Bacillus subtilis and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a Tibetan pig source bacillus subtilis, the classification name is bacillus subtilis Yirun2024, the Latin name is bacillus subtilis Yirun2024, the preservation number is CCTCC NO: M 20241752, the preservation date is August 7, 2024, the preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China. When the bacillus subtilis is fermented for 12 hours in a shake flask, the endo-cellulase activity, the exo-cellulase activity and the beta-glucosidase activity of the bacillus subtilis are respectively up to 2902.72 U / mL, 11026.33 U / mL and 2983.04 U / mL, which are far better than those of similar engineering bacteria. Meanwhile, the invention further provides application of the bacillus subtilis, the bacillus subtilis has important significance in the field of promotion of application of fiber resources, and meanwhile, the bacillus subtilis can keep a high survival rate in a high-temperature environment.

Description

Technical Field

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

[0002] Cellulose is a macromolecular polysaccharide composed of glucose. It is the main component of plant cell walls and the most widely distributed and highest-yielding renewable resource in nature. my country is a large agricultural country, and a large amount of high-fiber agricultural waste is produced every year, such as straw, rice stalks, and sugarcane bagasse. It is estimated that 741 million to 1.111 billion tons of rice straw are produced every year in the world. In 2022, the total amount of straw resources in my country has reached 856 million tons. 8 Tons. However, due to the low economic value of direct utilization and huge output, most high-fiber agricultural wastes are discarded or directly incinerated, causing environmental pollution and waste of resources.

[0003] Bioconversion is an important measure to improve the economic value of agricultural waste such as straw. The bioconversion of lignocellulose must undergo the degradation of cellulase and is widely used in the production of food, papermaking and feed. Cellulase is a complex enzyme system composed of endocellulase (endoglucanase), exocellulase (exoglucanase) and β-glucosidase. The complete degradation of cellulose must be completed through the synergistic action of the three cellulases. First, oligosaccharides are produced by random site hydrolysis of endocellulase, followed by cellobiose produced by exocellulase, and finally cellobiose is hydrolyzed into glucose by β-glucosidase. Therefore, the complete hydrolysis of cellulose must undergo the synergistic action of these three cellulases to hydrolyze the cellulose molecules that are not easily degraded into glucose.

[0004] Cellulase exists in animals, plants and microorganisms. Compared with animal cellulase, microbial cellulase has the advantages of easy operation, fast growth, simple production and low cost, and is currently the main source of industrial cellulase. However, compared with other microbial enzymes, the cost and yield of cellulase are still relatively low, which limits the industrial application of cellulase. Therefore, it is urgent to explore high-yield cellulase strains with rich enzyme systems and high activity to promote the development of industrial cellulase.

[0005] At present, the production and research of cellulase are mainly focused on molds, because molds have more types of enzymes and higher yields. However, molds have a longer enzyme production cycle, while bacteria have the advantages of faster growth and shorter enzyme production cycles. At the same time, bacteria are more adaptable to the environment, and there are more types of bacteria, wider sources, and they can produce more specific complex enzymes. Therefore, it is of great significance to develop bacteria with high cellulase production.

[0006] Chinese patent application CN202110425297.6 discloses a cellulase-producing Bacillus subtilis FMME ZK002, which was deposited in the China Center for Type Culture Collection on January 29, 2021, with a deposit number of CCTCC NO: M 2021168. The strain of this scheme can produce cellulase, and its cellulase activity is 118.67U / mL.

[0007] Further observation of this scheme shows that the cellulase tested in this scheme is carboxymethyl cellulase, i.e., endo-cellulase, but it does not show an effect on the activity of exo-cellulase and β-glucosidase, making it difficult to completely hydrolyze cellulose into glucose.

[0008] Chinese patent application CN202311261190.8 discloses a strain of Bacillus subtilis co-expressing multiple cellulase genes and its construction method and application. The deposited name of the genetically engineered recombinant strain of Bacillus subtilis is Bacillus subtilis C6-AEA3, which was deposited in the China Center for Type Culture Collection on August 31, 2023, with a deposit number of CCTCC NO: M20231579. The scheme knocks endoglucanase (endocellulase) genes, exoglucanase (exocellulase) genes and β-glucanase genes into Bacillus subtilis through the CRISPR / Cas9 system. The enzyme activities of endoglucanase, exoglucanase and β-glucosidase of the Bacillus subtilis engineered bacteria are 26.3U / mL, 9.8U / mL and 3.9U / mL, respectively, which are 3.1 times, 6.6 times and 3.0 times higher than the starting strain, respectively.

[0009] Further observation of the scheme shows that the Bacillus subtilis C6-AEA3 is a genetically engineered recombinant bacterium, the purpose of which is to increase the enzymatic activity of various cellulases, but the enzymatic activity of the strain still does not meet the expected requirements.

[0010] The technical problems existing in the prior art are that the cellulase activity of the naturally screened Bacillus subtilis strains is low and the enzyme system is incomplete. Improving the cellulase activity mainly relies on genetic modification, but it is still difficult to achieve industrial production expectations.

[0011] However, strains isolated from nature are superior to genetically engineered strains in terms of stability, safety, environmental adaptability, production costs and social acceptance.

[0012] At the same time, after analyzing the performance of Bacillus subtilis in the prior art, it was found that most of the Bacillus subtilis with environmental tolerance recorded in the prior art have a low survival rate under high temperature conditions (such as 80°C). For example, CN111808765A, a strain of Bacillus subtilis that efficiently degrades vomitoxin and its application, has a survival rate of about 90% at 50°C, but at 60°C and 80°C, the survival rate drops sharply to less than 20%. The high temperature resistance of Bacillus subtilis is an important performance indicator of fermentation. During the fermentation process, once the process control is not good enough, it is easy to exceed the temperature of the fermentation pile. If the fermentation bacteria can effectively adapt to the high temperature environment, the difficulty of fermentation process control will be reduced.

[0013] The technical problem to be solved in this case is: how to discover a naturally occurring strain of Bacillus subtilis that has a variety of cellulases with high activity and a certain environmental tolerance. Summary of the invention

[0014] The object of the present invention is to provide a Bacillus subtilis Yirun2024, which has a high cellulase production capacity. After fermentation in a shake flask for 12 hours, the activities of endocellulase, exocellulase and beta-glucosidase are as high as 2902.72 U / mL, 11026.33 U / mL and 2983.04 U / mL, respectively, which are far higher than similar engineered bacteria. At the same time, the bacteria can maintain a high survival rate in a high temperature environment.

[0015] Meanwhile, the invention also provides the application of the Bacillus subtilis.

[0016] To achieve the above object, the present invention provides the following technical solutions:

[0017] A strain of Bacillus subtilis Yirun2024, whose Latin name is Bacillus subtilis Yirun2024, whose preservation number is CCTCC NO:M 20241752, whose preservation date is August 7, 2024, whose preservation unit is China Center for Type Culture Collection, and whose preservation address is Wuhan University, Wuhan, China.

[0018] Preferably, the Bacillus subtilis is extracted from healthy Tibetan pig feces.

[0019] In addition, the invention also discloses the use of the Bacillus subtilis as a cellulase producing bacterium.

[0020] In addition, the invention also discloses the use of the Bacillus subtilis to ferment plant-derived raw materials containing plant fibers.

[0021] Preferably, the raw material is one or more combinations of grass, palm meal, soybean meal, corn meal, peanut meal, fruit peel, straw, and tree branches and leaves.

[0022] In addition, the present invention also discloses the use of the above-mentioned Bacillus subtilis for preparing feed additives.

[0023] Meanwhile, the present invention also discloses a feed, which is obtained by fermenting the plant-derived raw material containing plant fiber with the Bacillus subtilis as mentioned above.

[0024] In the above-mentioned feed, the raw materials are one or more combinations of grass, palm meal, soybean meal, corn meal, peanut meal, fruit peels, straw, and tree branches and leaves.

[0025] And, a fermentation bacterial agent containing the Bacillus subtilis as described above.

[0026] In some application scenarios of the present invention, the fermentation agent may also contain other fermentation bacteria, such as Bacillus coagulans, Lactobacillus plantarum, Saccharomyces cerevisiae, etc., in order to obtain better effects.

[0027] Finally, the present invention also discloses a feed additive containing the Bacillus subtilis as described above.

[0028] Preferably, it is used to be added to the feed of growing pigs and sows. During the feeding process of growing pigs and sows, the amount of Bacillus subtilis added to the feed is 10^8~10^9cfu / kg feed.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The Bacillus subtilis Yirun2024 provided by the present invention is separated from healthy Tibetan pigs, grows rapidly, and produces a high amount of cellulase. After fermentation in a shake flask for 12 hours, the activities of endocellulase, exocellulase and β-glucosidase are as high as 2902.72 U / mL, 11026.33 U / mL and 2983.04 U / mL, respectively, which are far superior to similar engineered bacteria, and can significantly improve the nutritional content and feeding value of high-fiber plant feed raw materials such as palm meal.

[0031] Another additional advantage of the present invention is that the strains screened by the present invention can maintain a survival rate of not less than 80% under high temperature conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the glucose standard curve;

[0033] Figure 2 is the phylogenetic tree of strain 3-1;

[0034] Figure 3 The colony morphology of Bacillus subtilis Yirun2024 in NA medium;

[0035] Figure 4 This is the Gram staining result of Bacillus subtilis Yirun2024;

[0036] Figure 5 is the growth curve of Bacillus subtilis Yirun2024;

[0037] Figure 6 is the survival rate of Bacillus subtilis Yirun2024 after treatment at different temperatures;

[0038] Figure 7 This is a photo of the initial screening culture results of high-producing cellulase strains. DETAILED DESCRIPTION

[0039] 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.

[0040] Product information:

[0041] Congo red, sodium carboxymethyl cellulose, salicin, microcrystalline cellulose, acetic acid and sodium acetate: Beijing Solaibao Technology Co., Ltd.

[0042] DNS reagent, bacterial genomic DNA extraction kit: Beijing Solebow Technology Co., Ltd.

[0043] Sodium chloride, concentrated hydrochloric acid: Sinopharm Chemical Reagent Co., Ltd.

[0044] Gram stain: Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.

[0045] Example 1: Isolation, screening and identification of high-producing cellulase bacteria

[0046] 1. Culture Media and Reagents

[0047] Nutrient broth (NB): peptone 10 g / L, beef extract powder 3.0 g / L, NaCl 5.0 g / L, pH 7.2±0.2, autoclave at 121°C for 15 min.

[0048] Nutrient agar medium (NA): peptone 10 g / L, beef extract powder 3.0 g / L, NaCI 5.0 g / L, agar 15.0 g / L, pH 7.3±0.1, high pressure sterilization at 121℃ for 15 min.

[0049] Cellulase enrichment medium: CMC-Na 10g / L, MgSO4 0.5g / L, NaNO3 3.0g / L, KCI 0.5g / L, K2HPO4 0.4g / L, FeSO4 0.01g / L, high pressure sterilization at 121℃ for 15min.

[0050] Isolation medium: CMC-Na 10 g / L, MgSO4 0.5 g / L, NaNO3 3.0 g / L, KCI 0.5 g / L, K2HPO4 0.4 g / L, FeSO4 0.01 g / L, agar 15 g / L, high pressure sterilization at 121°C for 15 min.

[0051] 2. Strain Isolation

[0052] Mix the fecal samples of 6 Tibetan pigs raised in the natural environment in equal mass, then weigh 5.0g of the mixed fecal sample and place it in a 250mL conical flask containing 100.0mL of cellulase enrichment medium, add glass beads and vortex thoroughly, and culture at 37℃, 200r / min, constant temperature for 24h. Take 1.0mL of bacterial solution and inoculate it into 100mL of cellulase enrichment medium, continue to culture at 37℃, 200r / min, constant temperature for 24h, repeat twice. Take 1.0mL of bacterial solution and add it to 9.0mL of normal saline, the dilution concentration is 10 -1 , and then diluted to 10 at a ratio of 1:10 -9 , take 100 μL of bacterial suspension from each dilution gradient and apply it to the separation medium, culture it at 37℃ for 24-72h until most colonies grow, pick colonies and streak them in NA medium until a single colony is purified. Then pick the purified strain and inoculate it in NB medium for amplification for 24h, and store it at 4℃ for later use.

[0053] 3. Screening of high-yield cellulase strains

[0054] 3.1. Initial screening

[0055] The activated strain was inoculated in the separation medium by three-point inoculation method and inverted at 37℃ for 48h. After the strain culture was completed, 10mL of 0.5% Congo red solution was slowly poured from the edge of the culture dish, and gently shaken to cover the entire plate with the Congo red solution. After standing for 10min, the liquid in the culture dish was poured out, and the plate was rinsed once with 1mol / L NaCl solution, and then 10mL of 1mol / L NaCl solution was added to the culture dish. After standing for "decolorization" for 15min, the liquid in the culture dish was poured out, and the transparent circle diameter (D) and colony diameter (d) on the culture medium plate were observed and measured, and the ratio of the transparent circle diameter (D) to the colony diameter (d) was calculated. The strains with a ratio of >4 were rescreened for cellulase determination.

[0056] Five strains with obvious hydrolysis circles were isolated from Tibetan pig feces, among which the diameter ratio of 3-1 and 1-1 was greater than 4 (Table 1, the hydrolysis circle of strain 3-1 was visible Figure 7 ) and cellulase activity was determined.

[0057] Table 1 Hydrolysis zone diameters of various strains

[0058] Strain number Hydrolysis circle diameter D(mm) Strain diameter d(mm) Diameter ratio D / d 3-1 29.07±1.35 4.95±0.34 5.89±0.26 1-1 26.98±0.88 5.44±0.44 4.97±0.30 XF-B 14.22±1.72 4.57±0.74 3.14±0.13 XF6-2 13.46±0.83 5.42±0.10 2.48±0.11 XF1-1-A 14.68±0.06 5.17±0.10 2.84±0.04

[0059] 3.2. Cellulase activity assay

[0060] 3.2.1. Preparation of glucose standard curve

[0061] Preparation of glucose standard stock solution: Weigh 1.0g analytical pure anhydrous glucose, dissolve it in buffer, and make up to 1000mL with a concentration of 1.0mg / mL. Add reagents according to the table below.

[0062] Table 2 Glucose standard curve solution preparation table

[0063] Serial number 0 1 2 3 4 5 6 7 Concentration (mg / mL) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Glucose standard stock solution (mL) 0 1 2 3 4 5 6 7 Buffer (mL) 10 9 8 7 6 5 4 3

[0064] After mixing the solutions in each tube, take 1.5 mL of glucose standard solution of different concentrations, add 2.0 mL of DNS solution to each test tube, boil for 10 min, cool and add distilled water to make up to 10 mL, measure the absorbance at 540 nm, and draw a standard curve using EXCEL software, with the actual mass of glucose as the horizontal axis and the absorbance value as the vertical axis.

[0065] Glucose standard curve Figure 1 As shown, with glucose content (mg) as the horizontal coordinate and OD540nm as the vertical coordinate, the equation of the glucose standard curve drawn is: y=0.1494x+0.0260, R2=0.9974, which meets the requirements of the standard curve.

[0066] 3.2.2. Cellulase activity assay

[0067] The strain obtained in the initial screening was activated and inoculated into a 250mL conical flask containing 100mL of cellulase enrichment medium at an inoculation rate of 3% (v / v). Three parallel tests were performed. After constant temperature cultivation at 37°C and 200r / min for 12h, the mixture was centrifuged at 10000rpm and 4°C for 10min. The supernatant was the crude enzyme solution.

[0068] Determination of endocellulase activity (carboxymethyl cellulase): After the crude enzyme solution is appropriately diluted, take 0.5 mL, add 1 mL of 1% sodium carboxymethyl cellulose buffer solution (pH 4.8, 0.2 mol / L acetic acid-sodium acetate buffer), react at 50°C for 30 min, quickly add 2 mL of DNS reagent, and then boil in water for 10 min. After cooling, add 6.5 mL of distilled water to make the final volume 10 mL, measure the absorbance value at 540 nm, and calculate the reducing sugar content produced by the reaction through the glucose standard curve.

[0069] Determination of exocellulase activity (microcrystalline cellulase): After the crude enzyme solution is appropriately diluted, 0.5 mL is taken, and 1 mL of a buffer solution (pH 4.8, 0.2 mol / L acetic acid-sodium acetate buffer) containing 1% microcrystalline cellulose is added. After reacting at 50°C for 30 minutes, 2 mL of DNS reagent is added to terminate the reaction, and the reaction is placed in a boiling water bath for 10 minutes. After cooling, 6.5 mL of distilled water is added to make the final volume 10 mL, and the absorbance value at 540 nm is measured.

[0070] Determination of β-glucosidase activity: 1 mL of 0.5% salicin solution (pH 4.8, 0.2 mol / L acetic acid-sodium acetate buffer) and 0.5 mL of appropriately diluted enzyme solution were added to a centrifuge tube in sequence, mixed evenly, reacted at 50°C for 30 min, then 2 mL of DNS reagent was added to terminate the reaction, and the reaction was in a boiling water bath for 10 min. After cooling, 6.5 mL of distilled water was added to make the final volume 10 mL, and the absorbance value at 540 nm was measured.

[0071] The above enzyme activity unit (U / ml) is defined as: the amount of enzyme required to degrade the substrate to produce 1ug of glucose in 1min under the above reaction conditions with 1mL of enzyme solution. The calculation formula of enzyme activity is as follows:

[0072]

[0073] Where: A is the content of glucose (mg)

[0074] N is the dilution factor

[0075] T is the time taken for enzymatic hydrolysis (min)

[0076] V is the amount of enzyme solution added (mL)

[0077] Cellulase activity is shown in Table 3. The activities of endocellulase, exocellulase and β-glucosidase of strain 3-1 were as high as 2902.72 U / mL, 11026.33 U / mL and 2983.04 U / mL, respectively. The enzyme activities were all higher than those of 1-1. Therefore, 3-1 was identified as a high-yield cellulase strain for subsequent experiments.

[0078] Table 3 Cellulase activity of strains

[0079]

[0080] 4. Identification of strains

[0081] The genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit produced by Beijing Solebow Technology Co., Ltd. The extracted DNA sample was appropriately diluted and used as a PCR template, and the universal primers of bacterial 16SrDNA were amplified using Qingke 1×TSE101 Gold Mix.

[0082] The 27F primer sequence is: AGAGTTTGATCMTGGCTCAG (SEQ ID No. 2);

[0083] The 1492R primer sequence is: GGTTACCTTGTTACGACTT (SEQ ID No. 3).

[0084] The components of the amplification system are shown in Table 4:

[0085] Table 4 Amplification system formula

[0086] 1×TSE101 Gold Mix 45uL 27F(10P) 2uL 1492R(10P) 2uL DNA template 1uL

[0087] The above amplification system was amplified according to the following amplification program, as shown in Table 5:

[0088] Table 5 Amplification program

[0089]

[0090]

[0091] After PCR amplification, the strain was identified by 2% agarose gel electrophoresis (2uL sample + 6uL bromophenol blue, 300V voltage for 12 minutes) and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The whole genome sequence of the strain is shown in SEQ ID No. 1; the sequence results were compared in the NCBI database, and the results showed that strain 3-1 was Bacillus subtilis ( Figure 2). The strain was named Bacillus subtilis Yirun2024, and was deposited in the China Center for Type Culture Collection on August 7, 2024, with the deposit number CCTCC NO:M 20241752, and the deposit address is Wuhan University, Wuhan, China.

[0092] Example 2 Biological characteristics of Bacillus subtilis Yirun 2024

[0093] 1. Colony morphology observation

[0094] The strain was inoculated on NA medium by the plate streak method, and the colony morphology was observed after inverted culture at 37°C for 16 to 18 hours, and the colonies were picked for Gram staining.

[0095] The colonies of Bacillus subtilis Yirun 2024 in NA culture are semi-transparent irregular circles with irregular edges and dry and wrinkled surfaces ( Figure 3 ). Further Gram staining was positive, and the bacteria were long rod-shaped ( Figure 4 ).

[0096] 2. Growth curve

[0097] After the strain is activated, adjust the bacterial solution to 1.0×10^8 cfu / mL, inoculate it into 100mL NB medium at a 3% (v / v) inoculation amount, and culture it in a shaking incubator at 30°C and 200r / min. Perform three parallel tests. Take out the strain every 2 hours and measure the absorbance at 600nm until there is no obvious growth. Draw a growth curve with the culture time as the horizontal axis and OD600 as the vertical axis.

[0098] The growth curve of Bacillus subtilis Yirun2024 is as follows Figure 5 As shown in the figure, Bacillus subtilis Yirun 2024 is in the lag phase of growth from 0 to 2 hours in fermentation culture, enters the logarithmic growth phase after 2 hours, and enters the stable phase after 16 hours.

[0099] 3. High temperature tolerance test

[0100] The bacterial liquid of the activated strain was adjusted to 1.0×10^8 cfu / mL and inoculated into 1 mL NB medium at a 3% (v / v) inoculation amount. The culture medium was placed in a water bath at 30°C, 60°C, and 80°C for 10 min, respectively. Three parallel tests were performed, followed by dilution and smear counting. The 30°C was used as the control group. The survival rate (%) = the number of colonies in the experimental group / the number of colonies in the control group × 100.

[0101] Depend on Figure 6 It can be seen that the survival rate of Bacillus subtilis Yirun2024 at 60°C is 89.76%, and the survival rate at 80°C is still 85.34%.

[0102] Example 3: Improvement of palm meal quality by fermentation with Bacillus subtilis Yirun 2024

[0103] 1. Preparation of Seed Solution

[0104] After the strain was activated, the bacterial solution was adjusted to 1.0×10^8 cfu / mL and inoculated into NB medium at an inoculum of 3% (v / v), and cultured at 37°C and 200 r / min for 12 hours to obtain the seed solution.

[0105] 2. Palm meal fermentation

[0106] The palm kernels were crushed and sieved, and 500.0 g was accurately weighed. Seed liquid was added at a ratio of 1%, 3%, 5% and 7% (v / w), with a material-water ratio of 1:1. The mixture was put into a fermentation bag with a valve, mixed evenly, and fermented at 30°C for 1d, 2d and 3d, respectively. Three parallel tests were conducted.

[0107] 3. Nutritional analysis of palm meal before and after fermentation

[0108] 100.0 g of unfermented and fermented palm meal were dried at 105 °C, passed through a 60-mesh sieve, and the sieve was collected. The crude protein and crude fiber of palm meal before and after fermentation were determined according to GB / T 6432-2018 and GB / T 6434-2022. The data were analyzed by independent sample t-test in SPSS26.0, and P<0.05 indicated significant differences.

[0109] As shown in Table 4, the crude fiber content of palm meal after fermentation was lower than that before fermentation, and the crude fiber content of palm meal fermented for 2 days with an inoculation amount of 3% decreased the most, decreasing by 32.67% compared with that before fermentation.

[0110] Table 4 Crude fiber content in palm meal before and after fermentation (dry matter basis)

[0111]

[0112] Through the above experiments, we can find that:

[0113] 1. The Bacillus subtilis Yirun2024 of the present invention is a strain naturally isolated from Tibetan pig feces, has a fast growth rate, and the cellulase in its fermentation product has an enzymatic activity that exceeds that of cellulase produced by other Bacillus subtilis and its genetically engineered strains;

[0114] It should be noted that in the literature in this field, some documents record enzyme activities that can exceed the above records, such as CN101643708B, a constitutive acidic endocellulase high-yielding strain, which detects the endocellulase after being purified 542.98 times. For the purified enzyme, its activity can be improved exponentially; in the relevant technical records in this field, no enzyme expressed by the strain and without purification has been found to achieve the enzyme activity effect of the present invention.

[0115] 2. The Bacillus subtilis Yirun2024 of the present invention has excellent high temperature resistance, can adapt to different harsh fermentation environments, and reduces the difficulty of fermentation process control.

[0116] 3. The palm meal fermented by the Bacillus subtilis Yirun 2024 of the present invention can significantly reduce the crude fiber content.

[0117] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A Bacillus subtilis strain, characterized in that: The classification name is Bacillus subtilis Yirun2024, the Latin name is Bacillus subtilis Yirun2024, the preservation number is CCTCC NO:M 20241752, the preservation date is August 7, 2024, the preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China.

2. The Bacillus subtilis according to claim 1, characterized in that The Bacillus subtilis is extracted from healthy Tibetan pig feces.

3. Use of the Bacillus subtilis according to any one of claims 1 to 2 as a production bacterium for producing at least one of endocellulase, exocellulase and β-glucosidase.

4. Use of the Bacillus subtilis according to any one of claims 1 to 2 to ferment a plant-derived raw material containing plant fiber.

5. The use according to claim 4, characterized in that The raw materials are one or more combinations of grass, palm meal, soybean meal, corn meal, peanut meal, fruit peels, straw, and tree branches and leaves.

6. Use of the Bacillus subtilis described in any one of claims 1-2 in preparing feed additives.

7. A feed, characterized in that: The product is obtained by fermenting a plant-derived raw material containing plant fiber with the Bacillus subtilis as claimed in claim 1 or 2.

8. The feed according to claim 7, characterized in that The raw materials are one or more combinations of grass, palm meal, soybean meal, corn meal, peanut meal, fruit peels, straw, and tree branches and leaves.

9. A fermentation agent, characterized in that: Contains the Bacillus subtilis according to claim 1 or 2.

10. A feed additive, characterized in that: Contains the Bacillus subtilis according to claim 1 or 2.

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