Bacillus subtilis capable of producing cellulase at high yield and application of bacillus subtilis

Abstract

The invention discloses bacillus subtilis capable of producing cellulase at high yield and application of the bacillus subtilis, and belongs to the technical field of biology, the bacillus subtilis is classified and named as bacillus subtilis KC7-2, the bacillus subtilis KC7-2 is preserved in China Center for Type Culture Collection on July 15, 2024, and the preservation number is CCTCC NO: M20241570. The bacillus subtilis provided by the invention shows excellent performance in the aspect of secretion of cellulase and hemicellulase, especially reaches an enzymatic activity peak in the early growth stage (24h) of a strain, and can maintain a certain level of enzymatic activity for a long time later. Due to the characteristics, the strain has important application value in the fields of forage feed improvement, biomass degradation, environmental protection and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Bacillus subtilis strain with high cellulase production and application thereof. Background Art

[0002] As an important feed source for herbivores, forage grass mainly consists of complex carbohydrates such as cellulose and hemicellulose. However, these components are complex in structure and difficult to degrade, which makes it impossible for animals to fully digest and absorb the nutrients in them. Especially for non-ruminants, their digestive systems lack sufficient endogenous cellulase and cannot effectively decompose cellulose and hemicellulose; and for ruminants, although rumen microorganisms can secrete a certain amount of cellulase, their production is limited and cannot fully meet the needs of efficient degradation. Therefore, the cellulose and hemicellulose resources in forage grass are often not reasonably utilized, resulting in great waste.

[0003] The commonly used method is to increase the degradation rate and nutritional value of forage and straw by adding exogenous cellulase. However, there are many limitations in the practical application of exogenous cellulase: first, its stability is poor and it is easy to be inactivated in a complex digestive environment; second, a single exogenous cellulase usually cannot cover the complete enzyme system and it is difficult to achieve comprehensive degradation of cellulose and hemicellulose; in addition, the high production cost limits its wide application in large-scale feed production.

[0004] In contrast, the use of cellulose-degrading bacteria has become a more economical and efficient solution. This type of microorganism can secrete a complete cellulase system, including multiple enzymes such as endoglucanase, exoglucanase and β-glucosidase, which act synergistically in the degradation process of cellulose. In addition, cellulose-degrading bacteria can not only pre-treat pasture and straw in vitro, but can also directly enter the animal body as a feed additive, further promoting the decomposition of cellulose and the absorption of nutrients.

[0005] Therefore, isolating and breeding a strain with high cellulase production is of great significance for improving the utilization rate of roughage for herbivorous animals. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a Bacillus subtilis with high cellulase production and applications thereof. The Bacillus subtilis disclosed in the present invention has relatively high cellulase and hemicellulase activities, and can degrade cellulose and hemicellulose in forage into soluble sugars such as glucose, thereby providing additional carbon sources and energy for livestock and poultry, promoting their growth and development, and making rational use of cellulose and hemicellulose resources in forage.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a Bacillus subtilis strain with high cellulase production. The Bacillus subtilis strain is classified and named as Bacillus subtilis KC7-2, which has been deposited in the China Center for Type Culture Collection on July 15, 2024, with a deposit number of CCTCC NO: M 20241570. The address of the deposit unit is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection.

[0009] The Bacillus subtilis described above is Gram-positive and has spores. After being cultured on an LB agar plate for 24 hours, it forms yellowish, round, rough and opaque colonies with irregular wrinkles on the edges.

[0010] The invention also discloses a microbial preparation containing the above-mentioned Bacillus subtilis.

[0011] The invention also discloses the use of the above-mentioned Bacillus subtilis or the above-mentioned microbial preparation in degrading cellulose or preparing cellulose degradation products.

[0012] The invention also discloses the use of the above-mentioned Bacillus subtilis or the above-mentioned microbial preparation in degrading hemicellulose or preparing hemicellulose degradation products.

[0013] The invention also discloses the application of the above-mentioned Bacillus subtilis or the above-mentioned microbial preparation in degrading cellulose or hemicellulose in forage.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The Bacillus subtilis provided by the present invention has high-efficiency cellulase and hemicellulase secretion ability, and provides good application potential for biomass degradation and feed improvement. Through the detection of the cellulase and hemicellulase activity secreted by the Bacillus subtilis disclosed in the present invention, it was found that the cellulase activity of the strain reached a maximum value (75U) when the strain grew for 24 hours, then slowly decreased, tended to be stable at 72 hours, and maintained at about 40U (such as Figure 5 The activity of hemicellulase of this strain also reached a peak value (300nmol / min / mL) at 24h, then slowly decreased, tended to be stable at 72h, and maintained at about 120nmol / min / mL (as shown in Figure 6 shown).

[0016] In summary, the Bacillus subtilis provided by the present invention exhibits excellent performance in the secretion of cellulase and hemicellulase, especially reaching a peak of enzyme activity in the early growth period (24h) of the strain, and then being able to maintain a certain level of enzyme activity for a long time. This characteristic makes it have important application value in the fields of forage feed improvement, biomass degradation and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A colony diagram of the Bacillus subtilis provided by the present invention on LB agar medium;

[0018] Figure 2 A Congo red staining test for cellulose degradation by Bacillus subtilis provided by the present invention;

[0019] Figure 3 Gram staining microscopic examination picture of Bacillus subtilis provided by the present invention;

[0020] Figure 4 To construct a phylogenetic tree of Bacillus subtilis provided by the present invention through BLAST comparison;

[0021] Figure 5 The cellulase activity of the Bacillus subtilis provided by the present invention at different time periods in the fermentation degradation test;

[0022] Figure 6 The hemicellulase activity of the Bacillus subtilis provided by the present invention at different time periods in a fermentation degradation test;

[0023] Figure 7 The cellulose content in the sample of the forage fermentation degradation test of Bacillus subtilis provided by the present invention for 96 hours;

[0024] Figure 8 The invention provides the hemicellulose content in the sample of the forage fermentation degradation test of Bacillus subtilis for 96 hours. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] The present invention provides a Bacillus subtilis strain with high cellulase production. The Bacillus subtilis strain is classified and named as Bacillus subtilis KC7-2, which has been deposited in the China Center for Type Culture Collection on July 15, 2024, with a deposit number of CCTCC NO: M 20241570. The address of the deposit unit is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection.

[0029] The above-mentioned Bacillus subtilis is Gram-positive and has spores. After being cultured on the LB agar plate for 24 hours, it forms yellowish, round, rough and opaque colonies with irregular wrinkles on the edges.

[0030] The invention also discloses a microbial preparation containing the Bacillus subtilis.

[0031] The invention also discloses the application of the Bacillus subtilis or the microbial preparation in degrading cellulose or preparing cellulose degradation products.

[0032] The invention also discloses the application of the Bacillus subtilis or the microbial preparation in degrading hemicellulose or preparing hemicellulose degradation products.

[0033] The invention also discloses the application of the Bacillus subtilis or the microbial preparation in degrading cellulose or hemicellulose in forage.

[0034] The solid and liquid culture medium formulas used in the following examples are as follows:

[0035] LB agar medium: tryptone 10g / L, yeast powder 5g / L, sodium chloride 5g / L, agar powder 15g / L.

[0036] Nutrient broth liquid culture medium: tryptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L.

[0037] Cellulase solid culture medium: peptone 10g / L, yeast powder 10g / L, sodium carboxymethyl cellulose 10g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 1g / L, agar powder 18g / L.

[0038] Forage fermentation medium: forage (alfalfa, oat grass 1:1) 10g / L, peptone 10g / L, beef meal 10g / L, yeast extract 5g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, sodium acetate 3g / L, magnesium sulfate 0.2g / L, manganese 0.04g / L, diammonium hydrogen citrate 2g / L.

[0039] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments:

[0040] Example 1

[0041] 1. Isolation, screening, identification and preservation of Bacillus subtilis

[0042] 1.1 Strain isolation and purification: The applicant collected rumen contents from the rumen of highland yaks in the Ali region for the isolation of Bacillus subtilis.

[0043] The specific operation method is: use tweezers to inoculate a small amount of sample into 3mL LB liquid culture medium in a sterile operating table, place it in a 37℃ constant temperature shaker for 12 hours, and set the shaker speed to 180r / min; after the culture is completed, use a sterile inoculation loop to inoculate the culture onto the LB solid culture medium using the three-zone streak method, and then place the culture medium in a 37℃ constant temperature box for further culturing for 12 hours. After the culture is completed, observe the morphological characteristics of the bacterial colonies, including color, size and surface characteristics, pick out irregular suspected colonies with rough and opaque surfaces, dirty white or slightly yellow, for Gram staining, and examine the staining results under a microscope. At the same time, use an inoculation loop again to inoculate the selected typical colonies onto the LB agar plate using the three-zone streak method for purification (such as Figure 1 As shown), purify for 3 to 4 generations until the colony size and morphology on the LB agar plate are uniform; then, select a single colony that is Gram-positive under microscopic examination from the purified colonies, inoculate it into a nutrient broth medium for expansion culture, and culture the culture medium in a 37°C constant temperature shaker for 12 hours, with the shaker speed still maintained at 180 r / min, to obtain an overnight cultured bacterial solution for use.

[0044] 1.2 Preliminary screening of cellulose-degrading bacteria:

[0045] The Congo red staining method was used to preliminarily screen the cellulose degradation ability of the purified strains. The specific operation is as follows: Use an inoculation loop to dip the mycelium of each purified strain separately, ensure that an equal amount is inoculated on the cellulase solid culture medium, and then put the inoculated culture medium into an incubator and culture it at 37°C for about 48 hours; after the culture is completed, add 1 mg / mL of Congo red dye to the culture medium for staining, the staining time is 10 to 15 minutes, then pour out the Congo red solution, and add 1 mol / mL of sodium chloride solution to wash away the unbound Congo red dye. The ability of the strain to produce cellulase is judged by observing the ratio of the hydrolysis circle diameter to the colony diameter. The larger the ratio, the stronger the cellulose degradation ability of the strain (such as Figure 2 According to the experimental results, the strain with the largest ratio of hydrolysis zone diameter to colony diameter was selected as the target strain, which has a strong cellulose degradation ability.

[0046] 2. Identification of strain taxonomy

[0047] The strains screened above were subjected to Gram staining and microscopic examination, and bluish purple, short rod-shaped, spore-like bacteria with spores in the middle of the bacteria were observed (such as Figure 3 As shown), a single purified colony was selected, genomic DNA was extracted using a DNA extraction kit, 16S rDNA universal primers were used to perform PCR amplification on the extracted genomic DNA, and the amplified product was then sequenced to obtain the 16S rDNA gene sequence of the strain. The measured 16S rDNA gene sequence was input into the NCBI database for BLAST comparison, and the parameter was set to Identify>95%. The top 30 16S rRNA sequences with the highest identity value in the comparison results were selected (if there were less than 30, all were selected), and these sequences were multiple aligned and sheared using MAFFT software to construct a phylogenetic tree (as shown in Figure 1). Figure 4 The isolated strain was found to have a very high similarity with Bacillus subtilis, and was determined to be Bacillus subtilis, classified and named Bacillus subtilis KC7-2, which has been deposited in China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20241570, and the address of the deposit unit is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Postal Code: 430072.

[0048] Example 2

[0049] This example is a detection of cellulase secretion by Bacillus subtilis provided by the present invention:

[0050] Preparation of fermentation broth samples: The Bacillus subtilis screened in the present invention is inoculated into the MRS liquid fermentation medium containing the forage sample. Three parallel samples are set up for the experiment, and the volume of the fermentation medium of each parallel sample is 1L. Subsequently, the fermentation system is placed in a constant temperature shaker at 37°C and 120r / min for 96h. During the fermentation process, samples are taken at 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h, respectively. After each sampling, the fermentation broth is fully stirred and the forage residue is filtered out with several layers of coarse gauze to obtain the filtered fermentation broth sample.

[0051] Preparation of cellulase extract: The fermentation broth sample was filtered through four layers of gauze to remove the residue, and the filtrate was treated according to the instructions of the cellulase detection kit (Shanghai Yuanye Biotechnology Co., Ltd.). The specific steps are as follows: the filtrate was centrifuged at 3000r / min for 10min, 10mL of the supernatant was added to a 50mL volumetric flask, and water was added to the scale to obtain the cellulase extract for the detection of the sample cellulase activity.

[0052] Cellulase activity detection: Yuanye cellulase detection kit (DNS microplate method) was used to detect the cellulase activity of the above samples. The specific steps are as follows: set up a test tube and a control tube respectively, and add 0.1 mL of cellulase extract to the test tube; add 0.1 mL of CES Assay buffer and 0.3 mL of CMC solution as substrates to the test tube and the control tube respectively; after mixing, place the test tube and the control tube in a 60°C water bath for incubation for 20 minutes to start the enzymatic reaction; after the reaction is completed, quickly add 0.3 mL of DNS reagent to each tube to terminate the reaction; add 0.1 mL of enzyme extract to the control tube as a negative control; after shaking, place the control tube and the test tube in a boiling water bath and heat for 5 minutes to allow the reducing sugar to react with the DNS reagent; after cooling, extract 280 μL from each tube in turn into a 96-well plate, adjust the "0" tube to zero, and use an enzyme reader to measure the absorbance value of each tube at a wavelength of 540 nm.

[0053] Preparation of standard curve: prepare a series of glucose standard solutions (such as 0μg / mL, 50μg / mL, 100μg / mL, 150μg / mL, 200μg / mL, 250μg / mL, 300μg / mL). According to the instructions of the kit, add an appropriate amount of DNS reagent to each tube of standard solution, mix well and heat in a boiling water bath for 5 minutes. After cooling, measure the absorbance at a wavelength of 540nm; draw a standard curve with glucose concentration (μg / mL) as the horizontal axis and absorbance value as the vertical axis.

[0054] According to the standard curve, the corresponding glucose concentrations C1 and C0 (unit: μg / mL) were calculated from the absorbance values ​​of the test tube and the control tube, respectively. According to the definition of the kit, the unit of cellulase activity is: at 60°C, 1 mL of enzyme extract catalyzes the hydrolysis of sodium carboxymethyl cellulose to produce 1 μg of glucose in 1 min, which is defined as 1 enzyme activity unit (U); calculate the cellulase activity in the sample.

[0055] The calculation formula is as follows:

[0056] U=k×(C1-C0) / t,

[0057] In the formula: U is the enzyme activity of the sample, in μg / (ml·min) or ug / (g·min); k is the sample dilution factor; C1 is the glucose concentration in the sample measurement tube, in μg / ml; C0 is the glucose concentration in the sample control tube, in μg / ml; t is the reaction time between the enzyme and the substrate, in min.

[0058] like Figure 5The figure shows the cellulase activity curve of Bacillus subtilis in different time periods in the fermentation degradation test. It can be seen from the figure that the cellulase activity of the strain reaches a maximum value of 75U when the strain grows for 24 hours, then slowly decreases, tends to be stable at 72 hours, and is maintained at around 40U; this indicates that the strain can quickly secrete cellulase in the early stage and efficiently degrade cellulose into glucose, and after 72 hours, the enzyme activity tends to be stable, indicating that the strain can maintain a certain enzyme activity for a long time, continue to play a role, and lay the foundation for subsequent biomass utilization.

[0059] Example 3

[0060] This example is the detection of hemicellulase activity secreted by Bacillus subtilis provided by the present invention:

[0061] Preparation of fermentation broth samples: The Bacillus subtilis screened in the present invention is inoculated into the MRS liquid fermentation medium containing the forage sample. Three parallel samples are set up for the experiment, and the volume of the fermentation medium of each parallel sample is 1L. Subsequently, the fermentation system is placed in a constant temperature shaker at 37°C and 120r / min for 96h. During the fermentation process, samples are taken at 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h, respectively. After each sampling, the fermentation broth is fully stirred and the forage residue is filtered out with several layers of coarse gauze to obtain the filtered fermentation broth sample.

[0062] Preparation of hemicellulase extract: The fermentation broth sample was centrifuged at 8000 g and 4° C. for 15 min, and the supernatant was collected after centrifugation to obtain the hemicellulase extract as the sample to be tested.

[0063] Hemicellulase activity detection: Use a hemicellulase activity assay kit (Shanghai Baishengyue Biotechnology Co., Ltd.) to detect the hemicellulase activity of the above-mentioned test samples. The specific steps are as follows: set up an assay tube and a control tube respectively, take 0.2 mL of the test sample and add it to the assay tube and the control tube, then add 0.3 mL of buffer as a substrate, add 0.1 mL of reagent 1 (containing xylan) in the kit to the assay tube, mix well, and incubate the reaction tube in a 50°C water bath for 15 minutes; add 0.1 mL of reagent 1 to the control tube as a negative control, and finally add 0.3 mL of DNS reagent to the reaction tube to terminate the reaction, and place the reaction tube in a boiling water bath and heat for 5 minutes to allow the reducing sugar to react with the DNS reagent; after cooling to room temperature, use a spectrophotometer to measure the absorbance (A) at a wavelength of 540 nm, and calculate ΔA=Aassay-Acontrol, and set a control tube for each assay tube.

[0064] The standard curve equation given in the manual is: y = 2.5554x-0.002, where: R 2 =0.9983.

[0065] Hemicellulase activity calculation formula: According to the definition of the kit, the hemicellulase activity unit is: under the conditions of 50°C and pH 4.8, the amount of enzyme required to decompose hemicellulose and produce 1 nmol reducing sugar per minute per milliliter of liquid sample is one hemicellulase activity unit.

[0066] The enzyme activity calculation formula is: hemicellulase activity (nmol / min / mL) = 435 × (ΔA + 0.002),

[0067] Here, ΔA is the absorbance of the sample minus the absorbance of the blank control.

[0068] like Figure 6 The figure shows the hemicellulase activity curve of Bacillus subtilis in different time periods in the fermentation degradation test. It can be seen from the figure that the hemicellulase activity of the strain also reaches a peak of 300nmol / min / mL at 24h, then slowly decreases, tends to be stable at 72h, and maintains at around 120nmol / min / mL; this indicates that Bacillus subtilis can quickly synthesize and secrete hemicellulase in the early growth stage, and efficiently decompose the hemicellulose components in the substrate. After 72h, the hemicellulase activity tends to be stable, which means that Bacillus subtilis can maintain a certain enzyme activity for a long time and continue to play a degradation role.

[0069] Example 4

[0070] This example is the application of the Bacillus subtilis of the present invention in the degradation of cellulose and hemicellulose of forage grass. The degradation capacity of cellulose and hemicellulose in the samples prepared below was tested and analyzed by using the differential method.

[0071] Preparation of fermentation broth samples: The strain screened above in the present invention was inoculated into MRS liquid fermentation medium added with forage samples, and 3 parallel samples were set up, and the volume of the medium for each parallel fermentation sample was 1L; the fermentation system was placed in a constant temperature shaker at 37°C and 120r / min for 96h, and samples were taken at the beginning (0h) and end (96h) of fermentation. Each time sampling was performed, the fermentation broth was fully stirred and evenly mixed, and the samples were collected for subsequent detection.

[0072] The cellulose content and hemicellulose content in the samples at 0 h and 96 h were detected using a cellulose content detection kit (Beijing Solebow Technology Co., Ltd.) and a hemicellulose content detection kit (Beijing Solebow Technology Co., Ltd.) according to the instructions.

[0073] (1) Determination of cellulose content:

[0074] Fermentation broth treatment: Filter the fermentation broth through filter paper or gauze to separate the solid fermentation substrate (residue) and the liquid part (filtrate), retain the solid fermentation substrate and discard the filtrate; place the separated residual fermentation substrate in a drying equipment at 60°C, dry to constant weight, take out the sample, cool it to room temperature, and properly store it as a sample to be tested for subsequent analysis.

[0075] The samples to be tested were processed as follows according to the preparation method described in the cellulose content detection kit (Beijing Solebow Technology Co., Ltd.).

[0076] Preparation of crude cell wall: weigh about 0.3g (W1) sample, add 1mL of extract solution 1 to the sample, quickly homogenize at room temperature, heat the sample in a 90℃ water bath for 20min (wrap the sealing film to prevent the lid from bursting), cool to room temperature, centrifuge at 6000g, 25℃ for 10min, and discard the supernatant. Wash the precipitate twice with 1.5mL of extract solution 1 and acetone respectively, in the order of extract solution 1-acetone-extract solution 1-acetone, then shake for 2min, centrifuge at 6000g, 25℃ for 10min, and discard the supernatant. The precipitate is the crude cell wall.

[0077] Preparation of cell wall material (CWM): add 1 mL of extract 2 to the crude cell wall, soak for 15 h, centrifuge at 6000 g, 25 °C for 10 min, and discard the supernatant; wash the precipitate twice with distilled water (vortex for about 2 minutes each time, then centrifuge at 6000 g, 25 °C for 10 min, and discard the supernatant), dry the precipitate at 60 °C to obtain cell wall material (CWM), weigh and record the mass as W2.

[0078] Extraction and determination of cellulose: Weigh 5 mg (W3) of dried CWM, add 0.5 mL of distilled water to the CWM and homogenize thoroughly; transfer the homogenate to an EP tube and dilute to 0.5 mL with distilled water; place the homogenate after distillation in an ice-water mixture, slowly add 0.75 mL of concentrated sulfuric acid, and slowly mix, let stand in an ice-water bath for 30 min, centrifuge at 8000 g and 4°C for 10 min, take the supernatant, dilute the supernatant 20 times with distilled water to obtain the test solution.

[0079] According to the instructions of the kit, a series of glucose standard solutions (such as 0.09 mg / mL, 0.08 mg / mL, 0.07 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, 0.00625 mg / mL) were prepared as standard tubes; assay tubes, standard tubes and blank tubes were set up respectively, 0.3 mL of the sample to be tested was added to the assay tube, 0.3 mL of the standard solution was added to the standard tube, and 0.3 mL of distilled water was added to the blank tube; 0.07 mL of working solution and 0.63 mL of concentrated sulfuric acid were added to all reaction tubes, mixed well and placed in a 95°C water bath for color development reaction, taken out and cooled to room temperature, and the absorbance (A) was measured at a wavelength of 620 nm using a spectrophotometer, and ΔA=Assay-Ablank, ΔAstandard=Astandard-Ablank were calculated; a control tube was set up for each assay tube.

[0080] Calculation of cellulose content: Draw a standard curve with glucose concentration (μg / mL) as the horizontal axis and absorbance value as the vertical axis. Measure the absorbance value of the diluted test solution and substitute it into the standard curve to calculate the corresponding glucose concentration x (mg / mL). Then, according to the cellulose content calculation formula as follows, calculate the cellulose content of the forage sample added with Bacillus subtilis of the present invention at 0h and 96h (such as Figure 7 ):

[0081] (1) Calculated by sample quality:

[0082] Cellulose (mg / g mass) = x × V 提 ×20×(W2-W3)÷W1÷1.11=22.52×x×W2÷W3÷W1.

[0083] like Figure 7 The figure shows the cellulose content in the samples of the forage fermentation degradation test of Bacillus subtilis of the present invention at 0h and 96h. It can be seen from the figure that at 0h (when the fermentation starts), the cellulose content in the forage sample is 166.41mg / g dry weight. After 96h of fermentation, the cellulose content in the forage sample drops to 80.27mg / g dry weight, which indicates that Bacillus subtilis significantly reduces the cellulose content in the forage in 96h, and the degradation rate is 51.76%; this shows that the Bacillus subtilis of the present invention has a high-efficiency cellulose degradation ability, and can significantly reduce the cellulose content in the forage in a relatively short time, thereby improving the nutritional value and digestibility of the forage.

[0084] (2) Determination of hemicellulose content:

[0085] The sample to be tested was processed according to the preparation method described in the hemicellulose content detection kit (Beijing Solebow Technology Co., Ltd.). 0.05 g of the sample to be tested and 1 mL of 80% ethanol were added to the test tube, mixed, 90°C water bath for 10 minutes, centrifuged at 8000 g for 10 minutes, and the precipitate was retained. 1 mL of distilled water was added, mixed, centrifuged at 8000 g for 10 minutes, and the supernatant was discarded. This step was repeated three times, and the precipitate was taken and dried to constant weight, which was the sample to be tested in the test tube.

[0086] Specific steps: Dry the sample to constant weight, grind it thoroughly, and pass it through a 30-50 mesh sieve.

[0087] According to the instructions, prepare a series of D-xylose standard solutions (such as 2.5 mg / mL, 2 mg / mL, 1 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL) as standard tubes; set up assay tubes, standard tubes and blank tubes respectively, take 0.5 mL of 80% ethanol and add it to the assay tubes and blank tubes, water bath at 90°C for 60 minutes, cool naturally to room temperature, add 0.5 mL of the extract solution 2 in the kit and mix well, centrifuge at 8000g for 10 minutes, and take the supernatant for testing. Add 0.125 mL of the corresponding supernatant to the assay tube and blank tube, and add 0.125 mL of the standard solution to the standard tube. Add 0.125 mL of reagent 2 and 0.75 mL of distilled water to all reaction tubes, vortex mix well, water bath at 90°C for 5 minutes, and cool naturally. The absorbance (A) was measured at a wavelength of 540 nm using a spectrophotometer, and ΔA=AAssay-AAblank, ΔAstandard=AAsstandard-AAblank were calculated. A control tube was set for each assay tube.

[0088] Calculation of hemicellulose content: Draw a standard curve with D-xylose concentration (y, mg / mL) as the horizontal axis and absorbance value as the vertical axis. Determine the absorbance value of the diluted test solution and substitute it into the standard curve to calculate the corresponding D-xylose concentration x (mg / mL). Then, according to the cellulose content calculation formula as follows, calculate the hemicellulose content of the forage sample added with Bacillus subtilis of the present invention at 0h and 96h:

[0089] Hemicellulose (mg / g dry weight) = y × V 样总 ÷W×F=y÷W×F,

[0090] V total sample: volume of extract added, 1 mL; W: sample mass, g; F: dilution factor.

[0091] like Figure 8The figure shows the hemicellulose content in the samples of the forage fermentation degradation test of Bacillus subtilis of the present invention at 0h and 96h. It can be seen from the figure that at 0h (when the fermentation starts), the hemicellulose content in the forage sample is 382.85mg / g dry weight. After 96h of fermentation, the hemicellulose content in the forage sample drops to 211.07mg / g dry weight, and the degradation rate is 44.87%. This shows that the Bacillus subtilis of the present invention has a high-efficiency hemicellulose degradation ability, can significantly reduce the hemicellulose content in forage in a relatively short time, and is helpful to improve the resource utilization efficiency in forage.

[0092] The above is a detailed introduction to a Bacillus subtilis strain with high cellulase production disclosed in the present invention and its application. Specific examples are used herein to illustrate the principles and implementation methods of the present invention, and the description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A Bacillus subtilis strain with high cellulase production, characterized in that: The Bacillus subtilis is classified and named Bacillus subtilis KC7-2, which was deposited in the China Center for Type Culture Collection on July 15, 2024, with a deposit number of CCTCC NO: M 20241570. The address of the depository unit is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection.

2. A microbial preparation containing the Bacillus subtilis according to claim 1.

3. Use of the Bacillus subtilis according to claim 1 or the microbial preparation according to claim 2 in degrading cellulose or preparing cellulose degradation products.

4. Use of the Bacillus subtilis according to claim 1 or the microbial preparation according to claim 2 in degrading hemicellulose or preparing hemicellulose degradation products.

5. Use of the Bacillus subtilis according to claim 1 or the microbial preparation according to claim 2 in degrading cellulose or hemicellulose in forage.

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