Bacillus subtilis and application thereof

Abstract

This application belongs to the field of cellulose-degrading strains, specifically disclosing a Bacillus subtilis strain and its applications. The Bacillus subtilis strain is classified as... Bacillus subtilis The name is Bacillus subtilis ( Bacillus subtilis B-03, was deposited on August 29, 2025, at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 66896. This application proposes a Bacillus subtilis strain with high cellulose and hemicellulose degradation rates. Bacillus subtilis B-03 has a cellulose degradation rate of 75.58% and a hemicellulose degradation rate of 97.24%, which is significantly higher than that of existing cellulose-degrading Bacillus subtilis bacteria. At the same time, it can be used in non-sterile straw fermentation scenarios, and has strong adaptability. Therefore, it has broad application prospects in industrial natural environments such as composting and biogas fermentation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cellulose-degrading strains, and specifically relates to a Bacillus subtilis and application thereof. BACKGROUND

[0002] With the development of agriculture, the yield of straw is increasing. If a large amount of straw cannot be effectively treated, it will not only cause resource waste, but also pollute the environment. At present, the main methods for treating straw are returning to field and burning, but these methods have certain limitations. Returning to field may lead to the breeding of pests and diseases, and burning may cause air pollution. Therefore, it is of great practical significance to find an efficient and environmentally friendly method for treating straw.

[0003] Cellulose is one of the main components of straw, and its degradation requires specific microorganisms. At present, there are some studies on cellulose-degrading bacteria in the existing field, but these strains still have some problems in practical application, such as low degradation efficiency and poor adaptability. Therefore, screening an efficient and adaptable cellulose-degrading bacterium is crucial for the resource utilization of straw. SUMMARY

[0004] The purpose of the present application is to solve at least one of the deficiencies of the prior art, promote the efficient resource utilization of agricultural waste (such as straw), and help the development of bioenergy and circular economy. Based on this, the present application provides a Bacillus subtilis in the first aspect, the taxonomic name of the Bacillus subtilis is Bacillus subtilis , and the name is Bacillus subtilis (B-03). It has been preserved in Guangdong Microbial Culture Collection Center on August 29, 2025, and the preservation number is GDMCC No: 66896. Bacillus subtilis

[0005] Preservation Note:

[0006] Strain Name: Bacillus subtilis

[0007] Taxonomic Name: Bacillus subtilis

[0008] Strain Number: B-03

[0009] Preservation Agency: Guangdong Microbial Culture Collection Center

[0010] Preservation Agency Abbreviation: GDMCC

[0011] Address: 5th Floor, Building 59, 100 Xianlie Middle Road, Guangzhou

[0012] Preservation Date: August 29, 2025

[0013] Preservation Center Registration Number: GDMCC No: 66896​

[0014] The Bacillus subtilis of this application ( Bacillus subtilis The cellulose degradation rate of B-03 can reach 75.58%, and the hemicellulose degradation rate is as high as 97.24%, significantly higher than existing cellulose-degrading Bacillus subtilis strains. Meanwhile, Bacillus subtilis (… Bacillus subtilis B-03 is suitable for non-sterile straw fermentation, which is significantly different from existing research on cellulose-degrading bacteria that require sterilized straw for fermentation. Therefore, the Bacillus subtilis strain described in this application... Bacillus subtilis B-03 has better application prospects in industrial natural environments such as composting and biogas fermentation.

[0015] Therefore, the Bacillus subtilis of this application ( Bacillus subtilis B-03, or its active ingredient as a microbial agent, can be better applied in the degradation of cellulose and / or hemicellulose, improving the degradation rate of cellulose and / or hemicellulose; and based on Bacillus subtilis... Bacillus subtilis B-03 forms a method for degrading cellulose and / or hemicellulose.

[0016] In the above-described method for degrading cellulose and / or hemicellulose, Bacillus subtilis is cultured in a fermentation medium. Preferably, the seed culture of Bacillus subtilis in the logarithmic growth phase is inoculated into the fermentation medium at an inoculum volume of 5%-10%. The fermentation medium comprises corn straw powder, peptone, sodium chloride, potassium dihydrogen phosphate, and magnesium sulfate. Specifically, the content of corn straw powder is 1.5 g / L-3 g / L, the content of peptone is 10 g / L-15 g / L, the content of sodium chloride is 4 g / L-5 g / L, the content of potassium dihydrogen phosphate is 0.5 g / L-1 g / L, and the content of magnesium sulfate is 0.5 g / L-1 g / L. If the peptone concentration is too high, it will lead to excessive cell growth and consumption of too much carbon source, reducing the resources required for product synthesis; it may also make the pH too alkaline, inhibiting the accumulation of acidic products. Conversely, if the concentration is too low, the bacterial count will be insufficient, resulting in a low product synthesis rate. Excessive sodium chloride significantly increases osmotic pressure and inhibits the growth of non-halophilic bacteria; while insufficient sodium chloride may lead to ion imbalance and affect membrane transport function. Potassium dihydrogen phosphate provides a phosphorus source (PO4). 3- ) and potassium ions (K + Excessive phosphorus (e.g., >2 g / L) can alter metabolic flux in certain fermentations; insufficient phosphorus inhibits cell growth and delays product synthesis. Magnesium sulfate primarily provides magnesium (Mg). 2+ ) and sulfur (SO4) 2- ), Mg 2+ It is an enzyme activator and stabilizes the ribosome structure. Sulfur is used to synthesize sulfur-containing amino acids and coenzymes, and a concentration of 0.5 g / L is sufficient.

[0017] Therefore, more preferably, the content of the above corn straw powder is 2 g / L, the content of the above peptone is 10 g / L, the content of the above sodium chloride is 5 g / L, the content of the above potassium dihydrogen phosphate is 1 g / L, and the content of the above magnesium sulfate is 0.5 g / L. The culture conditions are as follows: the culture time is 72 h, the culture temperature is 30 DEG C, the rotation speed is 180 r / min, and the initial pH value of the fermentation medium is 5.9-6.2.

[0018] The application has the following beneficial effects: the application provides a Bacillus subtilis (B-03) with high cellulose and hemicellulose degradation rates. Bacillus subtilis The cellulose degradation rate of the B-03 can reach 75.58%, and the hemicellulose degradation rate can reach 97.24%, which is significantly higher than that of the existing cellulose Bacillus subtilis degradation bacteria. Meanwhile, the B-03 can be applied to the non-sterilized straw fermentation scene, and has strong adaptability, so it has a broad application prospect in the industrial natural environment such as composting and biogas fermentation. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Fig. a shows the microbial colony diagram of the 30 strains of B-01 to B-30 grown on the culture medium; Fig. b shows the single colony of B-03 after preliminary separation and purification; Fig. c shows the effect diagram of the Congo red staining test on the B-03 strain in the preliminary screening stage; Fig. d shows the effect diagram of the Congo red staining test on the B-17 strain in the preliminary screening stage; and Fig. e shows the effect diagram of the Congo red staining test on the B-10 strain in the preliminary screening stage.

[0020] Figure 2 Fig. shows the growth curve diagram of the 8 Bacillus subtilis strains;

[0021] Figure 3 Fig. shows the standard curve diagram of p-nitrophenol when measuring the exoglucosidase activity;

[0022] Figure 4 Fig. shows the standard curve diagram of glucose when measuring the endoglucosidase activity;

[0023] Figure 5 Fig. shows the standard curve diagram of p-nitrophenol when measuring the beta-glucosidase activity;

[0024] Figure 6 Fig. shows the degradation rate column diagram of cellulose and hemicellulose of the 8 Bacillus subtilis strains. DETAILED DESCRIPTION

[0025] The concept and the generated technical effects of the present application will be described clearly and completely in combination with the embodiments and the drawings below, so as to fully understand the purpose, scheme and effect of the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0026] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0027] The Bacillus subtilis in the following examples Bacillus subtilis B-17, Bacillus subtilis Bacillus subtilis B-03, Bacillus subtilis Bacillus subtilis B-10, Bacillus subtilis Bacillus subtilis B-13, Bacillus subtilis Bacillus subtilis B-24, Bacillus subtilis Bacillus subtilis B-01, Bacillus subtilis Bacillus subtilis B-08, Bacillus subtilis Bacillus subtilis B-19, and Bacillus subtilis Bacillus subtilis The remaining strains in B-01 to B-30 are all from the soil samples collected from the flower field layer of the campus of Inner Mongolia University of Technology in Xincheng District, Hohhot, Inner Mongolia Autonomous Region.

[0028] The compositions of the culture medium / fluid used in the following examples are as follows, respectively:

[0029] Sodium carboxymethyl cellulose medium (g / L): sodium carboxymethyl cellulose 10.0, protein peptone 10.0, yeast powder 5.0, sodium chloride 10.0, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.2, agar 20.0.

[0030] LB medium (g / L): protein peptone 10.0, yeast powder 5.0, sodium chloride 10.0, manganese sulfate 0.005, agar 20.0.

[0031] Seed culture solution (g / L): protein peptone 10.0, yeast powder 5.0, sodium chloride 10.0, manganese sulfate 0.005.

[0032] Liquid fermentation medium (g / L): sodium carboxymethyl cellulose 10.0, protein peptone 10.0, yeast powder 5.0, sodium chloride 5.0, glucose 5.0, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.2.

[0033] Degradation fermentation medium (g / L): corn stalk powder 2.0, peptone 10.0, NaCl 5.0, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.5; the rest is deionized water.

[0034] The above medium / solution is made up with deionized water, and the pH is adjusted to 7.2-7.4, and sterilized at 121 ℃ for 20 min. Among them, the corn stalk powder is derived from corn stalk waste in agricultural production, which is a fine powder material processed through drying, crushing, screening and other processes after corn harvesting. Corn stalk, as the main stem part of corn plant, contains rich cellulose, hemicellulose and lignin and other components. In the following examples; the corn stalk powder is a standardized product treated by 40 mesh screening, which is uniform in particle size and beneficial to the attachment and degradation of microorganisms. Corn stalk powder, as a renewable resource, has wide application prospects in the fields of biomass energy, feed additives and organic fertilizers. The corn stalk in the following examples is from the experimental field of the National Modern Agricultural Demonstration Garden in Tumote Left Banner, Hohhot, Inner Mongolia Autonomous Region, and is ground into a powder by a crusher in the Green Biological Manufacturing Engineering Research Center of Inner Mongolia Autonomous Region.

[0035] The reagents involved in the following examples are prepared as follows:

[0036] (1) Preparation of DNS reagent: accurately weigh 6.3 g of 3,5-dinitrosalicylic acid, 21 g of sodium hydroxide particles, 182 g of potassium sodium tartrate, 5.0 g of phenol and 5.0 g of sodium sulfite. Put 3,5-dinitrosalicylic acid into a beaker, add 500 mL of distilled water, and water bath to slightly hot, stir with a glass rod during this period, and add the above medicines in turn. After dissolving, make up to 1000 mL. Filter the prepared DNS solution and transfer the filtrate into a brown reagent bottle, store in the dark, and use after 7 days, with a shelf life of one year.

[0037] (2) Phosphoric acid buffer solution with pH 7.0: prepare 320 mL of 0.2 mol / L sodium dihydrogen phosphate solution and 500 mL of 0.2 mol / L disodium hydrogen phosphate solution respectively, and adjust the pH after mixing.

[0038] (3) Preparation of glucose standard solution: accurately weigh 1 g of glucose, and make up to 1000 mL with deionized water.

[0039] (4) Preparation of pNP standard solution: accurately weigh 0.1 g of pNP, and make up to 1000 mL with deionized water.

[0040] (5) Preparation of 1 mg / mL pNPC + 1 mg / mL gluconolactone mixed solution: accurately weigh 0.1 g pNPC and 0.1 g gluconolactone, and dilute to 100 mL with phosphate buffer solution with pH 7.

[0041] (6) Preparation of 1 mg / mL pNPG solution: Accurately weigh 0.1 g pNPG and dilute to 100 mL with phosphate buffer solution with pH 7.0.

[0042] (7) Preparation of 0.5% Congo Red solution: Weigh 0.005 g of Congo Red and dilute to 100 mL with deionized water.

[0043] (8) Preparation of 0.5% NaCl solution: Weigh 0.005 g of sodium chloride and dilute to 100 mL with deionized water.

[0044] (9) Preparation of 1% sodium carboxymethyl cellulose solution: Accurately weigh 1 g of sodium carboxymethyl cellulose and dilute to 100 mL with phosphate buffer solution with pH 7.

[0045] The enzyme activity (U) used in the following examples is defined as follows: One unit of enzyme activity (U) is the amount of enzyme required to decompose pNPG to produce 1 μmol of pNP per minute from 1 mL of crude enzyme solution at 50 °C and pH 7.0. The calculation formula is as follows:

[0046] Enzyme activity (U / mL) =

[0047] In the formula: C is the glucose concentration calculated based on the measured absorbance value and the glucose standard curve; 25 is the volume of the colorimetric tube (mL); N is the dilution factor; T is the time (min) for enzymatic hydrolysis; and V is the amount of enzyme solution added (mL).

[0048] Example 1

[0049] I. Initial screening of bacterial strains

[0050] Bacillus subtilis was introduced using the spot inoculation method. Bacillus subtilis B-01 to B-30 were inoculated onto sodium carboxymethyl cellulose selection medium and incubated at 37 ℃ for 48 h. After colonies grew, the clear zone was observed by staining with 0.5% Congo red solution for 15 min and destaining with 5% sodium chloride solution for 15 min. Figure 1Figure a shows the growth of 30 strains of B-01 to B-30 on the medium, which are in accordance with the microbial colony chart of Bacillus subtilis; figure b shows the single colony of B-03 after preliminary separation and purification; figures c, d and e show the effect of Congo red staining test on B-03, B-17 and B-10 strains respectively in the preliminary screening stage. The ratio of the diameter of transparent circle to the diameter of colony on each selective medium is shown in Table 1.

[0051] Table 1: Results of transparent circle determination of cellulose-degrading bacteria in preliminary screening

[0052]

[0053] As shown in Table 1, the strains with higher HC values are Bacillus subtilis B-17, Bacillus subtilis B-03, Bacillus subtilis B-10, Bacillus subtilis B-13, Bacillus subtilis B-24, Bacillus subtilis B-01, Bacillus subtilis B-08 and Bacillus subtilis B-19 respectively. Bacillus subtilis B-17, Bacillus subtilis Bacillus subtilis B-03, Bacillus subtilis Bacillus subtilis B-10, Bacillus subtilis Bacillus subtilis B-13, Bacillus subtilis Bacillus subtilis B-24, Bacillus subtilis Bacillus subtilis B-01, Bacillus subtilis Bacillus subtilis B-08, Bacillus subtilis Bacillus subtilis B-19; it is preliminarily predicted that the eight strains have stronger cellulose-degrading ability and enter the secondary screening.

[0054] II. Screening of high cellulose-degrading strains

[0055] 1. Growth curve determination

[0056] (1) Activation of strains

[0057] Bacillus subtilis B-17, Bacillus subtilis B-03, Bacillus subtilis B-10, Bacillus subtilis B-13, Bacillus subtilis B-24, Bacillus subtilis B-01, Bacillus subtilis B-08 and Bacillus subtilis B-19 were activated respectively. Bacillus subtilis B-17, Bacillus subtilis Bacillus subtilis B-03, Bacillus subtilis Bacillus subtilis B-10, Bacillus subtilis Bacillus subtilis B-13, Bacillus subtilis Bacillus subtilis B-24, Bacillus subtilis Bacillus subtilis B-01, Bacillus subtilis Bacillus subtilis B-08, Bacillus subtilis Bacillus subtilis B-19 were activated respectively, and the activated bacterial liquid (OD 600nm=0.8) were inoculated into the seed culture medium at a 5% inoculum rate. After 24 h of inoculation, the inoculum was added to LB medium at a 2% inoculum rate and incubated at 37 ℃. Samples (3 mL) were taken from the bacterial culture at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, and 24 h, with uninoculated medium as a blank. The absorbance of the samples was measured at 600 nm using a UV-3200B UV-Vis spectrophotometer, and the number of bacteria was observed under a microscope. The bacterial growth curve was plotted with time on the x-axis and sample absorbance on the y-axis.

[0058] (2) Results of growth curve measurement

[0059] The growth curves of 8 Bacillus subtilis strains are shown in the figure. Figure 2 ,Depend on Figure 2 It can be seen that the growth trends of the eight Bacillus subtilis strains were basically consistent. Specifically, the first 0-5 hours after culture were the lag phase; from 5-15 hours, the cell number increased exponentially, indicating the entry into the logarithmic growth phase; and after 15 hours, the cell yield reached its peak, marking the stationary phase. During the logarithmic growth phase, the cells are well-nourished, grow rapidly, and have high metabolic activity. Furthermore, the cell properties are stable and uniform during this phase, making them more sensitive to physicochemical factors, prone to variation, and exhibiting good reproducibility. Therefore, cells in this growth phase are generally selected for treatment. Figure 2 It can be seen that, Bacillus subtilis B-03 Bacillus subtilis B-10 Bacillus subtilis B-19 showed significantly better growth than the other five strains. In the later stages, we should focus on the fiber degradation ability and cellulase activity of this strain. Taking into account factors such as cell biomass, we should select bacterial cells cultured for 12 hours for subsequent experiments.

[0060] 2. Determination of cellulase activity

[0061] Bacillus subtilis Bacillus subtilis B-17, Bacillus subtilis Bacillus subtilis B-03, Bacillus subtilis Bacillus subtilis B-10, Bacillus subtilis Bacillus subtilis B-13, Bacillus subtilis Bacillus subtilis B-24, Bacillus subtilis Bacillus subtilis B-01, Bacillus subtilis Bacillus subtilis B-08, Bacillus subtilis Bacillus subtilisB-19 were respectively inoculated into seed culture solution with 5% inoculation amount after activation, and cultured for 24 h, then inoculated into liquid fermentation culture medium with 5% inoculation amount, the culture temperature was 30 ℃, the shaking speed was 180 r / min, and samples were taken at 0 h, 8 h, 16 h, 24 h, 32 h, 40 h and 48 h.

[0062] The reaction substrates and reaction conditions were different when measuring the activities of several cellulases, and the specific measurement conditions are shown in Table 2.

[0063] Table 2 Cellulase activity determination conditions

[0064]

[0065] (1) The determination of exoglucosidase activity includes the following processes: 500 μL of fermentation liquor is taken as a crude enzyme solution, 50 μL of substrate (1 mg / mL of pNPC + 1 mg / mL of gluconolactone) is added, mixed, reacted at 50 ℃ for 30 min, 150 μL of 10% Na2CO3 is added to terminate the reaction. The amount of p-nitrophenol generated is measured at a wavelength of 405 nm to calculate the enzyme activity.

[0066] The standard curve of p-nitrophenol is shown as follows:

[0067] Prepare a standard solution of 0.1 mg / mL pNP. Add reagents according to Table 3 and mix well, boil in a water bath for 10 min, measure the OD value at 405 nm, take the pNP content (μg) as the abscissa, and the OD 405 value as the ordinate, draw the pNP standard curve. As shown in Figure 3 , the pNP standard curve equation is y=0.21371x+0.22371, where R 2 =0.99939.

[0068] Table 3 Reagent addition rules

[0069]

[0070] (2) The determination of endoglucosidase activity includes the following processes: 500 μL of fermentation liquor is taken as a crude enzyme solution, 1.5 mL of 1% CMC-Na solution is added, mixed, reacted at 50 ℃ for 30 min, 3 mL of DNS is added to terminate the reaction, boiled for 10 min, cooled after standing, diluted to 25 mL with distilled water, and mixed well, and the OD value is measured at a wavelength of 540 nm.

[0071] The preparation of glucose standard curve is shown as follows:

[0072] Take 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 mL of standard glucose solution in test tube, add distilled water to 2 mL, add DNS color reagent 1.5 mL, mix, boil in water bath for 10 min, cool to room temperature, add distilled water to 15 mL again, mix, measure the absorbance at OD 540nm 400, and draw the glucose standard curve. As shown in Figure 4 , the glucose standard curve equation is y = 0.3841x + 0.043, where R 2 = 0.9992.

[0073] (3) The determination of β-glucosidase activity includes the following processes: 100 μL of NPG (1 mg / mL) and 200 μL of crude enzyme solution are added to 300 μL of reaction system, 50 ℃ reaction for 30 min, 300 μL of 10% Na2CO3 is added to terminate the reaction, and the OD value is measured at wavelength 405 nm. p

[0074] The preparation of pNP standard curve is as follows:

[0075] Prepare 0.1 mg / mL pNP standard solution. Add reagents according to Table 4 and mix, boil in water bath for 10 min, measure the OD value at 405 nm, take the pNP content (μg) as the abscissa, and take the corresponding OD 405 value as the ordinate, draw the pNP standard curve. As shown in Figure 5 , the pNP standard curve equation is y = 0.1886x + 0.1939, where R 2 = 0.9995.

[0076] Table 4 Reagent addition rules

[0077]

[0078] (4) The same amount of substrate and crude enzyme solution (i.e. fermentation broth) is added to the control group and the experimental group, the difference is that the control group adds DNS reagent (or Na2CO3 solution) to inactivate the crude enzyme immediately after adding the substrate and the crude enzyme. The experimental group is to add DNS reagent (or Na2CO3 solution) to terminate the reaction after 50 ℃ water bath reaction for 30 min after adding the substrate and the crude enzyme.

[0079] The results of cellulase activity determination are shown in Table 5.

[0080] Table 5 Cellulase activity determination results

[0081]

[0082] ​According to the results of enzyme activity determination, the strains with high cellulase activity are B-03, B-10 and B-19.

[0083] 3. Determination of degradation rate

[0084] 100 mL of the degradation fermentation medium was placed in a 250 mL flask, and the seed liquid activated and inoculated into the seed culture medium to the logarithmic growth phase was inoculated into the degradation fermentation medium at a 5% inoculation amount, and cultured at 30°C and 180 r / min, and sampled at 72 h. The fermentation broth was centrifuged and dried to constant weight, and the mass change of corn stalk powder before and after fermentation was recorded.

[0085] 0.025 g of the sample dried to constant weight was accurately weighed, 250 μL of 74% H2SO4 solution was added to the sample, and the sample was shaken until it was fully mixed with the solution. Then, the sample was incubated in a 30°C water bath for 60 min. After the reaction was completed, the sample was quickly placed in an ice bath to terminate the reaction and 7 mL of deionized water was added.

[0086] Glucose loss rate sample preparation: 0.05 g of glucose was accurately weighed, 1 mL of 74% H2SO4 solution was added, and the mixture was reacted in a 30°C water bath for 60 min. The glucose was fully mixed with the sulfuric acid solution by constant stirring. After the reaction was completed, the sample was immediately placed in an ice bath to terminate the reaction, and 28 mL of deionized water was added for dilution.

[0087] The above sample solution was sterilized at 121°C and 0.1 MPa for 60 min. After the sterilization of the sample was completed, the monosaccharide (glucose) content in the sample was determined by high performance liquid chromatography, and the cellulose content was calculated according to the calculation method.

[0088] The conditions of high performance liquid chromatography are shown in Table 6.

[0089] Table 6 High performance liquid chromatography conditions

[0090]

[0091] The calculation method of cellulose content is as follows:

[0092] Acidolysis loss rate:

[0093] Mass of sugar in sample:

[0094] Mass of residual glucose:

[0095] Cellulose degradation rate in sample:

[0096] The results are shown in Table 7. Figure 6 As shown in Table 7, the cellulose degradation rate of strain B-03 was 48.6%, which was the highest among the three strains.Figure 6 It can be known that the bacillus subtilis strain of the application has the following advantages Bacillus subtilis The cellulose degradation ability of B-03 is the strongest, and the cellulose degradation rate can reach 75.78%, and the hemicellulose degradation rate can reach 97.24%.

[0097] To prove the strain screened by the application Bacillus subtilis The outstanding performance of B-03 is compared with 7 existing cellulose-degrading strains preserved in the laboratory, and all the strains are tested for degradation ability under the same culture conditions.

[0098] The results (see Table 7) show that under the same method of calculating cellulose and hemicellulose degradation rates, the cellulose degradation rate and the hemicellulose degradation rate of B-03 are significantly higher than those of all the comparison strains, reaching 75.58% and 97.24%, respectively. Bacillus subtilis The cellulose degradation rate and the hemicellulose degradation rate of B-03 are significantly higher than those of all the comparison strains, reaching 75.58% and 97.24%, respectively. Bacillus subtilis B-03 has improved the degradation efficiency of cellulose and hemicellulose by about 10.8% and 14.2%, respectively.

[0099] Table 7 Comparison of degradation rates of screened strains and existing strains

[0100]

[0101] The re-screening step breaks through the limitation of traditional single enzyme activity (such as CMCase) determination, and uses multi-enzyme synergistic determination (detects endoglucanase, exoglucanase and beta-glucosidase activity), and verifies the actual degradation rate under simulated industrial conditions with natural straw as the substrate. The screened strain can be better applied in industrial environments such as composting and biogas fermentation.

[0102] The above is only a preferred embodiment of the application, and the application is not limited to the above embodiments. As long as the same means achieve the technical effects of the application, they should belong to the protection scope of the application. The technical solutions and / or embodiments within the protection scope of the application can have various modifications and changes.

Claims

1. A Bacillus subtilis strain, characterized in that, The classification of Bacillus subtilis is named as follows: Bacillus subtilis The name is Bacillus subtilis ( Bacillus subtilis B-03 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 29, 2025, with accession number GDMCC No: 66896.

2. A microbial agent, characterized in that, The active ingredient of the bacterial agent includes Bacillus subtilis as described in claim 1.

3. The use of Bacillus subtilis according to claim 1 or the inoculant according to claim 2 in improving the degradation rate of cellulose and / or hemicellulose.

4. A method for degrading cellulose and / or hemicellulose, characterized in that, The method includes using Bacillus subtilis as described in claim 1 or the inoculum as described in claim 2.

5. The method according to claim 4, characterized in that, This includes culturing the Bacillus subtilis using a fermentation medium.

6. The method according to claim 5, characterized in that, The fermentation medium includes: corn stalk powder, peptone, sodium chloride, potassium dihydrogen phosphate, and magnesium sulfate.

7. The method according to claim 6, characterized in that, The content of the corn stalk powder is 1.5 g / L-3 g / L, the content of the peptone is 10 g / L-15 g / L, the content of the sodium chloride is 4 g / L-5 g / L, the content of the potassium dihydrogen phosphate is 0.5 g / L-1 g / L, and the content of the magnesium sulfate is 0.5 g / L-1 g / L.

8. The method according to claim 7, characterized in that, The content of the corn stalk powder is 2 g / L, the content of the peptone is 10 g / L, the content of the sodium chloride is 5 g / L, the content of the potassium dihydrogen phosphate is 1 g / L, and the content of the magnesium sulfate is 0.5 g / L.

9. The method according to any one of claims 5 to 8, wherein the culture conditions are: a culture time of 72 h, a culture temperature of 30 ℃, a rotation speed of 180 r / min, and an initial pH value of 5.9-6.2 in the fermentation medium.

10. The method according to claim 5, characterized in that, The seed culture of Bacillus subtilis in the logarithmic growth phase is inoculated into the fermentation medium at an inoculation rate of 5%-10%.

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