A marine-derived halophilic Staphylococcus nepalensis strain producing aminopeptidase and its application

Abstract

The invention relates to the field of microbial mutagenesis and fermentation technology, and discloses a marine halophilic Staphylococcus nepalensis strain with high aminopeptidase production and its application. The invention selects a strain with high aminopeptidase production from traditional fermented shrimp paste as a starting strain, and obtains a mutant halophilic bacteria with high safety performance, good fermentation performance and genetic stability through ARTP mutagenesis. The strain is named Staphylococcus nepalensis ( Staphylococcus nepalensis )T‑13, referred to as SN‑T13, is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20231090 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 Staphylococcus nepalensis strain with high aminopeptidase production and application thereof. Background Art

[0002] Traditional Chinese fermented aquatic products are popular among Asian consumers for their delicious taste and strong sauce flavor. Traditional fermented aquatic foods are not only a source of high-quality protein and unsaturated fatty acids, but also rich in functional factors that are beneficial to the human body, such as active peptides, astaxanthin, polysaccharides and trace elements, and they play an important role in the field of traditional fermented foods. At present, my country's traditional fermented aquatic products are still mainly natural fermentation. However, the traditional fermentation process has a long brewing cycle, high salinity, food safety hazards such as biogenic amines, and is prone to breeding spoilage bacteria. It has a heavy astringent and bitter taste. At the same time, workshop-style production and extensive management lead to unstable product quality, making it difficult to achieve precise and intensive production, and do not meet the requirements of modern standardized production and quality control. Although the purpose of reducing salinity and shortening the production cycle is achieved by adding strains and enzymes, the characteristic taste and aroma of traditional products cannot be obtained due to the lack of superior fermentation strain resources.

[0003] Halophiles refer to extreme microorganisms that can grow and metabolize in high-salt environments such as oceans or saline-alkali lakes. During the fermentation process, the halophilic bacteria, as the dominant microorganisms, can accelerate the decomposition and biotransformation of raw substrates by using the proteases, aminopeptidases and lipases they produce, and produce good flavor components and flavor precursors such as flavor peptides, free amino acids, nucleotides and organic acids, which together constitute a complex flavor compound system. By exploring the dominant flavor halophiles and using them for fermentation regulation of aquatic products, it is helpful to significantly improve the processing efficiency and quality of fermented aquatic products while maintaining the original flavor quality, which has important demonstration significance for protecting and promoting my country's traditional fermented foods. However, the current research on halophiles at home and abroad is mostly focused on the analysis of their physiological and biochemical characteristics, phylogenetic analysis and salt tolerance mechanisms, and the exploration of new resources of halophiles that can continuously metabolize and produce flavor enzymes in high-salt environments is extremely scarce.

[0004] Aminopeptidases are a type of exoproteinase that can gradually cleave amino acid residues from the N-terminus of a protein or polypeptide chain and produce free amino acids or shorter peptide chains. There are many types of aminopeptidases, and their hydrolysis degree is usually more thorough. The products are mostly free amino acids or flavor peptides, which are important substances that can improve the flavor and nutrition of food. Therefore, aminopeptidases are widely used in improving food flavor, removing bitterness from food, deep enzymatic hydrolysis of proteins, preparing functional active peptides, and shortening food fermentation cycles. Aminopeptidases have a wide range of sources and are commonly found in different types of organisms such as plants, animals, and microorganisms. Among them, the use of microorganisms to prepare aminopeptidases can not only overcome the disadvantages of extracting from animals and plants, but also has a simple extraction process, high enzyme activity, and is easy to preserve, which is particularly suitable for the industrial production of aminopeptidases.

[0005] At present, most of the aminopeptidase-producing microorganisms screened internationally are Aspergillus niger, yeast and Streptococcus, with an aminopeptidase production capacity of about 100 to 350 U / mL, and a few can exceed 800 U / mL. However, the enzyme production capacity of these microorganisms in extreme environments such as high salt, high heat or high acidity and alkali is only 1.35 to 60.58 U / mL. So far, halophiles that can efficiently produce aminopeptidases and exert their effects in high-salt environments are extremely scarce, making them difficult to apply industrially. At the same time, no relevant research has been found on the molecular mechanism of biotransformation in the complex metabolic system of "halophiles-aminopeptidase-flavor", which greatly limits the precise regulation of flavor quality by halophiles during shrimp paste fermentation and the industrial development of traditional fermented aquatic products in my country. 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 Staphylococcus nepalensis strain with high aminopeptidase production and application thereof.

[0007] In order to achieve the above-mentioned purpose, the technical solution of the present invention is: a marine halophilic Nepalese Staphylococcus with high aminopeptidase production, named Staphylococcus nepalensis T-13, Latin name Staphylococcus nepalensis T-13, referred to as SN-T13, preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation number is CCTCC NO: M 20231090, and the preservation date is June 25, 2023.

[0008] Furthermore, the 16S rRNA gene sequence of Staphylococcus nepalensis T-13 is shown in SEQ ID No: 1.

[0009] Another technical solution of the present invention is: the breeding method of Nepalese Staphylococcus T-13 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 aminopeptidase activity in the bacterial solution, and screening a strain with high aminopeptidase production as the starting strain, which is numbered J-37;

[0012] (3) ARTP mutagenesis: The starting strain J-37 was subjected to ARTP mutagenesis, and mutants with a lethality in the range of 95% to 99% corresponding to the mutagenesis time 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 aminopeptidase activity in the bacterial liquid was determined to screen out 7 mutant halophiles with high aminopeptidase activity. The safety performance, fermentation performance and genetic stability of the 7 mutant halophiles with high aminopeptidase activity were then characterized, and a mutant halophile with high safety performance, good fermentation performance and genetic stability was screened out, which was numbered J37-T13.

[0013] (4) The mutant halophilic bacterium J37-T13 was molecularly identified, named SN-T13, and preserved.

[0014] Furthermore, the Gibbons solid culture medium formula: 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 Staphylococcus nepalensis T-13.

[0017] Furthermore, the preparation method of the bacterial agent is as follows: the Nepalese Staphylococcus T-13 with a preservation number of CCTCC NO: M 20231090 is inoculated into a 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 then adjusted to 3.8×10 effective viable bacteria of the Nepalese Staphylococcus T-13 with physiological saline. 8 CFU / mL, and obtain the bacterial agent.

[0018] Furthermore, the Gibbons liquid culture medium formula: 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, adjust the pH to 7.4, and sterilize 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; the enriched bacterial solution is centrifuged at 8000r / min for 15 minutes at 4°C to obtain bacteria; the bacteria are fully washed 3 times with sterile saline.

[0020] Another technical solution of the present invention is: the use of the marine halophilic Staphylococcus nepalensis SN-T13 with high aminopeptidase production in industrial fermentation of aquatic products, preferably, in the preparation of fermented shrimp paste.

[0021] The beneficial effects of the present invention are as follows: the present invention selects a high-salt-resistant and high-aminopeptidase-producing strain from traditional fermented shrimp paste as a starting strain, and then performs ARTP mutagenesis on the starting strain to obtain a mutant halophilic bacterium SN-T13 with high safety performance, good fermentation performance, and genetic stability, which is resistant to high salt and high in aminopeptidase production. The ability of SN-T13 to produce aminopeptidase in a 15% high-salt environment is as high as 189.44 U / mL.

[0022] Due to its ability to produce high levels of aminopeptidase in a high-salt environment, the mutant halophilic bacteria SN-T13 of the present invention can be used for rapid industrial fermentation of aquatic products, which can significantly improve the fermentation degree and protein utilization rate of food, and improve the overall taste of shrimp paste by reducing bitter amino acids and increasing umami, sour and sweet amino acids. At the same time, the mutant halophilic bacteria SN-T13 can also increase the content of organic acids and flavor nucleotides in shrimp paste, which is beneficial to the improvement of the umami taste of shrimp paste. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a bar chart of the aminopeptidase production capacity of some halophilic bacteria;

[0024] Figure 2 This is the ARTP-induced lethality curve of moderately halophilic bacteria J-37;

[0025] Figure 3 Screening diagram of high-producing aminopeptidase mutants: (A) is the ability to produce aminopeptidase, (B) is the ability to form biofilm;

[0026] Figure 4 This is a diagram for evaluating the genetic stability of mutant strain J37-T13;

[0027] Figure 5This is the electrophoresis detection diagram of the PCR product after J37-T13 gene amplification;

[0028] Figure 6 The phylogenetic tree of the mutant halophile SN-T13 with high aminopeptidase production;

[0029] Figure 7 is a scanning electron micrograph of halophilic bacteria SN-T13;

[0030] Figure 8 The basic physical and chemical indicators of fermented shrimp paste are: (A) total colony count, (B) TVB-N, (C) histamine content, (D) AA-N;

[0031] Fig. 9 Radar chart for sensory evaluation of the taste of fermented shrimp paste;

[0032] Fig.10 This is an analysis chart of the free amino acid content of fermented shrimp paste;

[0033] Fig.11 The standard curve of organic acid content is as follows: (A) malic acid, (B) succinic acid, (C) citric acid, (D) tartaric acid;

[0034] Fig.12 Figure 2 shows the organic acid content of fermented shrimp paste: (A) malic acid, (B) succinic acid, (C) citric acid, and (D) tartaric acid.

[0035] Fig.13 The standard curves of flavor nucleotides are as follows: (A) 5'-AMP, (B) 5'-GMP, (C) 5'-IMP;

[0036] Fig.14 Analysis of the flavor nucleotide content of fermented shrimp paste: (A) 5'-AMP, (B) 5'-GMP, (C) 5'-IMP;

[0037] Fig.15 This is the equivalent umami evaluation diagram of fermented shrimp paste.

[0038] Note: Different letters in the bar graph represent significant differences (p < 0.05). DETAILED DESCRIPTION

[0039] 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.

[0040] The quantitative tests in the embodiments of the present invention were all repeated three times, and the results were averaged.

[0041] The formula of the culture medium used in the embodiment of the present invention is as follows:

[0042] 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;

[0043] 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.

[0044] 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.

[0045] Blood agar medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.;

[0046] The method for adjusting the concentration of the halophilic bacteria 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 in a constant temperature air bath shaker at 37°C and 200rpm for 13h 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 15min 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.

[0047] Embodiment 1:

[0048] 1. Screening of strains.

[0049] Step 1: Initial screening of strains

[0050] 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 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.

[0051] 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.

[0052] Table 1 Morphological characteristics of some halophilic bacteria in shrimp paste

[0053]

[0054]

[0055] Note: “+” indicates Gram-positive bacteria; “-” indicates Gram-negative bacteria

[0056] Step 2: Rescreening of halophilic bacteria with the ability to produce aminopeptidase

[0057] 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 aminopeptidase activity in the bacterial liquid was determined. The activity determination steps are as follows;

[0058] Use 50mM Tris-HCl buffer (pH=8.0) to prepare p-nitroaniline solutions of different concentrations (0mg / L, 0.5mg / L, 1.0mg / L, 1.5mg / L, 2.0mg / L, 2.5mg / L, 3.0mg / L, 3.5mg / L, 4.0mg / L), measure the absorbance at 405nm, and draw a standard curve with p-nitroaniline concentration as the horizontal axis and absorbance value as the vertical axis.

[0059] The enriched bacterial liquid was centrifuged at 4°C and 8000r / min for 10min to obtain a supernatant, and the supernatant was diluted 100 times; 0.5mL of the diluted bacterial supernatant was transferred to a glass tube, and 6mL of Tris-Hcl buffer solution (50mM, PH=8.0) was added to the glass tube, preheated at 40°C for 5min, 0.5mL of 26mM L-leucine-p-nitroaniline (LNA) solution (purchased from Sinopharm Chemical Reagent Co., Ltd.) was added, and the reaction was kept at 40°C for 10min, then ice-bathed for 5min to terminate the reaction; the absorbance was measured at 405nm, and the enzyme activity of the aminopeptidase produced by the halophilic bacteria was calculated by comparing with the standard curve.

[0060] The enzyme activity of aminopeptidase refers to the amount of enzyme consumed by a unit volume of enriched bacterial liquid to hydrolyze 1 μg of p-nitroaniline per unit time under the optimum conditions (40° C., pH=8.0), and the unit is U / mL.

[0061] A total of 8 halophilic bacteria with the ability to produce aminopeptidase were isolated, and the strain numbers were J-3, J-11, J-17, J-23, J-34, J-37, J-49 and J-55. Figure 1 Among them, the ability of J-37 to produce aminopeptidase in a 15% high-salt environment was significantly higher than that of other strains (P < 0.05), reaching 116.90U / mL, indicating that J-37 can produce halophilic aminopeptidase that tolerates a 15% high-salt environment through its own metabolism, and has the potential to promote the formation of special flavors in foods in a high-salt environment.

[0062] Step 3: ARTP mutagenesis of aminopeptidase-producing halophilic bacteria

[0063] There are large differences in the mortality of mutant strains at different mutagenesis times. A low mortality rate can easily lead to unsatisfactory mutation effects of microorganisms, while a high mortality rate can lead to an increase in the negative mutation rate of microorganisms. Therefore, in order to achieve the best mutagenesis effect on halophilic bacteria, J-37, a strain with strong aminopeptidase production, was used as the starting strain for ARTP mutagenesis.

[0064] 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-37 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-37 was used as a control to calculate the mutation lethality and draw a mutation lethality curve.

[0065] 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.

[0066] The mutagenic lethality curve is as follows Figure 2 As shown, within the mutagenesis time of 0 to 15 seconds, the mortality rate of bacteria J-37 increased sharply; when the mutagenesis time was 90 seconds, the mortality rate of bacteria J-37 was 93%; and when the mutagenesis time was 120 seconds, the mortality rate of bacteria J-37 was 98.5%, so the mutagenesis time was selected to be 120 seconds.

[0067] Step 4: Screening of mutant halophilic strains with high aminopeptidase production

[0068] 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;

[0069] A total of 78 mutant halophiles that can tolerate salt concentrations above 15% based on J-37 mutagenesis were screened and numbered. Table 2 shows the morphological characteristics of some mutant halophiles. Most of the mutants obtained by mutagenesis with J-37 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, J37-T9, J37-T13, J37-T46 and J37-T77 are the mutant numbers.

[0070] Table 2 Morphological characteristics of some mutants induced by ARTP

[0071]

[0072] Note: “+” indicates Gram-positive bacteria; “-” indicates Gram-negative bacteria.

[0073] 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 8CFU / 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 aminopeptidase activity in the bacterial solution according to the method for determining aminopeptidase activity in the second step. Screen mutant halophilic strains that can produce high aminopeptidase.

[0074] Twenty-one mutant halophilic strains that can tolerate 15% high-salinity environments and produce high aminopeptidase were screened. Among them, compared with the starting bacteria, the aminopeptidase production capacity of seven mutant halophilic strains (J37-T9, J37-T13, J37-T21, J37-T38, J37-T46, J37-T57 and J37-T63) was significantly increased (P < 0.05), such as Figure 3 As shown in (A), J37-T13 had the highest aminopeptidase production capacity, reaching 189.44 U / mL, which was 62.1% higher than that of the starting strain J-37.

[0075] 2. Identification of strains

[0076] The safety performance (hemolysis, biofilm formation ability and drug sensitivity test) of the above-mentioned 7 mutant halophilic bacteria with strong aminopeptidase production ability 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.

[0077] (1) Safety performance appraisal

[0078] 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.

[0079] 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.

[0080] Table 3 Hemolytic identification results of mutant halophilic bacteria

[0081]

[0082] 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 8 CFU / 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.

[0083] 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.

[0084] 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-37 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).

[0085] As shown in Table 4, J37-T13 and J37-T63 showed sensitivity to all 10 antibiotics tested, and all tested strains were sensitive to cephalosporins or between sensitive and insensitive.

[0086] Table 4 Results of drug sensitivity test of J-37 and 7 mutant halophilic bacteria

[0087]

[0088] Note: "S" stands for sensitive, "R" stands for insensitive, and "I" stands for between sensitive and insensitive.

[0089] (2) Fermentation performance identification: including salt tolerance, protease production and lipase production.

[0090] Identification of salt tolerance: The starting strain J-37 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 h, and the growth of the strains was observed.

[0091] 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-37 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.

[0092] 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-37 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.

[0093] As shown in Table 5, all 7 mutant halophilic bacteria have the ability to produce proteases and lipases and can survive in a 15% high salt environment. Compared with the starting strain J-37, the protease production ability of J37-T9, J37-T21, J37-T38 and J37-T63 was reduced, and the lipase production ability of J37-T21, J37-T38 and J37-T57 was also weakened to varying degrees. The protease production ability, lipase production ability and salt tolerance of J37-T13 were improved compared with the original strain. Combined with the aminopeptidase production ability of the mutant bacteria, safety assessment and fermentation performance analysis, it was found that J37-T13 can produce high aminopeptidase, can grow and metabolize in a high salt environment of 15-25%, the strain is safe, and the fermentation performance is excellent.

[0094] Table 5 Fermentation performance analysis of J-37 and 7 mutant halophilic bacteria

[0095]

[0096]

[0097] Note: “+” indicates that the microorganism can survive at this salt concentration; “-” indicates that the microorganism cannot survive at this salt concentration.

[0098] (3) Genetic stability identification

[0099] The mutant halophilic bacteria J37-T13 strain was streaked onto Gibbons solid medium (15% NaCl) and subcultured for 7 times, and the aminopeptidase production, protease production and fat production performance of the mutant bacteria at different subculture times were measured. Figure 4 As shown, after 7 subcultures, the ability of the mutant halophilic bacteria J37-T13 to produce aminopeptidase did not decrease significantly, and was always between 189.25 and 191.67 U / mL, with an average aminopeptidase production capacity of 190.33 U / mL. At the same time, its ability to produce protease and lipase did not change significantly with the increase in the number of subcultures, indicating that the mutant halophilic bacteria J37-T13 with high aminopeptidase production has good genetic stability.

[0100] (4) Physiological and biochemical identification of mutant halophilic bacteria

[0101] With reference to Bergey's Bacterial Identification Manual and Common Bacterial System Identification Manual, in a sterile environment and according to the operating requirements, the starting bacteria J-37 and the mutant halophilic bacteria J37-T13 were inoculated in bacterial microbiochemical identification tubes, and catalase activity tests, biogenic amine tests, gelatin liquefaction tests, VP tests, hydrogen sulfide gas production tests, and carbon source utilization tests were carried out.

[0102] As shown in Table 6, both J-37 and J37-T13 were catalase positive and had no ability to decompose lactose, but were able to ferment using glucose and arabinose as carbon sources and did not produce biogenic amines during the fermentation process.

[0103] Table 6

[0104]

[0105] Note: “+” indicates positive, “-” indicates negative.

[0106] 3. Molecular biological identification and whole genome analysis of J37-T13 strain

[0107] According to Thermo Fisher Scientific PicoPure TM The operation of the bacterial DNA extraction kit requires the extraction of total DNA;

[0108] The J37-T13 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;

[0109] 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;

[0110] Then, 2.5 μl of PCR product was taken for 2% agarose gel electrophoresis. Figure 5 As shown by Figure 5 The results of amplification and purification were good, and then molecular biological identification was performed. Figure 5 M represents Marker; 1 represents the PCR product after amplification.

[0111] Molecular biological identification: The J37-T13 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 6 As shown in Figure 1, strain J37-T13 and Staphylococcus nepalensis were in the same phylogenetic branch, and J37-T13 was determined to be Staphylococcus nepalensis T-13 (abbreviated as SN-T13). The bacterial morphology of SN-T13 (scanning electron microscope image) is shown in Figure 1. Figure 7 The 16S rRNA gene sequence of SN-T13 is shown in SEQ ID No: 1, see Table 7.

[0112] Table 7 16S rRNA gene sequence of SN-T13

[0113]

[0114]

[0115] Whole genome identification: Halophilic bacteria SN-T13 was sent to Shanghai Lingen Biotechnology Co., Ltd. for whole genome sequencing and data analysis. The results are shown in Table 8. The SN-T13 genome includes 1 circular chromosome and 3 circular plasmids, of which the chromosome size is 2889992bp, the 3 plasmid sizes are 33938bp, 29746bq and 3047bp respectively, and the overall genome size is 2956723bp. In addition, a total of 2861 genes were detected in the genome, with a total length of 2449389bp, accounting for 82.8% of the entire genome, an average length of 856bq, and a GC content of 34.1%. The genome also contains 60 tRNAs, 8 5S rRNAs, 7 16S rRNAs, and 7 23S rRNAs.

[0116] Table 8

[0117]

[0118]

[0119] The COG, NR and Swiss-Prot databases were used to jointly analyze the aminopeptidase genes in halophilic bacteria SN-T13. As shown in Table 9, a total of 7 aminopeptidase genes were mined, and their gene numbers were SN001106, SN001097, SN002103, SN001216, SN001222, SN001676 and SN000624, of which SN001106, SN001097 and SN001216 were retrieved in all three gene annotation libraries. From the functional annotation results, we can know that SN001106 is leucine aminopeptidase, SN001097 is methionine aminopeptidase, SN001676 is serine aminopeptidase, and SN000624 is glutamine aminopeptidase. From the genetic level, it shows that halophilic bacteria SN-T13 can effectively hydrolyze bitter amino acids such as leucine, methionine and serine at the end of the polypeptide chain, and can convert glutamine into umami amino acids such as glutamate, which has an important influence on the removal of bitterness and the enhancement of umami in food or protein.

[0120] Table 9

[0121]

[0122] Note: GeneID is the number of the gene in the SN-T13 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.

[0123] Staphylococcus nepalensis T13, referred to as SN-T13, is currently deposited in the China Center for Type Culture Collection, with the storage address at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The storage number is CCTCC NO: M 20231090, the storage date is June 25, 2023, and the storage period is 30 years.

[0124] Embodiment 2:

[0125] Preparation of SN-T13 bacterial agent: The SN-T13 bacteria with the deposit number of CCTCC NO: M 20231090 were inoculated into Gibbons liquid medium (15% NaCl) for enrichment and bacterial liquid concentration adjustment, so that the effective viable count of SN-T13 in the bacterial liquid reached 3.8×10 8 CFU / mL, the SN-T13 bacterial agent was obtained and placed in a 4°C refrigerator for later use.

[0126] Preparation of J-37 inoculum: The starting strain J-37 was inoculated into Gibbons liquid medium (15% NaCl) for enrichment and bacterial liquid concentration adjustment, so that the effective viable count of J-37 in the bacterial liquid reached 3.8×10 8 CFU / mL, and the J-37 bacterial agent was obtained and placed in a 4°C refrigerator for use.

[0127] 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:

[0128] Raw material group: wash and drain fresh hair shrimp raw materials, keep samples, and store in freezer;

[0129] In the blank group, no bacterial agent was inoculated, and the fermentation slurry was directly fermented at 37°C for 30 days;

[0130] The J-37 group was inoculated with J-37 bacteria at a 2% inoculum (v / w) and fermented rapidly at 37°C for 30 days;

[0131] The SN group was inoculated with SN-T13 at a 2% inoculum (v / w) and fermented rapidly at 37°C for 30 days;

[0132] The MIX group was inoculated with SN-T13 at a 4% inoculum (v / w) and fermented rapidly at 37°C for 30 days;

[0133] In the traditional fermentation group, no bacterial agent was inoculated, and the fermentation slurry was fermented in the natural environment of the original bacteria of the aquatic product, and the fermentation period was 6 months;

[0134] After fermentation was completed, basic physical and chemical indexes, taste sensory evaluation, free amino acid content analysis, organic acid analysis, flavor nucleotide analysis and equivalent umami evaluation were performed on the raw material group and 5 groups of fermented samples to evaluate the taste enhancement effect of rapidly fermented aquatic products under the metabolic regulation of the high-producing aminopeptidase halophilic bacteria SN-T13.

[0135] 1. Determination of basic physical and chemical indicators:

[0136] The total colony count of shrimp paste was determined with reference to GB 4789.2-2016; the volatile basic nitrogen (TVB-N) content of shrimp paste was determined with reference to GB 5009.228-2016; the amino acid nitrogen (AA-N) content of shrimp paste was determined with reference to GB 5009.235-2016; the histamine content of shrimp paste was determined with reference to GB 5009.208-2016.

[0137] Due to the particularity of the fermentation process, traditional fermented shrimp paste needs to be exposed to the sun and dew for a long time in the natural environment to form its strong fermentation aroma. Therefore, the fermentation cycle of traditional fermented shrimp paste is relatively long, and a large number of microorganisms are easily produced during the fermentation process. Figure 8 As shown in (A), the total colony count in the traditional fermentation group is 4.11lg CFU / g. According to the provisions of the Chinese Aquatic Condiment Hygiene Standard GB 10133-2014 for shrimp paste, the total colony count in shrimp paste should be less than 3.90lg CFU / g. Therefore, the shrimp paste of the traditional fermentation group usually needs to be sterilized before consumption or sale to reduce its total colony count. However, most sterilization methods will have adverse effects on the flavor and texture of shrimp paste, hindering the industrial development of shrimp paste products. Figure 8As shown in (A), the total colony counts of the SN group and the MIX group were significantly lower than those of the traditional fermentation group (P < 0.05), and they met the sanitary standards for aquatic condiments in my country, indicating that by inoculating the dominant flavor halophilic bacteria SN-T13 to shorten the fermentation cycle of shrimp paste, it is possible to obtain fermented shrimp paste with a lower total colony count while ensuring the flavor quality of the shrimp paste, which is conducive to the industrial production of fermented shrimp paste. TVB-N and histamine content are important evaluation indicators for the quality and safety of shrimp paste food. Combined with the provisions of the Chinese Aquatic Condiment Sanitation Standard GB10133-2014 and the Chinese Domestic Trade Industry Standard SB / T10525 on shrimp paste, the TVB-N content in shrimp paste should be less than 450 mg / 100 g, and the histamine content should be less than 50 mg / kg. Figure 8 As shown in Figures 8(B) and 8(C), all shrimp paste samples met the requirements of China's national safety standards. Meanwhile, the contents of TVB-N and histamine in the SN and MIX groups were significantly lower than those in the traditional fermentation group (P<0.05), which to some extent proved the advantages of fast fermentation products made by halophilic bacteria. Figure 8 As shown in (D), the AA-N content in the SN group and the MIX group was significantly higher than that in the hair shrimp raw material group (P < 0.05), and the AA-N content in the SN group and the MIX group was significantly higher than that in the raw material group and the traditional fermentation group (P < 0.05), indicating that the fermentation degree and protein utilization rate of food can be significantly improved under the regulation of halophilic bacteria SN-T13, which has an important impact on the taste of food.

[0138] 2. Taste sensory evaluation

[0139] Taste sensory evaluation method: Invite 10 professional tasters with rich taste sensory experience (aged 18-55 years old, with an equal ratio of men and women) to form a taste sensory panel. Organize the team members to participate in a 3-week training meeting (6 times a week, 2 hours each time) to identify, classify and memorize the taste attributes and taste intensity of fermented aquatic products. Through training and communication, a certain number of sensory descriptive words that can describe the taste characteristics of fermented aquatic products are finally determined. Subsequently, 10g of the sample to be evaluated is weighed with a sensory evaluation cup with uniform texture and complete consistency. In order to ensure the objectivity of the evaluation and scoring, the evaluation cup is randomly coded with four digits and distributed to the members of the trained sensory evaluation panel. The samples are sensory evaluated in an environment with independent compartments, no noise, no strong colors and constant temperature (25℃~28℃), and the sensory descriptors are scored according to the intensity. Each sensory descriptive word in the evaluation process is quantitatively evaluated by the digital intensity evaluation method, and the scoring standard is 0-10 points (0 points means that the taste is not obvious, and 10 points means that the taste is extremely strong).

[0140] like Fig. 9As shown in the results, there were significant differences in the five taste attributes of sweetness, sourness, umami, saltiness and astringency among the samples in each group (P < 0.05). Among them, the umami scores of the SN group and the MIX group were significantly higher than those of the raw material group, the blank group and the traditional fermentation group (P < 0.05), indicating that the fermentation of halophilic bacteria can not only significantly improve the umami degree of shrimp paste, but also its overall umami taste is significantly better than that of the traditional fermented shrimp paste. In addition, it was observed that the umami score of the SN group was significantly higher than that of the J-37 group before mutagenesis (P < 0.05), which may be because the halophilic bacteria SN-T13 has the ability to produce aminopeptidase more than J-37, which can release umami amino acids such as glutamic acid by cutting the glutamine at the end of the hair shrimp protein, thereby increasing the overall umami taste of the shrimp paste; the umami score of the MIX group was significantly better than that of the SN group and the J-37 group (P < 0.05), indicating that the fermentation effect of SN-T13 at a higher concentration is significantly better than that of the fermentation at a lower concentration. It is worth noting that the astringency score of the rapidly fermented shrimp paste inoculated with halophilic bacteria was significantly lower than that of the raw material group, blank group and traditional fermentation group (P < 0.05), indicating that halophilic bacteria fermentation can significantly reduce the astringency in the raw materials.

[0141] 3. Analysis of free amino acid content

[0142] Determination method of free amino acid content: weigh 3 g of sample, mix it with 20 mL of 0.1 mol / L trichloroacetic acid by shaking, let it stand for 30 minutes, centrifuge it at 4°C and 10000 r / min for 15 minutes to obtain the supernatant, then filter the supernatant through a 0.22 μm microporous filter membrane and use an amino acid automatic analyzer to determine the free amino acid content. The determination conditions are: wavelength 565 nm, buffer flow rate 0.3 mL / min; ninhydrin reaction solution flow rate 0.3 mL / min, reaction temperature 130°C, injection volume 10 μL.

[0143] Free amino acids are important flavor substances in shrimp paste. Fig.10As shown, the SN group and MIX group significantly increased the content of sweet amino acids and umami amino acids (P < 0.05), and their sweet amino acids and umami amino acids were significantly higher than those in the traditional fermentation group (P < 0.05). Halophilic bacteria fermentation significantly reduced the content of bitter amino acids in the raw materials, and the content of bitter amino acids in the SN group and MIX group was significantly lower than that in the J-37 group and the traditional fermentation group (P < 0.05), which may be related to the ability of halophilic bacteria SN-T13 to produce high aminopeptidase. In addition, the total free amino acid content in the raw material reached 5145.88 mg / kg, and the total free amino acid content of the blank group, SN, J-37, MIX and traditional fermented shrimp paste was 6596.52 mg / 100g, 7759.95 mg / 100g, 7371 mg / 100g, 7951.63 mg / 100g and 7703 mg / 100g, respectively, indicating that the fermentation of halophilic bacteria SN-T13 can significantly increase the total free amino acid content in the raw material of hair shrimp (P < 0.05). In summary, the rapid fermentation of shrimp paste by halophilic bacteria SN-T13 can improve the overall taste of shrimp paste by reducing bitter amino acids, increasing umami and sweet amino acids, and increasing the overall content of free amino acids, and its effect is better than that of traditional fermented shrimp paste.

[0144] 4. Organic acid analysis

[0145] Method for determining the content of organic acids: Weigh 5.00 g of sample and mix with 20 mL of 80% ethanol by volume and shake thoroughly. Centrifuge at 4°C and 8500 r / min for 15 min to collect the supernatant. Repeat the extraction of the precipitate 2 to 3 times using the same method, combine the supernatants and make up to 100 mL. 50 mL of the fixed-volume supernatant was placed in a rotary evaporator, and the mixture was evaporated at 40°C and 75 rpm for 15 min. After the ethanol was completely removed, the residue in the chicken heart bottle was transferred to a 25 mL volumetric flask with ultrapure water, 0.2 mL of 1 mol / L phosphoric acid was added, and the volume was fixed to 25 mL with ultrapure water. The fixed-volume liquid was then filtered through a 0.22 μm microporous filter membrane and used for HPLC analysis. The detection conditions were as follows: Hydrosphere C18 column (4.6 mm × 250 mm, 5 μm); injection volume 20 μL; UV detection wavelength 210 nm; column temperature 25°C; mobile phase 0.01 mol / L (NH4)2HPO4 (pH = 2.7). The flow rate was 0.9 mL / min.

[0146] The standard curves of tartaric acid, malic acid, succinic acid and citric acid standards are shown in Figure 2. Fig.11 As shown, by comparing the retention time and peak height of each group of samples with the organic acid standard, the organic acids in the samples were analyzed in a directional and quantitative manner. The results are shown in Fig.12As shown. The results showed that the contents of malic acid, tartaric acid, succinic acid and citric acid in the raw shrimp were significantly increased after fermentation (P < 0.05), and the malic acid content and citric acid content in the SN group were significantly higher than those in the blank group (P < 0.05), indicating that halophilic bacteria can significantly increase the contents of malic acid, citric acid and succinic acid in shrimp paste. In addition, in the MIX group, except for the significantly lower malic acid content than the traditional fermentation group, the contents of citric acid, tartaric acid and succinic acid were significantly higher than those in the traditional fermentation group (P < 0.05), indicating that the use of a higher concentration of halophilic bacteria SN-T13 fermentation can significantly increase the content of organic acids in shrimp paste (P < 0.05), thus having an important impact on the flavor of shrimp paste.

[0147] 5. Analysis of flavor nucleotides

[0148] Determination method of flavor nucleotides: weigh 5.00g sample and mix it with 20mL of 10% perchloric acid solution, then place it in a 4℃ constant temperature water bath for ultrasonic extraction for 10min, centrifuge it at 4℃ and 8500r / min for 10min and collect the supernatant, repeat the above steps for 2-3 times, combine the supernatants, then make up to 100mL with 10% perchloric acid solution, adjust the pH to 6.8 with 5mol / L KOH solution, let it stand at 4℃ for 30min, then centrifuge it at 8500r / min to collect the supernatant, make up to 50mL with ultrapure water, filter it through a 0.22μm microporous filter membrane and perform HPLC analysis, the detection conditions are as follows: HydrosphereC18 chromatographic column (4.6mm×250mm, 5μm); mobile phase A is 0.05mol / L An equal proportion mixed solution of KH2PO4 and K2HPO4 (pH = 6.5), mobile phase B is methanol solution; isocratic elution; detection wavelength is 254nm; column temperature is 28°C; injection volume is 10μL.

[0149] Flavor nucleotides contribute significantly to the overall flavor of food. They not only give shrimp paste a unique umami flavor, but also increase the umami sensory experience exponentially through the synergistic effect with free amino acids and organic acids. They are important flavor components in shrimp paste. The standard curves of 5,-IMP, 5,-GMP and 5,-AMP standards are shown in Figure 1. Fig.13 As shown in the figure, the flavor nucleotides in the shrimp paste sample were analyzed by comparing the retention time and peak height with those of the flavor nucleotide standard. The results are shown in Fig.14As shown. The results showed that the contents of flavor nucleotides in the blank group, SN group, J-37 group, MIX group and traditional fermentation group were significantly higher than those in the raw material group (P < 0.05), indicating that the content of flavor nucleotides in hair shrimp increased significantly after fermentation, and the overall taste was improved. Among them, 5, -IMP, 5, -GMP and 5, -AMP in the MIX group were significantly higher than those in the traditional fermentation group, indicating that a higher concentration of SN-T13 can produce more flavor nucleotide substances, which is beneficial to the improvement of the umami taste of shrimp paste.

[0150] 6. Evaluation of umami effect

[0151] Determination method of effective umami evaluation: The calculation method of equivalent umami concentration value (EUC) is shown in the following formula:

[0152] EUC (g MSG / 100g) = ∑a i b i +1218(∑a i b i )(∑a j b j )

[0153] Among them, a i Refers to the concentration of umami amino acids (glutamic acid, aspartic acid) (g / 100g); a j Refers to the concentration of 5' umami nucleotides (5'-AMP, 5'-IMP, 5'-GMP) (g / 100g); b i It refers to the relative freshness coefficient of umami amino acids relative to monosodium glutamate (MSG) (aspartic acid is 0.077, glutamic acid is 1); b j It refers to the relative freshness coefficient of flavor nucleotides relative to 5'-IMP (5'-AMP is 0.18, 5'-IMP is 1, and 5'-GMP is 2.3); 1218 = synergistic constant.

[0154] Studies have shown that the equivalent umami evaluation is to use the amount of MSG to represent the total amount of umami substances in food, which can intuitively express the synergistic umami effect of umami amino acids and flavor nucleotides and reflect the overall umami degree of the sample. Fig.15As shown in the figure, the EUC values ​​in the SN group, J-37 group and MIX group were significantly higher than those in the raw material group, blank group and traditional fermentation group (P < 0.05), indicating that the inoculation of halophilic bacteria J-37 and SN-T13 can significantly increase the overall umami taste of shrimp paste, and the overall umami taste of shrimp paste fermented by halophilic bacteria J-37 and SN-T13 is higher than that of traditional fermented shrimp paste. At the same time, the EUC value of the SN group was significantly higher than that of the J-37 group (P < 0.05), indicating that the mutated halophilic bacteria SN-T13 has a stronger ability to enhance the umami taste than the original bacteria J-37, which is consistent with the results of sensory evaluation. In addition, the EUC value of the MIX group was significantly higher than that of the SN and J-37 groups (P < 0.05), indicating that a higher concentration of halophilic bacteria SN-T13 can significantly enhance the umami taste of shrimp paste, and is more significant in improving the overall umami taste level of rapidly fermented shrimp paste.

[0155] 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 Staphylococcus nepalensis strain producing aminopeptidase, characterized in that: The name is Staphylococcus nepalensis T-13, the Latin name is Staphylococcus nepalensis T-13, abbreviated as SN-T13, and it is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation number is CCTCCNO: M 20231090, and the preservation date is June 25, 2023.

2. The marine-derived halophilic Staphylococcus nepalensis producing aminopeptidase according to claim 1, characterized in that: The 16S rRNA gene sequence of Staphylococcus nepalensis T-13 is shown in SEQ ID No:

1.

3. A bacterial agent prepared from the marine halophilic Staphylococcus nepalense producing aminopeptidase according to claim 1.

4. The bacterial agent according to claim 3, characterized in that The preparation method comprises the following steps: inoculating Nepalese Staphylococcus aureus T-13 with a preservation number of CCTCC NO: M20231090 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 Nepalese Staphylococcus aureus T-13 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 Staphylococcus nepalensis producing aminopeptidase according to claim 1 in industrial fermentation of aquatic products.

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