Bacillus amyloliquefaciens with high yield of acid protease and application thereof

Abstract

This invention discloses a strain of *Bacillus amyloliquefaciens* B3 that produces a high amount of acidic protease, the protease isolated from this strain, and its applications, belonging to the field of bioengineering fermentation technology. The *Bacillus amyloliquefaciens* B3 was deposited on January 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33456, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. This strain exhibits a high capacity for producing acidic protease, achieving an enzyme activity of 277 U / mL in shake-flask conditions at pH 5.0, and demonstrating good stability under acidic conditions. The application of the protease produced by this strain in the fermentation process of soy sauce can not only increase the content of amino acid nitrogen and free amino acids in the product, but also enhance the types and content of volatile flavor compounds, significantly improving the quality of soy sauce.

Description

Technical Field

[0001] This invention relates to a strain of Bacillus amyloliquefaciens that produces a high amount of acidic protease and its applications, belonging to the field of bioengineering technology. Background Technology

[0002] In the production of fermented soybean products such as soy sauce and fermented soybean paste, proteases secreted by microorganisms play a crucial role. For example, during the koji-making and fermentation processes, proteases degrade the raw soybean protein into amino acids and oligopeptides. Some of these amino acids provide nitrogen sources and flavor precursors for the growth and metabolism of the microbial community during fermentation, while others remain in the fermented soybean products, contributing to the accumulation of amino acid nitrogen. In the production of fermented soybean products, the fermented mash or paste is typically acidic; therefore, microbial strains with high acid protease production capabilities are essential for improving product quality and flavor. Currently, microbial strains screened for fermented soybean products generally exhibit high neutral and alkaline protease activities, while their acid protease activities are relatively low. Introducing microbial strains with high acid protease production or exogenous acid proteases can help accelerate the fermentation efficiency of fermented soybean products, while simultaneously improving product quality and flavor.

[0003] Bacillus is a genus of microorganisms with a strong protease-producing ability. Among them, *Bacillus amyloliquefaciens*, also known as *Bacillus amyloliquefaciens*, is an FDA-recognized safe microorganism and typically possesses a certain protease production capacity. *Bacillus amyloliquefaciens* belongs to the domain Bacteria, kingdom Bacteria, phylum Firmicutes, class Bacillus, order Nucleochaetes, family Bacillusaceae. It is an aerobic, spore-forming, Gram-positive bacterium. In the inventors' previous research, a strain of *Bacillus amyloliquefaciens*, BS5582 (CGMCC accession number: 1314), with a strong protease production capacity, was screened, showing an acidic protease activity of 191 U / mL in shake flasks.

[0004] Further improving its acidic protease activity will be more conducive to its application in the acidic environment of fermented soybean products. Summary of the Invention

[0005] To address the existing problems, this invention provides a Bacillus amyloliquefaciens B3 that produces a high level of acidic protease.

[0006] The first aspect of the present invention provides a strain of Bacillus amyloliquefaciens B3, which was deposited on January 17, 2025 by the China General Microbiological Culture Collection Center (CMCC) with accession number CMCC No. 33456, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

[0007] Furthermore, the Bacillus amyloliquefaciens B3 was obtained by mutagenesis screening using Bacillus amyloliquefaciens BS5582 as the starting strain, through ambient pressure room temperature plasma (ARTP) technology.

[0008] In some embodiments, the present invention provides a method for culturing Bacillus liquefactionus B3: the Bacillus liquefactionus B3 is inoculated into a nutrient broth liquid culture medium and cultured at 37°C and 180-220 rpm for 10-12 h with shaking until the mid-to-late logarithmic growth stage, to obtain Bacillus liquefactionus B3 seed culture.

[0009] In some preferred embodiments, in the method for culturing Bacillus liquefactionus B3 provided by the present invention, Bacillus liquefactionus B3 is inoculated into a nutrient broth liquid culture medium at an inoculation amount of 1%, and cultured at 37°C and 180 rpm for 10 h with shaking.

[0010] Furthermore, the present invention also provides a fermentation method for Bacillus liquefaction of starch B3, wherein the seed culture of Bacillus liquefaction of starch B3 is inoculated into corn flour-soybean meal liquid culture medium at an inoculation amount of 10-16%, and cultured at 35°C and 200 rpm for 58 h with shaking.

[0011] A second aspect of the invention provides a microbial agent containing the aforementioned Bacillus liquefactionus B3.

[0012] Furthermore, the microbial agent contains live cells of the Bacillus liquefaction B3 bacterial cells, freeze-dried Bacillus liquefaction B3 bacterial cells, immobilized Bacillus liquefaction B3 cells, liquid Bacillus liquefaction B3 bacterial agent, solid Bacillus liquefaction B3 bacterial agent, culture broth and fermentation broth obtained by culturing or fermenting Bacillus liquefaction B3, or Bacillus liquefaction B3 strain and its culture broth or fermentation broth existing in other forms.

[0013] In some embodiments, the microbial agent is a liquid agent prepared from the fermentation broth of Bacillus liquefactionus B3.

[0014] In some preferred embodiments, the microbial agent is prepared from the supernatant of the fermentation broth obtained by the fermentation method described above.

[0015] In some preferred embodiments, the microbial agent has an acidic protease activity ≥277 U / mL.

[0016] In some preferred embodiments, the microbial agent, at pH 6.0, exhibits a protease activity of 2534 U / mL using casein as a substrate.

[0017] A third aspect of the invention provides three proteases isolated from the aforementioned *Bacillus liquefactionus* B3, said proteases being Bpr, Bpn', and Mpr. These proteases are isolated after fermentation from the aforementioned *Bacillus liquefactionus* B3 or the aforementioned microbial inoculant.

[0018] Further, the protease is: (a) a protein consisting of the amino acid sequences shown in SEQ ID No. 1 to SEQ ID No. 3; or (b) a protein derived from (a) having one or more amino acids substituted, deleted, or added to the amino acid sequence in (a) and possessing protease activity.

[0019] Furthermore, the gene sequences encoding the proteases Bpr, Bpn', and Mpr are shown in SEQ ID No. 4 to SEQ ID No. 6.

[0020] A fourth aspect of the present invention provides the application of the Bacillus liquefactionus B3, or the microbial agent, or the protease in the food industry; in some embodiments, it is applied in the production of fermented legume foods.

[0021] In some embodiments, the microbial agent or the protease is applied to the fermentation production of sauce products; the sauce products include, but are not limited to, soy sauce, broad bean paste, and soybean paste.

[0022] In some embodiments, the microbial agent or the protease is applied in the fermentation production of soy sauce; the application method is as follows: the microbial agent or the protease is added in the early stage of soy sauce fermentation to increase the content of amino acid nitrogen and free amino acids in soy sauce and improve the flavor of soy sauce.

[0023] In some preferred embodiments, an enzyme solution diluted to a casein activity of 100-120 U / mL is added during the fermentation process of fermented legume foods.

[0024] In some embodiments, the present invention also provides a seasoning containing the aforementioned Bacillus liquefactionus B3, the aforementioned microbial inoculant, or the aforementioned protease. The seasoning includes, but is not limited to, soy sauce, broad bean paste, and soybean paste.

[0025] Beneficial effects

[0026] This invention discloses a strain of Bacillus amyloliquefaciens B3 that produces a high amount of acidic protease, and a microbial inoculum containing this strain. The protease activity of this strain reaches 2534 U / mL at pH 6.0 and 277 U / mL at pH 5.0, and it has good stability under acidic conditions.

[0027] The present invention also provides three proteases isolated from this strain; among which Bpr has a wide pH tolerance range and good salinity tolerance, and the protease can still retain 80% of its activity at pH 5.0-9.0; Bpn' can retain 50% of its protease activity at pH 7.0-8.0; and Mpr can retain 50% of its protease activity at pH 6.0-8.0.

[0028] This invention further develops the application of this strain or the protease isolated from this strain in the food industry; when applied to soy sauce fermentation, it can effectively increase the amino acid nitrogen content of soy sauce, and also improve the types and contents of volatile flavor substances; it can significantly improve the quality of soy sauce.

[0029] Preservation of biological materials

[0030] Bacillus amyloliquefaciens B3 was deposited on January 17, 2025, by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33456. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0031] The accompanying drawings are provided to further illustrate the present application and form part of the specification. They are used together with the embodiments of the present application to explain the present application, but do not constitute a limitation thereof; in the drawings:

[0032] Figure 1 Colony morphology and microscopic images of Bacillus amyloliquefaciens B3;

[0033] Figure 2 Growth curves and enzyme production curves of Bacillus amyloliquefaciens B3;

[0034] Figure 3 Enzymatic properties of protease produced by Bacillus amyloliquefaciens B3; (A) Optimal temperature; (B) Optimal pH; (C) pH tolerance; (D) Salinity tolerance;

[0035] Figure 4 The changes in physicochemical parameters of Bacillus liquefaction B3 protease during soy sauce fermentation: (A) amino acid nitrogen; (B) total nitrogen; (C) total acid; (D) pH.

[0036] Figure 5 The metabolite changes during soy sauce fermentation were influenced by the protease produced by Bacillus liquefaction B3; (A) free amino acids; (B) organic acids; (C) types of volatile flavor compounds; (D) content of volatile flavor compounds.

[0037] Figure 6 Flavor sensory radar diagram of the application of Bacillus liquefaction B3 protease in soy sauce fermentation process;

[0038] Figure 7 SDS-PAGE images of important proteases produced by Bacillus liquefaction B3 after protein purification;

[0039] Figure 8 Enzymatic analysis of important proteases produced by Bacillus liquefactionus B3; (A) Optimal temperature; (B) Optimal pH; (C) pH tolerance; (D) Salinity tolerance. Detailed Implementation

[0040] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them, and do not limit the claims of the present invention in any way. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described, unless otherwise specified, are all commercially available.

[0041] The Bacillus amyloliquefaciens BS5582 involved in the following examples is deposited by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1314, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0042] The culture media involved in the following examples are as follows:

[0043] Seed liquid culture medium: 10 g·L -1 5 g·L of beef extract -1 Sodium chloride 5 g·L -1 pH 7.0.

[0044] LB solid medium: 10 g / L peptone -1 5 g·L yeast powder -1 Sodium chloride 10 g·L -1 20 g / L agar -1 .

[0045] Fermentation medium: 50.0 g·L⁻¹ corn flour -1 40.0 g·L soybean meal -1 CaCl2 0.75 g·L -1 (NH4)2SO4 2.0 g·L -1 MgSO4 1.8 g·L -1 3.0 g·L KH2PO4 -1 And Na2HPO4·12H2O 6.0 g·L -1 pH 6.5.

[0046] The protease activity assay methods used in the following examples refer to the Folin method for the determination of protease activity in the enzyme preparation quality requirements standard document GB / T23527.1-2023. The enzyme activity assay in soy sauce refers to the Folin method in SB / T 10317-1999, "Determination of Protease Activity".

[0047] A sodium acetate buffer solution with a pH of 5.0 was prepared for determining the activity of acidic proteases, a phosphate buffer solution with a pH of 7.0 was prepared for determining the activity of neutral proteases, and a borate buffer solution with a pH of 10.5 was prepared for determining the activity of alkaline proteases.

[0048] Example 1: Mutagenesis and Screening of Bacillus amyloliquefaciens with High Production of Acidic Protease

[0049] Casein solid culture medium: casein 4 g·L -1 Na₂HPO₄·7H₂O 1.07 g·L⁻¹ -1 KH2PO4 0.36 g·L -1 Solid culture medium supplemented with 20 g·L⁻¹ agar powder -1 .

[0050] Seed culture medium: 5.0 g·L -1 10.0 g·L peptone -1 NaCl 5.0 g·L -1 pH 7.0.

[0051] Fermentation medium: 50.0 g·L⁻¹ corn flour -1 40.0 g·L soybean meal -1 Na₂HPO₄·12H₂O 6.0 g·L⁻¹ -1 KH2PO4 3.0 g·L -1 (NH4)2SO4 2.0 g·L -1 MgSO4 1.8 g·L -1 CaCl2 0.75 g·L -1pH 6.0.

[0052] LB medium: 10 g / L tryptone -1 Yeast extract 5 g·L -1 NaCl 10 g·L -1 .

[0053] Select strains cultured to 10 h (logarithmic growth phase) for mutagenesis. B. amyloliquefaiens BS5582 was inoculated onto seed culture medium and cultured at 37℃ and 180 r / min on a rotary shaker for 10 h to reach the logarithmic growth phase. A bacterial suspension was prepared using 10% glycerol sterile physiological saline, and ARTP mutagenesis was induced at 0 s, 30 s, 60 s, 75 s, 90 s, 105 s, 120 s, 150 s, 180 s, and 210 s, respectively. Lethality was calculated.

[0054] Lethality (%) = (Number of viable bacteria before mutation - Number of viable bacteria after mutation) / Number of viable bacteria before mutation * 100%

[0055] When the lethality rate of the strain mutagenesis is between 80-90%, the probability of positive mutation is higher. In the embodiments of the present invention, the mutagenesis time is selected as 90 s.

[0056] The diluted bacterial suspension was spread onto acidic casein plates (pH 5.0) and incubated at 37°C for 12 h. The ratio of the clear hydrolysis zone to the colony diameter (H / C) was measured. Colonies with a higher H / C value than BS5582 were screened, cultured, and preserved. These colonies were then inoculated into seed culture medium and cultured at 37°C with shaking at 180 r / min for 10 h. A 10% inoculum was then added to the fermentation medium, and the culture was continued at 35°C with 200 r / min for 48 h to obtain the fermentation broth. The crude enzyme solution was obtained by centrifugation at 10000 r / min for 5 min at 4°C, and the acidic protease activity was determined using the Folin-Ciocalteu method. Strains with significantly different enzyme activities obtained from the secondary screening, along with BS5582, were streaked in three zones on LB solid medium and incubated at 37°C for 10 h. Newly activated single colonies were picked and streaked in three zones on fresh LB solid medium. This process was repeated 18 times. Single colonies from each generation of solid medium were picked and cultured into seed culture for fermentation, and the acidic protease activity was measured. A microbial strain with high casein protease activity and stable inheritance was selected and designated as B3.

[0057] Example 2: Colony morphology analysis of Bacillus amyloliquefaciens B3

[0058] Bacillus amyloliquefaciens B3 was spread onto LB solid medium and incubated at 37°C to form single colonies. Figure 1As shown in Figure A, its colonies are round, with a rough, raised surface and irregular edges; the colonies are pale yellow and opaque; further observation of their morphology under a microscope reveals, for example... Figure 1 As shown in Figure B, it is found that it is mainly in the form of a straight rod.

[0059] Example 3 Enzyme production analysis of Bacillus amyloliquefaciens B3

[0060] The seed culture of *Bacillus amyloliquefaciens* B3 obtained in Example 1 was inoculated at a rate of 10% into corn flour-soybean meal liquid medium (initial pH 6.0) and cultured at 35°C with shaking at 200 rpm. Its growth and enzyme production curves were determined (under pH 5.0 and pH 7.5 conditions). Figure 2 As shown, this strain entered the logarithmic growth phase at 4 hours of culture and reached its peak at 20 hours, after which it gradually declined. The total protease activity of Bacillus liquefaction B3 began to rise slowly at the beginning of fermentation, rose significantly at 36 hours of culture, and reached its peak at 58 hours of fermentation (pH 5.0: 277 U / mL; pH 7.5: 3484 U / mL).

[0061] The protease activity under different pH conditions is as follows: 202 U / mL under alkaline conditions (pH 10.5), 3484 U / mL under neutral conditions (pH 7.5), and 277 U / mL under acidic conditions (pH 5.0).

[0062] Example 4 Enzymatic Properties Analysis of Bacillus amyloliquefaciens B3

[0063] Using casein as a substrate, the protease activity and enzymatic properties of proteases produced by *Bacillus amyloliquefaciens* B3 and the unmutated strain BS5582 were determined and compared. At pH 5.0, the protease activity of strain B3 reached 277 U·mL⁻¹. -1 Compared to the unmutated strain, it increased by 45.03%.

[0064] The protease activity was measured at reaction temperatures of 20, 25, 30, 35, 40, 45, 50, 55, 60, and 70 °C, and the relative enzyme activity was calculated. The highest value was recorded as 100%, and the remaining enzyme activities were expressed as residual enzyme activity percentages. Each point was repeated three times, and the corresponding temperature was taken as the optimum temperature. Figure 3 As shown in A, the optimal temperature for B3 to produce protease is 40℃. At this temperature, the enzyme activity lasts the longest, and 97.28% of the protease activity can still be retained after 1 hour.

[0065] Enzyme activity was determined using the Folin-Ciocalteu method in buffer solutions with pH values ​​ranging from 5.0 to 10.0. For pH 5.0-7.5, a 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was used; for pH 8.0-9.0, a 20 mM Tris-HCl buffer was used; and for pH 10.0, a 20 mM borate-sodium borate buffer was used. Relative enzyme activity was calculated with maximum enzyme activity as 100% to determine the optimal pH. Each assay was repeated three times. Figure 3 As shown in B, the optimal pH for B3 to produce protease is pH 6.

[0066] The enzyme solutions were placed in the above pH buffer system and treated at 40°C for 60 min. The B3 protease activity was then measured at the optimal temperature. The remaining relative enzyme activity was calculated as the ratio of the enzyme activity after treatment to the untreated enzyme activity under the same pH conditions. Figure 3 As shown in C, the B3-produced protease still retains more than 90% of its residual enzyme activity after incubation at pH 5.5-7.0 for 1 h, and 74.52% of the protease activity remains after incubation at pH 5.0 for 1 h, indicating that the B3-produced protease has good acid resistance.

[0067] Example 5: Analysis of the application of Bacillus starch liquefaction B3 in the fermentation process of soy sauce

[0068] Soybean meal and wheat bran were mixed in a 3:2 ratio (by weight), with 100% water content. The mixture was cooked at 121°C for 20 minutes, then cooled to below 40°C. 6 Aspergillus oryzae strain 3.042 was inoculated into the material at an inoculum size of spores / g and cultured at 30℃ and 95% humidity for 72 h to obtain koji (fermentation starter). Brine was added to the koji at a ratio of 1:3 (w / w), resulting in a final salinity of 140 g / kg. Fermentation was carried out at room temperature for 90 days. The fermented mash was filtered, and the filtrate was analyzed. On day 2 of fermentation, 100 U / (per g of koji) of BS5582 protease, B3 protease, and commercially available protease were added as experimental groups, while no protease was added as a control.

[0069] The changes in physicochemical indicators and metabolites during the fermentation process of soy sauce were measured, such as... Figure 4 As shown in A and 4C, the amino acid nitrogen content of soy sauce increased rapidly in the first 30 days of fermentation, and then increased slowly thereafter. At 90 days of fermentation, the amino acid nitrogen content in the soy sauce mash with added B3 protease reached 0.85 g / 100 g. The total acid content increased rapidly from 0 to 60 days of soy sauce fermentation, and then gradually leveled off after 60 days. Figure 4 As shown in D, the pH of the mash gradually changed from neutral to acidic during the soy sauce fermentation process. After 90 days of fermentation, the amino acid nitrogen content in the soy sauce mash with added B3 protease reached 0.85 g / 100 g, the total acid content was 13.94 g / kg, and the total nitrogen content was 16.92 mg / mL. Figure 4 B) The pH is 5.03.

[0070] like Figure 5 As shown in Figure A, the main free amino acids in soy sauce are glutamic acid, arginine, aspartic acid, and leucine, which contribute to its umami flavor. The main organic acids in soy sauce are lactic acid and acetic acid. Figure 5 B), the content of B3 protease in soy sauce is 0.13 g / 100 g and 0.42 g / 100 g, respectively.

[0071] The volatile flavor compounds in soy sauce samples fermented for 90 days were determined using HS-SPME-GC-MS. Figure 5 As shown in C and 5D, the levels of esters, alcohols, and acids in the four groups of soy sauces with added B3 protease were slightly increased. The soy sauce with added B3 protease had the highest content of esters and alcohols, at 1969.82 µg / kg and 5243.63 µg / kg, respectively, which were 161.88% and 209.09% higher than the soy sauce without added protease, and 52.31% and 45.16% higher than the soy sauce with added BS5582 protease, respectively.

[0072] Example 6: Isolation and purification of important B3 protease from Bacillus starch liquefaction

[0073] The fermentation broth obtained in Example 3 was centrifuged at 4°C and 10,000 rpm for 10 min, and the supernatant was collected after discarding the bacterial cells. A fractional salting-out method was used: first, (NH4)2SO4 was added to a saturation of 30%, and after standing, the supernatant was retained by centrifugation. Then, the supernatant was slowly added to a saturation of 80%, and the protein precipitate was collected by centrifugation. The protein precipitate was then reconstituted in 20 mmol·L⁻¹ solution. -1 In a pH 8.5 Tris-HCl buffer solution, add the ammonium sulfate-precipitated sample to the dialysis bag and place it in a 20 mmol·L⁻¹ solution. -1 Dialyze in pH 8.5 Tris-HCl buffer at 4°C.

[0074] The protease was purified using a strong anion exchange Q column and a hydrophobic HiPrep butyl column. After dialyzing and centrifugation with a molecular weight cutoff of 10 kDa, Subtilisin (Bpn') was obtained, with a molecular weight of approximately 27.5 kDa. Figure 7 );

[0075] The protease was purified using a HiPrep DEAE FF ion exchange column (1.6 cm × 10 cm) and a HiPrep Butyl FF hydrophobic chromatography column. The collected active peak was processed through an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, and then eluted using a Superdex 200 10 / 300GL gel filtration chromatography column with 0.15 mol / L NaCl solution (dissolved in 0.02 mol / L pH 7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer) to obtain extracellular neutral metalloprotease (Mpr) with a molecular weight of approximately 44 kDa.

[0076] The protease was purified using a HiPrep DEAE FF ion exchange column (1.6 cm × 10 cm) and a Superdex200 10 / 300GL gel filtration chromatography column. The collected active peak was processed through an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa to obtain Bacillopeptidase F (Bpr), with a molecular weight of approximately 50 kDa.

[0077] Example 7 Enzymatic Properties Analysis of Important B3 Proteases in Bacillus Amyloliquefaciens

[0078] Reaction temperatures of 30, 35, 40, 45, 50, 55, 60, 65, and 70°C were used. After preheating for 2 minutes, enzyme activity was measured and relative enzyme activity was calculated. The highest value was recorded as 100%, and the remaining enzyme activities were expressed as residual enzyme activity percentages. Each point was repeated three times, and the corresponding temperature was the optimum temperature. Figure 8 As shown in (A), the optimal temperatures for Bpr, Bpn' and Mpr are 40℃, 60℃ and 50℃, respectively.

[0079] Enzyme activity was determined using the national standard Folin-Ciocalteu method in buffer solutions with different pH ranges from 5.0 to 10.0. For pH 5.0-7.5, a 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was used; for pH 8.0-9.0, a 20 mM Tris-HCl buffer was used; and for pH 10.0, a 20 mM borate-sodium borate buffer was used. Relative enzyme activity was calculated with the maximum enzyme activity as 100%. Each assay was repeated three times to determine the optimal pH. Figure 8 As shown in (B), the optimal pH values ​​for Bpr, Bpn' and Mpr are pH 5.0, pH 8.0 and pH 7.0, respectively.

[0080] The enzyme solutions were placed in the above pH buffer system and treated at 40°C for 60 min. Enzyme activity was then measured at the optimal temperature. The remaining relative enzyme activity was calculated as the ratio of the enzyme activity after treatment to the untreated enzyme activity under the same pH conditions. Figure 8As shown in (C), Bpr has a wide pH tolerance range, and the protease can still retain 80% of its activity at pH 5.0-9.0; Bpn' can retain 50% of its protease activity at pH 7.0-8.0; and Mpr can retain 50% of its protease activity at pH 6.0-8.0.

[0081] The protease activity was determined using the national standard Folin-Ciocalteu method in 8%, 12%, 14%, 16%, and 20% NaCl concentration buffers, respectively. Figure 8 As shown in (D), the activities of all three proteases decreased with increasing salinity, with Bpr showing better salinity tolerance compared to the other two proteases. When the NaCl concentration reached 20%, the activities of the three proteases Bpr, Bpn', and Mpr were 20 U / mL, 14 U / mL, and 17 U / mL, respectively.

[0082] In summary, this invention discloses a strain of *Bacillus amyloliquefaciens* B3 that produces a high amount of acidic protease, a microbial inoculum containing this strain, and three proteases isolated from this strain; and further develops its application in the food industry. This strain exhibits a high capacity for producing acidic protease, with an enzyme activity of 277 U / mL at pH 5.0 in shake flask conditions, and good stability under acidic conditions. At pH 6.0, its protease activity reaches 2534 U / mL. When applied to soy sauce fermentation, it can effectively increase the amino acid nitrogen content of soy sauce, and also enhance the types and content of volatile flavor compounds, significantly improving the quality of soy sauce.

[0083] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens B3, characterized in that, It was deposited on January 17, 2025 by the China General Microbiological Culture Collection Center (CMCC), with accession number CMCCNo.33456. The deposit address is: No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. A microbial agent containing Bacillus liquefactionus B3 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent contains live cells of the Bacillus amyloliquefaciens B3 bacterial cells.

4. The microbial agent according to claim 2, characterized in that, The microbial agent contains freeze-dried Bacillus liquefaction B3 dry cells or immobilized Bacillus liquefaction B3 cells.

5. The microbial agent according to claim 2, characterized in that, The microbial agent contains a liquid agent of Bacillus liquefaction B3, a solid agent of Bacillus liquefaction B3, or a culture broth and fermentation broth obtained by culturing or fermenting Bacillus liquefaction B3.

6. The microbial agent according to claim 2, characterized in that, The microbial agent produces acidic protease with an enzyme activity ≥277 U / mL through fermentation.

7. The microbial agent according to claim 2, characterized in that, The microbial inoculant produces Bpr, Bpn' and Mpr proteases through fermentation, and their amino acid sequences are shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively.

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