Bacillus amyloliquefaciens J1 with high yield of cellulase and application of bacillus amyloliquefaciens J1

Abstract

The invention discloses a bacillus amyloliquefaciens J1 for high yield of cellulase, which belongs to the field of microbial fermentation engineering and is preserved in China Center for Type Culture Collection, the preservation address is No. 299 in eight road, Wuchang District, Wuhan City, Hubei Province, the preservation date is January 6, 2025, the preservation number is CCTCC NO: M 2025045, and the bacillus amyloliquefaciens J1 is classified and named as bacillus amyloliquefaciens J1. The strain solves the problem that a wild strain lacks high enzyme activity, has the advantages of high growth speed, short metabolic cycle, low requirements on culture conditions and enzyme production conditions, high thermal stability of produced cellulase and the like, and has good probiotic and stress-resistant characteristics. When the method is applied to the distiller's grains, the content of neutral detergent fibers in the distiller's grains can be remarkably reduced, the content of reducing sugar is increased, and the utilization efficiency of the distiller's grains is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of microbial fermentation engineering, and particularly relates to a Bacillus amyloliquefaciens J1 with high cellulase productivity and its application. Background Art

[0002] Distillers' grains contain some undigested nutrients and various functional components produced during the fermentation process. Among them, using distillers' grains as animal feed is the main form of current development of distillers' grains, which not only solves the problem of resource waste but also optimizes the feed source. The main factor affecting the wide application of distillers' grains is their high cellulose content. Therefore, pretreatment and processing of distillers' grains to improve the nutrient utilization efficiency focuses on the degradation of cellulose components. The main cellulose-degrading strains selected for biological treatment are fungi, bacteria, and actinomycetes. Fungi are highly adaptable to acidic environments and show significant advantages in degrading cellulose with their unique biological characteristics. However, their growth cycle is long, and they will produce secondary metabolites harmful to livestock and poultry during the process of degrading cellulose. Actinomycetes have strong tolerance to harsh conditions, but their growth rate is slow, the treatment efficiency is low, and the demand for nutrients is high. Moreover, most of the existing microorganisms that can effectively degrade cellulose in distillers' grains are non-probiotics (molds), and they often produce secondary metabolites harmful to livestock and poultry during the process of degrading cellulose. Compared with fungi and actinomycetes, bacteria have successfully entered the public eye due to their short metabolic cycle, wide tolerance range to temperature, pH, etc., easy cultivation, and large elasticity to environmental pressure. Therefore, it is of great application prospect and practical significance to provide a probiotic strain capable of high cellulase production, apply it to distillers' grains, reduce the fiber content in distillers' grains, improve the feeding value of distillers' grains, and increase the utilization rate of distillers' grains. Summary of the Invention

[0003] Based on the above problems, the purpose of the present invention is to provide a Bacillus amyloliquefaciens J1 with high cellulase productivity, which solves the problem of the lack of high enzyme activity in wild strains and improves the utilization efficiency of distillers' grains.

[0004] The technology adopted by the present invention is as follows: A Bacillus amyloliquefaciens J1 with high cellulase productivity, which is preserved in the China Center for Type Culture Collection, preservation address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, preservation date: January 6, 2025, preservation number: CCTCC NO: M 2025045, taxonomic name: Bacillus amyloliquefaciens J1.

[0005] Another object of the present invention is to provide a cultivation method for the above-mentioned Bacillus amyloliquefaciens J1 with high cellulase productivity, as follows: Take OD 600The activated bacterial liquid with an OD of 1 was inoculated into the LB medium at an inoculation amount of 1% by volume, at a temperature of 20 - 50 °C, a pH of 5 - 9, and cultured with shaking in a shaker for 4 - 24 hours to obtain a seed liquid.

[0006] Another object of the present invention is to provide a method for fermenting and producing enzymes of Bacillus amyloliquefaciens J1 with high cellulase productivity as described above: Take the seed liquid with an OD of 600 = 1.7 and cultured with shaking in LB for 9 hours, and inoculate it into the enzyme-producing fermentation medium at an inoculation amount of 2% by volume, at a temperature of 25 °C - 50 °C, a pH of 4.5 - 7, and culture with shaking in a shaker for 36 hours. The formula of the enzyme-producing fermentation medium is: sodium carboxymethylcellulose 10 g, peptone 3 g, yeast extract powder 0.5 g, sodium chloride 5 g, calcium chloride 0.1 g, ammonium sulfate 2.0 g, potassium dihydrogen phosphate 1.0 g, magnesium sulfate heptahydrate 0.5 g, distilled water 1 L, and the pH is natural.

[0007] Another object of the present invention is to provide the application of Bacillus amyloliquefaciens J1 with high cellulase productivity as described above as a fermentation agent in the solid-state fermentation of distillers' grains.

[0008] Furthermore, for the above-mentioned application, according to the solid-liquid ratio, 2.5 ml of the bacterial agent and 37.5 ml of sterile water are added to 25 g of distillers' grains, and fermented at a constant temperature of 37 °C for 5 days, which can significantly reduce the content of neutral detergent fiber in the distillers' grains and increase the content of reducing sugar.

[0009] Advantages and beneficial effects of the present invention: The strain J1 of the present invention is a Gram-positive bacterium, and the cell shape is short rod-shaped. On the LB solid medium, it shows a rough surface, with bulges, irregular edges, milky white and opaque, fast growth rate, can enter the rapid growth phase in 2 hours, short metabolic cycle, has low requirements for culture conditions and enzyme production conditions, can grow well at 30 °C - 50 °C and a pH of 5 - 9, and can produce cellulase with high activity at 25 °C - 50 °C and a pH of 4.5 - 7. The produced cellulase can maintain high enzyme activity within the range of 25 - 70 °C, has good thermal stability. And the Bacillus amyloliquefaciens J1 provided by the present invention has a viable bacteria rate higher than 50% in the artificial gastric juice environment with a pH of 3 and 4, and can tolerate 0.3% bile salts, and has an inhibitory effect on Escherichia coli and Staphylococcus aureus in vitro, and has good probiotic and stress resistance characteristics. Applying it to distillers' grains can significantly reduce the content of neutral detergent fiber in the distillers' grains and increase the content of reducing sugar, greatly improving the utilization efficiency of distillers' grains. Description of the Drawings

[0010] Figure 1 It is the Gram staining diagram of the strain J1 of the present invention;

[0011] Figure 2 It is the phylogenetic tree diagram of the strain J1 of the present invention;

[0012] Figure 3 Growth curve of strain J1 of the present invention;

[0013] Figure 4 Growth curve of strain J1 of the present invention at different temperatures;

[0014] Figure 5 Growth curve of strain J1 of the present invention at different pH values;

[0015] Figure 6 Enzyme production curve of strain J1 of the present invention with the change of initial temperature; wherein (A) is the curve of filter paper enzyme activity with the change of initial temperature, and (B) is the curve of endoglucanase with the change of initial temperature;

[0016] Figure 7 Enzyme production curve of strain J1 of the present invention with the change of initial pH; wherein (A) is the curve of filter paper enzyme activity with the change of initial pH, and (B) is the curve of endoglucanase with the change of initial pH;

[0017] Figure 8 Thermal stability detection curve of cellulase produced by strain J1 of the present invention; wherein (A) is the curve of filter paper enzyme activity with the change of reaction temperature, and (B) is the curve of endoglucanase with the change of reaction temperature;

[0018] Figure 9 pH tolerance detection curve of cellulase produced by strain J1 of the present invention; wherein (A) is the curve of filter paper enzyme activity with the change of reaction pH, and (B) is the curve of endoglucanase with the change of reaction pH;

[0019] Figure 10 Antibacterial performance diagram of strain J1 of the present invention against pathogenic bacteria; wherein (A) is the antibacterial effect against Staphylococcus aureus, and (B) is the antibacterial effect against Escherichia coli;

[0020] Figure 11 Comparison diagram of neutral detergent fiber before and after fermentation of distillers' grains by strain J1 of the present invention;

[0021] Figure 12 Comparison diagram of reducing sugar before and after fermentation of distillers' grains by strain J1 of the present invention;

[0022] Figure 13 Scanning electron micrograph before and after fermentation of distillers' grains by strain J1 of the present invention; wherein (A) is the electron micrograph of rice husk before fermentation, (B) is the electron micrograph of sorghum before fermentation, (C) is the electron micrograph of rice husk after fermentation, and (D) is the electron micrograph of sorghum after fermentation. Detailed implementation manners

[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] Example 1: Isolation and screening of cellulose-degrading bacteria

[0025] Experimental method:

[0026] Culturable microorganisms were screened using the cornfield soil collected from the Acheng Base of Northeast Agricultural University. Immediately after sample collection, 10 g of soil was taken and placed in a 250 mL conical flask containing 90 mL of sterile normal saline, shaken and mixed evenly at 37 °C for 30 minutes to prepare a bacterial suspension. 2 mL of the bacterial suspension was inoculated into a 500 mL conical flask containing 200 mL of enrichment medium and cultured at 37 °C and 220 r / min for 3 d to prepare an enrichment culture solution. The enrichment culture solution was aspirated and serially diluted with sterile normal saline to 10 -3 、10 -4 、10 -5 、10 -6 , spread on the primary screening medium plate, with three replicates for each dilution factor, and cultured at 37 °C for 72 hours. An appropriate amount of 0.3% congo red solution was added to the culture dish for staining for 30 min, and then the staining solution was discarded. The plate was rinsed once with 4 - 5 mL of 1 mol / L NaCl solution, and then 8 - 10 mL of 1 mol / L NaCl solution was added to the culture dish. After standing for "decolorization" for 30 min, the liquid in the culture dish was poured out, and the diameters of the colonies and hydrolysis zones were observed. The colonies with obvious transparent zones were selected and streaked and purified on the LB plate to obtain single colonies. The obtained single colonies were respectively determined for cellulase activity for re-screening, and the strain with the best cellulose degradation effect was named J1.

[0027] 1. The method for determining cellulase activity is as follows:

[0028] (1) Preparation of crude enzyme solution: The activated culture solution was adjusted to OD 600 = 1.7, which was the seed solution for the experiment. Then the seed solution was inoculated into a 500 mL conical flask containing 150 mL of enzyme-producing medium at an inoculation amount of 2.0%, and fermented and cultured at 37 °C and 220 r / min for 36 h to prepare a fermentation broth. The fermentation broth was centrifuged at 6000 r / min and 4 °C for 10 min, and the supernatant was the crude enzyme solution.

[0029] (2) Preparation of glucose standard curve: Pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 mL of standard glucose solution (1 mg / mL) into clean test tubes respectively, make up to 2 mL with distilled water, then add 1.5 mL of DNS color reagent, mix well. After boiling water bath for 10 min, quickly cool to room temperature, add distilled water to 10 mL again, mix well, measure the absorbance at OD 540 , and draw the glucose standard curve.

[0030] (3) Determination of filter paper enzyme activity (FPase): Take 4 test tubes and number them in sequence. Use Whatman NO.1 filter paper strip as the reaction substrate (about 50 mg), fold it into a small roll, put it into 4 test tubes respectively, then add 1.5 mL of citric acid - sodium citrate buffer solution with pH = 4.8 to each test tube, try to wet the filter paper, incubate at 50 °C for 5 min. Then, add 0.5 mL of crude enzyme solution to test tubes 1 - 3, and add inactivated crude enzyme solution to test tube 4. Place all the above test tubes in a 50 °C water bath for reaction for 1 h. After taking out, immediately add 1.5 mL of DNS solution to terminate the reaction. Boil in boiling water bath for 10 min. Then quickly cool to room temperature, add distilled water to make the volume up to 10 mL, mix well, measure the absorbance at a wavelength of 540 nm. Zero with test tube 4 as the control group, measure the absorbance values of test tubes 1 - 3, and record the absorbance values.

[0031] (4) Determination of endoglucanase activity (CMCase): Take 4 test tubes respectively and number them in sequence. Add 0.5 mL of crude enzyme solution to test tubes 1 - 3, and add 0.5 mL of high - temperature inactivated crude enzyme solution to test tube 4. Add 1.5 mL of 1% CMC - Na citrate buffer solution to each test tube. Place the above test tubes in a 50 °C water bath for reaction for 30 min. After taking out, immediately add 1.5 mL of DNS solution to terminate the reaction. Boil in boiling water bath for 10 min. Then quickly cool to room temperature, add distilled water to make the volume up to 10 mL, shake well, measure the absorbance at a wavelength of 540 nm, zero with test tube 4 as the control group, measure the absorbance values of test tubes 1 - 3, and record the absorbance values.

[0032] (5) Definition of enzyme activity: The amount of enzyme required for 1 mL of enzyme solution to catalyze the hydrolysis of substrate to generate 1 μg of glucose per minute is 1 unit (U) of cellulase activity.

[0033] In the present invention, the cellulase activity of the strain J1 was measured, and it was found that the FPase of the culture solution of strain J1 at 36 h was 3.87 U / mL, and the CMCase was 10.53 U / mL.

[0034] 2. The above - mentioned culture medium components:

[0035] Enrichment medium (g / L): Distillers grains powder 10 g, sodium chloride 5 g, calcium chloride 0.1 g, ammonium sulfate 2.0 g, potassium dihydrogen phosphate 1.0 g, magnesium sulfate heptahydrate 0.5 g, distilled water 1 L, pH natural.

[0036] Primary screening medium: Sodium carboxymethyl cellulose 10 g, sodium chloride 5 g, calcium chloride 0.1 g, ammonium sulfate 2.0 g, potassium dihydrogen phosphate 1.0 g, magnesium sulfate heptahydrate 0.5 g, agar 20 g, distilled water 1 L, pH natural.

[0037] Fermentation medium for enzyme production (g / L): Sodium carboxymethyl cellulose 10 g, peptone 3 g, yeast extract powder 0.5 g, sodium chloride 5 g, calcium chloride 0.1 g, ammonium sulfate 2.0 g, potassium dihydrogen phosphate 1.0 g, magnesium sulfate heptahydrate 0.5 g, distilled water 1 L, pH natural

[0038] Example 2: Identification of the strain

[0039] The Gram staining result of strain J1 is as Figure 1 shown. It is a Gram-positive bacterium, and the cell shape is short rod-shaped. On LB, it presents colonies with a rough surface, raised, irregular edges, and milky white and opaque appearance. The full length of the 16S rDNA sequence of strain J1 is 1436 bp, and the sequence is as shown in SEQ ID NO.1. Using the BLAST function of NCBI, the sequenced sequence was retrieved and aligned for homology in the GeneBank database, and the homology with Bacillus amyloliquefaciens was above 99%; using MEGA13 software to draw a phylogenetic tree, as Figure 2 shown. Finally, it was determined that strain JI is Bacillus amyloliquefaciens.

[0040] Example 3: Determination of strain growth performance

[0041] 1. Growth curve determination

[0042] Inoculate strain J1 into a 50 ml Erlenmeyer flask containing 15 mL of LB medium and culture for 9 h. Use an ultraviolet spectrophotometer and sterilized LB medium to adjust the absorbance value of the activated bacterial liquid to OD 600 = 1.0. Then, inoculate the adjusted bacterial liquid into a 500 ml Erlenmeyer flask containing 120 mL of LB medium at an inoculation amount of 1% by volume. Set three replicates and continuously culture in a constant temperature shaker at 37 °C and 220 r / min. Take 3 mL every 2 h in a laminar flow hood and measure the absorbance value of the bacterial liquid at OD 600 until the bacteria show decline, and draw a growth curve.

[0043] As Figure 3As shown, the strain grew rapidly within 2 - 12 h and entered the stationary growth phase at 12 h.

[0044] 2. Determination of the Optimal Temperature and Optimal pH Value

[0045] Determination of the optimal temperature: The activated seed liquid was inoculated into the LB medium at an inoculation amount of 1% by volume. The temperature of the constant temperature shaker was set at 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, and cultured for 9 h under the condition of 220 r / min. The absorbance value at 0D600 was measured by an ultraviolet spectrophotometer. Determination of the optimal pH value: The pH value of the LB medium was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 using 1 mo1 / L HC1 and NaOH solutions. The activated seed liquid was transferred to the LB medium with different pH values at an inoculation amount of 1% and cultured overnight at 37°C and 220 r / min, and its 0D 600 absorbance value was measured, and the growth curve of strain J1 under different temperature and pH conditions was plotted.

[0046] As Figure 4 shown, the strain could grow within the temperature range of 15 - 55°C, and the optimal growth temperature was 30 - 50°C; as Figure 5 shown, the growth pH of the strain was 3 - 9, and the optimal growth pH was 5 - 9.

[0047] Example 4: Effects of Different Culture Conditions on Enzyme Production by Strain J1

[0048] 1. Effects of different initial temperatures on enzyme production by strain J1: The activated seed liquid was inoculated into the enzyme production fermentation medium at an inoculation amount of 2% by volume. The temperature of the constant temperature shaker was set at 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, and FPase and CMCase were measured.

[0049] 2. Effects of different initial pH values on enzyme production by strain J1: The pH value of the enzyme production fermentation medium was adjusted to 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 using 1 mo1 / L HC1 and NaOH solutions. The activated seed liquid was transferred to the enzyme production fermentation medium with different pH values at an inoculation amount of 2% and cultured for 36 h, and FPase and CMCase were measured. The influence diagram of different initial temperatures and pH conditions on enzyme production by strain J1 was plotted.

[0050] Effects of different initial temperatures on enzyme production by strain J1: The enzyme production curve of strain J1 with the change of the starting temperature is as Figure 6As shown in the figure. Between 25°C and 50°C, strain J1 can produce relatively high cellulase activity. At 37°C, both CMCase and FPase reach their maximum values, which are 3.87 U / mL and 10.53 U / mL respectively. Therefore, the starting temperature of 37°C is the optimal temperature for enzyme production by strain J1. At 50°C, CMCase can still maintain more than 80% of the optimal temperature, and at 45°C, FPase can still maintain more than 75% of the optimal temperature.

[0051] Effect of different initial pH values on enzyme production by strain J1 during fermentation: The enzyme production curve of strain J1 with the change of the starting pH value is as Figure 7 shown. When the starting pH value is between 4.5 and 7, the FPase of the strain is relatively stable. When the pH is between 5.5 and 6.5, the CMCase increases rapidly and reaches the highest value of 21.85 U / mL at pH = 6.5. Considering comprehensively, the starting pH of 6.5 is the optimal starting pH value for enzyme production by Bacillus amyloliquefaciens J1.

[0052] Example 5: Study on the enzymatic properties of the cellulase produced by strain J1

[0053] 1. Detection of the thermal stability of the cellulase produced by strain J1: Inoculate the activated seed liquid into the fermentation enzyme production medium at an inoculation amount of 2% by volume, and culture it at 37°C and 220 r / min for 36 h to prepare a crude enzyme solution. Adjust the temperature of the water bath to 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C respectively, and measure FPase and CMCase at different water bath temperatures.

[0054] 2. Detection of the pH tolerance of the cellulase produced by strain J1: Inoculate the activated seed liquid into the fermentation enzyme production medium at an inoculation amount of 2% by volume, and culture it at 37°C and 220 r / min for 36 h to prepare a crude enzyme solution. Adjust the pH value of the citric acid-sodium citrate buffer solution to 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0, and measure FPase and CMCase in buffer solutions with different pH values, and draw a graph showing the effects of different temperatures and pH values on the enzyme activity of the cellulase produced by strain J1.

[0055] Results of the detection of the thermal stability of the cellulase produced by strain J1: The change curve of the reaction temperature is as Figure 8 shown. Between 25 - 55°C, FPase is relatively stable. When the temperature rises to 70°C, its enzyme activity is still more than 85% of the enzyme activity at the optimal temperature. Between 25 - 60°C, CMCase is relatively stable. When the temperature rises to 80°C, its enzyme activity is still about 85% of the enzyme activity at the optimal temperature. CMCase plays a major role in cellulose degradation, indicating that the cellulase produced by strain J1 has good thermal stability.

[0056] (2) Detection results of the pH tolerance of cellulase produced by strain J1: The change curves of cellulase production by Bacillus amyloliquefaciens J1 in this embodiment with respect to pH are as shown in Figure 9 . When the pH is 6, the highest values of FPase and CMCase are reached, which are 6.74 U / mL and 24.06 U / mL respectively.

[0057] Example 6: Determination of probiotic properties and stress resistance

[0058] 1. Determination of antibacterial performance

[0059] Separate sterile cotton swabs were dipped into Escherichia coli and Staphylococcus aureus with a concentration of 1×10 8 cfu / mL, and repeatedly spread on LB plates. A 6-mm diameter puncher was used to punch holes in the LB plates, and 100 μL of the fermentation supernatant of the isolated strain and 0.2 mg / mL of polymyxin b were respectively added into the holes. After culturing at 37 °C for 24 h, the antibacterial effects of the isolated strain on pathogenic bacteria were observed. The experimental results are as shown in Figure 10 . Strain J1 has inhibitory effects on both Escherichia coli and Staphylococcus aureus.

[0060] Preparation of fermentation supernatant: The seed liquid of the isolated strain (including 4 screened control bacteria with fiber degradation effect) was inoculated into 100 mL of LB liquid medium at an inoculation amount of 1%, cultured overnight with shaking at 37 °C, centrifuged at 12000 rpm for 15 min to separate the cells and the supernatant, and the supernatant was filtered through a filter membrane (filter membrane diameter is 0.22 μm).

[0061] 2. Determination of the performance of resistance to artificial gastric juice

[0062] The artificial gastric juice was prepared with reference to the preparation method in the 2010 edition of the Pharmacopoeia of the People's Republic of China. Take 16.4 mL of dilute hydrochloric acid, add about 800 mL of water and 10 g of pepsin, shake well, then dilute with water to 1000 mL, and adjust the pH values to 2, 3, and 4 respectively, and sterilize through a filter membrane (filter membrane diameter is 0.22 μm). Take 2 mL of the overnight cultured J1 bacterial suspension, centrifuge at 6000 r / min for 10 min to collect the cell precipitate and resuspend it with the above-mentioned artificial gastric juice with adjusted pH values. Another portion was resuspended with sterile water (pH = 7) as a blank control. Incubate at 37 °C and 220 r / min with constant shaking for 120 min, and then perform colony counting and calculate the survival rate. Strain survival rate = number of viable bacteria after 2 h of different treatments / number of viable bacteria after 2 h of control treatment × 100%.

[0063] Test results: The isolated strains were subjected to the artificial gastric juice tolerance test. The results showed (see Table 1) that the viable cell rate of strain J1 was 40.23% in artificial gastric juice with pH = 2, and the viable cell rate was higher than 50% in environments with pH = 3 and 4.

[0064] Table 1 Determination results of the artificial gastric juice tolerance performance of strain J1

[0065] pH value Colony count Survival rate 7 348±9.17 100％ 4 203±6.81 58.33％ 3 175±5.03 50.29％ 2 140±4.73 40.23％

[0066] 3. Determination of bile salt tolerance

[0067] Weigh 2.5 g of porcine bile salt and dissolve it in 50 mL of deionized water to prepare a 5% bile salt solution, and filter it with a filter membrane (0.22 μm). Then, add 0 μL, 200 μL, 600 μL, and 1000 μL of the 5% bile salt solution to 10 mL, 9.8 mL, 9.4 mL, and 9 mL of sterilized LB broth respectively to prepare LB broth media containing 0%, 0.1%, 0.3%, and 0.5% bile salt.

[0068] Take the J1 bacterial solution that tolerated artificial gastric juice with pH = 2 and inoculate it into LB broth containing 0%, 0.1%, 0.3%, and 0.5% bile salt at a ratio of 1% respectively. After culturing at 37 °C and 220 r / min for 5 h, colony counting was carried out. Calculate the survival rate: the same as the artificial gastric juice tolerance test.

[0069] Test results: The isolated strains were subjected to the bile salt tolerance test. The results showed (see Table 2) that after treatment at a bile salt concentration of 0.5% for 5 h, the survival rate was 82.59%, and after treatment at a bile salt concentration of 0.3% for 5 h, the survival rate was above 85%, indicating that it could tolerate bile salt.

[0070] Table 2 Determination results of the bile salt tolerance performance of strain J1

[0071] Bile salt concentration Colony count Survival rate 0 149.33±7.57 100％ 0.1 146±3.61 97.77％ 0.3 128.67±4.16 86.16％ 0.5 123.33±4.93 82.59％

[0072] Example 7: Preparation of Bacillus amyloliquefaciens J1 bacterial agent

[0073] In this example, the cryopreserved strain of Bacillus amyloliquefaciens J1 was used to prepare the bacterial agent. The specific operation method was as follows: Inoculate strain J1 into LB for overnight activation culture, and then transfer the activated culture to LB at an inoculation ratio of 1%. The culture conditions were: temperature 37 °C, pH = 7. After shaking flask culture for 9 h, the J1 bacterial agent could be obtained.

[0074] Example 8: Application of strain J1 in solid-state fermented distillers' grains

[0075] The present invention selects distillers' grains as raw materials for solid-state fermentation, and the method is as follows: Weigh 25 g of distillers' grains with an electronic balance into a 500 mL conical flask, and additionally add 37.5 mL of distilled water. Sterilize at 121 °C for 15 min and let it stand overnight. Inoculate the Bacillus amyloliquefaciens J1 bacterial agent prepared above into the distillers' grains at an addition amount of 10%, stir well, seal with a breathable membrane, and place it in a constant temperature and humidity incubator at 37 °C for fermentation for 5 days. The blank control is the same operation, but the bacterial agent is replaced with an equal amount of sterile LB medium. After 5 days, take it out, dry it in an oven at 65 °C until constant weight, measure the contents of neutral detergent fiber and reducing sugar before and after fermentation, and observe the morphological changes of the distillers' grains before and after fermentation.

[0076] Test results: As Figure 11 - 12 shown, compared with the blank group, the neutral detergent fiber in the distillers' grains of the J1 group decreased from 50.82% to 41.56%, and the degradation rate was 18.22%; the content of reducing sugar increased from 1.48% to 2.35%, an increase of 58.78%, indicating that adding the bacterial agent J1 can significantly improve the nutritional value of distillers' grains. The SEM images ( Figure 13 ) show the surface microstructure of rice husk and sorghum in the distillers' grains before and after fermentation. In the unfermented distillers' grains, the surfaces of rice husk and sorghum are relatively flat and dense, and the internal structure cannot be seen. After fermentation, the structures of rice husk and sorghum in the distillers' grains are damaged, and the surfaces are rough and fluffy, which helps the further degradation of the distillers' grains.

Claims

1. A strain of Bacillus amyloliquefaciens J1 with high cellulase production, deposited in China Center for Type Culture Collection, deposit address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: January 6, 2025, deposit number: CCTCC NO: M2025045, classification name: Bacillus amyloliquefaciens amyloliquefaciens J1.

2. The method for culturing a high-yield cellulase-producing Bacillus amyloliquefaciens J1 according to claim 1, characterized in that: The method is as follows: Take OD 600 = 1 activated bacterial solution is inoculated into LB medium at a volume ratio of 1%, the temperature is 20-50°C, the pH is 5-9, and the shaking culture is carried out on a shaker for 4-24 hours to obtain a seed solution.

3. The method for fermenting and culturing a high-yield cellulase-producing Bacillus amyloliquefaciens J1 according to claim 1, characterized in that: The method is as follows: Take OD 600 =1.7, the seed liquid cultured with LB shaking for 9 hours is inoculated into the fermentation enzyme production medium at a volume ratio of 2%, the temperature is: 25°C-50°C, pH: 4.5-7, and the shaking culture is carried out on a shaking table for 36 hours. The fermentation enzyme production medium is formulated according to the solid-liquid ratio: 10g sodium carboxymethyl cellulose, 3g peptone, 0.5g yeast extract powder, 5g sodium chloride, 0.1g calcium chloride, 2.0g ammonium sulfate, 1.0g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 1L distilled water, and natural pH.

4. Use of the high-cellulase-producing Bacillus amyloliquefaciens J1 according to claim 1 as a fermentation agent in solid-state fermentation of white wine grains.

5. The use according to claim 4, characterized in that: According to the solid-liquid ratio, add 2.5 ml of bacterial agent and 37.5 ml of sterile water to 25 g of white wine lees, and ferment at a constant temperature of 37°C for 5 days.

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