Bacillus amyloliquefaciens with high yield of acid protease and application of bacillus amyloliquefaciens

Abstract

The invention discloses bacillus amyloliquefaciens B3 with high yield of acid protease, protease separated from the bacillus amyloliquefaciens B3 and application of the bacillus amyloliquefaciens B3, and belongs to the technical field of bioengineering fermentation. The bacillus amyloliquefaciens B3 is preserved in the China General Microbiological Culture Collection Center on January 17, 2025, the preservation number is CGMCC No.33456, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing. The strain has high acid protease production capacity, the shake flask enzyme activity reaches 277U / mL under the condition that the pH value is 5.0, and the strain has good stability under the acid condition. The protease produced by the strain is applied in the soy sauce fermentation process, so that the content of amino acid nitrogen and free amino acid in the product can be increased, the variety and content of volatile flavor substances are also increased, and the quality of soy sauce can be remarkably improved.

Description

Technical Field

[0001] The invention relates to a starch liquefying bacillus capable of producing high acidic protease and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] In the production process of fermented bean foods such as soy sauce and bean paste, proteases secreted by microorganisms play an important role. For example, during the koji making and fermentation process, proteases will degrade raw bean proteins into amino acids and oligopeptides, part of which provide nitrogen sources and flavor precursors for the growth and metabolism of microbial communities in the fermentation process, and the other part remains in the fermented bean foods to help the accumulation of amino acid nitrogen in the products. In the production process of fermented bean foods, fermented mash or mash is usually acidic, so microbial strains with high acid protease production ability play an important role in improving product quality and flavor. At present, the microbial strains screened in fermented bean foods usually have high neutral protease and alkaline protease activities, while acid proteases are relatively low. The introduction of microbial strains with high acid protease production or exogenous acid proteases can help accelerate the fermentation efficiency of fermented bean foods, while improving product quality and flavor.

[0003] Bacillus is a genus of microorganisms with strong protease production ability. Among them, Bacillus amyloliquefaciens, also known as Bacillus amyloliquefaciens, is a safe microorganism recognized by the FDA and usually has a certain protease production ability. Bacillus amyloliquefaciens belongs to the domain of bacteria, kingdom of bacteria, phylum Firmicutes, class Bacillus, order Nucleocotyles, family Bacillaceae, and is a type of aerobic, spore-forming Gram-positive bacteria. In the inventor's previous research, a strain of Bacillus amyloliquefaciens BS5582 (CGMCC preservation number: 1314) with strong protease production ability has been screened, and its shake flask acid protease activity is 191U / mL.

[0004] Further improving the activity of its acidic protease will be more conducive to its application in the acidic environment of bean fermented foods. Summary of the invention

[0005] In order to solve the existing problems, the present invention provides a starch liquefaction Bacillus (Bacillus amyloliquefaciens) B3 with high acid protease production.

[0006] The first aspect of the present invention provides a strain of Bacillus amyloliquefaciens B3, which was deposited by the General Microbiology Center of the China Microorganism Culture Collection Administration on January 17, 2025, with a deposit number of CGMCC No. 33456, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

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

[0008] In some embodiments, the present invention provides a method for culturing Bacillus amyloliquefaciens B3: inoculating the Bacillus amyloliquefaciens B3 into a nutrient broth liquid culture medium, and culturing at 37° C. and 180-220 rpm for 10-12 hours until the middle and late stages of logarithmic growth to obtain a Bacillus amyloliquefaciens B3 seed solution.

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

[0010] Furthermore, the present invention also provides a fermentation method of Bacillus liquefacting B3, inoculating a 10-16% inoculum of Bacillus liquefacting B3 seed liquid into a corn flour-soybean cake powder liquid culture medium, and shaking and culturing at 35° C. and 200 rpm for 58 hours.

[0011] The second aspect of the present invention provides a microbial agent containing the amyloliquefaciens Bacillus B3.

[0012] Furthermore, the microbial agent contains living cells of the amyloliquefaciens Bacillus B3 bacteria, dry amyloliquefaciens Bacillus B3 bacteria obtained by freeze-drying, immobilized amyloliquefaciens Bacillus B3 cells, liquid agent of the amyloliquefaciens Bacillus B3, solid agent of the amyloliquefaciens Bacillus B3, culture liquid and fermentation liquid obtained by culturing or fermenting the amyloliquefaciens Bacillus B3, or the amyloliquefaciens Bacillus B3 strain in other forms and its culture liquid or fermentation liquid.

[0013] In some embodiments, the microbial agent is a liquid agent prepared from the fermentation broth of Bacillus amyloliquefaciens 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 acid protease activity of the microbial agent is ≥277U / mL.

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

[0017] The third aspect of the present invention provides three proteases separated from the amyloliquefaciens Bacillus sp. B3, wherein the proteases are Bpr, Bpn' and Mpr, which are separated and obtained by fermentation of the amyloliquefaciens Bacillus sp. B3 or the microbial agent described above.

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

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

[0020] The fourth aspect of the present invention provides the use of the starch liquefying Bacillus B3, or the microbial agent, or the protease in the food industry; in some embodiments, it is used in the production of fermented bean food.

[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, bean paste and soybean paste.

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

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

[0024] In some embodiments, the present invention also provides a condiment containing the starch liquefying Bacillus B3, the microbial agent or the protease. The condiment includes but is not limited to soy sauce, bean paste and soybean paste.

[0025] Beneficial Effects

[0026] (1) The present invention discloses a starch liquefying Bacillus B3 with high acid protease production and a microbial agent containing the strain; the protease activity of the strain reaches 2534 U / mL at pH 6.0; the protease activity reaches 277 U / mL at pH 5.0, and the strain has good stability under acidic conditions.

[0027] (2) The present invention also provides three proteases isolated from the strain; among them, Bpr has a wide tolerance range for pH and good tolerance to salinity, 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] (3) The present invention further develops the application of the strain or the protease isolated from the strain in the food industry; when applied to soy sauce fermentation, it can effectively increase the amino acid nitrogen content of the soy sauce, and the types and contents of volatile flavor substances are also increased; and the quality of the soy sauce can be significantly improved.

[0029] Biomaterial Deposit

[0030] Bacillus amyloliquefaciens, classified and named Bacillus amyloliquefaciens B3, was deposited by the General Microbiology Center of the China Culture Collection Administration on January 17, 2025, with the deposit number CGMCCNO.33456. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings are used to provide a further understanding of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the present application and do not constitute a limitation of the present application. In the accompanying drawings:

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

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

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

[0035] Figure 4 Changes of physical and chemical indicators in the process of soy sauce fermentation when protease produced by Bacillus amyloliquefaciens B3 was used; (A) amino acid nitrogen; (B) total nitrogen; (C) total acid; (D) pH;

[0036] Figure 5 Metabolite changes in soy sauce fermentation process using protease produced by Bacillus amyloliquefaciens B3; (A) free amino acids; (B) organic acids; (C) types of volatile flavor substances; (D) content of volatile flavor substances;

[0037] Figure 6 Flavor sensory radar chart of protease produced by Bacillus amyloliquefaciens B3 used in soy sauce fermentation process;

[0038] Figure 7 SDS-PAGE profile of protein purification of important proteases produced by Bacillus amyloliquefaciens B3;

[0039] Figure 8 Analysis of enzymatic properties of important proteases produced by Bacillus amyloliquefaciens B3; (A) Optimum temperature; (B) Optimum pH; (C) pH tolerance; (D) Salinity tolerance. DETAILED DESCRIPTION

[0040] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and do not limit the claims of the present invention in any way. The experimental methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0041] The starch liquefaction Bacillus (Bacillus amyloliquefaciens BS5582) involved in the following examples is deposited by the General Microbiological Center of China Microorganism Culture Collection Committee with a deposit number of CGMCC No.1314 and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0042] The culture medium involved in the following examples is as follows:

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

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

[0045] Fermentation medium: corn flour 50.0 g·L -1 , bean cake powder 40.0g·L -1 , CaCl 2 0.75g·L -1 , (NH 4 ) 2 SO 4 2.0g·L -1 , MgSO 4 1.8 g L -1 , KH 2 PO 4 3.0g·L -1 and Na 2 HPO 4 12H 2 O 6.0g·L -1 ; pH6.5.

[0046] The protease activity determination method involved in the following examples refers to the protease activity determination - Folin method in the enzyme preparation quality requirement standard document GB / T23527.1-2023. The enzyme activity determination in soy sauce refers to the Folin method in SB / T 10317-1999 "Protease Activity Determination Method".

[0047] Sodium acetate buffer at pH 5.0 was prepared for determining the activity of acidic proteases, phosphate buffer at pH 7.0 was prepared for determining the activity of neutral proteases, and boric acid buffer at pH 10.5 was prepared for determining the activity of alkaline proteases.

[0048] Example 1 Mutagenesis and screening of Bacillus starch liquefacti with high acid protease production

[0049] Casein solid medium: casein 4g·L -1 , Na 2 HPO 4 7H 2 O 1.07 g·L -1 , KH 2 PO 4 0.36 g L -1 , add agar powder 20g·L to solid culture medium -1 .

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

[0051] Fermentation medium: corn flour 50.0 g·L -1 , bean cake powder 40.0g·L -1 , Na2 HPO 4 12H 2 O 6.0g·L -1 , KH 2 PO 4 3.0g·L -1 , (NH 4 ) 2 SO 4 2.0g·L -1 , MgSO 4 1.8 g L -1 , CaCl 2 0.75g·L -1 , pH 6.0.

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

[0053] The strains cultured for 10 hours (logarithmic phase) were selected for mutagenesis. B. amyloliquefaiens BS5582 was inoculated into seed culture medium and cultured in a rotary shaker at 37°C and 180 r / min for 10 hours until the logarithmic growth phase, and a bacterial suspension was prepared with sterile saline containing 10% glycerol. ARTP mutagenesis was performed at 0s, 30s, 60s, 75s, 90s, 105s, 120s, 150s, 180s, and 210s, and the lethality was calculated.

[0054] Lethality rate (%) = (number of live bacteria before mutagenesis - number of live bacteria after mutagenesis) / number of live bacteria before mutagenesis * 100%

[0055] When the lethality rate of strain mutation is between 80% and 90%, the probability of positive mutation is higher. In the embodiment of the present invention, the mutagenesis time is selected to be 90s.

[0056] The diluted bacterial suspension was spread on an acid casein plate (pH 5.0), and after constant temperature culture at 37°C for 12 hours, the ratio of the transparent hydrolysis zone to the colony diameter (H / C) was measured, and the colonies with a larger H / C value than BS5582 were screened for culture and preservation, inoculated into a seed culture medium, and cultured at 37°C and 180r / min for 10 hours, and inoculated into a fermentation medium with a 10% inoculum, and continued to be cultured at 35°C and 200r / min for 48 hours to obtain a strain fermentation liquid, and centrifuged at 4°C and 10000r / min for 5 minutes to obtain a crude enzyme liquid, and its acid protease activity was determined by the Folin phenol method. The strains with significant differences in enzyme activity obtained by repeated screening and BS5582 were three-zoned on LB solid culture medium, cultured at 37°C for 10 hours, and the newly activated single colonies were picked and three-zoned on new LB solid culture medium, and repeated 18 times in sequence, and the single colonies on each generation of solid culture medium were picked and cultured into seed liquid, fermented and cultured, and the acid protease activity was determined. A microbial strain with high caseinase activity and stable inheritance was selected and numbered B3.

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

[0058] The starch liquefaction Bacillus B3 was spread on LB solid medium and cultured at 37°C to form single colonies. Figure 1 As shown in A, the colony is round, with a rough surface, ridges, and irregular edges; the colony is light yellow and opaque; further observation of its morphology under a microscope, such as Figure 1 As shown in B, it was found that it was mainly in the form of a straight rod.

[0059] Example 3 Analysis of enzyme production of starch liquefying Bacillus B3

[0060] The seed liquid of Bacillus amyloliquefaciens B3 obtained in Example 1 was inoculated into a corn flour-soybean cake powder liquid culture medium (the initial pH of the culture medium was 6.0) at a 10% inoculation rate and cultured at 35°C and 200 rpm. Its growth and enzyme production curves (under pH 5.0 and pH 7.5 conditions) were measured. Figure 2 As shown in the figure, the strain entered the logarithmic growth phase at the 4th hour of culture, reached the highest value at the 20th hour of culture, and then began to gradually decline. The total protease activity of Bacillus amyloliquefaciens B3 began to rise slowly at the beginning of fermentation, and began to rise significantly at the 36th hour of culture, and reached the highest value at the 58th hour of fermentation (pH5.0: 277U / mL; pH7.5: 3484U / mL).

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

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

[0063] The protease activity and enzymatic properties of proteases produced by Bacillus amyloliquefaciens B3 and unmutated strain BS5582 were determined and compared using casein as substrate. At pH 5.0, the protease activity of strain B3 reached 277 U·mL -1 , an increase of 45.03% compared to the non-mutagenized strain.

[0064] The protease activity was measured at 20, 25, 30, 35, 40, 45, 50, 55, 60, and 70°C as reaction temperatures, and the relative activity was calculated. The highest 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 optimal temperature. Figure 3 As shown in A, the optimum temperature for B3 protease production is 40°C, at which the enzyme activity lasts longest, and 97.28% of the protease activity can still be retained after 1 hour.

[0065] The enzyme activity was determined by the national standard Folin method in different buffers of pH 5.0-10.0. 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was used for pH 5.0-7.5, 20 mM Tris-HCl buffer was used for pH 8.0-9.0, and 20 mM boric acid-sodium borate buffer was used for pH 10.0. The relative enzyme activity was calculated with the maximum enzyme activity as 100% to determine the optimal pH. Each point was repeated three times. Figure 3 As shown in B, the optimum pH for B3 protease production is pH 6.

[0066] The enzyme solution was placed in the above pH buffer system, and treated at 40°C for 60 min. The protease activity of B3 was measured at the optimal temperature, and the residual relative enzyme activity was calculated by the ratio of the enzyme activity after treatment to the enzyme activity without treatment under the same pH conditions. Figure 3 As shown in C, the protease produced by B3 can still maintain more than 90% of the residual enzyme activity after incubation at pH 5.5-7.0 for 1 hour, and 74.52% of the protease activity remains after incubation at pH 5.0 for 1 hour, indicating that the protease produced by B3 has good acid resistance.

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

[0068] Mix soybean meal and bran in a ratio of 3:2 (mass ratio), moisten with 100% water, cook at 121℃ for 20min, cool to below 40℃, and heat for 10min. 6 The material was inoculated with Aspergillus oryzae 3.042 strains at a spore inoculation rate of 100 / g, and the temperature was controlled at 30℃ and the humidity was 95% for 72h to obtain the finished koji. Salt water was added to the finished koji at a ratio of 1:3 (w / w), and the final salinity of the sauce mash was 140g / kg. The fermentation was carried out at room temperature for 90d, and the sauce mash was filtered and the filtrate was analyzed in the next step. On the second day of fermentation, 100U / (per g koji) of BS5582 protease, B3 protease and commercial protease were added as the test group, and no protease was added as the control.

[0069] The changes in physical and chemical indicators and metabolites during soy sauce fermentation were determined, 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. At 90 days of fermentation, the amino acid nitrogen content in the soy sauce mash with the addition of B3 protease reached 0.85g / 100g. The total acid content increased rapidly from 0 to 60 days of soy sauce fermentation, and gradually leveled off after 60 days. Figure 4 As shown in Figure D, the pH of the mash gradually changed from neutral to acidic during the fermentation of soy sauce. At 90 days of fermentation, the amino acid nitrogen content in the mash produced by adding B3 protease reached 0.85g / 100g, the total acid content was 13.94g / kg, and the total nitrogen content was 16.92mg / mL ( Figure 4 B) pH is pH 5.03.

[0070] like Figure 5 As shown in A, the free amino acids in soy sauce are mainly glutamic acid, arginine, aspartic acid, and leucine, which provide the umami taste of soy sauce. The organic acids in soy sauce are mainly lactic acid and acetic acid ( Figure 5 B), the contents of B3 protease-producing soy sauce were 0.13g / 100g and 0.42g / 100g respectively.

[0071] HS-SPME-GC-MS was used to determine the volatile flavor substances in soy sauce samples after 90 days of fermentation. Figure 5 As shown in C and 5D, the ester, alcohol and acid substances in the four groups of soy sauces added with B3 protease increased slightly, and the ester and alcohol contents in the soy sauce added with B3 protease were the highest, which were 1969.82μg / kg and 5243.63μg / kg, respectively, which were 161.88% and 209.09% higher than those in the soy sauce without protease added, and 52.31% and 45.16% higher than those in the soy sauce added with BS5582 protease.

[0072] Example 6 Isolation and purification of important proteases from Bacillus amyloliquefaciens B3

[0073] The fermentation broth obtained in Example 3 was centrifuged at 4°C and 10,000 rpm for 10 min, and the cells were discarded and the supernatant was collected. The fractionated salting-out method was adopted, and (NH 4 ) 2 SO 4 The saturation was 30%, and the supernatant was retained after standing. The supernatant was then slowly added until the saturation was 80%, and the protein precipitate was collected by centrifugation. The protein precipitate was redissolved in 20 mmol·L -1 , pH 8.5 Tris-HCl buffer, add the sample precipitated with ammonium sulfate into the dialysis bag and place it in 20mmol·L -1 The protein was dialyzed in Tris-HCl buffer, pH 8.5, at 4°C.

[0074] The protease was purified by using a strong anion Q column and a hydrophobic chromatography HiPrep butyl column, dialyzed and centrifuged using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa to obtain Subtilisin (Bpn') with a molecular weight of approximately 27.5 kDa ( Figure 7 );

[0075] The protease was purified using HiPrep DEAE FF ion exchange column (1.6 cm × 10 cm) and hydrophobic chromatography column HiPrep Butyl FF. The collected active peak was treated with an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, and then eluted using a gel filtration chromatography column Superdex 200 10 / 300GL with 0.15 mol / L NaCl solution (dissolved in 0.02 mol / L sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.0) 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 gel filtration chromatography column Superdex 20010 / 300GL. 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 Analysis of the enzymatic properties of important proteases of Bacillus amyloliquefaciens B3

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

[0079] The enzyme activity was determined by the national standard Folin method in different buffers of pH 5.0-10.0. 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was used for pH 5.0-7.5, 20 mM Tris-HCl buffer was used for pH 8.0-9.0, and 20 mM boric acid-sodium borate buffer was used for pH 10.0. The relative enzyme activity was calculated with the maximum enzyme activity as 100%. Each point was repeated three times to determine the optimal pH. Figure 8 As shown in (B), the optimal pH values ​​of Bpr, Bpn' and Mpr are pH 5.0, pH 8.0 and pH 7.0, respectively.

[0080] The enzyme solution was placed in the above pH buffer system, and treated at 40°C for 60 min. The enzyme activity was measured at the optimum temperature, and the remaining relative enzyme activity was calculated by the ratio of the treated enzyme activity to the untreated enzyme activity under the same pH conditions. Figure 8 As shown in (C), Bpr has a wide tolerance range for pH, 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 by the national standard Folin method in 8%, 12%, 14%, 16%, and 20% NaCl buffer. Figure 8 As shown in (D), the activities of the three proteases decreased with the increase of salinity, among which Bpr had better tolerance to salinity than the other two proteases. When the NaCl concentration reached 20%, the activities of Bpr, Bpn' and Mpr were 20U / mL, 14U / mL and 17U / mL respectively.

[0082] In summary, the present invention discloses a strain of Bacillus amyloliquefaciens B3 with high acid protease production, a microbial agent containing the strain, and three proteases isolated from the strain; and further develops its application in the food industry. The strain has a high ability to produce acidic proteases, and its shake flask enzyme activity reaches 277U / mL under pH 5.0 conditions, and has good stability under acidic conditions. At pH 6.0, its protease activity reaches 2534U / mL. When applied to soy sauce fermentation, it can effectively increase the amino acid nitrogen content of soy sauce, and the types and contents of volatile flavor substances are also improved, which can significantly improve 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 rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

[0084] The sequences used in the present invention are as follows:

[0085] SEQ ID No.1Bpr amino acid sequence

[0086] MKKKTRKRWTGSVLSAIVVSSLLFPGTAGANSTPGAVSFTKDLSASKSIQHKISDSVKKRFEKSDKVTFLIK

[0087] FKEKANTKKAVKEAEKNAKSQSLSVAKTEYQKRSAVISSLKVAAHDAQQNMKTYLEKQKKKGKADHIH

[0088] SYYIVNGMAVTASKEVMEKAASFPEVEKVLPNEKRQLTQSKAPFQMKKKQKEIKAKGGIEWNISQIDAPK

[0089] AWASGYDGTGTVVASIDTGVQWDHPALKEKYRGYDPKNPAVPNHEMNWYDAVAKKDAPYDDLEHGT

[0090] HVTGTMTGSEPDGSNQIGVAPGAKWIAVKAFSDDGGTDADILDAGEWVLAPKDKNGTPHPEMAPDVVN

[0091] NSWAGGSGIDEWYRDMVNAWRAAGIFPEFSAGNVDLFTPGGPGSIANPANYPEAFATGATDSQKKLADF

[0092] SLQGPPSYHETKPDISAPGVNIRSSIPGGTYEGGWNGTSMAGPHVAATAALLRQANASITVDEMEDVLTRT

[0093] AEKLTDSDFPESPNNGYGHGLVNAFDAVSAVTDGIGSIEGKVSSAGEDHNPPSWHHEPVSEAYKGANLPL

[0094] TVTAEDDVSVTEVLLSYQFDKGEWKTIAAVRKSGDEKKGTYQADIPHVTGSTVSYKWTIKDFGGHSAES

[0095] DTYRADVKPSITAGYKEDFESQPAGWFSYGTHDQWEWGIPSSGPGAAFSGDKVYATNLSGPYADSANMN

[0096] LVMPPIQVPDSGRLFLQFKSWHKLEEFFDYGYVFVLPEGKSNWEQAAVYNGDSAGWNDEEADLSAYKG

[0097] QNIKLMFNMQSDEVLNEDGWYIDDVRLSSSSLGKAAEKRKSNRQTTPGHLKKKAVSPKEAKPAVKSPEK

[0098] TVKRETNLLPLRAQISVAETGKSVYSDPATGSYSLSHKAGNYTLKAEAYGFKPAVKQVSIQSDKTAQADF

[0099] TLEQMPSGTLKGTITNQSTGEPVEGAVLYVVEDAAIEPAVTNDKGEYSLQAYEGSYTIKAAAKGFYNSEFS

[0100] VDIKGDAEKNAKLKPYYEGYEIAYDNGTEEAALSYFKAGSKSAVKMTLKDGKEHGMLTGGLFKFWGA

[0101] DWPDPGGTEFQAEVYDASGPDGAPGSKIAGPFQAEALRNGEWTKVDLSSKGIAVGKDFYLVFRQTKPNP

[0102] YSPALSSDDGSPYSNRNWEYLDGSWSSKADKSDGNFMIRALVNYEAIVPDITSPDRSFTNKKKTITVKGTAS

[0103] PGTAVRLTNNGKTAAETKAGADGNFQADVTLRKDANRLTAASVTDRGSTDESRPVTVILDQDKPDVTIDS

[0104] PANGDKTNKEAVTVKGKAYDAHLKEVKVNGKKAEVNNGSYQARILLENGSNEIKVTASDEAGNKTTKK

[0105] TVIDVNYNAPVISGLVPGADKELKAGESVKIAFSSGKKLDATFVIRLPLTNARAGSQNATELPLREISPGRY

[0106] ESYWTATSSIKA

[0107] SGAKIDVIVRDDYGNETRQTAKGKLYIND

[0108] SEQ ID No.2 Bpn' amino acid sequence

[0109] MRGKKVWISLLFALALIFTMAFGSTSSAQAAGKSNGEKKYIVGFKQTMSTMSAAKKKDVISEKGGKVQK

[0110] QFKYVDAASATLNEKAVKELKKDPSVAYVEEDHVAHAYAQSVPYGVSQIKAPALHSQGYTGSNVKVAVI

[0111] DSGIDSSHPDLKVAGGASMVPSETNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADG

[0112] SGQYSWIINGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPG

[0113] KYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHP

[0114] NWTNTQVRSSLENTTTKLGDSFYYGKGLINVQAAAQ

[0115] SEQ ID No.3 Mpr amino acid sequence

[0116] MSLGLSPDTARRQPAWDDSINSAAESSFILQNARLLVYYNNISFHYFQKGGFIVGLGKKLSVAVAASFMSL

[0117] TISLPGVQAAENPQLKENLTNFVPKHSLVQSELPSVSDKAIKQYLKQNGKVFKGNPSERLKLIDQTTDDLG

[0118] YKHFRYVPVVNGVPVKDSQVIIHVDKSNNVYAINGELNNDVSAKTANSKKLSANQALDHAYKAIGKSPE

[0119] AVSNGTVANKNKAELKAAATKDGKYRLAYDVTIRYIEPEPANWEVTVDAETGKILKKQNKVEHAATTGT

[0120] GTTLKGKTVSLNISSESGKYVLRDLSKPTGTQIITYDLQNREYNLPGTLVSSTTNQFTTSSQRAAVDAHYNL

[0121] GKVYDYFYQKFNRNSYDNKGGKIVSSVHYGSRYNNAAWIGDQMIYGDGDGSFFSPLSGSMDVTAHEMT

[0122] HGVTQETANLNYENQPGALNESFSDVFGYFNDTEDWDIGEDITVSQPALRSLSNPTKYGQPDNFKNYKNL

[0123] PNTDAGDYGGVHTNSGIPNKAAYNTITKIGVNKAEQIYYRALTVYLTPSSTFKDAKAALIQSARDLYGSQD

[0124] AASVEAAWNAVGL

[0125] SEQ ID No.4Bpr nucleotide sequence

[0126] TTGAAGAAAAAAACGAGAAAAAGATGGACAGGCTCCGTTTTAAGCGCGATTGTCGTCAGTTCACTGC

[0127] TGTTTCCGGGTACGGCCGGAGCGAACAGCACACCGGGAGCGGTTTCTTTCACCAAAGATCTTTCTGCC

[0128] TCTAAGAGCATTCAGCATAAGATTTCCGATTCAGTCAAAAAGCGTTTTGAAAAAAGTGATAAAGTCA

[0129] CGTTTCTCATCAAATTTAAAGAGAAAGCCAATACGAAAAAGCAGTTAAAGAAGCAGAAAAAAACG

[0130] CCAAGTCTCAATCGCTTTCCGTGGCAAAAACCGAATATCAAAAGCGGTCAGCCGTCATCTCTTCTTTA

[0131] AAAGTGGCGGCTCATGATGCGCAGCAAAATATGAAAACATATTTGGAAAAACAAAAAAAGAAAGGC

[0132] AAGGCGGATCATATCCATTCCTATTATATTGTCAACGGAATGGCAGTCACTGCTTCGAAAGAGGTAAT

[0133] GGAAAAAGCCGCCTCATTTCCTGAAGTAGAAAAAGTTCTTCCGAATGAAAAGCGTCAGCTTACTCAA

[0134] TCAAAAAGCTCCTTTTCAAATGAAGAAAAAACAAAAAGAGATCAAAGCAAAGGGAGGCATTGAATGG

[0135] AACATCAGTCAAATTGACGCGCCGAAGGCATGGGCCTCAGGGTACGACGGCACCGGTACGGTTGTCG

[0136] CCTCCATTGATACAGGGGTTCAGTGGGATCATCCGGCGCTTAAGGAAAAATACCGGGGGTACGATCC

[0137] TAAAAACCCCGCAGTGCCGAACCATGAAATGAACTGGTATGATGCCGTGGCTAAAAAAGACGCGCCC

[0138] TACGATGACCTTGAACATGGCACTCATGTAACAGGCACCATGACGGGTTCAGAACCTGACGGCTCAA

[0139] ATCAAATCGGTGTGGCCCCTGGCGCGAAATGGATCGCAGTAAAAGCATTTTCGGATGATGGAGGAAC

[0140] CGATGCCGACATTTTGGATGCCGGTGAATGGGTGCTTGCGCCAAAAGATAAAAACGGTACTCCTCAT

[0141] CCCGAGATGGCGCCGGATGTTGTCAATAATTCCTGGGCAGGCGGCTCAGGAATCGATGAGTGGTACC

[0142] GCGATATGGTGAATGCATGGAGAGCAGCAGGGATTTTTCCGGAATTCTCTGCGGGAAATGTAGACTT

[0143] GTTTACACCGGGGGGGCCCGGTTCAATCGCAAATCCGGCCAATTATCCGGAAGCATTTGCGACAGGT

[0144] GCGACCGACAGCCAAAAGAAACTCGCTGATTTCTCACTTCAAGGCCCCTCACCGTATCATGAAACAA

[0145] AACCGGATATTTCGGCACCGGGAGTGAATATCCGTTCCTCTATTCCTGGGGGAACATATGAAGGCGG

[0146] CTGGAACGGAACTTCAATGGCCGGACCGCATGTGGCAGCGACGGCGGCTTTGCTACGGCAGGCGAAC

[0147] GCTTCTATTACGGTCGATGAAATGGAAGACGTTTTGACGCGCACCGCCGAAAAGCTGACAGACTCCG

[0148] ATTTTCCGGAATCACCGAATAACGGTTACGGCCACGGCTTAGTCAATGCTTTTGACGCTGTTTCTGCT

[0149] GTAACAGATGGAATAGGAAGCATAGAAGGCAAGGTATCCTCTGCGGGTGAAGATCACAATCCGCCCA

[0150] GCTGGCATCATGAACCCGTCTCAGAAGCTTACAAAGGTGCAAACCTCCCGTTAACGGTTACGGCGGA

[0151] AGATGATGTAAGTGTGACGGAGGTGTTACTTTCCTATCAGTTTGATAAAGGAGAGTGGAAAACGATC

[0152] GCCGCCGTTCGAAAAAGCGGTGATGAAAAAAAGGGAACATACCAGGCGGACATTCCGCACGTAACG

[0153] GGCAGCACGGTAAGTTATAAATGGACAATAAAAGATTTCGGAGGTCATTCCGCAGAATCTGATACGT

[0154] ACCGGGCTGACGTAAAACCAAGCATTACGGCGGGGTACAAAGAGGATTTTGAATCACAGCCGGCCGG

[0155] CTGGTTCAGTTACGGGACACATGACCAATGGGAATGGGGAATCCCGAGTTCAGGGCCAGGCGCTGCA

[0156] TTTTCCGGAGATAAAGTATATGCGACGAACCTGTCCGGACCTTATGCCGATTCAGCCAATATGAATTT

[0157] GGTGATGCCTCCGATCCAGGTTCCGGATTCAGGAAGGTTGTTTTTGCAATTTAAAAGCTGGCACAAGC

[0158] TCGAAGAATTCTTTGATTACGGGTATGTGTTCGTTCTTCCGGAAGGAAAATCAAATTGGGAGCAGGCG

[0159] GCTGTTTACAATGGTGACTCCGCCGGATGGAACGAT

[0160] GAAGAAGCAGATTTATCTGCGTATAAAGGCCAAAACATAAAGCTTATGTTTAATATGCAGTCAGACG

[0161] AAGTACTCAATGAAGACGGATGGTATATCGATGATGTCCGGCTTTCAAGCAGCTCTCTCGGCAAAGC

[0162] AGCCGAAAAAAGGAAATCCAATAGACAAACAACGCCCGGTCATCTGAAGAAAAAGCTGTCAGTCC

[0163] GAAAGAGCTAAACCGGCGGTGAAATCACCCGAGAAAACGGTGAAACGTGAAAACCAATCTCCTGCC

[0164] GCTCAGAGCGCAAATCAGTGTCGCCGAGACGGGGAAATCCGTGTATTCCGACCCTGCAACCGGAAGC

[0165] TACAGCCTGTCTCATAAAGCGGGAAACTACACGCTGAAAGCTGAGGCTTACGGATTTAAGCCAGCTG

[0166] TGAAACAAGTTTCCATTCAATCGACAAAAACCGCACAGGCAGACTTTACCTTGGAGCAAATGCCGTC

[0167] CGGTACGTTAAAAGGAACCATCACCAATCAGTCAACAGGCGAACCGGTGGAAGGGGCTGTGCTTTAT

[0168] GTTGTAGAGGATGCCGCAATTGAGCCGCGGTGACAAATGATAAAGGGGAATATTCGCTTCAGGCGT

[0169] ACGAGGGCTCCTATACAATCAAAGCAGCCGCAAAAGGCTTTTACAACAGTGAATTTTCTGTTGATATC

[0170] AAAGGGATGCTGAAAAAACGCCAAGCTTAAGCCGTATATCGGCTATGAAGGGGAAATTGCCTATG

[0171] ATAACGGGACCGAAGAAGCCGCGCTGTCATATTTTAAGGCAGGCAGCAAATCCGCGGTCAAGATGAC

[0172] GCTTAAAGACGGAAAAGAACACGGTATGCTGACAGGAGGCCTCTTTAAATTTTGGGGCGCGGACTGG

[0173] CCTGATCCGGGCGGCACCGAATTTCAAGCGGAAGTTTACGATGCATCCGGACCTGACGGGGCCCCGG

[0174] GCAGCAAAATTGCCGGCCCGTTCCAAGCTGAAGCGCTTCGAAACGGAGAATGGACGAAAGTGGATCT

[0175] CAGCTCAAAAGGAATCGCAGTCGGAAAGGATTTTTATCTAGTGTTCCGCCAGACGAAACCCAATCCTT

[0176] ATTCACCGGCCCTGTCATCTGATGACGGCAGTCCGTATTCGAACAGAAATTGGGAATATCTCGACGGC

[0177] AGCTGGTCAAAAGCCGATAAATCAGACGGAAACTTTATGATCCGGGCTCTGGTCAACTACGAAGCTA

[0178] TTGTGCCTGACATTACGTCACCTGATGACCGGTCGTTTACAAACAAGAAGACAATCACGGTAAAAGG

[0179] AACGGCCTCTCCGGGAACCGCTGTCCGCCTTACCAATAACGGTAAAACCGCCGCTGAAACGAAAGCC

[0180] GGTGCAGACGGCAATTTCCAGGCGGATGTCACACTCCGTAAAGATGCCAATCGGCTGACGGCGGCAT

[0181] CTGTGACAGACAGGGGATCAACAGATGAGTCACGCCCTGTCACGGTCATATTGGACCAGGATAAGCC

[0182] GGATGTGACGATTGACAGTCCGGCAAACGGAGATAAAACAAATAAAGAAGCAGTGACCGTAAAAGG

[0183] AAAAGCTTATGACGCCCATTTAAAAGAGGTAAAGGTGAACGGCAAAAAAGCAGAAGTGAATAACGG

[0184] CTCTTATCAAGCAAGAATCCTTCTGGAAAACGGCAGCAATGAGATCAAGGTTACGGCCTCAGACGAA

[0185] GCGGGTAACAAAACAACGAAAAAGACGGTAATCGATGTCAATTACAACGCTCCCGTCATTTCGGGGC

[0186] TGGTTCCCGGAGCGGATAAAGAATTAAAAGCCGGAGAATCAGTGAAAATCGCCTTTTCAAGCGGGAA

[0187] GAAATTAGACGCAACCTTCGTTATCCGTCTGCCTCTGACAAACGCGCGGGCGGGGAGCCAAAACGCA

[0188] ACGGAGCTTCCGCTGAGAGAAATTT

[0189] CTCCGGGAAGATATGAGAGTTACTGGACGGCGACCTCTTCTATCAAAGCAAGCGGAGCAAAAATTGA

[0190] CGTTATCGTAAGAGATGACTACGGAAATGAAACGAGACAGACGGCAAAAGGAAAACTTTATATTAAC

[0191] GATTAA

[0192] SEQ ID No.5 Bpn’ nucleotide sequence

[0193] GTGAGAGGCAAAAAAGTATGGATCAGTTTGCTGTTTGCTTTAGCGTTAATCTTTACGATGGCGTTCGG

[0194] CAGCACATCCTCTGCCCAGGCGGCAGGGAAATCAAACGGGGAAAAGAAATATATTGTCGGGTTTAAA

[0195] CAGACAATGAGCACGATGAGCGCCGCTAAGAAGAAAGATGTCATTTCTGAAAAAGGCGGGAAAGTG

[0196] CAAAAGCAATTCAAATATGTAGACGCAGCTTCAGCTACATTAAACGAAAAAGCTGTAAAAGAATTGA

[0197] AAAAAGACCCGAGCGTCGCTTACGTTGAAGAAGATCACGTAGCACATGCGTACGCGCAGTCCGTGCC

[0198] TTACGGCGTATCACAAATTAAAGCCCCTGCTCTGCACTCTCAAGGCTACACTGGATCAAATGTTAAAG

[0199] TAGCGGTTATCGACAGCGGTATCGATTCTTCTCATCCTGATTTAAAGGTAGCAGGCGGAGCCAGCATG

[0200] GTTCCTTCTGAAACAAATCCTTTCCAAGACAACAACTCTCACGGAACTCACGTTGCCGGCACAGTTGC

[0201] GGCTCTTAATAACTCAATCGGTGTATTAGGCGTTGCGCCAAGCGCATCACTTTACGCTGTAAAAGTTC

[0202] TCGGTGCTGACGGTTCCGGCCAATACAGCTGGATCATTAACGGAATCGAGTGGGCGATCGCAAACAA

[0203] TATGGACGTTATTAACATGAGCCTCGGCGGACCTTCTGGTTCTGCTGCTTTAAAAGCGGCAGTTGATA

[0204] AAGCCGTTGCATCCGGCGTCGTAGTCGTTGCGGCAGCCGGTAACGAAGGCACTTCCGGCAGCTCAAG

[0205] CACAGTGGGCTACCCTGGTAAATACCCTTCTGTCATTGCAGTAGGCGCTGTTGACAGCAGCAACCAAA

[0206] GAGCATCTTTCTCAAGCGTAGGACCTGAGCTTGATGTCATGGCACCTGGCGTATCTATCCAAAGCACG

[0207] CTTCCTGGAAACAAATACGGGGCGTACAACGGTACGTCAATGGCATCTCCGCACGTTGCCGGAGCGG

[0208] CTGCTTTGATTCTTTCTAAGCACCCGAACTGGACAAACACTCAAGTCCGCAGCAGTTTAGAAAACACC

[0209] ACTACAAAACTTGGTGATTCTTTCTACTATGGAAAAGGGCTGATCAACGTACAGGCGGCAGCTCAGT

[0210] AA

[0211] SEQ ID No.6 Mpr nucleotide sequence

[0212] GTGTCTCTCGGCTTATCCCCTGACACCGCCCGCCGACAGCCCGCATGGGACGATTCTATCAATTCAGC

[0213] CGCGGAGTCTAGTTTTATATTGCAGAATGCGAGATTGCTGGTTTATTATAACAATATAAGTTTTCATTA

[0214] TTTTCAAAAAGGGGGATTTATTGTGGGTTTAGGTAAGAAATTGTCTGTTGCTGTCGCCGCTTCCTTTAT

[0215] GAGTTTAACCATCAGTCTGCCGGGTGTTCAGGCCGCTGAGAATCCTCAGCTTAAAGAAAACCTGACG

[0216] AATTTTGTACCGAAGCATTCTTTGGTGCAATCAGAATTGCCTTCTGTCAGTGACAAAGCTATCAAGCA

[0217] ATACTTGAAACAAAACGGCAAAGTCTTTAAAGGCAATCCTTCTGAAAGATTGAAGCTGATTGACCAA

[0218] ACGACCGATGATCTCGGCTACAAGCACTTCCGTTATGTGCCTGTCGTAAACGGTGTGCCTGTGAAAGA

[0219] CTCTCAAGTCATTATTCACGTCGATAAATCCAACAACGTCTATGCGATTAACGGTGAATTAAACAACG

[0220] ATGTTTCCGCCAAAACGGCAAACAGCAAAAAATTATCTGCAAATCAGGCGCTGGATCATGCTTATAA

[0221] AGCGATCGGCAAATCACCTGAAGCCGTTTCTAACGGAACCGTTGCAAACAAAAACAAAGCCGAGCTG

[0222] AAAGCAGCAGCCACAAAAGACGGCAAATACCGCCTCGCCTATGATGTAACCATCCGCTACATCGAAC

[0223] CGGAACCTGCAAACTGGGAAGTAACCGTTGATGCGGAAACAGGAAAAATCCTGAAAAAGCAAAACA

[0224] AAGTGGAGCATGCCGCCACAACCGGAACAGGTACGACTCTTAAAGGAAAAACGGTCTCATTAAATAT

[0225] TTCTTCTGAAAGCGGCAAATATGTGCTGCGCGATCTTTCTAAACCTACCGGAACACAAATTATTACGT

[0226] ACGATCTGCAAAACCGCGAGTATAACCTGCCGGGCACACTCGTATCCAGCACCACAAACCAGTTTAC

[0227] AACTTCTTCTCAGCGCGCTGCCGTTGATGCGCATTACAACCTCGGCAAAGTGTATGATTATTTCTATCA

[0228] GAAGTTTAATCGCAACAG

[0229] CTACGACAATAAAGGCGGCAAGATCGTATCCTCCGTTCATTACGGCAGCAGATACAATAACGCAGCC

[0230] TGGATCGGCGACCAAATGATTTACGGTGACGGCGACGGTTCATTCTTCTCACCTCTTTCCGGTTCAAT

[0231] GGACGTAACCGCTCATGAAATGACACATGGCGTTACACAGGAAACAGCCAACCTGAACTACGAAAAT

[0232] CAGCCGGGCGCTTTAAACGAATCCTTCTCTGATGTATTCGGGTACTTCAACGATACTGAGGACTGGGA

[0233] TATCGGTGAAGATATTACGGTCAGCCAGCCGGCTCTCCGCAGCTTATCCAATCCGACAAAATACGGA

[0234] CAGCCTGATAATTTCAAAAATTACAAAAACCTTCCGAACACTGATGCCGGCGACTACGGCGGCGTGC

[0235] ATACAAACAGCGGAATCCCGAACAAAGCCGCTTACAATACGATTACAAAAATCGGCGTGAACAAAG

[0236] CGGAGCAGATTTACTATCGTGCTCTGACGGTATACCTCACTCCGTCATCAACTTTTAAAGATGCAAAA

[0237] GCCGCTTTGATTCAATCTGCGCGGGACCTTTACGGCTCTCAAGATGCTGCAAGCGTAGAAGCTGCCTG

[0238] GAATGCAGTCGGATTGTAA

Claims

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

2. A microbial agent containing the starch liquefying Bacillus B3 according to claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial agent contains living cells of the amyloliquefaciens Bacillus B3 bacteria, dry amyloliquefaciens Bacillus B3 bacteria obtained by freeze-drying, immobilized amyloliquefaciens Bacillus B3 cells, liquid agent of the amyloliquefaciens Bacillus B3, solid agent of the amyloliquefaciens Bacillus B3, culture solution and fermentation solution obtained by culturing or fermenting the amyloliquefaciens Bacillus B3, or the amyloliquefaciens Bacillus B3 strain in other forms and its culture solution or fermentation solution.

4. The microbial agent according to claim 3, characterized in that: The microbial agent is a liquid agent, which is prepared from the supernatant of the fermentation broth of Bacillus amyloliquefaciens B3; And / or, the acid protease activity of the microbial agent is ≥277U / mL.

5. The protease isolated from the amyloliquefaciens Bacillus B3 of claim 1, characterized in that: The proteases are Bpr, Bpn' and Mpr.

6. The protease according to claim 5, characterized in that (a) a protein consisting of the amino acid sequence shown in SEQ ID No.1 to SEQ ID No.3; or, (b) A protein derived from (a) in which the amino acid sequence in (a) is substituted, deleted or added with one or more amino acids and has protease activity.

7. The protease according to claim 6, characterized in that The coding gene sequences of the proteases Bpr, Bpn' and Mpr are shown in SEQ ID No.4 to SEQ ID No.

6.

8. Use of the starch liquefying Bacillus B3 according to claim 1 / or the microbial agent according to any one of claims 2 to 4 / or the protease according to any one of claims 5 to 7 in the food industry.

9. The use according to claim 8, characterized in that: Applied to the fermentation production of sauce products; And / or, the sauce products include but are not limited to soy sauce, bean paste and soybean paste.

10. The use according to claim 9, characterized in that: Applied in soy sauce fermentation production; The application method is: adding the microbial agent or the protease in the early stage of soy sauce fermentation to increase the amino acid nitrogen and free amino acid content of the soy sauce and improve the flavor of the soy sauce.

Images