Salt-tolerant bacillus velezensis capable of efficiently degrading protein and biogenic amine and application of salt-tolerant bacillus velezensis

Abstract

The invention discloses a salt-tolerant Bacillus velezensis strain capable of efficiently degrading protein and biogenic amine and an application of the salt-tolerant Bacillus velezensis strain. The bacillus velezensis is a strain FJNU-SIO1, is classified and named as bacillus velezensis, is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation date is September 26, 2025, the preservation number is CGMCC No.36086, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The strain FJNU-SIO1 is wide in growth adaptation range, can tolerate the salt concentration of 1%-10%, and can still efficiently degrade protein and nine common biogenic amines including putrescine, cadaverine, histamine and tyramine under the high salt concentration of 10%, the degradation rate of the protein can reach 47.68%, and the degradation rate of the total biogenic amines can reach 32.77%. The strain FJNU-SIO1 provided by the invention has the outstanding advantages of simplicity and convenience in operation, low cost, wide applicability and the like when being applied to degradation treatment of proteins and biogenic amines, and has an important value for guaranteeing safe production of fermented foods.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a salt-tolerant Bacillus velezensis with high efficiency in degrading proteins and biological amines and application thereof. BACKGROUND

[0002] Fish sauce is a very popular traditional fermented aquatic product seasoning, and its unique flavor and umami core are derived from the microbial degradation of proteins in raw materials. This complex biochemical process eventually generates a large amount of flavor substances such as free amino acids and small molecule peptides, which constitute the basis of the rich and delicious flavor of fish sauce. However, the traditional fermentation mode faces the technical bottlenecks of prominent biological amine safety hazards, excessively long fermentation period, and lack of multifunctional starter suitable for specific salinity environment, which restricts the modernization development and quality improvement of the industry.

[0003] Biological amines (BAs) are toxic substances produced by microbial amino acid decarboxylase acting on free amino acids. In the complex natural fermentation system of fish sauce, a variety of inherent or extraneous contaminating microorganisms will produce such decarboxylase in the metabolic process. They use tyrosine, histidine and other precursors to catalyze the generation of corresponding tyramine, histamine, etc. Among them, cadaverine and histamine have significant physiological toxicity to the human body, and can cause a series of adverse reactions such as headache, allergy, abnormal blood pressure, etc. Due to the openness and complexity of the traditional fermentation system, the generation pathway of biological amines is difficult to be effectively inhibited, resulting in generally high content of biological amines in the final product, which not only directly endangers the health of consumers, but also becomes the main technical barrier for product market competition.

[0004] The formation of fish sauce flavor is essentially the process of protein being enzymatically hydrolyzed into small molecule peptides and free amino acids. In natural fermentation, this process relies on the joint action of fish autolytic enzymes and salt-tolerant microbial secreted protease system. However, the abundance and activity of high-efficiency protein-degrading bacteria in the natural flora are often insufficient, and their enzyme activity is inhibited by a certain salinity environment, resulting in an undesirable protein conversion rate. Low protein degradation efficiency directly leads to an unusually long fermentation period of fish sauce. Therefore, accelerating this process by exogenous addition of high-efficiency protein-degrading bacteria is a key breakthrough direction recognized in the field.

[0005] Current researches are mostly focused on the screening and application of single functional strains. For example, Bacillus subtilis (CN115161222B), Saccharomyces cerevisiae (CN113943665B) and lactic acid bacteria (CN114395512B) have certain biogenic amine degradation ability, but they have poor environmental adaptability and little contribution to shortening the fermentation period. Patent application CN116686969A discloses a fish sauce preparation method, which shortens the fermentation period of fish sauce through continuous fermentation by adding enzymes and bacteria, but the high salt environment during fermentation can easily lead to a decrease in enzyme activity and strain stability, limiting its application effect in high-salt foods. Strains that have efficient protein and biogenic amine degradation activity and are resistant to high-salt environments are particularly scarce. Therefore, it is of great significance to separate and screen microbial strains that have good salt tolerance, high protein degradation activity and efficient broad-spectrum BA degradation ability to improve the controllability and safety of the production process of traditional fermented foods. SUMMARY

[0006] The purpose of the present application is to provide a salt-tolerant Bacillus velezensis strain that efficiently degrades proteins and biogenic amines and its application.

[0007] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0008] A salt-tolerant and efficient protein and biogenic amine-degrading strain FJNU-SIO1, which is classified as Bacillus velezensis, was deposited with the China General Microbiological Culture Collection Center (CGMCC) on September 26, 2025, and has the accession number CGMCC No. 36086 and the address of the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Beijing City, Chaoyang District, 3, China.

[0009] The nucleotide sequence of the 16S rDNA of the strain FJNU-SIO1 is shown in SEQ ID No. 1.

[0010] The strain FJNU-SIO1 can be used to degrade biogenic amines or proteins. The biogenic amines include tryptamine, phenethylamine, putrescine, cadaverine, histamine, octopamine, tyramine, spermine or spermidine, etc.

[0011] Further, the application is carried out in a salt-containing environment. The application fields include food fermentation, aquatic product processing or high-protein wastewater treatment, etc.

[0012] The present application also provides a method for simultaneously degrading proteins and biogenic amines in a salt-containing environment, which uses the strain FJNU-SIO1 and the reaction is carried out in an environment with a salt concentration of 1%-13%.

[0013] The application also provides a microbial agent containing the strain FJNU-SIO1.

[0014] Further, the microbial agent is a liquid or solid preparation (dry powder), which can be used in combination or alone for degrading biological amines and proteins.

[0015] The strain FJNU-SIO1 is isolated from a fermented fish sauce sample of Fuzhou Min Tian Food Industry Park Co., Ltd. The main characteristics of the strain are strong salt tolerance, capable of growing in a 1%-10% NaCl concentration range; outstanding protein degradation capability, with a degradation rate of up to 47.68% at 10% salt concentration in 72h; and broad-spectrum biological amine degradation capability, with degradation effects on putrescine, cadaverine, histamine, tyramine, tryptamine, phenylethylamine, octopamine and spermidine, etc., and a total biological amine degradation rate of up to 32.77% at 10% salt concentration. In addition, the application covers a microbial agent containing the strain FJNU-SIO1 and a preparation method thereof, which can be prepared into a liquid fermenting agent, a freeze-dried powder or a high-purity enzyme preparation, etc.

[0016] Compared with the prior art, the Bacillus velezensis FJNU-SIO1 provided by the application not only has good salt tolerance and strong protein degradation capability, but also has high-efficiency degradation capability on various biological amines, and maintains excellent degradation performance in a high-salt environment, thus having good application prospect and promotion value. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a single colony morphology of the strain FJNU-SIO1 by plate streaking.

[0018] Figure 2 is a phylogenetic evolution tree constructed based on the 16S rRNA sequence of the strain FJNU-SIO1.

[0019] Figure 3 is a temperature tolerance curve (A), an acid tolerance curve (B) and a salt tolerance curve (C) of the strain FJNU-SIO1.

[0020] Figure 4 is a growth curve of the strain FJNU-SIO1.

[0021] Figure 5 is a protein hydrolysis circle of the strain FJNU-SIO1 in 24h.

[0022] Figure 6 is a protein degradation rate of the strain FJNU-SIO1 under different NaCl concentrations in 24h, 48h and 72h.

[0023] Figure 7The degradation rate of total biogenic amines of the strain FJNU-SIO1 under different NaCl concentrations for 24h. DETAILED DESCRIPTION

[0024] The application will be further described below in conjunction with the accompanying drawings and examples.

[0025] The raw materials and reagents in the application are commercially available unless otherwise specified.

[0026] Example 1 Isolation, screening and identification of the strain

[0027] 1. Isolation and screening of the strain

[0028] 1.1 Materials: fermented fish sauce samples from Fujian Fuzhou Min Tian Food Industrial Park Co., Ltd.

[0029] 1.2 Test medium

[0030] (1) LB liquid medium: 10.0 g / L of tryptone, 5.0 g / L of yeast powder, 10.0 g / L of sodium chloride, fully stirred and uniformly, and then sterilized at 121℃ for 20 min.

[0031] (2) LB solid medium: 10.0 g / L of tryptone, 5.0 g / L of yeast powder, 10.0 g / L of sodium chloride, 20.0 g / L of agar, fully stirred and uniformly, and then sterilized at 121℃ for 20 min.

[0032] (3) Different NaCl concentration LB liquid medium: 10.0 g / L of tryptone, 5.0 g / L of yeast powder, 10.0, 40.0, 70.0, 100.0, 130.0 g / L of sodium chloride, fully stirred and uniformly, and then sterilized at 121℃ for 20 min.

[0033] (4) NA solid medium: 10.0 g / L of tryptone, 3.0 g / L of beef extract powder, 5.0 g / L of sodium chloride, 15.0 g / L of agar, sterilized at 121℃ for 20 min.

[0034] (5) 2216E solid medium: 5.0 g / L of tryptone, 1 g / L of yeast extract, 0.1 g / L of ferric citrate, 19.45 g / L of sodium chloride, 5.98 g / L of magnesium chloride, 3.24 g / L of sodium sulfate, 1.8 g / L of calcium chloride, 0.55 g / L of potassium chloride, 0.16 g / L of sodium carbonate, 0.08 g / L of potassium bromide, 0.034 g / L of strontium chloride, 0.022 g / L of boric acid, 0.004 g / L of sodium silicate, 0.0024 g / L of sodium fluoride, 0.0016 g / L of sodium nitrate, 0.008 g / L of disodium hydrogen phosphate, 15 g / L of agar, sterilized at 121℃ for 20 min.

[0035] (6) MRS solid medium: Proteose peptone 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 5.0 g / L, glucose 20.0 g / L, potassium phosphate dibasic 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L, agar 15 g / L, sterilized at 121 ℃ for 20 min.

[0036] (7) Casein hydrolysis medium: Tryptone 5.0 g / L, beef extract 1.0 g / L, sodium chloride 5.0 g / L, casein 1.0 g / L, agar 20 g / L, sterilized at 121 ℃ for 20 min after fully stirring.

[0037] (8) Protein-based fermentation medium: Casein 10.0 g / L, glucose 7.5 g / L, yeast extract 2.0 g / L, magnesium sulfate heptahydrate 0.1 g / L, sodium chloride 10.0, 30.0, 50.0, 70.0, 100.0 g / L, sterilized at 121 ℃ for 20 min. (9) Biogenic amine fermentation LB-B medium: Tryptone 10.0 g / L, yeast powder 5.0 g / L, sodium chloride 10.0, 30.0, 50.0, 70.0, 100.0 g / L, biogenic amine (tryptamine, phenylethylamine, putrescine, cadaverine, histamine, octopamine, tyramine, spermine, and spermidine) 0.1 g / L each, sterilized at 121 ℃ for 20 min.

[0038] 1.3 Isolation of strains

[0039] Take 1 mL of fish sauce fermentation sample for gradient dilution (10 -2 to 10 -6 ), shake well, and then take 100 μL of the diluent to spread on LB solid medium, NA solid medium, 2216E solid medium, and MRS solid medium, and plate in a 30 ℃ incubator. After colony formation, target colonies are picked and repeatedly streaked on the corresponding medium plates three times according to the differences in colony phenotypes. The purified strain FJNU-SIO1 is inoculated with a single colony on LB liquid medium, cultured at 30 ℃, 220 rpm for 12 h, and the bacterial solution is preserved in a -80 ℃ refrigerator with 50% glycerol (v:v = 1:1) for further analysis.

[0040] 1.4 DNA extraction and PCR amplification of strains

[0041] Bacterial cultures incubated overnight at 30°C were collected, and genomic DNA was extracted from strain FJNU-SIO1 using a bacterial genomic DNA extraction kit (Beijing Qingke Biotechnology Co., Ltd.). The extracted bacterial DNA was stored at -20°C for later use. Using the genome as a template, bacterial 16S rDNA was amplified using universal primers 27F and 1492R, which were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0042] The PCR amplification reaction system consisted of a 50.0 μL system: 1.0 μL DNA template, 1.0 μL each of primers 27F and 1492R (10 μmol / L), 25.0 μL 2×San Taq PCR Mix (Sangon Biotech (Shanghai) Co., Ltd.), and ddH2O to complete the reaction. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles; a final extension at 72℃ for 10 min, and storage at 4℃. The amplified products were subjected to Sanger sequencing performed by Sangon Biotech (Shanghai) Co., Ltd., and the sequencing results were analyzed by BLAST comparison in the NCBI database.

[0043] 2. Identification of strains

[0044] The isolated strains were activated by streaking on LB solid medium. Single colonies were picked up with an inoculation stick, and the colony morphology was described. After slide preparation, the microscopic morphology was measured and described, as follows:

[0045] Morphological description of strain FJNU-SIO1: The morphology of this bacterium was observed and data recorded after 1 day of cultivation on LB solid medium. The colony morphology was irregular, white, opaque, flat, and dry on the front. Figure 1 ).

[0046] Based on the 16S rDNA sequence of the strain, BLAST alignment analysis was performed, and a phylogenetic tree was constructed using MEGA-12 with the neighbor-joining method. The strain FJNU-SIO1 showed high homology with *Bacillus velezensis*. Figure 2 ).

[0047] The 16S rDNA sequence BLASTn alignment result shows that the similarity of strain FJNU-SIO1 to Bacillus velezensis can reach 98.80%. Based on the morphological characteristics and molecular data, strain FJNU-SIO1 is identified as Bacillus velezensis. Bacillus velezensis is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No. 36086, and the preservation date is September 26, 2025.

[0048] 3. Preservation of the strain

[0049] Strain FJNU-SIO1 was activated by streaking on LB medium plates and incubated at 30°C for 2 days. After that, single colonies were purified by streaking three times. The purified strain was inoculated into LB liquid medium, and incubated at 30°C, 220 rpm overnight. Under sterile conditions, the culture was taken and added to a sterile 50% glycerol (v:v = 1:1) cryovial, which was stored in a -80°C ultra-low temperature freezer, and the survival of the strain was checked regularly.

[0050] Example 2 Temperature tolerance curve, acid tolerance curve and salt tolerance curve of strain FJNU-SIO1

[0051] Strain FJNU-SIO1 was incubated overnight and inoculated into LB liquid medium at a 10% inoculation amount and incubated at 220 rpm to analyze the effects of temperature, pH and NaCl concentration on the growth of the strain. The strain was inoculated into LB liquid medium at temperatures of 25, 30, 35, 40 and 45°C, pH of 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, and NaCl concentration of 1%, 4%, 7%, 10% and 13% at a 10% inoculation amount, and sampled at 24 h to measure the OD 600 ( Figure 3 ).

[0052] The experimental results show that the biomass of strain FJNU-SIO1 is the highest at a temperature of 35°C, and decreases with increasing temperature; the biomass is the highest at a pH of 6.0, and decreases with increasing pH. The biomass of the strain is the highest at a NaCl concentration of 1%, and decreases with increasing NaCl concentration.

[0053] Example 3 Growth curve of strain FJNU-SIO1

[0054] The strain FJNU-SIO1 was cultured overnight, and then inoculated into LB liquid medium with NaCl concentration of 1%, 4%, 7%, 10%, 13% at 10% inoculation amount, and cultured at 35°C, 220 rpm, and the optical density (OD) of the bacterial suspension was measured at 600 nm every 2 hours. 600 The biomass of the strain FJNU-SIO1 was represented by OD Figure 4 .

[0055] The strain showed a faster growth rate and a shorter lag phase, and a higher final biomass under low salt concentration (1%, 4%). The growth under 1% salt concentration was close to the optimal condition (if there was no other limiting factor). The growth rate decreased and the lag phase extended under medium salt concentration (7%, 10%), indicating that the effect of salt stress appeared, but the strain could still grow to a certain extent. There was almost no obvious growth under high salt concentration (13%), and the OD value remained almost unchanged, indicating that the high salt environment severely inhibited the growth of the strain.

[0056] Example 4: Determination of the protein degradation ability of the strain FJNU-SIO1

[0057] (1) Hydrolysis circle of the strain FJNU-SIO1 on casein plate

[0058] The strain was inoculated on the casein hydrolysis medium and cultured at 35°C for 48 hours. The diameter of the hydrolysis circle (D) and the diameter of the colony (d) were measured, and the D / d value was calculated. Figure 5

[0059] The results showed that the diameter of the hydrolysis circle was 3.00 mm, the diameter of the colony was 1.10 mm, and the D / d value was 2.73 after 48 hours of culture.

[0060] (2) Protein degradation rate of the strain FJNU-SIO1

[0061] The strain FJNU-SIO1 was cultured overnight, and then inoculated into protein-based fermentation medium with different salt concentrations (1%, 3%, 5%, 7%, 10%, 13%) at 2% inoculation amount, and placed in a 35°C, 220 r / min shaker for shaking culture. At 24h, 48h and 72h, 0.5mL of the fermentation broth was centrifuged at 10000 r / min for 3 min, and then 50.0μL of the supernatant was taken into a test tube. The protein degradation ability of the strain was determined by the Coomassie brilliant blue method. Figure 6

[0062] The results showed that the protein degradation rate of the strain increased with time, and there was no significant difference in the degradation rate at 48h and 72h, indicating that the protein degradation ability of the strain reached a stable state. The protein degradation rate of the strain decreased with increasing salt concentration, but the degradation rate under 10% salt concentration still reached 47.68% at 72h.​​

[0063] Example 5 Determination of the ability of strain FJNU-SIOl to degrade biogenic amines

[0064] 1. Determination of the ability to degrade biogenic amines: the strain FJNU-SIOl was cultured overnight and inoculated into LB-B medium with different salt concentrations (1, 3, 5, 7, 10%) at a 2% inoculation amount, and cultured at 35°C, 220 rpm for 24 h. The supernatant was collected by centrifugation at 10,000 rpm for 10 min, and 1 mL of the supernatant was analyzed for BAs content.

[0065] 2. Detection of biogenic amines in fermentation broth:

[0066] Pre-treatment of fermentation broth: 2 mL of the collected culture broth was centrifuged at 10,000 rpm for 10 min, and 1 mL of the supernatant was collected to prepare the sample to be tested. The sample to be tested was derivatized and then the content of biogenic amines was determined by high performance liquid chromatography.

[0067] (1) Pre-column derivatization of biogenic amines

[0068] 1 mL of the mixed standard BAs solution or sample supernatant was mixed with 1 mL of saturated NaHCO3 solution, 100 μL of NaOH solution (1 mol / L), 250 μL of internal standard solution, and 1 mL of derivatizing agent (10 mg / mL). The reaction mixture was placed in a water bath at 60°C for 15 min. Then 0.5 mL of ammonia water was added to terminate the reaction, and the mixture was placed in a water bath at 60°C for 15 min. The sample was extracted with 3 mL of diethyl ether, and then the supernatant was collected in a 10 mL test tube and dried. Finally, the residue was redissolved in 1 mL of acetonitrile, filtered through a 0.22 μm filter, and analyzed by high performance liquid chromatography.

[0069] (2) Preparation of biogenic amine standard curve

[0070] 100 mg / L of the mixed standard BAs solution was diluted with 0.1 mol / L HCl solution to prepare a concentration gradient of 1, 2.5, 5, 10, 15, 25, 50, and 100 mg / L. The standard curve was prepared with the peak area of each BAs / peak area of the internal standard as the y-axis and the concentration gradient of each BAs as the x-axis.

[0071] (3) High performance liquid chromatography conditions

[0072] Chromatographic separation conditions: HPLC-UV (Waters e2695-2998, Waters, USA) and reverse phase C18 chromatographic column (Waters Sunfire, 5 μm, 4.6 x 250 mm) were used to detect the derivatized BAs. The gradient elution rate of acetonitrile (mobile phase A) and water (mobile phase B) was 0.8 mL / min; the ultraviolet detection wavelength was 254 nm, and the gradient elution program is shown in Table 1.

[0073] Table 1 Elution program for RP-HPLC determination of biogenic amines

[0074]

[0075] 3. Results: After 24 h of culture, the degradation rates of total biogenic amines under different salt concentrations were 18.15%, 17.95%, 20.58%, 27.10%, 32.77% (from low to high salt concentration), respectively. Figure 7 The test proved that Bacillus velezensis FJNU-SIO1 has excellent biogenic amine degradation ability, and the ability of Bacillus velezensis to degrade biogenic amines increases with increasing salt concentration.

[0076] Example 7 Preparation of a degradation agent based on strain FJNU-SIO1

[0077] (1) After streaking and activating strain FJNU-SIO1 on LB plates, single colonies were picked and inoculated into test tubes containing LB liquid medium, and cultured to the exponential growth phase as the first-stage seed liquid; then inoculated into LB liquid medium at a 1% (v / v) inoculation amount, and shaken and cultured to the exponential growth phase to obtain a fermentation seed liquid.

[0078] (2) The fermentation seed liquid prepared above was inoculated into the culture medium in the production tank at 5-15% (v / v), and the pH was adjusted to 5.0-7.0 during the culture process, the aeration amount of sterile air was 1.0-1.5 vvm, the stirring rate was 200-300 rpm, the culture temperature was 30-37°C, and the culture was fermented for 24-48 h. The culture liquid after fermentation was directly packaged into a liquid degradation agent, or the bacterial cells were dried to prepare a powder, or a high-purity preparation was prepared through separation and purification steps. The culture medium in the production tank was LB liquid medium.

[0079] The above results show that strain FJNU-SIO1 has good salt tolerance and can efficiently degrade proteins and biogenic amines.

[0080] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification, equivalent change and modification of the above embodiment without departing from the technical solution content of the present application and according to the technical essence of the present application still belongs to the scope of the technical solution of the present application.

Claims

1. A salt-tolerant bacterial strain FJNU-SIO1 that degrades proteins and biogenic amines, characterized in that, The strain is classified as Bacillus belesiensis (B. belesiensis). Bacillus velezensis The accession number is CGMCC No. 36086, and it is deposited at the China General Microbiological Culture Collection Center on September 26, 2025.

2. The salt-tolerant strain FJNU-SIO1 that degrades proteins and biogenic amines according to claim 1, characterized in that, The nucleotide sequence of the 16S rDNA of strain FJNU-SIO1 is shown in SEQ ID No.

1.

3. A method for simultaneously degrading proteins and biogenic amines in a saline environment, characterized in that, The method uses the strain FJNU-SIO1 described in claim 1.

4. The method for degrading proteins and biogenic amines in a saline environment according to claim 3, characterized in that, The reaction is carried out in an environment with a salt concentration of 1%-13%.

5. A method for degrading proteins or biogenic amines, characterized in that, The method uses the strain FJNU-SIO1 described in claim 1.

6. The application of strain FJNU-SIO1 as described in claim 1 in the degradation of biogenic amines or proteins.

7. The application according to claim 6, characterized in that, The application is carried out in a saline environment and the fields of application include food fermentation, aquatic product processing, or high-protein wastewater treatment.

8. The application according to claim 6, characterized in that, The biogenic amines include tryptamine, phenylethylamine, putrescine, cadaverine, histamine, octopamine, tyramine, spermine, or spermidine.

9. A microbial inoculant, characterized in that, It contains the strain FJNU-SIO1 as described in claim 1.

10. The microbial inoculant according to claim 9, characterized in that, The microbial agent is a liquid or solid preparation.

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