A strain of Bacillus amyloliquefaciens phb03 and its application

Abstract

The present invention relates to the technical field of industrial microorganisms, and specifically discloses a strain of Bacillus amyloliquefaciens phb03 and its application. The present invention provides a strain of Bacillus amyloliquefaciens phb03, which has stress resistance, can tolerate 2M salt, grows well at 42°C, and can achieve a degradation rate of 42.26% for trypsin inhibitors; the protease produced by the strain also has the characteristics of salt and high temperature resistance, and can still maintain 57% relative enzyme activity after being stored at 50°C for 180 minutes, and can still achieve a degradation rate of 40% for trypsin inhibitors at a salt concentration of 4M. 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 increase the amino acid nitrogen and total acid content in 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 amyloliquefaciens PHB03 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 amyloliquefaciens (Bacillus amyloliquefaciens) phb03, which is stress-resistant, salt-resistant and high-temperature-resistant, and has high-efficiency degradation activity on 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 the first aspect, an embodiment of the present application provides a strain of Bacillus amyloliquefaciens phb03, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 27, 2024. The address of the depository unit is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64808.

[0007] In a second aspect, the present invention provides a fermentation agent, which comprises the above-mentioned Bacillus amyloliquefaciens PHB03. The fermentation agent can be a liquid agent or a solid agent, and can be prepared by conventional technical means and by adding auxiliary materials permitted in the field of microbial preparations.

[0008] In a third aspect, the present invention provides the use of the above-mentioned Bacillus amyloliquefaciens phb03 or fermentation agent in degrading trypsin inhibitors.

[0009] In a fourth aspect, the present invention provides the use of the above-mentioned Bacillus amyloliquefaciens PHB03 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 amyloliquefaciens phb03 of claim 1 or the fermentation agent of 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 treated using the Bacillus amyloliquefaciens phb03 of claim 1 or the fermentation agent of 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 amyloliquefaciens PHB03 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 amyloliquefaciens phb03 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 42.26% for trypsin inhibitors.

[0018] 2. The protease produced by Bacillus amyloliquefaciens phb03 provided by the present invention also has the characteristics of salt and high temperature resistance; after being stored at 50°C for 180 minutes, it can still maintain 57% relative enzyme activity; after being stored under 4M high salt conditions for 11 days, it can still maintain 66% relative enzyme activity; at a salt concentration of 4M, the degradation rate of trypsin inhibitor can still reach 40%.

[0019] 3. The Bacillus amyloliquefaciens PHB03 provided by the present invention can process soybeans or soybean-derived products by means of synergistic fermentation or preparation of enzyme preparations, degrade trypsin inhibitors in the raw materials, and improve the utilization rate of raw material protein.

[0020] 4. The Bacillus amyloliquefaciens phb03 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 and total acid content 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 The colony morphology of Bacillus amyloliquefaciens phb03;

[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 The growth curves of Bacillus amyloliquefaciens phb03 at different temperatures;

[0025] Figure 5 is the growth curve of Bacillus amyloliquefaciens phb03 under different salt concentrations;

[0026] Figure 6 The tolerance of the protease produced by Bacillus amyloliquefaciens phb03 under different salt concentrations;

[0027] Figure 7 The change trend of protease produced by Bacillus amyloliquefaciens phb03 in degrading STI under different salt concentrations;

[0028] The Bacillus amyloliquefaciens phb03 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 the collection number is GDMCC No: 64808; the strain was received and registered by the collection center on June 27, 2024, and was detected as a surviving 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 0.5 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, 4 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] Strain No. Species Degradation STI 3 Bacillus amyloliquefaciens + 5 Bacillus velezensis + 7 Bacillus subtilis + 8 Bacillus tequilensis +

[0059] 2. Rescreening of STI-degrading strains:

[0060] Single colonies of the four 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 3 had the strongest ability to degrade STI, belonged to Bacillus amyloliquefaciens, and had application potential. This strain was named phb03. 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 GDMCC No: 64808.

[0061] 3. Verification of STI degradation strain performance:

[0062] The activated Bacillus amyloliquefaciens phb03 strain 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.

[0063] Depend on Figure 3 The results showed that after the supernatant of Bacillus amyloliquefaciens PHB03 was stored at 55°C for 24 hours, the relative activity of the protease still remained at 57.5%, and the relative activity of the STI-degrading enzyme still remained at 58.2%, proving that the protease produced by Bacillus amyloliquefaciens PHB03 has a certain temperature resistance, and after being treated at 55°C, it can still maintain a high enzyme activity and has a high ability to degrade STI.

[0064] Example 2 Stability of Bacillus amyloliquefaciens phb03

[0065] 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.

[0066] The activated Bacillus amyloliquefaciens phb03 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.

[0067] Table 2: Stability of PHB03 passages

[0068] Generation Number Protease (U / mL) Generation 1 1015 5th Generation 1094 10th Generation 1137

[0069] According to the results in Table 2, after subculture, the enzyme production capacity of the 10th generation strain of Bacillus amyloliquefaciens PHB03 was not significantly reduced compared with the 1st generation strain, which proved that the subculture performance of the PHB03 strain was stable.

[0070] Example 3 Growth characteristics test of Bacillus amyloliquefaciens phb03

[0071] 1. Test of the growth of phb03 at different temperatures

[0072] A single colony of Bacillus amyloliquefaciens phb03 was selected and inoculated into LB liquid medium and cultured at 37°C overnight. The inoculation volume was transferred to LB liquid medium at 1%, and cultured at 25°C, 37°C, 42°C, 50°C and 60°C respectively. The bacterial solution concentration (OD 600 ). Each test point was repeated 3 times, and the average value was taken to draw the growth curve of phb03 at different temperatures. The results were recorded in Figure 4 middle.

[0073] according to Figure 4 The results show that phb03 grows well at 42℃ and can maintain a certain growth activity at 50℃. Therefore, Bacillus amyloliquefaciens phb03 has a certain heat resistance and can withstand a relatively high temperature of 42℃. It grows well in liquid culture at 42℃.

[0074] 2. Growth test of phb03 under different salt concentrations

[0075] A single colony of Bacillus amyloliquefaciens phb03 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 divided into 3 parallels, and the average value was taken to draw the growth curve of pHB03 under different salt concentrations. The results were recorded in Figure 5 middle.

[0076] according to Figure 4 The results showed that phb03 could maintain a certain growth activity when grown at 2M salt concentration for 9 days (216h), and its growth was significantly inhibited at 3M salt concentration, which proved that Bacillus amyloliquefaciens phb03 could tolerate 2M salt.

[0077] Example 4 Test on the properties of protease produced by Bacillus amyloliquefaciens phb03

[0078] 1. Salt resistance test of protease produced by phb03

[0079] The activated phb03 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.

[0080] Depend on Figure 6The results show that after the phb03 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 phb03 has strong salt tolerance.

[0081] 2. Test on the ability of protease produced by phb03 to degrade STI at different salt concentrations

[0082] (1) The activated phb03 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.

[0083] (2) Add solid NaCl to the supernatant obtained from the pHB03 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, 3 M, 3.5 M, and 4 M, respectively, and store at 4°C overnight.

[0084] (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.

[0085] Depend on Figure 7 The results show that the relative residual activity of STI in phb03 supernatant treated with different salt concentrations presents a V-shaped distribution. The ability to degrade STI is the best at 2M salt concentration, with only 9.5% relative activity remaining and 90.5% degradation of STI; the relative residual activity of STI at 0M salt concentration is 30.0%, and the ability of phb03 supernatant to degrade STI slightly decreases at 0.5M salt concentration, and then the ability to degrade STI gradually increases from 0.5M to 2M salt concentration, the relative residual activity of STI decreases from 35.6% to 9.5%, and the ability to degrade STI increases from 64.4% to 90.5%. After the salt concentration exceeds 2M, the ability to degrade STI begins to decrease, and the relative residual activity of STI at 2.5M salt concentration is 22.9%; the relative residual activity of STI at 3M salt concentration is 57.8%. At 3M-4M salt concentrations, the ability of enzymes to degrade STI is basically the same. This shows that the protease produced by phb03 has a significant advantage in degrading STI at a salt concentration of 2M, with a degradation rate of more than 90%. At the same time, although the ability to degrade STI decreases when the salt concentration is greater than 2M, it can still degrade 40% of STI at a high salt concentration of 4M, proving that the protease produced by phb03 still has a high ability to degrade STI at a salt concentration of 4M.

[0086] Example 5 Effect of Bacillus amyloliquefaciens phb03 on soy sauce fermentation

[0087] (1) Strain activation: Activate phb03 and strain 8 (the Bacillus strain with poor STI degradation ability obtained in the initial screening) and transfer them to LB medium. Culture them at 37°C, 200 r / min, in a constant temperature air shaker for 2 days.

[0088] (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 (1‰ of the dry material) is added. 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.

[0089] (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 inoculation amount 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.

[0090] Table 3: Indicators of the inoculated phb03 strain during soy sauce fermentation

[0091] Group Amino acid nitrogen (g / 100mL) Total acid (g / 100mL, calculated as lactic acid) Phb03 Group 0.848 0.915 Strain Group 8 0.727 0.830 Control group 0.693 0.803

[0092] As shown in Table 3, the amino acid nitrogen content and total acid content of soy sauce in the phb03 group increased significantly, with the amino acid nitrogen content increasing by 22.4% and the total acid content increasing by 13.9%, which was better than the No. 8 strain with poor STI degradation ability. This shows that phb03 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.

[0093] Example 6 Effect of crude enzyme solution prepared by Bacillus amyloliquefaciens phb03 on soy sauce fermentation

[0094] Preparation of crude enzyme solution: The phb03 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.

[0095] 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.

[0096] 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 phb03 crude enzyme solution (the crude enzyme solution addition was calculated based on the koji material quality) was added respectively, and commercial protease, No. 8 strain supernatant 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 sauce mash were recorded in Table 4.

[0097] Table 4: Amino acid nitrogen test results during soy sauce fermentation

[0098]

[0099]

[0100] The results are shown in Table 4. The amino acid nitrogen content of fermented soy sauce increased significantly after using crude enzyme solution PHB03 during the fermentation process. The amino acid nitrogen in the crude enzyme solution group with 750U / g PHB03 increased by 22.5% after 30 days, and the effect was better than that of commercial protease and supernatant of strain 125. This shows that crude enzyme solution PHB03 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.

[0101] 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 amyloliquefaciens ( Bacillus amyloliquefaciens ) PHB03, characterized in that, The deposit number of the Bacillus amyloliquefaciens phb03 is GDMCC No: 64808.

2. A fermentation agent, characterized in that: The fermentation agent comprises the Bacillus amyloliquefaciens phb03 as claimed in claim 1.

3. Use of the Bacillus amyloliquefaciens phb03 as claimed in claim 1 or the fermentation agent as claimed in claim 2 in degrading trypsin inhibitors.

4. Use of the Bacillus amyloliquefaciens PHB03 according to claim 1 or the fermentation agent according to claim 2 in the processing of soybeans or soybean-derived products, wherein the use is for the fermentation of soy sauce.

5. The use according to claim 3 or 4, characterized in that: The application is to inoculate the Bacillus amyloliquefaciens phb03 described in claim 1 or the fermentation 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 using the Bacillus amyloliquefaciens phb03 of claim 1 or the fermentation agent of claim 2.

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