Bacillus subtilis and application thereof

Abstract

The invention belongs to the field of cellulose degradation strains, and particularly discloses bacillus subtilis and application thereof. The bacillus subtilis is classified and named as bacillus subtilis, the name of the bacillus subtilis is bacillus subtilis B-03, the bacillus subtilis is preserved in Guangdong Microbial Culture Collection Center on August 29, 2025, and the preservation number of the bacillus subtilis is GDMCC No: 66896. The invention provides bacillus subtilis B-03 with high cellulose and hemicellulose degradation rate, the cellulose degradation rate of the bacillus subtilis B-03 can reach 75.58%, the hemicellulose degradation rate of the bacillus subtilis B-03 is as high as 97.24%, and the cellulose degradation rate and the hemicellulose degradation rate of the bacillus subtilis B-03 are obviously higher than those of existing cellulose bacillus subtilis degrading bacteria; meanwhile, the method can be suitable for a non-sterilization straw fermentation scene and is high in adaptability, so that the method has a wide application prospect in industrial natural environments such as composting and biogas fermentation.

Description

Technical Field

[0001] This application belongs to the field of cellulose-degrading strains, specifically relating to a Bacillus subtilis strain and its applications. Background Technology

[0002] With agricultural development, straw production is constantly increasing. If large amounts of straw are not effectively managed, it will not only waste resources but also pollute the environment. Currently, straw treatment methods mainly include returning straw to the field and burning, but these methods have certain limitations. Returning straw to the field may lead to the breeding of pests and diseases, while burning will cause air pollution. Therefore, finding an efficient and environmentally friendly straw treatment method is of significant practical importance.

[0003] Cellulose is one of the main components of straw, and its degradation requires specific microorganisms. Currently, there is some research on cellulose-degrading bacteria in the field, but these strains still have some problems in practical applications, such as low degradation efficiency and poor adaptability. Therefore, screening out a highly efficient and adaptable cellulose-degrading bacterium is crucial for the resource utilization of straw. Summary of the Invention

[0004] The purpose of this application is to address at least one deficiency of existing technologies, promote the efficient resource utilization of agricultural waste (such as straw), and contribute to the development of bioenergy and the circular economy. Based on this, the first aspect of this application provides a Bacillus subtilis strain, which is classified as... 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.

[0005] Preservation instructions: Bacterial strain name: Bacillus subtilis Classification and naming: Bacillus subtilis Strain number: B-03 Preservation Institution: Guangdong Provincial Center for Microbial Culture Collection Abbreviation for depository institution: GDMCC Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou Deposit date: August 29, 2025 Collection Center Registration Number: GDMCC No.: 66896 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 can be applied to the fermentation of non-sterilized straw, which is quite different from the related cellulose-degrading bacteria researches in the prior art which need to use sterilized straw for fermentation. Therefore, the B. subtilis (B-03) of the present application has a better application prospect in the industrial natural environment such as composting and biogas fermentation. Bacillus subtilis ) B-03 can be applied to the fermentation of non-sterilized straw, which is quite different from the related cellulose-degrading bacteria researches in the prior art which need to use sterilized straw for fermentation. Therefore, the B. subtilis (B-03) of the present application has a better application prospect in the industrial natural environment such as composting and biogas fermentation.

[0006] Therefore, the B. subtilis (B-03) of the present application, or the microbial agent thereof as an active ingredient, can be better applied in degrading cellulose and / or hemicellulose, and improve the degradation rate of cellulose and / or hemicellulose; and based on the B. subtilis (B-03), a method for degrading cellulose and / or hemicellulose is formed. Bacillus subtilis ) B-03 can be applied to the fermentation of non-sterilized straw, which is quite different from the related cellulose-degrading bacteria researches in the prior art which need to use sterilized straw for fermentation. Therefore, the B. subtilis (B-03) of the present application has a better application prospect in the industrial natural environment such as composting and biogas fermentation. Bacillus subtilis B-03 can be applied to the fermentation of non-sterilized straw, which is quite different from the related cellulose-degrading bacteria researches in the prior art which need to use sterilized straw for fermentation. Therefore, the B. subtilis (B-03) of the present application has a better application prospect in the industrial natural environment such as composting and biogas fermentation.

[0007] In the above method for degrading cellulose and / or hemicellulose, the B. subtilis is cultured in a fermentation medium; preferably, the seed liquid of the B. subtilis in the logarithmic growth phase is inoculated into the fermentation medium at an inoculation amount of 5%-10%. The fermentation medium comprises corn straw powder, peptone, sodium chloride, potassium dihydrogen phosphate and magnesium sulfate. Specifically, the content of the corn straw 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. If the concentration of the peptone is too high, the excessive growth of the bacteria will consume too much carbon source, reducing the resources needed for product synthesis; at the same time, it may make the pH alkaline, inhibiting the accumulation of acidic products; on the contrary, if the concentration of the peptone is too low, the amount of the bacteria is insufficient, and the product synthesis rate is low. Excessive sodium chloride will significantly increase the osmotic pressure, inhibiting the growth of non-salt-tolerant bacteria; and too low may lead to ion imbalance, affecting the membrane transport function. Potassium dihydrogen phosphate provides phosphorus source (PO4 3- ) and potassium ion (K + ), if the phosphorus is excessive (such as >2 g / L), it will change the metabolic flow in a specific fermentation; and if the phosphorus is insufficient, the growth of the bacteria is hindered, delaying the product synthesis period. Magnesium sulfate mainly provides magnesium (Mg 2+ ) and sulfur (SO4 2- ), Mg 2+ is an enzyme activator and stabilizes the ribosome structure, and the sulfur element is used for synthesizing sulfur-containing amino acids and coenzymes, and the concentration of 0.5 g / L has met the demand.

[0008] Therefore, more preferably, the content of the above corn stalk powder is 2 g / L, the content of the above protein 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.

[0009] The beneficial effects of the present application are that the present application proposes 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

[0010] Figure 1 The figure shows the microbial colony diagram of the 30 strains of B-01 to B-30 growing on the culture medium; b shows the single colony of B-03 after preliminary separation and purification; c shows the effect diagram of the Congo red staining test on the B-03 strain in the preliminary screening stage; d shows the effect diagram of the Congo red staining test on the B-17 strain in the preliminary screening stage; e shows the effect diagram of the Congo red staining test on the B-10 strain in the preliminary screening stage; Figure 2 The figure shows the growth curve diagram of the 8 strains of Bacillus subtilis; Figure 3 The figure shows the standard curve diagram of p-nitrophenol when measuring the exoglucosidase activity; Figure 4 The figure shows the standard curve diagram of glucose when measuring the endoglucosidase activity; Figure 5 The figure shows the standard curve diagram of p-nitrophenol when measuring the beta-glucosidase activity; Figure 6 The figure shows the degradation rate column diagram of cellulose and hemicellulose of the 8 strains of Bacillus subtilis. DETAILED DESCRIPTION

[0011] The concept and the technical effects produced by the present application will be described clearly and completely in combination with the embodiments and the drawings below, so as to fully understand the purposes, schemes and effects 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.

[0012] 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, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0013] 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 soil samples collected from the flowerbed layer in the campus of Inner Mongolia University of Technology in Xincheng District, Hohhot, Inner Mongolia Autonomous Region.

[0014] The compositions of the culture media / liquids used in the following examples are as follows, respectively: 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.

[0015] LB medium (g / L): protein peptone 10.0, yeast powder 5.0, sodium chloride 10.0, manganese sulfate 0.005, agar 20.0.

[0016] Seed culture solution (g / L): protein peptone 10.0, yeast powder 5.0, sodium chloride 10.0, manganese sulfate 0.005.

[0017] 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.

[0018] Degradation fermentation medium (g / L): corn straw powder 2.0, protein peptone 10.0, NaCl 5.0, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.5; the rest is deionized water.

[0019] The above-mentioned culture medium / fluid 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 straw powder is derived from corn straw waste in agricultural production, which is a fine powder material processed through drying, crushing, screening and other processes after corn harvesting. As the main stem part of corn plants, corn straw contains rich cellulose, hemicellulose and lignin and other components. In the following examples; the corn straw powder is a standardized product treated by 40 mesh screening, which is uniform in particle size and is beneficial to the attachment and degradation of microorganisms. As a renewable resource, corn straw powder has a wide application prospect in the fields of biomass energy, feed additives and organic fertilizers. The corn straw in the following examples comes 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 at the Green Biological Manufacturing Engineering Research Center of Inner Mongolia Autonomous Region.

[0020] The reagents involved in the following examples are prepared as follows: (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 then add the above-mentioned drugs 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.

[0021] (2) Phosphoric acid buffer solution with pH value of 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, mix well and adjust the pH value.

[0022] (3) Preparation of glucose standard solution: accurately weigh 1 g of glucose, and make up to 1000 mL with deionized water.

[0023] (4) Preparation of pNP standard solution: accurately weigh 0.1 g of pNP, and make up to 1000 mL with deionized water.

[0024] (5) Preparation of 1 mg / mL pNPC+1 mg / mL gluconolactone mixed solution: accurately weigh 0.1 g of pNPC and 0.1 g of gluconolactone, and make up to 100 mL with pH 7 phosphoric acid buffer solution.

[0025] (6) Preparation of 1 mg / mL pNPG solution: accurately weigh 0.1 g of pNPG, and make up to 100 mL with pH 7.0 phosphoric acid buffer solution.

[0026] (7) Preparation of 0.5% Congo red solution: weigh 0.005 g of Congo red, and dilute to 100 mL with deionized water.

[0027] (8) Preparation of 0.5% NaCl solution: weigh 0.005 g of sodium chloride, and dilute to 100 mL with deionized water.

[0028] (9) Preparation of 1% sodium carboxymethyl cellulose solution: accurately weigh 1 g of sodium carboxymethyl cellulose, and dilute to 100 mL with pH 7 phosphate buffer solution.

[0029] The enzyme activity (U) involved in the following examples is defined as follows: the amount of enzyme required to decompose 1 μmol of pNP per minute per 1 mL of crude enzyme solution at 50°C and pH 7.0 is one enzyme activity unit (U), and the calculation formula is as follows: Enzyme activity (U / mL) =

[0030] In the formula, C is the glucose concentration calculated according to the measured absorbance value and the glucose standard curve; 25 is the constant volume of the colorimetric tube (mL); N is the dilution multiple; T is the time for enzyme hydrolysis (min); and V is the amount of enzyme solution added (mL).

[0031] Example 1 I. Strain preliminary screening The B-01 to B-30 of Bacillus subtilis were respectively inoculated on the sodium carboxymethyl cellulose screening medium by point inoculation method, and incubated at 37°C for 48 h. After the colonies grew, 0.5% Congo red solution was added dropwise for dyeing for 15 min, and 5% sodium chloride solution was added for decolorization for 15 min to observe the transparent circle. Among them, Bacillus subtilis is a strain colony map; wherein a shown is the 30 strains of B-01 to B-30 growing on the medium in accordance with the microbial colony map of Bacillus subtilis; b shown is the single colony of B-03 after preliminary isolation and purification; c, d and e shown are the effect pictures of Congo red dyeing test on B-03, B-17 and B-10 strains respectively in the preliminary screening stage. The ratio of the diameter of the transparent circle to the diameter of the colony on each selection medium was counted, and the results are shown in Table 1. Figure 1 Table 1 Results of transparent circle determination of cellulose-degrading bacteria in preliminary screening

[0032] As shown in Table 1, the strains with larger HC values are Bacillus subtilis B-17, Bacillus subtilis B-03, and Bacillus subtilis B-10.

[0033] Bacillus subtilis 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; Preliminary predictions indicate that these 8 strains have strong cellulose degradation capabilities, and they will proceed to secondary screening.

[0034] II. Screening of high-cellulose-degrading strains: 1. Growth curve determination (1) Activation of strain 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 subtilis B-19 was activated separately, and the activated bacterial solution (OD) was then used to further activate the bacterial solution. 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.

[0035] (2) Results of growth curve measurement 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.

[0036] 2. Determination of cellulase activity 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 subtilis After activation, B-19 was inoculated into the seed culture medium at a rate of 5%. After culturing for 24 h, it was transferred to the liquid fermentation medium at a rate of 5%. The culture temperature was 30 ℃ and the shaking speed was 180 r / min. Samples were taken at 0 h, 8 h, 16 h, 24 h, 32 h, 40 h, and 48 h.

[0037] The reaction substrates and reaction conditions differ when determining the activity of several cellulases. The specific determination conditions are shown in Table 2.

[0038] Table 2. Conditions for Cellulase Activity Assay

[0039] (1) The determination of exoglucanase activity includes the following steps: Take 500 μL of fermentation broth as crude enzyme solution, add 50 μL of substrate (1 mg / mL pNPC + 1 mg / mL glucono-delta-lactone), mix well, react at 50 ℃ for 30 min, and add 150 μL of 10% Na2CO3 to terminate the reaction. Calculate the enzyme activity by measuring the amount of p-nitrophenol produced at a wavelength of 405 nm.

[0040] The standard curve for p-nitrophenol is shown below: Prepare a 0.1 mg / mL pNP standard solution. Add reagents according to Table 3 and mix well. Incubate in a boiling water bath for 10 min, and measure the OD value at 405 nm. Plot the pNP content (μg) on ​​the x-axis, corresponding to the OD value. 405 Plot the pNP standard curve with the ordinate as the vertical axis. For example... Figure 3 As shown, the standard curve equation for pNP is y = 0.21371x + 0.22371, where R0 2=0.99939.

[0041] Table 3 Reagent Addition Rules

[0042] (2) The determination of endoglucanase activity includes the following steps: Take 500 μL of fermentation broth as crude enzyme solution, add 1.5 mL of 1% CMC-Na solution, mix well, react at 50 ℃ for 30 min, add 3 mL of DNS to terminate the reaction, boil for 10 min, let stand and cool, add distilled water to make up to 25 mL, shake well, and measure the OD value at a wavelength of 540 nm.

[0043] The glucose standard curve is plotted as shown below: Pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mL of standard glucose solution into test tubes, respectively. Add distilled water to a final volume of 2 mL, then add 1.5 mL of DNS colorimetric reagent. Mix well, incubate in a boiling water bath for 10 min, cool to room temperature, and then add distilled water again to a final volume of 15 mL. Mix well and measure the OD value. 540nm Measure the absorbance at the specified location and plot a glucose standard curve. For example... Figure 4 As shown, the equation for the glucose standard curve is y = 0.3841x + 0.043, where R0 2 =0.9992.

[0044] (3) The determination of β-glucosidase activity includes the following steps: 100 μL of [unspecified ingredient] is added to a 300 μL reaction system. p NPG (1 mg / mL) and 200 μL of crude enzyme solution were reacted at 50 °C for 30 min. The reaction was terminated by adding 300 μL of 10% Na2CO3. The OD value was measured at a wavelength of 405 nm.

[0045] The standard curve for p-nitrophenol is shown below: Prepare a 0.1 mg / mL pNP standard solution. Add reagents according to Table 4 and mix well. Incubate in a boiling water bath for 10 min, and measure the OD value at 405 nm. Plot the pNP content (μg) on ​​the x-axis, corresponding to the OD value. 405 Plot the pNP standard curve with the ordinate as the vertical axis. For example... Figure 5 As shown, the standard curve equation for pNP is y = 0.1886x + 0.1939, where R0 2 =0.9995.

[0046] Table 4 Reagent Addition Rules

[0047] (4) The control group and the experimental group are added with the same amount of substrate and crude enzyme liquid (i.e. fermentation liquid), the difference is that the control group is added with DNS reagent (or Na2CO3 solution) to inactivate the crude enzyme immediately after the addition of the substrate and the crude enzyme liquid. The experimental group is added with DNS reagent (or Na2CO3 solution) to terminate the reaction after 30 min of reaction in a 50 ℃ water bath.

[0048] The results of the determination of the cellulase activity are shown in Table 5.

[0049] Table 5 Results of determination of cellulase activity

[0050] According to the results of the determination of the enzyme activity, the strains with higher cellulase activity are B-03, B-10 and B-19.

[0051] 3. Determination of degradation rate 100 mL of fermentation medium for degradation is placed in a 250 mL flask, and the seed liquid activated and inoculated into the seed culture liquid and cultured to the logarithmic growth phase is inoculated into the fermentation medium for degradation at a 5% inoculation amount, and cultured at 30 ℃ and 180 r / min, and sampled at 72 h. The fermentation liquid is centrifuged and dried to constant weight, and the mass change of the corn straw powder before and after fermentation is recorded.

[0052] 0.025 g of the sample dried to constant weight is accurately weighed, 250 μL of 74% H2SO4 solution is added to the sample, and the sample is shaken until it is fully mixed with the solution. The mixture is then incubated in a 30 ℃ water bath for 60 min. After the reaction is completed, the mixture is quickly cooled in an ice bath to terminate the reaction and 7 mL of deionized water is added.

[0053] Glucose loss rate sample preparation: 0.05 g of glucose is accurately weighed and added to 1 mL of 74% H2SO4 solution. The mixture is then incubated in a 30 ℃ water bath for 60 min, and the glucose is fully contacted with the sulfuric acid solution by constant stirring. After the reaction is completed, the mixture is quickly cooled in an ice bath to terminate the reaction, and 28 mL of deionized water is added for dilution.

[0054] The above sample solution is sterilized at 121 ℃ and 0.1 Mpa for 60 min. After the sterilization of the sample is completed, the monosaccharide (glucose) content in the sample is determined by high performance liquid chromatography, and the cellulose content is calculated according to the calculation method.

[0055] The conditions of high performance liquid chromatography are shown in Table 6.

[0056] Table 6 Conditions of high performance liquid chromatography

[0057] The calculation method of the cellulose content is as follows: Acid hydrolysis loss rate:

[0058] Mass of sugar in the sample:

[0059] The mass of remaining glucose:

[0060] Cellulose degradation rate in the sample:

[0061] The results are as follows Figure 6 As shown. By Figure 6 It is known that the Bacillus subtilis of this application Bacillus subtilis B-03 exhibits the strongest cellulose degradation ability, with a cellulose degradation rate of up to 75.78% and a hemicellulose degradation rate as high as 97.24%.

[0062] To demonstrate the strains obtained through screening in this invention Bacillus subtilis B-03's superior performance led to a parallel comparative experiment with seven existing cellulose-degrading strains preserved in the laboratory. All strains were tested for degradation ability under the same culture conditions.

[0063] The results (see Table 7) show that, under the condition that the methods for measuring the degradation rates of cellulose and hemicellulose are consistent, the strains Bacillus subtilis The cellulose degradation rate and hemicellulose degradation rate of B-03 were significantly higher than those of all control strains, reaching 75.58% and 97.24%, respectively. Bacillus subtilis B-03 improved the degradation efficiency of cellulose and hemicellulose by approximately 10.8% and 14.2%, respectively.

[0064] Table 7 Comparison of degradation rates between screened strains and existing strains

[0065] The secondary screening process breaks through the limitations of traditional single enzyme activity (such as CMCase) determination. It uses multi-enzyme synergistic determination (detecting endoglucanase, exoglucanase and β-glucosidase activities), and verifies the actual degradation rate under simulated industrial conditions using natural straw as a substrate. The screened strains can be better applied in industrial environments such as composting and biogas fermentation.

[0066] The above description is merely a preferred embodiment of this application. This application is not limited to the above-described embodiments. Any embodiment that achieves the technical effect of this application using the same means should fall within the protection scope of this application. Within the protection scope of this application, the technical solutions and / or implementation methods can have various modifications and variations.

Claims

1. A Bacillus subtilis, characterized in that, The taxonomic name of the said Bacillus subtilis is Bacillus subtilis Bacillus subtilis (Bacillus subtilis) Bacillus subtilis B-03, which was preserved in Guangdong Microbial Culture Collection Center on August 29, 2025, with the preservation number of GDMCC No: 66896.

2. An inoculant characterized in that, The active ingredient of the bacterial agent comprises the Bacillus subtilis of claim 1.

3. The Bacillus subtilis of claim 1 or the bacterial agent of claim 2 is used for improving the degradation rate of cellulose and / or hemicellulose.

4. A method for degrading cellulose and / or hemicellulose, characterized by, The method comprises using the Bacillus subtilis of claim 1 or the bacterial agent of claim 2.

5. The method of claim 4, wherein, The method comprises culturing the Bacillus subtilis in a fermentation medium.

6. The method of claim 5, wherein, The fermentation medium comprises corn stalk powder, peptone, sodium chloride, potassium dihydrogen phosphate and magnesium sulfate.

7. The method of claim 6, wherein, 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 of claim 7, wherein, 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 of any one of claims 5-8, wherein the culture conditions are as follows: the culture time is 72 h, the culture temperature is 30 ℃, the rotation speed is 180 r / min, and the initial pH value of the fermentation medium is 5.9-6.

2.

10. The method of claim 5, wherein, The seed liquid of the Bacillus subtilis cultured to the logarithmic growth phase is inoculated into the fermentation medium for culture at an inoculation amount of 5%-10%.

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