A strain of Bacillus velez phb02 and its application

Abstract

The present invention relates to the technical field of industrial microorganisms, and specifically discloses a strain of Bacillus velezensis phb02 and its application. The present invention provides a strain of Bacillus velezensis phb02, which has stress resistance, can tolerate 2M salt, grows well at 42°C, and can achieve a degradation rate of 59.34% for trypsin inhibitors; the protease produced by the strain also has the characteristics of salt and high temperature resistance, and at a salt concentration of 3M, the degradation rate of trypsin inhibitors can still reach 35%. The strain can process soybeans or soybean-derived products by means of coordinated fermentation or preparation of enzyme preparations, degrade trypsin inhibitors in raw materials, improve the utilization rate of raw material protein, can be used in the production of fermented condiments such as soy sauce, significantly improve the ammonia nitrogen level of the product, and improve product quality.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus velez phb02 and an application thereof. Background Art

[0002] Soybean trypsin inhibitor (STI) refers to proteins or peptides that can inhibit the activity of trypsin, and is mainly found in plants of the Gramineae and Leguminosae families. Trypsin inhibitors react with trypsin and chymotrypsin in the animal body to inhibit the activity of trypsin, chymotrypsin, etc., reduce protein digestibility and utilization, and lead to exogenous nitrogen loss. In addition, STI will also hinder the application of trypsin in the deep processing of beans, so it is very necessary to study how to eliminate trypsin inhibitors.

[0003] At present, the main methods for inhibiting the activity of STI are physical, chemical and biological methods. The physical method generally uses heat treatment, but long-term high heat will reduce the nutritional value of the raw protein, and there are disulfide bonds in the STI structure, which has high thermal stability, so traditional processing techniques such as heating and boiling cannot effectively remove it. The disadvantage of chemical methods, such as adding sulfates, urea, choline, etc., is that they cannot be accurately quantified and are accompanied by chemical residues, and are rarely used in food processing. The biological method mainly inactivates trypsin inhibitors by adding enzymes or strains with the function of degrading STI. The application of biotechnology to degrade STI has the advantages of being green and environmentally friendly, safe and efficient without residue, and having little damage and impact on the nutritional components of the raw materials. At present, most of the research on biological degradation of STI is to study the optimal conditions for directly adding exogenous proteases, or to screen strains to inhibit STI. For example, in patent CN 110452833 A, a strain of Bacillus pumilus that can efficiently degrade the activity of trypsin inhibitors was screened from red beans using the sole carbon source method. However, there are currently no reports of strains that can still function effectively under high-salt conditions, and condiment fermentation is usually a high-salt system. Therefore, studying a strain that can still efficiently degrade STI activity in a high-salt environment is of great significance and application potential. Summary of the invention

[0004] Based on the above technical problems, the main purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and provide a strain of Bacillus velezensis phb02, which is stress-resistant, salt-resistant and high-temperature-resistant, and has high-efficiency degradation activity for trypsin inhibitors. It can process soybeans or soybean-derived products by synergistic fermentation or preparation of enzyme preparations to improve the utilization rate of raw protein.

[0005] To achieve the above objectives, the inventors conducted in-depth research and completed the solution of the present invention after repeated research and demonstration, which is as follows:

[0006] In a first aspect, an embodiment of the present application provides a strain of Bacillus velezensis phb02, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on June 27, 2024, with a deposit number of GDMCC No: 64807.

[0007] In a second aspect, the present invention provides a fermentation agent, which contains the above-mentioned Bacillus Velez subtilis phb02. The fermentation agent can be a liquid agent or a solid agent, and can be prepared by conventional technical means and adding auxiliary materials allowed in the field of microbial preparations.

[0008] In a third aspect, the present invention provides the use of the above-mentioned Bacillus Velezii phb02 or fermentation agent in degrading trypsin inhibitors.

[0009] In a fourth aspect, the present invention provides the use of the above-mentioned Bacillus Velezii phb02 or fermentation agent in the processing of soybeans or soybean-derived products.

[0010] The soybean-derived products include, but are not limited to, soybean flour, soybean protein isolate, soybean protein concentrate, soybean oil, soybean milk, tofu, soybean dregs, soybean meal, and soy sauce.

[0011] Furthermore, the application is to inoculate the Bacillus Velez subtilis phb02 described in claim 1 or the fermentation agent described in claim 2 into the sample to be processed for fermentation culture.

[0012] Furthermore, the application is for fermentation of soy sauce.

[0013] In a fifth aspect, the present invention provides a method for degrading trypsin inhibitors, the method comprising the step of treating a sample to be processed using the Bacillus Velez subtilis phb02 described in claim 1 or the fermentation agent described in claim 2.

[0014] In a sixth aspect, the present invention provides a crude enzyme solution, which is obtained by filtering and concentrating the fermentation liquid of the Bacillus Velez subtilis phb02 described in claim 1 or the fermentation agent described in claim 2.

[0015] In a seventh aspect, the present invention provides the use of the crude enzyme solution in the processing of soybeans or soybean-derived products, preferably in soy sauce fermentation.

[0016] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0017] 1. The Bacillus Velezii phb02 provided by the present invention has stress resistance, can tolerate 2M salt, grows well at 42°C, and can efficiently degrade trypsin inhibitors, with a degradation rate of 59.34% for trypsin inhibitors.

[0018] 2. The protease produced by the Bacillus Velezii phb02 provided by the present invention is also salt-resistant and high-temperature-resistant; after being stored at 50°C for 180 minutes, it can still maintain 53% relative enzyme activity; after being stored at 4M high salt conditions for 11 days, it can still maintain 66% relative enzyme activity; at a salt concentration of 3M, the degradation rate of trypsin inhibitor can still reach 35%.

[0019] 3. The Bacillus Velezii phb02 provided by the present invention can be used to treat soybeans or soybean-derived products by means of synergistic fermentation or preparation of enzyme preparations, thereby degrading trypsin inhibitors in the raw materials and improving the utilization rate of raw material protein.

[0020] 4. The Bacillus Velezii phb02 provided by the present invention can be used for the production of fermented condiments such as soy sauce, can degrade trypsin inhibitors in raw materials, improve the utilization rate of raw material protein, significantly increase the ammonia nitrogen level of the product, and improve product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The inhibition of STI by different strains;

[0022] Figure 2 This is the colony morphology of Bacillus velez phb02;

[0023] Figure 3 The relative activities of protease and STI degrading enzyme and the relative residual activity of STI at different temperatures are shown;

[0024] Figure 4 is the growth curve of Bacillus velez phb02 at different temperatures;

[0025] Figure 5 is the growth curve of Bacillus velez phb02 under different salt concentrations;

[0026] Figure 6 The tolerance of the protease produced by Bacillus Velezii phb02 under different salt concentrations;

[0027] Figure 7 The degradation trend of STI by protease produced by Bacillus velez phb02 under different salt concentrations;

[0028] The Bacillus velezensis phb02 provided by the present invention has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 27, 2024, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and a collection number of GDMCC No: 64807; the strain was received and registered by the collection center on June 27, 2024, and was detected as a viable strain by the collection center on June 27, 2024. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0031] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional food-grade reagents, methods and equipment in the art.

[0032] The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0033] The detection method used in the following examples is as follows:

[0034] Protease activity determination method: refer to the national standard GBT23527.1-2023 Determination of protease activity.

[0035] Trypsin inhibitor activity assay method:

[0036] Trypsin can react with benzoyl-L-arginine-p-nitroaniline (L-BAPA) to generate p-nitroaniline (yellow), which has characteristic absorption at 410nm. Trypsin inhibitors in soy products will inhibit the reaction and reduce the absorbance value, and the degree of reduction is proportional to the activity of trypsin. The absorbance value before and after the reaction can be measured by a spectrophotometer at 410nm to quantitatively determine the activity of trypsin inhibitors.

[0037] (1) 950 μL of sample solution + 50 μL of STI solution (160 BAEE u / mL), in a water bath at a certain temperature for a period of time;

[0038] (2) Take 400 μL of the solution after the water bath in the previous step, add 2 mL of the substrate benzoyl-L-arginine-p-nitroaniline hydrochloride (L-BAPA) and 800 μL of distilled water, and incubate at 37°C in a water bath for 10 min;

[0039] (3) Add 400 μL of trypsin solution and incubate in a 37°C water bath for 10 min;

[0040] (4) Add 400 μL of 30% glacial acetic acid to terminate the reaction;

[0041] (5) After the reaction was terminated, the solution was centrifuged at 8000 rpm for 5 min and the absorbance was measured at 410 nm.

[0042] Note: No sample and STI were added to the standard solution, which was replaced by distilled water, and the measurement results were expressed as "ΔA pancreas";

[0043] The control sample (CK) was not added with sample solution, but distilled water was used instead. The rest of the operations were the same, and the measurement results were expressed as "ΔA pairs";

[0044] The blank sample is prepared by first adding glacial acetic acid to terminate the reaction, and the rest of the operation is the same. The measurement result is expressed as "ΔA blank"

[0045] express;

[0046] The measurement results of the sample solution are expressed as "ΔA sample".

[0047] Activity of STI in the control sample (%): i = (ΔA pancreas - ΔA pair) / ΔA pancreas * 100%

[0048] Relative inactivation rate of STI after sample solution treatment (%): d = (ΔA sample - ΔA pair) / ΔA pancreas * 100%

[0049] Relative residual activity of STI after sample solution treatment (%): r = id

[0050] STI degradation enzyme activity in sample solution (U / mL): e = (ΔA sample - ΔA pair) / (10*0.001*sample solution volume (mL));

[0051] The definition of one STI degradation enzyme activity is: under the experimental conditions, an increase of 0.001 in Δ410nm per minute is one degradation enzyme activity unit.

[0052] Example 1 Screening of strains

[0053] 1. Initial screening of strains that degrade STI:

[0054] (1) Collect various high-salt soybean fermentation products, including traditional fermentation products such as soybean dregs, fermented soybean paste, fermented black beans, etc.

[0055] (2) Grind 5 g of the collected sample and transfer it to enrichment medium (KH2PO4 0.05%, MgSO4·7H2O 0.02%, NaCl 0.1%, NH4Cl 0.1%, yeast extract 0.1%), and culture it at 37°C, 220 r / min shaking for 5 days; dilute the cultured bacterial solution in a gradient manner, and take 100 μL of the bacterial solution with different dilution multiples to apply it to STI medium (STI as the only carbon source) and culture it for 48 hours until a single colony grows. Take the single colony with different growth morphology on STI medium and streak it onto LB solid medium and culture it for 24 hours until a single colony grows and is numbered.

[0056] (3) The single colony after streaking was inoculated into LB-STI medium (LB medium was supplemented with STI to make the final concentration of STI reach 1 mg / mL) for subculture at 37°C, 220 r / min, and cultured for 48 h. The supernatant after culture was taken for SDS-PAGE to check the degradation of STI. Finally, 12 strains with STI degradation activity were obtained. The 16S sequence was amplified using 16S universal primers and then sequenced to determine the species of the colonies. The results are recorded in Table 1. "+" indicates the phenomenon of STI degradation.

[0057] Table 1: Preliminary screening of STI-degrading strains

[0058]

[0059]

[0060] 2. Rescreening of STI-degrading strains:

[0061] Single colonies of the 12 strains obtained in the initial screening were picked and cultured in LB liquid medium at 37°C for 48 hours. The culture supernatant was mixed with the STI standard and incubated for 2 hours to evaluate the inhibition rate of different strains on STI. The STI standard was used as a control, and the relative residual activity of STI was calculated. The results were recorded in Figure 1 Finally, it was concluded that strain 118 had the strongest ability to degrade STI, belonged to Bacillus Velezii, and had application potential. This strain was named phb02. The colony morphology of this strain on LB solid medium is as follows Figure 2 As shown, it was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 27, 2024, with the deposit number GDMCCNo: 64807.

[0062] 3. Verification of STI degradation strain performance:

[0063] The activated Bacillus Velezii phb02 was inoculated into LB liquid culture medium at a rate of 1.5%, and cultured at 37°C with shaking for 24 hours. After centrifugation of the bacterial solution, the supernatant was incubated at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C for 24 hours, and then STI solution was added and incubated for 30 minutes. Samples were taken to determine the relative activities of protease, STI degradation enzyme, and remaining STI, and the results were recorded in Figure 3 middle.

[0064] Depend on Figure 3 The results showed that after the supernatant of Bacillus Velez subtilis phb02 was stored at 55°C for 24 hours, the relative activity of the protease still remained at 59.6%, and the relative activity of the STI-degrading enzyme still remained at 73.7%, which proved that the protease produced by Bacillus Velez subtilis phb02 had a certain temperature resistance. After being treated at 55°C, it could still maintain a high enzyme activity and had a high ability to degrade STI.

[0065] Example 2: Stability of passage of Bacillus velez phb02

[0066] According to Example 1 Figure 3 The results showed that the relative activity change trends of proteases and STI-degrading enzymes at different temperatures were basically the same. With the increase of temperature, the relative activities of proteases and STI-degrading enzymes decreased, while the relative residual activity of STI increased. This indicated that the ability of the strain to degrade STI was positively correlated with the protease activity. Subsequent experiments used protease activity to characterize the stability of strain generation.

[0067] The activated Bacillus Velez phb02 strain was inoculated into LB solid medium and cultured at 37°C for 24 hours, and then transferred to LB solid medium and cultured at 37°C for 24 hours, and so on to obtain strains of different generations to determine the stability of strain generation. After activating the strains from the 1st, 5th and 10th generation culture medium, the strains were inoculated into LB liquid medium at a 1% inoculation amount, and the culture supernatant was taken after shaking culture at 37°C for 48 hours to determine the protease activity, and the results are recorded in Table 2.

[0068] Table 2: Stability of PHB02 passages

[0069] Generation Number Protease (U / mL) Generation 1 715 5th Generation 749 10th Generation 782

[0070] According to the results in Table 2, after subculture, the enzyme production capacity of the 10th generation strain of Bacillus Velezii PHB02 was not significantly reduced compared with the 1st generation strain, which proved that the subculture performance of the PHB02 strain was stable.

[0071] Example 3 Growth characteristics test of Bacillus velez phb02

[0072] 1. Test of the growth of phb02 at different temperatures

[0073] Pick a single colony of Bacillus Velezii phb02 and inoculate it into LB liquid medium and culture it at 37°C overnight. Then transfer it to LB liquid medium at a 1% inoculum volume and culture it at 25°C, 37°C, 42°C, 50°C and 60°C respectively. Take samples at fixed time to measure the concentration of bacterial solution (OD 600 ). Each test point was repeated 3 times, and the average value was taken to draw the growth curve of phb02 at different temperatures. The results were recorded in Figure 4 middle.

[0074] according to Figure 4 The results show that phb02 grows well at 42℃ and can maintain a certain growth activity at 50℃. Therefore, Bacillus velez phb02 has a certain heat resistance and can tolerate a relatively high temperature of 42℃. It grows well in liquid culture at 42℃.

[0075] 2. Growth test of phb02 under different salt concentrations

[0076] A single colony of Bacillus Velezii phb02 was selected and inoculated into LB liquid medium and cultured at 37°C overnight. The inoculation volume was transferred to LB liquid medium containing 0M, 1M, 2M and 3M sodium chloride at 1% and cultured at 37°C with shaking. The bacterial solution concentration (OD 600 ). Each salt was tested in parallel for 3 times, and the average value was taken to draw the growth curve of pHB02 under different salt concentrations. The results were recorded in Figure 5 middle.

[0077] according to Figure 4 The results showed that phb02 had a good growth trend at 2M salt concentration, and its growth was significantly inhibited at 3M salt concentration, proving that Bacillus velez phb02 could tolerate 2M salt.

[0078] Example 4 Test of the properties of the protease produced by Bacillus velez phb02

[0079] 1. Salt resistance test of protease produced by phb02

[0080] The activated phb02 strain was inoculated into LB liquid culture medium at 1% inoculation volume, and cultured at 37°C with shaking for 24 hours. The bacterial solution was centrifuged at 8000r / min for 5 minutes, and the supernatant was placed in 0M, 3M, and 4M salt concentrations at 4°C for a certain period of time. Samples were taken every 2 days to measure the relative activity of the protease and the results were recorded. Figure 6 middle.

[0081] Depend on Figure 6The results show that after the phb02 protease was stored under 4M high salt conditions for 11 days, the relative activity of the enzyme still remained at 66%, proving that the protease produced by phb02 has strong salt tolerance.

[0082] 2. Test on the ability of protease produced by phb02 to degrade STI at different salt concentrations

[0083] (1) The activated phb02 strain was inoculated into LB liquid culture medium at a 1% inoculum amount, and cultured at 37°C with shaking for 24 hours. The bacterial solution was centrifuged at 8000 r / min for 5 minutes and the supernatant was collected for later use.

[0084] (2) Add solid NaCl to the supernatant obtained from the pHB02 culture and fully dissolve it to make the final salt concentrations 0 M, 0.5 M, 1 M, 1.5 M, 2 M, 2.5 M, and 3 M, respectively, and store at 4°C overnight.

[0085] (3) Take the supernatant from the previous step and mix it with STI for 30 minutes to measure the relative residual activity of STI. The results are recorded in Figure 7 middle.

[0086] Depend on Figure 7 The results show that the protease produced by phb02 has the best ability to degrade STI at 0M salt concentration, with only 37% relative activity remaining and 63% degraded STI; at 0.5M-1.5M salt concentration, the ability to degrade STI is also relatively stable, and the relative remaining activity of STI is controlled within 49%; with the increase of salt concentration, the protease produced by phb02 can still degrade STI, but the degradation ability is not as strong as that at low salt concentration, until it can still degrade 35% of STI at a high salt concentration of 3M, proving that the protease produced by phb02 still has a high ability to degrade STI at a salt concentration of 3M.

[0087] Example 5 Effect of Bacillus Velezii phb02 on soy sauce fermentation

[0088] (1) Strain activation: The phb02 and 125 strains (the Bacillus velezensis strains with poor STI degradation ability obtained in the initial screening) were activated and transferred to LB medium and cultured at 37°C, 200 r / min, in a constant temperature air shaker for 2 days.

[0089] (2) Soy sauce koji making: Soybeans are washed, soaked, and steamed, then cooled to room temperature and mixed with wheat flour at a dry material mass ratio of 7:3. 3.042 koji essence is added at 1‰ of the dry material. The koji is incubated at 32°C for 12 h, then turned over and incubated at 28°C until the surface of the soybeans is covered with yellow-green spores.

[0090] (3) Soy sauce fermentation: Take 250 g of the koji and place it in a 900 mL sauce bottle. Add 500 g of 18% (M / V) saline and mix thoroughly. 8 The inoculum of CFU / mL was inoculated into the mash, and the non-inoculated group was used as the control. The fermentation was carried out in a constant temperature room at 30°C for 30 days, and the amino acid nitrogen and total acid indexes in the mash were measured. The results are recorded in Table 3.

[0091] Table 3: Indicators of the inoculated phb02 strain during soy sauce fermentation

[0092]

[0093]

[0094] As shown in Table 3, the amino acid nitrogen content of soy sauce in the phb02 group increased significantly, while the total acid content was not significantly affected. The amino acid nitrogen increased by 21.8%, which was better than the 125 strain with poor STI degradation ability. This shows that phb02 has an inhibitory effect on trypsin inhibitors, allowing the protease in the fermentation system to hydrolyze soybeans more efficiently and fully, which has an improved effect on the decomposition of soybean nutrients and improves the utilization rate of soybean nutrients.

[0095] Example 6 Effect of crude enzyme solution prepared by Bacillus Velez PHB02 on soy sauce fermentation

[0096] Preparation of crude enzyme solution: The phb02 Bacillus strain was cultured in a solid medium of tryptone glucose yeast (TGY) at 37°C for 16 hours to obtain a single colony, and a single colony was selected and inoculated in a liquid TGY medium at 37°C for 16 hours. Then, the inoculation amount was 1.5%, inoculated in a liquid TGY medium, and cultured at 37°C for 24 hours to obtain a seed solution. Then, the seed solution was inoculated into a fermenter at an inoculation amount of 1%, and fermented at 37°C for 20 hours. During this period, a defoamer and sugar were added as appropriate. The fermenter speed was 200-800rpm, the tank pressure was 0.05Mpa, and the ventilation volume was 0.54Nm3 / h. After the fermentation was completed, diatomaceous earth was added and stirred for 30 minutes, and then the fermented liquid was passed through a plate frame to obtain a supernatant, and concentrated with a fiber ultrafiltration membrane with a molecular weight cutoff of 3kDa, and sterilized through a 0.22um filter membrane, and finally a crude enzyme solution was obtained, and its protease activity was measured.

[0097] Koji making for soy sauce: After washing, soaking and steaming, the soybeans are cooled to room temperature and mixed with wheat flour at a dry material mass ratio of 7:3. Add 3.042 koji essence at 1‰ of the dry material, culture at 32℃ for 12h, turn the koji over, and then transfer to 28℃ for further culture until the surface of the soybeans is covered with yellow-green spores.

[0098] Soy sauce fermentation: 250g of koji was taken, placed in a 900mL sauce bottle, 500g of 18% (M / V) saline was added, fully mixed, 0U / g, 250U / g, 500U / g, 750U / g of phb02 crude enzyme solution (the crude enzyme solution addition amount was calculated based on the koji material quality) was added respectively, and commercial protease, 125 strain supernatants and no enzyme solution group were added as a control group at 750U / g, and then fermentation was continued in a 30°C constant temperature chamber according to routine. After fermentation to 15 and 30 days, the amino acid nitrogen results of the koji mash were recorded in Table 4.

[0099] Table 4: Amino acid nitrogen test results during soy sauce fermentation

[0100]

[0101]

[0102] The results are shown in Table 4. The amino acid nitrogen content of fermented soy sauce increased significantly after using crude PHB02 enzyme solution during the fermentation process. The amino acid nitrogen in the crude PHB02 enzyme solution group increased by 10.8% after 30 days of adding 750U / g crude PHB02 enzyme solution, and the effect was better than that of commercial protease and 125 strain supernatant. This shows that crude PHB02 enzyme solution can effectively reduce the activity of trypsin inhibitors, allowing the protease in the fermentation system to hydrolyze soybeans more efficiently and fully, thereby improving the utilization rate of soybean nutrients.

[0103] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A strain of Bacillus velezinsis ( Bacillus velezensis ) phb02, characterized in that The deposit number of the Bacillus velez phb02 is GDMCC No:64807.

2. A bacterial agent, characterized in that: The bacterial agent comprises the Bacillus Velez subtilis phb02 as claimed in claim 1, and the Bacillus Velez subtilis phb02 in the bacterial agent is in a live bacterial state.

3. Use of the Bacillus Velezii phb02 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product for degrading trypsin inhibitors.

4. Use of the Bacillus Velezii phb02 according to claim 1 or the bacterial agent according to claim 2 in soy sauce fermentation.

5. The use according to claim 4, characterized in that: The application is to inoculate the Bacillus Velez subtilis phb02 described in claim 1 or the bacterial agent described in claim 2 into the sample to be processed for fermentation culture.

6. A method for degrading trypsin inhibitors, characterized in that: The method comprises the step of treating the sample to be treated with the Bacillus Velez subtilis phb02 of claim 1 or the bacterial agent of claim 2, and the method is used for soy sauce fermentation.

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