditional technologies such as ultraviolet radiation or chemical mutagenesis, ARTP is faster and more convenient, with controllable conditions, and can effectively increase the mutation sites of DNA, with a high success rate and easier to obtain target strains with excellent traits. At the same time, because of its excellent genetic stability, it has been widely used in industrial microbial breeding and has become a hot spot in the field of microbial breeding. However, at this stage, no relevant research has been found on the use of ARTP mutagenesis technology to screen halophilic microorganisms with high-yield branched-chain amino acid transaminases.

[0005] At present, there is a shortage of fermentation strains with high branched-chain amino acid transaminase production in my country. There is a large gap between China and foreign countries in the selection and comprehensive utilization of halophilic microbial resources with high branched-chain amino acid transaminase production. Most halophilic strains with excellent fermentation performance have long been monopolized by international companies, and there is a shortage of enzyme-producing halophilic strains with independent intellectual property rights, which has greatly restricted the modernization of traditional fermented aquatic products. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a marine-derived halophilic Bacillus Velezii strain with high branched-chain amino acid transaminase production and application thereof.

[0007] In order to achieve the above-mentioned purpose, the technical solution of the present invention is: a marine-derived halophilic Bacillus velezensis with high production of branched-chain amino acid transaminase, characterized in that: its name is Bacillus velezensis T-11, its Latin name is Bacillus velezensis T-11, abbreviated as BV-T11, and it is deposited in the China Center for Type Culture Collection, the storage address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the storage number is CCTCC NO: M 20231091, and the storage date is June 25, 2023.

[0008] Furthermore, the 16S rRNA gene sequence of Bacillus velez T-11 is shown in SEQ ID No: 1.

[0009] Another technical solution of the present invention is: the breeding method of the Bacillus Velez T-11 comprises the following steps:

[0010] (1) Initial screening of starting strains: The traditional fermented shrimp paste was cultured on Gibbons solid medium to screen out halophilic bacteria that can grow and metabolize in a salt concentration of ≥15%;

[0011] (2) Rescreening of the starting strain: enriching the halophilic bacteria screened in step (1), and determining the branched-chain amino acid transaminase activity in the bacterial solution, and screening a strain with high branched-chain amino acid transaminase production as the starting strain, which is numbered J-58;

[0012] (3) ARTP mutagenesis: The starting strain J-58 was subjected to ARTP mutagenesis, and mutants with a mutagenesis time corresponding to a lethality in the range of 95% to 99% were selected. The mutants were cultured on Gibbons solid medium to screen out mutant halophiles that can grow and metabolize in a salt concentration of ≥15%. The mutant halophiles were then enriched and the branched-chain amino acid transaminase activity in the bacterial liquid was determined to screen out 7 mutant halophiles with high branched-chain amino acid transaminase production. The safety performance (hemolytic activity, biofilm formation ability, drug sensitivity), fermentation performance (salt tolerance, protease production performance, lipase production performance) and genetic stability of the 7 mutant halophiles with high branched-chain amino acid transaminase production were then characterized to screen out a mutant halophile with high safety performance, good fermentation performance and genetic stability, which was numbered J58-T11.

[0013] (4) The mutant halophilic bacterium J58-T11 was molecularly identified, named BV-T11, and preserved.

[0014] Further; the Gibbons solid medium: 10.0g of hydrolyzed casein, 2.5g of sodium citrate, 2.0g of potassium chloride, 0.5g of cobalt chloride, 7.0g of peptone, 15g of magnesium sulfate heptahydrate, 2g of glucose, 5g of yeast extract, 100g-300g of sodium chloride, 25g of agar powder, 1000mL of distilled water, adjust the pH to 7.4, and sterilize at 121°C for 15min.

[0015] Furthermore, the mutagenesis parameters are: mutagenesis power 100W, gas flow 10L / min, irradiation distance 2mm, and treatment temperature 38°C.

[0016] Another technical solution of the present invention is: a bacterial agent prepared by the aforementioned Bacillus Velez T-11.

[0017] Furthermore, the preparation method is as follows: Bacillus Velez T-11 with a preservation number of CCTCC NO: M 20231091 is inoculated into Gibbons liquid medium for activation, and an enriched bacterial solution is obtained after activation, and the enriched bacterial solution is centrifuged to obtain bacterial cells, and the bacterial cells are fully washed and adjusted to an effective viable count of Bacillus Velez T-11 to 3.8×10 8 CFU / mL, and obtain the bacterial agent.

[0018] Further; the Gibbons liquid medium: 10.0g of hydrolyzed casein, 2.5g of sodium citrate, 2.0g of potassium chloride, 0.5g of cobalt chloride, 7.0g of peptone, 15g of magnesium sulfate heptahydrate, 2g of glucose, 5g of yeast extract, 100g-300g of sodium chloride, 1000mL of distilled water, adjusted to pH 7.4, and sterilized at 121°C for 15min.

[0019] Furthermore, the activation conditions are: enrichment culture in a constant temperature air bath shaker at 37°C and 200rpm for 12 to 14 hours.

[0020] Furthermore, the enriched bacterial solution was centrifuged at 8000 r / min for 15 min at 4°C to obtain bacterial cells; the bacterial cells were fully washed 3 times with sterile saline (0.85%).

[0021] Another technical solution of the present invention is: the application of the marine-derived halophilic Bacillus Velez subtilis BV-T11 with high branched-chain amino acid transaminase production in industrial fermentation of aquatic products, preferably, in the preparation of fermented shrimp paste.

[0022] The beneficial effects of the present invention are as follows: the present invention selects a strain that is resistant to high salt and has a high yield of branched-chain amino acid transaminase from traditional fermented shrimp paste as a starting strain, and then performs ARTP mutagenesis on the starting strain to obtain a mutant halophilic bacterium BV-T11 that is resistant to high salt and has a high yield of branched-chain amino acid transaminase, has high safety performance, good fermentation performance, and is genetically stable. The ability of BV-T11 to produce branched-chain amino acid transaminase in a 15% high-salt environment is as high as 121.84 U / mL.

[0023] Due to its high production capacity of branched-chain amino acid transaminase, the mutant halophilic bacteria BV-T11 of the present invention can be used for rapid industrial fermentation and aroma quality improvement of aquatic products. It can produce volatile components that are closer to traditional shrimp paste in a shorter period of time, and can significantly reduce the amine taste and fishy smell of the shrimp paste during the fermentation process, and increase the fermented aroma, fruity aroma and plant aroma of the shrimp paste. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a bar chart showing the ability of some halophilic bacteria to produce branched-chain amino acid transaminases;

[0025] Figure 2 This is the ARTP-induced lethality curve of halophilic bacteria J-58;

[0026] Figure 3 Screening diagram of high-yield branched-chain amino acid transaminase mutants: (A) is the ability to produce branched-chain amino acid transaminase; (B) is the ability to form biofilm;

[0027] Figure 4This is the electrophoresis detection diagram of the PCR product after J58-T11 gene amplification;

[0028] Figure 5 This is the phylogenetic tree of halophile BV-T11;

[0029] Figure 6 is a scanning electron micrograph of halophilic bacteria BV-T11;

[0030] Figure 7 This is a radar chart for the sensory evaluation of the aroma of fermented shrimp paste;

[0031] Figure 8 The radar chart and principal component analysis chart of the electronic nose of fermented shrimp paste: (A) is the radar chart; (B) is the principal component analysis chart;

[0032] Fig. 9 The fingerprint of volatile components of fermented shrimp paste based on GC-IMS: (A) is a three-dimensional topographic map of volatile components; (B) is a two-dimensional map of volatile component difference comparison;

[0033] Fig.10 GC-IMS fingerprint of fermented shrimp paste: (A) is the common volatile component; (B) is the characteristic volatile component;

[0034] Note: Different letters in the bar graph represent significant differences. "★" in the radar chart indicates that there is a significant difference between samples, that is, P < 0.05; "★★" indicates that there is an extremely significant difference between samples, that is, P < 0.01. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The quantitative tests in the embodiments of the present invention were performed three times and the results were averaged.

[0037] The formula of the culture medium used in the embodiment of the present invention is as follows:

[0038] Gibbons liquid medium (15% NaCl): hydrolyzed casein 10.0 g, sodium citrate 2.5 g, potassium chloride 2.0 g, cobalt chloride 0.5 g, peptone 7.0 g, magnesium sulfate heptahydrate 15 g, glucose 2 g, yeast extract 5 g, sodium chloride 150 g, distilled water 1000 mL, adjust pH to 7.4, and sterilize at 121°C for 15 min;

[0039] Gibbons solid medium (15% NaCl): 10.0 g of hydrolyzed casein, 2.5 g of sodium citrate, 2.0 g of potassium chloride, 0.5 g of cobalt chloride, 7.0 g of peptone, 15 g of magnesium sulfate heptahydrate, 2 g of glucose, 5 g of yeast extract, 150 g of sodium chloride, 25 g of agar powder, 1000 mL of distilled water, adjust the pH to 7.4, and sterilize at 121°C for 15 min.

[0040] The difference between the formula of Gibbons solid medium with NaCl concentration of 10%, 20%, 25% and 30% and Gibbons solid medium (15% NaCl) is that the mass of sodium chloride is 100g, 200g, 250g and 300g respectively.

[0041] Blood agar medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.;

[0042] The method for enriching halophilic bacteria and adjusting the concentration of bacterial solution of the present invention is as follows: the halophilic bacteria are transferred to Gibbons liquid culture medium (15% NaCl) in a sterile environment, and enriched and cultured for 13 hours in a constant temperature air bath shaker at 37°C and 200rpm to obtain an enriched bacterial solution. The concentration of the bacterial solution is adjusted by ultraviolet spectrophotometry: the enriched bacterial solution is centrifuged at 8000r / min for 15 minutes at 4°C to obtain bacterial bodies, the bacterial bodies are fully washed three times with sterile saline (0.85%, w / v), and then 3mL of sterile saline is added, and the absorbance value at 460nm is measured with sterile saline as a control, and the absorbance value of the bacterial solution is diluted to 0.45 by adding sterile saline, and the concentration of the bacterial solution is 3.8×10 8 CFU / mL.

[0043] Embodiment 1:

[0044] 1. Screening of strains.

[0045] Step 1: Initial screening of strains

[0046] In a sterile environment, 25 g of traditional fermented shrimp paste (purchased from Kaiping Road Farmers' Market in Qingdao) was weighed and mixed with 225 mL of sterilized saline (0.85%, w / v). After vibrating for 30 min, the bacterial suspension was obtained. The bacterial suspension was diluted to 10 -1 , 10 -2 , 10 -3 200 μL of bacterial suspensions from three dilution gradients were taken and spread on Gibbons solid medium (15% NaCl) respectively. After culturing in a 37°C constant temperature biochemical incubator for 72 h, culture dishes with uniform colony distribution were selected. Based on the differences in colony morphology and growth characteristics in the culture dishes, halophiles with different characteristics were screened and numbered.

[0047] A total of 68 strains of moderate halophiles that can grow and metabolize in a salt concentration of ≥15% were screened. As shown in Table 1, most halophiles are Gram-positive bacteria, showing a spherical or rod-shaped morphology under microscopic examination, with a white or yellow color, and their colony characteristics vary greatly.

[0048] Table 1 Morphological characteristics of some halophilic bacteria in shrimp paste

[0049]

[0050]

[0051] Note: “+” indicates Gram-positive bacteria; “-” indicates Gram-negative bacteria

[0052] Step 2: Rescreening of halophilic bacteria capable of producing branched-chain amino acid transaminase

[0053] The concentration of the moderately halophilic bacteria screened in the first step was uniformly adjusted to 3.8×10 8 CFU / mL. 200 μL of each was inoculated into 50 mL of sterilized Gibbons liquid medium (15% NaCl), and cultured at 37°C, 200 rpm for 36 hours to obtain enriched bacterial liquid, and the branched-chain amino acid transaminase activity in the bacterial liquid was determined. The activity determination steps are as follows;

[0054] First, different concentrations of α-ketoisocaproic acid (0 mg / L, 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, 2.0 mg / L, 2.5 mg / L, 3.0 mg / L, 3.5 mg / L, 4.0 mg / L) were mixed evenly with 0.2 g / L 2,4-dinitrophenylhydrazine color developer, the absorbance was measured at 520 nm and a standard curve was drawn.

[0055] The enriched bacterial liquid was centrifuged at 4°C and 10000r / min for 15min to obtain a supernatant, and the supernatant was diluted 100 times; 0.5mL of the diluted solution was transferred to a glass tube, and 0.5mL of 0.1mmol / L 5'-pyridoxal phosphate, 0.5mL of 0.1mol / L leucine, 0.5mL of 0.1mol / L α-ketoglutaric acid, and 3mL of 0.1mol / L phosphate buffer (pH=7.4) were added in sequence, and the mixture was mixed well and placed in a 37°C constant temperature water bath for reaction for 30min, and finally 5mL of 10% trichloroacetic acid was added to terminate the reaction;

[0056] Subsequently, 0.2 g / L of 2,4-dinitrophenylhydrazine was used to react with the reaction system to develop a color reaction, and the absorbance was measured at 520 nm and compared with the standard curve to calculate the branched-chain amino acid transaminase activity.

[0057] The enzyme activity of branched-chain amino acid transaminase refers to the amount of enzyme required to catalyze 1 mL of leucine to produce 1 μmol of α-ketoisocaproic acid per unit time at 37°C, with the unit of U / mL.

[0058] A total of 7 halophilic bacteria with strong ability to produce branched-chain amino acid transaminase were isolated, and the strain numbers were J-9, J-15, J-26, J-43, J-58, J-63 and J-68. Figure 1 Among them, the ability of J-58 to produce branched-chain amino acid transaminase in a 15% high-salt environment was significantly higher than that of other strains (P < 0.05), reaching 54.92 U / mL, indicating that it has the potential to decompose branched-chain amino acids in a high-salt environment.

[0059] Step 3: ARTP mutagenesis of halophilic bacteria producing branched-chain amino acid transaminase

[0060] There are large differences in the mortality of mutant strains at different mutagenesis times. A too low mortality rate can easily lead to unsatisfactory mutation effects of microorganisms, while a too high mortality rate can lead to an increase in the negative mutation rate of microorganisms. Therefore, in order to achieve the best mutagenesis effect for halophilic bacteria, J-58, a strain with strong branched-chain amino acid transaminase production, was used as the starting strain for ARTP mutagenesis.

[0061] In a sterile environment, 10 μL of the bacterial solution prepared in step 2 was taken out to a concentration of 3.8×10 8 The starting strain J-58 with a CFU / mL was smeared on a metal slide and the samples were induced for 15s, 30s, 45s, 60s, 75s, 90s, 105s and 120s respectively on the ARTP operating table. The induced mutant bacterial solution was spread on Gibbons solid medium (15% NaCl) and cultured at 37°C for 72h. The total number of colonies was calculated and the starting strain J-58 was used as a control to calculate the mutation lethality and draw a mutation lethality curve.

[0062] The mutagenesis parameters were set as follows: mutagenesis power 100 W, gas flow 10 L / min, irradiation distance 2 mm, and treatment temperature 38 °C.

[0063] The mutagenic lethality curve is as follows Figure 2 As shown, within the mutagenesis time of 0 to 45 seconds, the lethality of bacteria J-58 increased sharply. When the mutagenesis time was 90 seconds, the lethality was 92%. When the mutagenesis time increased to 120 seconds, the lethality was 99.5%, close to 100%. Therefore, the mutagenesis time of 120 seconds was selected.

[0064] Step 4: Screening of mutant halophilic strains with high branched-chain amino acid transaminase production

[0065] The mutant bacterial solution selected in the third step after 120s of mutagenesis was directly transferred to 1mL of sterile saline in a sterile environment, shaken and mixed, and diluted to 10 -1 , 10 -2 , 10 -3 , then spread the diluted mutant bacterial solution on Gibbons solid medium (15% NaCl) and culture at 37°C for 72 hours; select the culture dishes with uniform colony distribution in the culture medium, and number the halophilic bacteria with different characteristics based on the differences in colony morphology and growth characteristics in the culture dishes, so as to screen the mutant strains that can tolerate salinity above 15% by taking salinity as an indicator;

[0066] A total of 82 mutant halophiles that can tolerate salt concentrations above 15% based on J-58 mutagenesis were screened and numbered. Table 2 shows the morphological characteristics of some mutant halophiles. Most of the mutants obtained by mutagenesis with J-58 as the starting strain are Gram-positive bacteria, which are spherical under microscopic examination, with white colonies, and most of the colonies are smooth, slightly convex circles with neat edges. In Table 2, J58-T3, J58-T11, J58-T23 and J58-T72 are the mutant numbers.

[0067] Table 2 Morphological characteristics of some mutants induced by ARTP

[0068] Strain number color Colony characteristics Microscopic morphology Gram J-58 milky Round, raised, smooth, with clean edges, flat Short rod + J58-T3 milky Round, neatly edged, slightly convex, wrinkled, dry Short rod + J58-T11 White Round, neatly edged, raised, smooth, slightly dry Short rod + J58-T23 White Round, neatly edged, raised, slightly transparent, moist Short rod + J58-T72 White Round, clean-edged, raised, wrinkled, mucous Short rod +

[0069] Note: “+” indicates Gram-positive bacteria; “-” indicates Gram-negative bacteria.

[0070] Then, the mutant strains that could tolerate salinity above 15% were selected and placed in physiological saline. The concentration of each mutant strain was uniformly adjusted to 3.8×10 8 CFU / mL, then take 200 μL and inoculate into 50 mL of Gibbons liquid medium (15% NaCl), culture at 37°C, 200 rpm for 36 h to obtain enriched bacterial solution, and then detect the branched-chain amino acid transaminase activity in the bacterial solution according to the branched-chain amino acid transaminase activity determination method in the second step. Screening mutant halophilic strains that can produce high branched-chain amino acid transaminase.

[0071] Eighteen mutant halophilic strains that can tolerate 15% high salt environment and produce high branched-chain amino acid transaminases were screened. Compared with the starting bacteria, the branched-chain amino acid transaminases production capacity of seven mutant halophilic strains (J58-T3, J58-T11, J58-T23, J58-T35, J58-T47, J58-T55 and J58-T65) was significantly increased (P < 0.05), such as Figure 3As shown in (A), J58-T11 had the highest capacity of producing branched-chain amino acid transaminase, reaching 121.84 U / mL, which was 121% higher than that of the starting strain J-58.

[0072] 2. Identification of strains

[0073] The safety performance (hemolysis, biofilm formation ability and drug sensitivity test) of the above-mentioned 7 mutant halophilic bacteria with enhanced ability to produce branched-chain amino acid transaminase were identified, and the mutant strains that can be used for food fermentation and are safe and harmless were selected; the fermentation performance was identified, and the mutant strains with good fermentation performance were selected; the genetic stability was identified, and the mutant strains with excellent genetic stability were selected.

[0074] (1) Safety performance appraisal

[0075] Identification of hemolytic activity of strains: The 7 mutant halophilic bacteria were streaked onto blood agar medium under a sterile environment and cultured anaerobically in a constant temperature biochemical incubator at 37°C for 24 hours. The presence of transparent areas around the colonies was observed, and the hemolytic phenomenon of Staphylococcus aureus and Escherichia coli on blood agar medium was used as a positive control.

[0076] The toxicity of pathogenic microorganisms is related to hemolytic activity. According to the hemolytic ring of the strain on the blood plate, it is divided into α hemolytic (semi-transparent grass green hemolytic ring), β hemolytic (transparent hemolytic ring) and γ hemolytic (non-hemolytic). Most hemolytic strains are pathogenic. As shown in Table 3, with Escherichia coli and Staphylococcus aureus as positive controls, it was observed that the hemolytic activity of the 7 mutant halophilic bacteria was γ hemolytic, which met the food safety requirements.

[0077] Table 3 Hemolytic identification results of mutant halophilic bacteria

[0078] Mutant strain number Hemolytic J58-T3 γ J58-T11 γ J58-T23 γ J58-T35 γ J58-T47 γ J58-T55 γ J58-T65 γ Control bacteria: Escherichia coli α Control bacteria: Staphylococcus aureus β

[0079] Bacteria that can produce biofilms have strong drug resistance and can escape the body's immune response. Therefore, the biofilm formation ability was identified to determine safety; the concentration of the seven mutant halophilic bacteria was adjusted to 3.8×10 8CFU / mL, 20 μL of bacterial solution and 2 mL of Gibbons liquid culture were placed in a sterile 96-well plate, cultured at 37°C for 24 hours, the culture medium and wells were rinsed with sterile saline, 100 μL of methanol solution was used to fix at room temperature for 20 minutes, the methanol was aspirated and allowed to air dry, 100 μL of crystal violet stain (0.2%, w / v) was added to stain for 5 minutes, the stain and wells were rinsed with sterile saline, 150 μL of glacial acetic acid (33%, w / v) was added, and the absorbance value at 590 nm was measured after culture at 37°C for 30 minutes. The sample absorbance value was compared with the critical value to judge its biofilm formation ability. Among them, the critical value refers to twice the absorbance value of Gibbons liquid culture medium (15% NaCl) without halophilic bacteria inoculation. The results are as follows Figure 3 As shown in (B), the OD values ​​of the seven mutant halophiles did not exceed the limit value of 0.087, indicating that the seven mutant halophiles had no biofilm-forming ability and were safe.

[0080] In addition, since some drug-resistant genes in bacteria can be transferred to pathogens, leading to increased drug resistance of pathogens, it is necessary to conduct drug sensitivity tests on each mutant halophile to understand its drug sensitivity spectrum.

[0081] Prepare drug sensitivity papers for vancomycin, amikacin, streptomycin, tetracycline, ceftriaxone, cefazolin, cephalexin, erythromycin, gentamicin and penicillin for antibiotic sensitivity identification; adjust the concentration of starting bacteria J-58 and 7 mutant halophilic bacteria to 3.8×10 8 CFU / mL, aspirate 100 μL of bacterial suspension and spread it on Gibboons solid medium (15% NaCl), use sterile tweezers to stick each drug-susceptibility paper sheet to the medium, let it stand at 4°C for 2 h to allow the antibiotic to diffuse, then culture it at 37°C for 24 h, measure the diameter of the transparent circle, and determine the antibiotic susceptibility with reference to the National Committee for Clinical Laboratory Standards (NCCLS) standards provided by the World Health Organization (WHO).

[0082] As shown in Table 4, J58-T13, J58-T11 and J58-T55 were sensitive to the 10 antibiotics tested, and all the tested strains were sensitive to cephalosporins or between sensitive and insensitive.

[0083] Table 4 Results of drug sensitivity test of J-58 and 7 mutant halophilic bacteria

[0084]

[0085]

[0086] Note: "S" stands for sensitive, "R" stands for insensitive, and "I" stands for between sensitive and insensitive.

[0087] (2) Fermentation performance identification: including salt tolerance, protease production and lipase production.

[0088] Identification of salt tolerance: The starting strain J-58 and 7 mutant halophilic bacteria were spread on Gibbons solid medium with different NaCl concentrations (10%, 15%, 20%, 25% and 30%, w / v), cultured at 37°C for 72 hours, and the growth of the strains was observed.

[0089] Protease production performance verification: Add 5% (w / v) skim milk powder to Gibbons solid medium (15% NaCl), sterilize at 121℃ for 15min to obtain protease verification medium. Use a microinoculation needle to take out the J-58 and 7 mutant halophilic bacteria samples and then inoculate them in the protease verification medium, culture at 37℃ for 48h, observe whether a transparent circle is formed around the colony, and record the ratio of the transparent circle diameter to the colony diameter.

[0090] To verify the lipase production performance, 2% (w / v) ultrasonically emulsified tributyrin was added to Gibbons solid medium (15% NaCl), and sterilized at 121°C for 15 minutes to obtain a lipase verification medium. The J-58 and 7 mutant halophilic bacteria samples were taken out with a microinoculation needle and then inoculated into the lipase verification medium, and cultured at 37°C for 48 hours. Whether a transparent circle was formed around the colony was observed, and the ratio of the transparent circle diameter to the colony diameter was recorded.

[0091] As shown in Table 5, all 7 mutant halophiles have the ability to produce proteases and lipases and can survive in a 15% high-salt environment. Compared with the starting strain J-58, only J58-T35 and J58-T55 have reduced protease production capabilities, while J58-T11, J58-T35, J58-T55 and J58-T65 have increased lipase production capabilities. Combined with the determination of the ability of mutants to produce branched-chain amino acid transaminases, safety assessment and fermentation performance analysis, it was found that compared with other mutant halophiles of J-58, the mutant halophile J58-T11 not only has a significantly high ability to produce branched-chain amino acid transaminases, but can also grow and metabolize in a 15-30% high-salt environment. The strain is safe and has good protease and lipase production capabilities.

[0092] Table 5 Fermentation performance analysis of J-58 and 7 mutant halophilic bacteria

[0093]

[0094]

[0095] Note: “+” indicates that the microorganism can survive at this salt concentration; “-” indicates that the microorganism cannot survive at this salt concentration.

[0096] (3) Genetic stability identification

[0097] The mutant halophilic bacteria J58-T11 strain was streaked into Gibbons solid medium (15% NaCl) and subcultured for 7 times, and the branched-chain amino acid transaminase production performance, protease production performance and fat production performance of the mutant bacteria at different subculture times were determined. After 7 subcultures, the ability of the mutant halophilic bacteria J58-T11 to produce branched-chain amino acid transaminase did not change significantly, and was always between 117.32 and 123.93 U / mL. The average ability to produce branched-chain amino acid transaminase reached 120.43 U / mL, and its ability to produce protease and lipase in a high-salt environment did not change significantly with the increase in the number of subcultures, indicating that the mutant halophilic bacteria J58-T11 with high branched-chain amino acid transaminase production has good genetic stability.

[0098] (4) Physiological and biochemical identification

[0099] With reference to Bergey's Bacterial Identification Manual and Common Bacterial System Identification Manual, the starting strain J-58 and mutant halophilic bacteria J58-T11 were inoculated in bacterial microbiochemical identification tubes according to the operating requirements in a sterile environment, and catalase activity test, biogenic amine test, gelatin liquefaction test, VP test, hydrogen sulfide gas production test, and carbon source utilization test were carried out.

[0100] The test results are shown in Table 6. Both J-58 and J58-T11 are catalase positive and have the ability to decompose glucose and arabinose.

[0101] Table 6

[0102]

[0103] Note: “+” indicates positive, “-” indicates negative.

[0104] 3. Molecular biological identification and whole genome analysis of J58-T11 strain

[0105] According to Thermo Fisher Scientific PicoPure TM The operation requirements of the bacterial DNA extraction kit are to extract total DNA;

[0106] The J58-T11 strain gene was amplified by PCR based on the bacterial universal primer 27F-1492R. The amplification system was a 50 μl reaction system: 5×FastPfu Buffer (10 μl), 2.5 mM dNTPs (2 μl), 5 μM Forward Primer (1 μl), 5 μM Reverse Primer (1 μl), FastPfu Polymerase (0.5 μl), Template DNA (10 ng), supplemented with ddH2O to 50 μl;

[0107] The entire amplification system was pre-denatured at 98°C for 5 min in PCR, followed by denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 45 s for 27 cycles, and a final extension at 72°C for 10 min;

[0108] Then, 2.5 μl of PCR product was taken for 2% agarose gel electrophoresis. Figure 4 As shown by Figure 4 The results of amplification and purification were good, and then molecular biological identification was performed. Figure 4 M represents Marker; 1 represents the PCR product after amplification.

[0109] Molecular biological identification: The J58-T11 strain was sent to Shanghai Lingen Biotechnology Co., Ltd. for DNA sequencing; sequence homology analysis was performed using the NCBI website (www.ncbi.nlm.gov. / blast); a phylogenetic tree was constructed and calculated using the Neighbor-Joining method, which was constructed using MEGA7. Figure 5 As shown in the figure, the J58-T11 strain is in the same phylogenetic branch as Bacillus velezensis and has a high homology. It is determined that J58-T11 belongs to the genus Bacillus velezensis and is named Bacillus velezensis T-11, abbreviated as BV-T11. The bacterial morphology of BV-T11 (scanning electron microscope image) is shown in Figure 6 The 16S rRNA gene sequence of BV-T11 is shown in SEQ ID No: 1, see Table 7.

[0110] Table 7 16S rRNA gene sequence of BV-T11

[0111]

[0112]

[0113] Whole genome identification: The halophilic bacterium BV-T11 was sent to Shanghai Lingen Biotechnology Co., Ltd. for whole genome sequencing and data analysis. The results are shown in Table 8. The genome of halophilic bacterium BV-T11 contains only one circular chromosome of 3973979 bp. In addition, a total of 4093 genes were detected in the genome, with a total length of 3574218 bp, accounting for 89.9% of the entire genome, an average length of 873 bp, a GC content of 47.2%, 1984 positive chain genes, and 2109 negative chain genes. It also contains 86 tRNAs, 9 5S rRNAs, 9 16S rRNAs and 9 23S rRNAs.

[0114] Table 8

[0115] Genomic features Analyze the results Number of genes 4093 Total gene length (bp) 3574218 Average gene length (bp) 873 Gene percentage (kb) 1.029 GC content (%) 47.2 Gene length / genome length (%) 89.9 tRNA 86 5S rRNA 9 16S rRNA 9 23S rRNA 9

[0116] The COG, NR and Swiss-Prot databases were used to jointly analyze the branched-chain amino acid transaminase genes in the genome of halophilic bacteria BV-T11. The results are shown in Table 9. A total of 9 genes related to the production of branched-chain amino acid transaminases by BV-T11 were screened out. Among them, gene annotation marked BV003827, BV000242, BV000554, BV003177, BV003334, BV003372, BV001639, BV001037 and BV002721 as pyridoxal phosphate-dependent branched-chain amino acid transaminases, while BV002850 and BV002872 were annotated as important components of the branched-chain amino acid transport system, indicating that halophilic bacteria BV-T11 has complete branched-chain amino acid transport and metabolic coordination, proving its ability to produce high branched-chain amino acid transaminase activity at the genetic level.

[0117] Table 9

[0118]

[0119] Note: GeneID is the number of the gene in the BV-T11 genome, COG ID refers to the gene name annotated in the COG database, NR ID refers to the gene name annotated in the NR database, Swiss-Prot ID refers to the gene name annotated in the Swiss-Prot database, annotation function refers to the gene function annotated in the COG, NR and Swiss-Prot databases, and nd means that the gene is not annotated in this gene library.

[0120] Bacillus velezensis T11, referred to as BV-T11, is currently preserved in the China Center for Type Culture Collection, with the preservation address at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The preservation number is CCTCC NO: M 20231091, the preservation date is June 25, 2023, and the preservation period is 30 years.

[0121] Embodiment 2:

[0122] Preparation of BV-T11 bacterial agent: BV-T11 with the deposit number of CCTCC NO: M 20231091 was inoculated into Gibbons liquid medium (15% NaCl) for enrichment and bacterial liquid concentration adjustment to make the effective viable count of BV-T11 in the bacterial liquid reach 3.8×10 8 CFU / mL, the BV-T11 bacterial agent was obtained and placed in a 4°C refrigerator for use.

[0123] Preparation of J-58 inoculum: The starting strain J-58 was inoculated into Gibbons liquid medium (15% NaCl) for enrichment and bacterial liquid concentration adjustment, so that the effective viable count of J-58 in the bacterial liquid reached 3.8×10 8 CFU / mL, and the J-58 bacterial agent was obtained and placed in a 4°C refrigerator for use.

[0124] Aquatic product fermentation: Fresh shrimp purchased from the farmers' market on Kaiping Road in Qingdao were washed and drained (not sterilized, with the original bacterial phase of aquatic products) and added to an industrial fermentation tank. Coarse sea salt was weighed at a mass ratio of 15:100 to fresh shrimp. The coarse sea salt was crushed and added to the industrial fermentation tank. A propeller was used to fully stir the salt particles and the fresh shrimp raw materials to form a fermentation slurry. The bacterial agent was added to the fermentation tank and fermented at a constant temperature of 37°C to obtain fermented shrimp paste. The fermented aquatic products were divided into six groups according to the inoculation amount of the bacterial agent:

[0125] Raw material group: wash and drain fresh hair shrimp raw materials, keep samples, and store in freezer;

[0126] In the blank group, no bacterial agent was inoculated, and the fermentation slurry was directly fermented at 37°C for 30 days;

[0127] The J-58 group was inoculated with J-58 at a 2% inoculum (v / w) and fermented rapidly at 37°C for 30 days;

[0128] The BV group was inoculated with BV-T11 at a 2% inoculum (v / w) and fermented rapidly at 37°C for 30 days;

[0129] The MIX group was inoculated with BV-T11 at a 4% inoculum (v / w) and fermented rapidly at 37°C for 30 days;

[0130] The traditional shrimp paste group was not inoculated with bacterial agents, and the fermentation slurry was fermented in the natural environment of the original bacteria of aquatic products, with a fermentation period of 6 months;

[0131] After fermentation was completed, aroma sensory evaluation, electronic nose analysis and gas chromatography-ion mobility spectrometry (GC-IMS) were performed on the six groups of samples to evaluate the aroma quality improvement effect of rapidly fermented aquatic products under the metabolic regulation of BV-T11.

[0132] Aroma sensory evaluation method:

[0133] Ten professional tasters with rich aroma sensory experience (aged 18 to 55 years old, with an equal ratio of men and women) were invited to form an aroma sensory panel. The team members were organized to participate in a 3-week training meeting (6 times a week, 2 hours each time) to identify, classify and memorize the aroma attributes and intensity of fermented shrimp paste. Through training and communication, a certain number of sensory descriptive words that can describe the aroma characteristics of fermented shrimp paste were finally determined. Subsequently, 10g of the sample to be evaluated was weighed with a sensory tasting cup with uniform texture and complete consistency. In order to ensure the objectivity of the evaluation and scoring, the tasting cups were randomly coded with four-digit numbers and distributed to the members of the trained sensory tasting panel. The samples were sensory evaluated in an environment with independent compartments, no noise, no strong colors and constant temperature (25°C), and the sensory descriptors were scored according to intensity. Each sensory descriptive word in the evaluation process was quantitatively evaluated using the digital intensity evaluation method, and the scoring standard was 0-10 points (0 points means that the smell is not obvious, and 10 points means that the smell is extremely strong). The results are as follows Figure 7 As shown in the figure, there were significant differences in the six aroma attributes of fermentation aroma, plant aroma, fruit aroma, shrimp aroma, amine smell and fishy smell among the samples in each group (P<0.05). Among them, the fishy smell scores of the J-58 group, BV group and MIX group were significantly lower than those of the raw material group, blank group and traditional shrimp paste group (P<0.05), indicating that the fermentation of halophilic bacteria BV-T11 can significantly reduce the fishy smell in shrimp paste; at the same time, the fermentation aroma, plant aroma and fruity aroma scores of the J-58 group, BV group and MIX group were significantly higher than those of the raw material group and blank group (P<0.05), indicating that the fermentation of halophilic bacteria BV-T11 increased the fermentation aroma, fruity aroma and plant aroma in shrimp paste. It is worth noting that the BV group had significantly higher scores in fermentation aroma and fruity aroma than the traditional group (P < 0.05), indicating that the shrimp paste inoculated with halophilic bacteria BV-T11 is superior to traditional Chinese shrimp paste in fermentation aroma and fruity aroma, while the MIX group had significantly higher scores in fermentation aroma, plant aroma and fruity aroma than the traditional shrimp paste group, J-58 group and BV group (P < 0.05), and significantly lower scores in amine smell and fishy smell than the traditional shrimp paste group, J-58 group and BV group (P < 0.05), indicating that the overall flavor of shrimp paste inoculated with a higher concentration of halophilic bacteria BV-T11 is significantly better than that of traditional shrimp paste, showing greater application potential in terms of aroma attributes.

[0134] Electronic nose (E-nose) analysis:

[0135] Weigh 20g of each sample into a 50mL headspace sampling bottle and incubate at 45°C for 10min to enrich the volatile components in the headspace bottle. Then insert the injection needle and gas replenishment needle into the headspace bottle, and use the PEN-3 electronic nose of Airsense of Germany to detect the volatile components of fermented aquatic products. The parameters are as follows: gas flow rate is 150mL / min, pre-sampling time is 10s, and sampling time is 150s. In this process, the volatile components react chemically with the coating materials of 10 specific metal oxide sensors of the electronic nose to generate electrical signals, forming a sample volatile aroma spectrum detection based on multidimensional data. Among them, the response relationship between the electronic nose sensor and the volatile components is shown in Table 10.

[0136] Table 10 Response relationship between electronic nose sensor and volatile components

[0137]

[0138]

[0139] like Figure 8 As shown in (A), the response values ​​of the samples in each group to the W1W, W2S, W2W, W3S, W1C, W5S and W3C sensors were significantly different (P < 0.05), indicating that the difference in aroma between the samples in each group was mainly caused by the presence of sulfur compounds, alcohols, aromatic compounds, alkane compounds and nitrogen compounds. In order to deeply analyze the differences in volatile components of hair shrimp raw materials, shrimp paste fermented by halophilic bacteria BV-T11 and traditional shrimp paste, PCA analysis was performed based on the response values ​​of the electronic nose sensor. The results are shown in Figure 2. Figure 8 As shown in (B), the spatial region of the shrimp paste samples shows that the raw material group and the blank group are close to each other, indicating that the volatile components of the fermented shrimp paste without halophilic bacteria inoculation are similar to those of the raw material at the fermentation cycle of 1 month. This may be because in the early stage of fermentation, with the sudden increase in salt content, the activity of some halophilic bacteria in the raw shrimp material decreased and some non-halophilic bacteria died, and it took a long time to gradually restore the overall activity of the fermentation microbial community. In addition, the J-58 group and the traditional shrimp paste group are close to each other, indicating that the aroma of the shrimp paste inoculated with halophilic bacteria J-58 is similar to that of the traditional shrimp paste; at the same time, the MIX group is the farthest from the other five groups of shrimp paste, indicating that in the fermentation process, the inoculation of high concentrations of BV-T11 halophilic bacteria has the greatest impact on the flavor of the shrimp paste.

[0140] GC-IMS analysis:

[0141] 8g of each sample and 50μl of internal standard (2,4,6-trimethylpyridine, 1000ppm) were weighed and placed in a headspace sample bottle to enrich the volatile components. The incubator parameters were set as follows: temperature 45℃, time 10min, and shaking speed 500rpm / min. After headspace enrichment, the autosampler delivered 0.5mL of headspace gas to the GC-IMS injection port (MXT-WAX polar column, 15m×0.53mmID×1.0μm df) for separation and identification of volatile components, and the sequence design software Library Editor was used to set the program: 2mL / min for 3min, 20mL / min for 3min, 50mL / min for 3min, 100mL / min for 3min, and 130mL / min for 15min. Among them, ultrapure nitrogen (purity ≥99.99%) was used as the carrier gas, and the front temperature of the IMS migration tube was set to 80℃ and the rear temperature was set to 50℃. Finally, the flow rate was set to 150 mL / min to clean the instrument after detection. The Library search and LAV-plugin plug-ins attached to GC-IMS were used to perform qualitative, quantitative and characteristic fingerprint analysis of volatile components.

[0142] Studies have shown that the three-dimensional spectrum and two-dimensional top view of GC-IMS can reflect the overall information of volatile components in each group of samples. Fig. 9 As shown in (A), the samples of the raw material group, blank group, J-58 group, BV group, MIX group and traditional shrimp paste group have great differences in the position, height and number of chromatographic peaks. In order to gain a deeper understanding of the differences in volatile components in different samples, the raw material group was used as a reference to construct a difference comparison chart of each group of samples based on the LAV Reporter plug-in. In the difference comparison mode, if the content of volatile components in the sample is higher than that in the reference raw material group, it will appear red in the chart. The darker the color, the greater the difference in content. Otherwise, it will appear blue. Fig. 9As shown in (B), the types and contents of volatile components in the blank group, halophilic fermented shrimp paste groups (J-58 group, BV group and MIX group) and traditional shrimp paste groups were significantly different from those in the raw material group. At the same time, the volatile components of the J-58 group, BV group and MIX group were significantly different from those of the blank group and traditional shrimp paste group, indicating that halophilic fermentation can produce more volatile components. In-depth analysis found that among the three groups of halophilic fermented shrimp paste, the MIX group produced the most differential volatile components, followed by the J-58 group. In order to further study the differences in volatile components, the volatile compounds of the samples were determined by retention index, retention time and drift time based on the IMS library. As shown in Table 11, a total of 115 typical volatile compounds were identified, including 33 esters, 11 ketones, 13 acids, 14 aldehydes, 11 phenols, 22 alcohols, 1 nitrogen-containing compound and 1 sulfur-containing compound. At the same time, the Gallery plug-in is used to extract all the peaks to form a characteristic aroma fingerprint, such as Fig.10 As shown, we can more intuitively and specifically understand the change pattern and relative content of volatile compounds in shrimp paste samples fermented by halophilic bacteria.

[0143] In the characteristic fingerprint of each group of samples, each row represents a different sample, each column represents a volatile component (corresponding one to one with the number of the volatile component in Table 11), and the color of the squares in each column represents the content of the volatile component. The darker the color, the higher the content. Fig.10 (A) shows the common volatile components in the raw material group, blank group, J-58 group, BV, MIX, and traditional shrimp paste groups, including aldehyde, ester, ketone, phenol and alcohol compounds such as n-valeraldehyde, ethyl cinnamate, methyl benzoate, 2-heptanone, 2-acetylpyrazine, propionic acid, 5-methylfuranaldehyde, 2-methylbutanal, ethanol, 2-acetylpyrrole, cyclohexanone, nerol, α-ionone, ethyl pyruvate, propionaldehyde, carveol and guaiacol, which are the main sources of the sweet, fruity and plant aroma of shrimp paste. Fig.10As shown in (B), the raw material group, blank group, J-58 group, BV group and traditional shrimp paste group all contained high levels of eucalyptol, methyl butyrate, octanol, 2-nonyl alcohol, 1-octen-3-ol, ethyl pentadecanoate, benzothiazole, furanone and 2,3-butanediol, but these volatile components were not detected in the IMX group, which was consistent with the classification trend of PCA; in addition, n-valeric acid, α-pinene and methyl oleate were only present in the raw material of hair shrimp, which may escape or be converted into other volatile components during the fermentation of shrimp paste. It is worth noting that the MIX group contains more characteristic volatile components, mainly including phenylethanol, 2,6-dimethylphenol, 2,3-pentanedione, heptaldehyde, dodecanol, indole, lauric acid, phenylacetic acid and eugenol, which is the main reason why MIX shrimp paste has a strong fruity and fermented aroma. In summary, the fermentation regulation of halophilic bacteria BV-T11 can produce volatile components that are closer to traditional shrimp paste within a shorter fermentation cycle, and has significant advantages in improving the overall volatile components and aroma quality of shrimp paste.

[0144] Table 11 Volatile compounds and aroma properties

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] Corresponding serial number: The serial number in this table is Fig.10 The numbers correspond to the volatile components detected by GC-IMS.

[0152] Aroma attributes: retrieved from ChemicalBook website (https: / / www.chemicalbook.com), “nd” means not found.

[0153] The above-described embodiments are only preferred solutions of the present invention and are not intended to limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solutions described in the claims.

Claims

1. A marine-derived halophilic Bacillus Velezii strain producing branched-chain amino acid transaminase, characterized in that: The name is Bacillus Velez T-11, and the Latin name is Bacillus velezensis T-11, abbreviated as BV-T11, is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO: M 20231091, and the deposit date is June 25, 2023.

2. The marine-derived halophilic Bacillus Velezii producing branched-chain amino acid transaminase according to claim 1, characterized in that: The 16S rRNA gene sequence of Bacillus velez T-11 is shown in SEQ ID No:

1.

3. The bacterial agent prepared by the marine halophilic Bacillus Velezii producing branched-chain amino acid transaminase according to claim 1.

4. The bacterial agent according to claim 3, characterized in that The preparation method comprises the following steps: inoculating Bacillus Velez T-11 with a preservation number of CCTCC NO: M20231091 into Gibbons liquid medium for activation, obtaining an enriched bacterial solution after activation, centrifuging the enriched bacterial solution to obtain bacterial cells, washing the bacterial cells thoroughly, and adjusting the effective viable count of Bacillus Velez T-11 to 3.8×10 8 CFU / mL, and obtain the bacterial agent.

5. The bacterial agent according to claim 4, characterized in that: The Gibbons liquid culture medium comprises: 10.0 g of hydrolyzed casein, 2.5 g of sodium citrate, 2.0 g of potassium chloride, 0.5 g of cobalt chloride, 7.0 g of peptone, 15 g of magnesium sulfate heptahydrate, 2 g of glucose, 5 g of yeast extract, 100 g to 300 g of sodium chloride, and 1000 mL of distilled water. The pH value is adjusted to 7.4, and the medium is sterilized by high pressure at 121° C. for 15 min.

6. The bacterial agent according to claim 4, characterized in that: The activation conditions are as follows: enrichment culture in a constant temperature air bath shaker at 37°C and 200rpm for 12 to 14h; centrifuging the enriched bacterial solution at 8000r / min for 15min at 4°C to obtain bacterial cells; and washing the bacterial cells three times with sterile physiological saline.

7. Use of the marine halophilic Bacillus Velezii producing branched-chain amino acid transaminase according to claim 1 in industrial fermentation of aquatic products.

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