Bacillus licheniformis capable of producing alkaline protease and application of bacillus licheniformis

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus licheniformis mutant strain for high yield of alkaline protease and application of the bacillus licheniformis mutant strain. The bacillus licheniformis is specifically bacillus licheniformis LKT2025-01, and the preservation number of the bacillus licheniformis LKT2025-01 is CGMCC (China General Microbiological Culture Collection Center) No.36047. The bacillus licheniformis LKT2025-01 and the bacillus licheniformis Alkaline protease produced through liquid fermentation of the strain has a good degradation effect on antigen protein in soybean meal, the degradation rate of glycinin reaches 90%, the degradation rate of beta-conglycinin reaches 80%, toxic and side effects on livestock and poultry are effectively reduced, immunity is enhanced, the health level is improved, and then the growth performance of the livestock and poultry is improved. In addition, the strain is high in temperature resistance, the enzyme activity retention rate is about 85% after the strain is treated at the high temperature of 85 DEG C for 3 min, and the strain can be applied to the feed industry and the livestock and poultry breeding industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a bacillus licheniformis mutant strain with high yield of alkaline protease and application thereof. BACKGROUND

[0002] As a byproduct of soybean processing, soybean meal is rich in protein and contains a balanced proportion of amino acids, and has high nutritional value, and is a high-quality plant-derived protein. The protein content in soybean meal is about 40-50%, and it has good palatability, so it is highly praised by users as a protein fermentation raw material or protein feed raw material.

[0003] However, there are two long-term unresolved pain points behind its wide application: the problem of anti-nutritional substances and the limitations of traditional processing technology. Among the anti-nutritional substances, heat-stable antigenic proteins, including glycinin and beta-conglycinin, cannot be eliminated by simple heating methods. Animal diets can cause allergic reactions, especially in young animals, which can directly lead to diarrhea, intestinal mucosal hyperplasia and other adverse reactions, and further cause the decline of livestock and poultry immune function and growth performance, resulting in increased production costs for enterprises. Traditional processing technology has high energy consumption, has the risk of excessive heating, greatly reduces the nutritional value of soybean meal, and still cannot eliminate the immunogenicity of antigenic proteins. With the promotion of the global trend of "antibiotic ban" and "antibiotic reduction" in feed and breeding, it is essential to find a nutritional solution that can maintain the health of the animal gut and replace the growth-promoting effect of antibiotics. How to reduce the intestinal burden of livestock and poultry animals and prevent diarrhea through raw material pretreatment methods has become the focus of industry research.

[0004] Enzymatic soybean meal, through the precise catalysis of biological enzymes, realizes the "value reconstruction" of soybean meal. It not only overcomes the defects of traditional thermal processing in technology, but more importantly, the products produced can directly address the "antibiotic-free" challenge and "high efficiency" demand currently faced by the livestock industry. Protease, without a doubt, is the key to enzymatic soybean meal technology. Its core role is to degrade anti-nutritional factors (glycinin and beta-conglycinin) and eliminate antigenicity; cut peptide bonds to hydrolyze macromolecular proteins into small peptides and amino acids, thereby improving the nutritional value of soybean meal; destroy plant cell walls to release embedded nutrients; produce bioactive peptide components with antibacterial, antioxidant, and immune-enhancing properties; and finally improve the palatability of feed. Therefore, obtaining an efficient, stable, and targeted protease is the key to the success of enzymatic soybean meal technology. It can greatly reduce the digestive burden of livestock and poultry in the later stages, improve the health level of livestock and poultry, enhance the immune function of the animal body, and thus improve the utilization efficiency of feed and animal growth performance, thereby achieving the goal of reducing production costs and increasing enterprise efficiency.

[0005] This application aims to use ambient temperature pressure plasma (ARTP)-lithium chloride (LiCl) combined mutagenesis technology to treat the original strains that produce alkaline protease, and to selectively screen superior strains with strong enzyme production capacity, high antigen protein degradation efficiency, and strong genetic stability through genetic modification, shake-flask enzyme activity and antigen protein content detection. Summary of the Invention

[0006] This invention provides a mutant strain producing alkaline protease and its application in submerged liquid fermentation. This technology overcomes the problems of low livestock and poultry production performance, impaired immune function, and increased production costs caused by the high content of antigenic proteins in soybeans or soybean meal used by livestock and poultry farming enterprises. It provides a new technology and opens up new avenues for increasing revenue and reducing costs for livestock and poultry farming enterprises, enzyme preparation and feed production enterprises.

[0007] To achieve the above objectives, the technical approach adopted by the present invention is as follows: One of the technical solutions provided by this invention is a strain of Bacillus licheniformis that produces a high amount of alkaline protease, specifically Bacillus licheniformis (… Bacillus licheniformis Strain LKT2025-01 was obtained by targeted screening of the original starting strain L-01 after ARTP-LiCl combined mutagenesis. Currently, Bacillus licheniformis LKT2025-01 has been deposited on September 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 36047.

[0008] The second technical solution provided by this invention is Bacillus licheniformis ( Bacillus licheniformis Application of LKT2025-01; Furthermore, it has applications in the production of alkaline protease; Furthermore, it is used in the preparation of enzymatically hydrolyzed soybean meal.

[0009] The third technical solution provided by this invention is a method for producing alkaline protease using Bacillus licheniformis LKT2025-01 as described in one of the technical solutions; Furthermore, the method is specifically as follows: Seed culture: Select a single colony of LKT2025-01 and inoculate it into the culture medium for seed culture. The culture conditions are: temperature 38℃, stirring speed 220 r / min, culture time 36-38 h, and seed culture is completed. Seed tank expansion culture: Inoculate the seed liquid into the seed tank culture medium at an inoculation rate of 4-6% for seed tank expansion culture. The culture conditions are: temperature 36-38℃, stirring speed 220-240 r / min, tank pressure 0.05-0.07 MPa, aeration rate 1.1-1.4 vvm, and culture time 40 h. Fermentation tank cultivation: inoculum size 5-7% (v / v), temperature 36-38℃, rotation speed 230-250 r / min, ventilation rate 1.4-1.6 vvm, tank pressure 0.06-0.08 MPa, pH 9.0-9.2. When the pH is below 9.0, add ammonia to adjust the pH to 9.0-9.2. When the pH is above 9.2, adjust the pH to 9.0-9.2 by adding feed. After 48-52 hours of fermentation, the enzyme activity in the fermentation broth increases slowly, and the pH rises significantly. At this point, the cells begin to autolyze, so stop the culture and remove the broth from the container.

[0010] The composition of the culture medium used in the above liquid fermentation process is as follows: Seed culture medium (g / L): peptone 10, glucose 12, yeast extract 10, calcium chloride 0.6, sodium chloride 0.2, magnesium sulfate heptahydrate 0.2, the remainder is deionized water, pH 9.0; Seed tank culture medium (g / L): peptone 10-12, glucose 12-15, yeast extract 10-12, calcium chloride 0.6, sodium chloride 0.2-0.3, magnesium sulfate heptahydrate 0.2-0.3, the remainder is deionized water, pH 8.8-9.0; Fermentation tank culture medium (g / L): soybean meal powder 50-55, glucose 10-12, yeast extract 13-15, ammonium phosphate 10-12, magnesium sulfate heptahydrate 0.2, calcium chloride 0.5, Tween-80 0.5, the remainder is water, pH 8.8-9.0; Feeding medium (g / L): yeast extract 20, peptone 12, glucose 60, magnesium sulfate heptahydrate 1.0, Tween-80 0.3, the remainder being water, pH 8.7-8.9.

[0011] The fourth technical solution provided by the present invention is an alkaline protease produced by Bacillus licheniformis LKT2025-01 as described in one of the technical solutions.

[0012] The fifth technical solution provided by the present invention is the application of the alkaline protease described in the fourth technical solution in the preparation of enzymatically hydrolyzed soybean meal.

[0013] The alkaline protease prepared by liquid submerged fermentation using the mutant strain LKT2025-01 has the following properties: 1. The optimal reaction temperature is 40℃, and the operating temperature is wide.

[0014] 2. The optimal reaction pH is 10.5, and the relative enzyme activity is above 85% when the pH is between 9.0 and 12.0.

[0015] 3. It has strong temperature resistance. After being treated at 85℃ for 3 minutes, the enzyme activity retention rate is about 85%, which can be applied to the feed industry.

[0016] 4. The enzyme activity fermentation level reaches over 94,000 U / mL, which is 50-59% higher than the enzyme production capacity of the original strain.

[0017] 5. When 800 U / g is added to soybean meal, the degradation rate of soy globulin is about 90%, and the degradation rate of β-conglycinin is about 80%.

[0018] Beneficial effects: 1. This invention uses ARTP-LiCl combined mutagenesis to obtain a mutant strain LKT2025-01 that produces high alkaline protease by targeted screening through shaking flask enzyme activity and antigen protein content detection.

[0019] 2. This invention provides a liquid fermentation method for producing high-yield alkaline protease, specifically using a mutant strain LKT2025-01 to produce alkaline protease via liquid fermentation. This method for producing alkaline protease utilizes readily available raw materials, is simple in process, achieves high fermentation efficiency, and is low in cost, playing a crucial role in increasing revenue and reducing costs for enzyme preparation manufacturers.

[0020] 3. The alkaline protease produced by strain LKT2025-01 through liquid fermentation exhibits excellent degradation effects on antigenic proteins in soybean meal at around 37℃, with a degradation rate of 90% for daidzein and 80% for β-congaidzein. This effectively reduces toxic side effects on livestock and poultry, enhances immunity, improves health, and consequently improves growth performance. Furthermore, it demonstrates strong temperature resistance; after treatment at 85℃ for 3 minutes, the enzyme activity retention rate remains at approximately 85%, making it suitable for application in the feed industry and livestock farming. Attached Figure Description

[0021] Figure 1 Degradation rate of soybean globulin by alkaline protease; Figure 2 Degradation rate of β-conglycin by alkaline protease; Figure 3 The optimal reaction temperature curve for alkaline protease; Figure 4 The optimal pH curve for alkaline protease reaction; Figure 5 Comparison of relative enzyme activities after temperature resistance. Detailed Implementation

[0022] The present invention will be described in detail below through specific embodiments. Unless otherwise specified, the technical methods used in this invention are all methods well known to those skilled in the art. Furthermore, the methods in the embodiments should be understood as illustrative and not as limiting the scope of the invention. The essence and scope of this invention are defined only by the corresponding claims. For those skilled in the art, various changes or improvements to the material composition and dosage in these embodiments without departing from the essence and scope of the invention are all within the protection scope of this invention.

[0023] Example 1: Screening of strains by combined mutagenesis 1. ARTP mutagenesis: Take a fresh and mature slant culture, pick a single colony of the original Bacillus licheniformis L-01, and incubate it in an inoculum bottle. The incubation conditions are 37℃ and 220 r / min. The bacterial concentration is monitored during incubation until the OD value is reached. 600 When the value reaches 0.68-0.8, stop the culture (about 36 h of culture). The bacterial suspension at this time is used as ARTP mutagenesis solution.

[0024] Take 10 μL of the above bacterial suspension, place it in the center of a mutagenesis-specific slide, and spread it evenly. Then, using sterile forceps, place the slide in the corresponding hole position on the rotating stage of the ARTP system operating chamber. Adjust the irradiation distance to 2 mm, power to 100 W, and gas flow rate to 10 L / min for mutagenesis. The mutagenesis times are set to 0 s, 30 s, 60 s, 90 s, 120 s, 150 s, and 180 s, respectively. After mutagenesis, use sterile forceps to transfer the slide to 100 μL of physiological saline to prepare the bacterial solution for lithium chloride mutagenesis, for later use.

[0025] 2. ARTP-LiCl combined mutagenesis: The ARTP-mutated bacterial suspensions were diluted at different gradients and spread on screening plates containing different concentrations of lithium chloride, specifically 0%, 0.4%, 0.8%, 1.2%, 1.6%, and 2.0%. The plates were incubated at 37°C for 36 hours, and the colonies were observed and counted at the end of the incubation period.

[0026] Single colonies with good growth were selected for subculturing and shake-flask culture. After multiple shake-flask verifications, the target bacterium LKT2025-01, which showed significantly improved enzyme production capacity and stable heritability, was screened out. Then, Bacillus licheniformis (… Bacillus licheniformis LKT2025-01 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 23, 2025, with accession number CGMCC No. 36047.

[0027] 3. Culture medium: The culture media used in the above-mentioned combined mutagenesis screening process are as follows: Screening plate culture medium (g / L): peptone 5, yeast extract 5, skim milk powder 10, sodium chloride 5, agar 13, the remainder is deionized water, pH 8.8-9.0; Slant culture medium (g / L): peptone 5, yeast extract 5, sodium chloride 5, agar 13, the remainder is deionized water, pH 8.8-9.0; Culture medium for seed bottles (g / L): peptone 10, glucose 15, yeast extract 5, beef extract 8, sodium chloride 5, the remainder is deionized water, pH 8.8-9.0; Shake flask culture medium (g / L): peptone 10, glucose 15, yeast extract 5, beef extract 8, potassium chloride 0.5, calcium chloride 0.5, magnesium sulfate heptahydrate 0.2, Tween-80 0.3, the remainder being deionized water, pH 8.8-9.0.

[0028] 4. Validation test of the genetic traits of the mutant strain LKT2025-01: Parent strain L-01 and mutant strain LKT2025-01 were inoculated onto screening plates and cultured for 36 h. After culture, well-grown single colonies were picked and activated on slant agar for 36 h. After activation, an appropriate amount of cells was inoculated into a seed bottle and cultured at 37℃ and 220 r / min for 36 h. The cells were then transferred to shake flasks and fermented for 72 h. The fermentation supernatant was collected by centrifugation, and enzyme activity was measured. The mutant strain LKT2025-01 was subcultured for 8 generations, and the results are shown in Table 1 below.

[0029] Table 1. Alkaline protease activity of mutant strain LKT2025-01 after 8 generations

[0030] The results in the table show that after 8 generations of inheritance, the ability of the mutant strain LKT2025-01 to produce alkaline protease remains stably inherited, and is more than 70% higher than that of the parent strain L-01. It is believed that this strain can be applied to industrial fermentation production.

[0031] Example 2: Method for Assaying Alkaline Protease Activity 1. Definition of alkaline protease activity in this invention: One unit of enzyme activity is defined as 1 g or 1 mL of enzyme that, at 40°C and pH 10.50, hydrolyzes casein for 1 min to produce 1 μg of tyrosine. This activity is expressed as U / g (U / mL).

[0032] 2. Principle of alkaline protease activity assay in this invention: Alkaline protease hydrolyzes casein substrate under certain conditions (e.g., 40℃, pH 10.50) to produce tyrosine containing phenolic groups. Under alkaline conditions, Folin-Ciocalteu reagent is reduced to produce molybdenum blue and tungsten blue. The absorbance of the solution is measured at a wavelength of 680 nm using a spectrophotometer. Enzyme activity is proportional to absorbance, and thus the enzyme activity of the product can be calculated.

[0033] 3. Plotting the standard curve: Prepare L-tyrosine standard solutions of different concentrations: 0, 10, 20, 30, 40, and 50 μg / mL. Take 1.00 mL of each standard solution and place it in a test tube. Add 5 mL of 0.4 mol / L sodium carbonate solution and 1 mL of Folin-Ciocalteu reagent to each solution. Incubate in a water bath at (40±0.2)℃ for 20 min. Measure the absorbance at 680 nm. Using the tyrosine-free tube "0" as a blank, plot the absorbance value A on the ordinate and the tyrosine concentration c on the abscissa to construct a standard curve (this curve passes through zero). Calculate the amount of tyrosine (μg) at an absorbance of 1 using the graph or a regression equation; this is the absorbance constant K. The K value should be within the range of 95–100.

[0034] 4. Enzyme activity assay method: First, preheat the casein solution (10.0 g / L) in a water bath at (40±0.2)℃ for 5 min. Add 1 mL of diluted enzyme solution to each of four test tubes and preheat the tubes in the water bath at (40±0.2)℃ for 2 min. Then, add 1 mL of preheated casein solution to three of the test tubes. Simultaneously, add 2 mL of 0.4 mol / L trichloroacetic acid solution to the remaining test tube (blank tube) containing 1 mL of enzyme solution. Shake well and time the reaction for 10 min. After the reaction is complete, add 2 mL of 0.4 mol / L trichloroacetic acid to each of the three sample test tubes to terminate the reaction, shake well, and simultaneously add 1 mL of preheated casein solution to the blank tube and shake well. Take out four test tubes, let them stand for 10 min, filter (using slow qualitative filter paper), take 1 mL of the filtrate and add it to a test tube that has been pre-filled with 5 mL of 0.4 mol / L sodium carbonate solution, then add 1 mL of Folin reagent, and develop the color in a constant temperature water bath at (40±0.2)℃ for 20 min. After the color development is complete, use a 10 mm cuvette to measure the absorbance value at a wavelength of 680 nm.

[0035] 5. Formula for calculating alkaline protease activity:

[0036] Where: U—enzyme activity of the sample, U / mL; A—average absorbance of parallel tests of the sample; K—absorption constant; 4—total volume of the reaction; 10—reaction time, 10 min, calculated as 1 min; n—dilution factor.

[0037] Example 3: Production of alkaline protease by liquid fermentation of strain LKT2025-01 1. Liquid fermentation method for strains L-01 and LKT2025-01: Single colonies of L-01 and LKT2025-01 were picked and inoculated into 300 mL of culture medium for seed culture. The culture conditions were 38℃, stirring speed 220 r / min, and culture time 36 h. After the seed culture was completed, 5% of the seed culture was transferred to a seed tank for scale-up culture. The culture conditions were 37℃, stirring speed 240 r / min, tank pressure 0.05 MPa, aeration rate 1.2 vvm, and culture time 40 h. After 40 h, fermentation was carried out in a fermenter. The culture conditions were 6% inoculum, 37℃, stirring speed 240 r / min, aeration rate 1.5 vvm, tank pressure 0.06 MPa, and pH 9.0-9.2. When the pH was below 9.0, ammonia was added to adjust it to 9.0-9.2. When the pH was above 9.2, the pH was adjusted by feeding to control it at 9.0-9.2. After about 50 hours of fermentation, the enzyme activity in the fermentation broth increases slowly, and the pH rises significantly. At this point, the cells undergo autolysis, so stop the culture and remove the broth from the container.

[0038] 2. Culture medium used in the fermentation process: Seed culture medium (g / L): peptone 10, glucose 12, yeast extract 10, calcium chloride 0.6, sodium chloride 0.2, magnesium sulfate heptahydrate 0.2, the remainder is deionized water, pH 9.0; Seed tank culture medium (g / L): peptone 10, glucose 10, yeast extract 10, calcium chloride 0.6, sodium chloride 0.25, magnesium sulfate heptahydrate 0.3, the remainder is deionized water, pH 9.0; Fermentation tank culture medium (g / L): soybean meal powder 53, glucose 12, yeast extract 14, ammonium phosphate 10, magnesium sulfate heptahydrate 0.2, calcium chloride 0.5, Tween-80 0.5, the remainder is water, pH 9.0; Feeding medium (g / L): yeast extract 20, peptone 12, glucose 60, magnesium sulfate heptahydrate 1.0, Tween-80 0.3, the remainder being water, pH 8.7-8.9.

[0039] 3. Results of liquid fermentation experiment: The original strain L-01 and the mutant strain LKT2025-01 were used for liquid deep fermentation in a 60 L fermenter. After fermentation, the fermentation broth was centrifuged and the supernatant was used as an alkaline protease sample. The enzyme activity test results are shown in Table 2.

[0040] Table 2. Fermentation Experiment Results

[0041] After eight consecutive fermentations, the alkaline protease activity of strain LKT2025-01 consistently exceeded 94,000 U / mL, representing a more than 50% increase compared to the original strain. This indicates that the mutant strain LKT2025-01 exhibits significantly enhanced alkaline protease production and possesses stable genetic characteristics, making it suitable for large-scale industrial fermentation.

[0042] Example 4 Enzymatic hydrolysis experiment of antigenic protein in soybean meal 1. Enzymatic hydrolysis of soybean meal: Weigh 100 g (accurate to ±0.0002 g) of soybean meal (sampling of soybean meal according to feed sampling standard GB / T 14699-2023) into a breathing bag, and add deionized water at a feed-to-water ratio of 5:4 (m / v). Simultaneously, centrifuge the fermentation broths L-01 and LKT2025-01 from Example 3, and use the supernatant as the alkaline protease sample for enzymatic hydrolysis of soybean meal. Add alkaline protease at dosages of 200 U / g, 400 U / g, 600 U / g, 800 U / g, 1000 U / g, 1200 U / g, 1400 U / g, and 1600 U / g (enzyme activity / soybean meal mass) to the breathing bags respectively, stir evenly with a glass rod, and seal. Place the breathing bags in a 37℃ incubator for enzymatic hydrolysis for 24 h. After enzymatic hydrolysis, open the breathing bag, stir the material evenly, transfer it to a clean petri dish, and place the petri dish in a 65℃ forced-air drying oven to dry at a constant weight. Crush the enzymatically hydrolyzed soybean meal sample after constant weight, sieve it (60 mesh), mix it thoroughly, and store it for testing.

[0043] 2. Detection of antigen protein content in enzymatically hydrolyzed soybean meal: The detection kit (purchased from Tianjin Longke Ark Biotechnology Co., Ltd.) uses an indirect competitive ELISA method. The micro-well strips of the ELISA plate are pre-coated with soy globulin antigen. The soy globulin in the sample competes with the pre-coated antigen for soy globulin antibody. After adding the enzyme-labeled secondary antibody, the sample is developed with TMB substrate. The absorbance value of the sample is negatively correlated with the content of soy globulin. The content of soy globulin in the sample can be obtained by comparing with the standard curve.

[0044] Weigh 0.30 g of the enzymatically hydrolyzed soybean meal sample from step 1 into a 50 mL centrifuge tube, add 30 mL of sample extraction working solution (test kit tape), and extract at 37℃ in a water bath at 200 rpm for 1 h. After extraction, centrifuge at 4000 rpm for 5 min. Dilute the supernatant 140 times with sample diluent (test kit tape), and use this supernatant as the test sample.

[0045] Add 50 μL of calibrator (from the test kit) and 50 μL of the sample to be tested to different wells of the test plate, and record the position. Then add 50 μL of antibody working solution to each well, gently shake to mix, cover with the cover film, and react in a microplate reader at 37℃ for 10 min. After the reaction, forcefully tap off the solution in the wells of the test plate and fill the wells with washing working solution to wash, repeating the washing 4 times, and pat dry on filter paper. After patting dry, add 100 μL of enzyme-labeled reagent to each well of the test plate, cover with the cover film, and continue to react at 37℃ for 10 min. After the reaction, forcefully tap off the solution in the wells of the test plate, fill the wells with washing working solution to wash, repeating the washing 4 times, and pat dry on filter paper. After patting dry, add 100 μL of colorimetric solution to each well, cover with the cover film, and react at 37℃ in the dark for 10 min. After the reaction, add 100 μL of stop solution to each well to stop the reaction, and read the absorbance values ​​at dual wavelengths of 450 and 630 nm. The antigen protein content can be obtained by substituting the measured absorbance value into the dedicated calculation software provided with the kit.

[0046] It should be noted that the detection methods for the two antigen proteins, glycinin and β-conglycinin, are the same; the difference lies in the reagent kits used.

[0047] 3. Formula for calculating the degradation rate of antigenic proteins in soybean meal:

[0048] In the formula, X—antigen protein degradation rate, %; E—antigen protein content in soybean meal, mg / g; E1—antigen protein content in enzymatically hydrolyzed soybean meal, mg / g.

[0049] 4. Experimental Results The antigenic protein in soybean meal was enzymatically hydrolyzed according to experimental step 1 above. The hydrolyzed samples were then tested according to steps 2 and 3 respectively. The enzymatic hydrolysis effect of the antigenic protein is shown below. Figure 1 , Figure 2 As shown.

[0050] like Figure 1As shown, the degradation rate of soy globulin in soybean meal increased with the increase of alkaline protease addition. At 800 U / g, the degradation rate of soy globulin by LKT2025-01 alkaline protease reached 90%, more than 70% higher than that of L-01. This indicates that adding 800 U / g of the novel alkaline protease to soybean meal can significantly improve the anti-nutritional properties of soy globulin, thereby alleviating its toxic side effects on livestock and poultry.

[0051] like Figure 2 As shown, the degradation rate of β-conglycinin in soybean meal gradually increased with the increase of alkaline protease addition. At 800 U / g, the alkaline protease produced by LKT2025-01 achieved a degradation rate of 80%, which was 60% higher than that of L-01. Adding 800 U / g of the novel alkaline protease produced by LKT2025-01 to soybean meal resulted in the degradation of 80% of β-conglycinin, significantly reducing its side effects, increasing livestock and poultry immunity, thereby improving livestock and poultry growth performance and increasing enterprise profitability.

[0052] Example 5: Other characteristics of alkaline protease production by strain LKT2025-01 1. Optimal reaction temperature The fermentation broth of LKT2025-01 from Example 3 was centrifuged, and the supernatant was used as an alkaline protease sample. Enzyme activity was measured at 30, 35, 40, 45, and 50°C under pH 10.50 conditions. Relative enzyme activity was calculated with the enzyme activity at 40°C as 100%. The results are as follows: Figure 3 As shown in the figure, the optimal reaction temperature for the alkaline protease produced by LKT2025-01 is 40℃, and its operating temperature range is wide, with relative enzyme activity exceeding 85% at 30-50℃.

[0053] 2. Optimal reaction pH The fermentation broth of LKT2025-01 from Example 3 was centrifuged, and the supernatant was used as an alkaline protease sample. The enzyme activity was measured at pH 5.0, 6.0, 7.0, 8.0, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, and 12.0 at 40°C. The relative enzyme activity was calculated with the enzyme activity at pH 10.5 as 100%. The results are as follows: Figure 4 As shown, the optimal reaction pH for LKT2025-01 alkaline protease production is 10.5, and the relative enzyme activity is above 85% when the pH is between 9.0 and 12.0.

[0054] 3. Temperature resistance The fermentation broths produced by strains L-01 and LKT2025-01 in Example 3 were prepared into powder products using pressure spray drying technology. Samples were weighed and wrapped in filter cloth (300-400 mesh). The filter cloth containing the samples was then placed in an autoclave at 80℃ (controlled at 80℃±0.5℃) and 85℃ (controlled at 85℃±0.5℃) for 3 minutes (the preparation time for feed pelleting is 20-45 seconds). After the autoclaving was completed, the samples were immediately removed, and the enzyme activity of the samples before and after the autoclaving was measured. The enzyme activity of the samples before the autoclaving was taken as 100%, and the relative enzyme activity was calculated. The results are as follows: Figure 5 As shown, the temperature resistance of strain LKT2025-01 is significantly improved compared to that of L-01. After treatment at 85℃ for 3 min, the relative enzyme activity can still be maintained at about 85%.

[0055] The above-described embodiments are merely examples of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this invention patent. It should be noted that those skilled in the art can make similar variations and improvements to the above embodiments without departing from the central idea of ​​this invention patent, and these variations or modifications all fall within the protection scope of this invention patent. Therefore, the protection scope of this patent should be determined by the claims.

Claims

1. A strain of Bacillus licheniformis, characterized in that, Specifically, Bacillus licheniformis ( Bacillus licheniformis LKT2025-01, accession number CGMCC No.36047.

2. The Bacillus licheniformis as described in claim 1 ( Bacillus licheniformis Application of LKT2025-01 in the production of alkaline protease.

3. The Bacillus licheniformis as described in claim 1 ( Bacillus licheniformis Application of LKT2025-01 in the preparation of enzymatically hydrolyzed soybean meal.

4. A method for producing alkaline protease, characterized in that, Bacillus licheniformis ( Bacillus licheniformis LKT2025-01 Fermentation preparation.

5. The method as described in claim 4, characterized in that, Specifically, inoculate the seed culture into the fermenter culture medium at an inoculation rate of 5-7%, maintain a temperature of 36-38℃, a rotation speed of 230-250 r / min, an aeration rate of 1.4-1.6 vvm, a tank pressure of 0.06-0.08 MPa, and a pH of 9.0-9.

2. When the pH is below 9.0, add ammonia water to adjust the pH to 9.0-9.

2. When the pH is above 9.2, adjust the pH to 9.0-9.2 by adding feed. Fermentation continues until the enzyme activity in the fermentation broth increases slowly and the pH rises significantly. At this point, the cells begin to autolyze, so stop the culture and remove the product from the container.

6. The method as described in claim 5, characterized in that, The fermenter culture medium, in g / L, consists of: soybean meal powder 50-55 g, glucose 10-12 g, yeast extract 13-15 g, ammonium phosphate 10-12 g, magnesium sulfate heptahydrate 0.2 g, calcium chloride 0.5 g, Tween-80 0.5 g, and the remainder is water, with a pH of 8.8-9.

0. The feed medium, in g / L, consists of: yeast extract 20 g, peptone 12 g, glucose 60 g, magnesium sulfate heptahydrate 1.0 g, Tween-80 0.3 g, and the remainder is water, with a pH of 8.7-8.

9.

7. An alkaline protease prepared by the method according to any one of claims 4-6.

8. The application of the alkaline protease according to claim 7 in the preparation of enzymatically hydrolyzed soybean meal.

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