A Bacillus subtilis strain and its applications

Abstract

The present invention belongs to the field of biotechnology, and discloses a Bacillus subtilis strain from Tibetan pigs, with the classification name of Bacillus subtilis Yirun2024, the Latin name of Bacillus subtilis Yirun2024, the preservation number of CCTCC NO: M 20241752, the preservation date of August 7, 2024, the preservation unit of China Center for Type Culture Collection, and the preservation address of Wuhan University, Wuhan, China. This Bacillus subtilis has high cellulase production. After 12 h of fermentation in a shake flask, the activities of endoglucanase, exoglucanase and β-glucosidase are respectively as high as 2902.72 U / mL, 11026.33 U / mL and 2983.04 U / mL, far exceeding those of similar engineering bacteria. At the same time, the present invention also provides the application of this Bacillus subtilis, which is of great significance in promoting the application field of fiber resources. At the same time, this strain can maintain a relatively high survival rate in a high-temperature environment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a Bacillus subtilis strain and its application. Background Art

[0002] Cellulose is a macromolecular polysaccharide composed of glucose, which is the main component of plant cell walls and the most widely distributed and highest-yielding renewable resource in nature. China is a large agricultural country, and a large amount of high-fiber agricultural waste is generated every year, such as straw, rice straw, bagasse, etc. It is estimated that 741 million - 1.111 billion tons of rice straw are produced globally every year. In 2022, the total straw resource in China has reached 8.56×10 8 tons. However, due to the low direct economic value and huge output, most high-fiber agricultural wastes are discarded or directly burned, causing environmental pollution and resource waste.

[0003] Bioconversion is an important measure to improve the economic value of agricultural wastes 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 endoglucanase (endo - glucanase), exoglucanase (exo - glucanase), and β - glucosidase. The complete degradation of cellulose must be completed through the synergistic action of three cellulases. First, random - site hydrolysis by endoglucanase produces oligosaccharides, then cellobiose is produced by the action of exoglucanase, and finally cellobiose is hydrolyzed to glucose by β - glucosidase. Therefore, the complete hydrolysis of cellulose must go through the synergistic action of these three cellulases to hydrolyze the indigestible cellulose molecules into glucose.

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

[0005] At present, due to the relatively large number of enzyme - producing species and high yields of molds, the production and research of cellulase mainly focus on molds. However, the enzyme - producing cycle of molds is relatively long, while bacteria have the advantages of faster growth rate and shorter enzyme - producing cycle. At the same time, bacteria have stronger environmental adaptability, more species, wider sources, and can produce more specific composite enzymes. Therefore, the development of high - yield cellulase - producing bacteria is of great significance.

[0006] Chinese Patent Application CN202110425297.6 discloses a Bacillus subtilis FMME ZK002 that produces cellulase. It was deposited at the China Center for Type Culture Collection on January 29, 2021, with the deposit number CCTCC NO: M 2021168. The strain in this solution can produce cellulase, and its cellulase activity is 118.67 U / mL.

[0007] Further observing this solution, it can be seen that the measured cellulase in this solution is carboxymethyl cellulase, that is, endocellulase, but it does not show the activity effects on exocellulase and β-glucosidase, and it is difficult to completely hydrolyze cellulose into glucose.

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

[0009] Further observing this solution, it can be seen that Bacillus subtilis C6-AEA3 belongs to a genetically engineered recombinant strain, and its purpose is to improve the enzyme activities of various cellulases, but the enzyme activity of this strain still fails to meet the expected requirements.

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

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

[0012] Meanwhile, after analyzing the performance of Bacillus subtilis in the prior art, it is found that most of the Bacillus subtilis with environmental tolerance recorded in the prior art have relatively low survival rates under high-temperature conditions (such as 80 °C). For example, in CN111808765A, a Bacillus subtilis with high efficiency in degrading vomitoxin and its application, its survival rate reaches about 90% at 50 °C, but when the temperature reaches 60 °C and 80 °C, the survival rate drops sharply to less than 20%. The high-temperature tolerance of Bacillus subtilis is an important performance index for fermentation. During the fermentation process, once the process control is not good enough, it is easy to occur the phenomenon that the temperature of the heap exceeds the standard. If the fermentation bacteria can effectively adapt to the high-temperature environment, it will reduce the difficulty of fermentation process control.

[0013] The technical problem to be solved in this case is: how to discover a naturally occurring Bacillus subtilis with a large number of cellulase types and high activity and certain environmental tolerance. Summary of the Invention

[0014] The object of the present invention is to provide a Bacillus subtilis Yirun2024, which has high cellulase production. The activities of endoglucanase, exoglucanase and β-glucosidase in the shake flask fermentation for 12 h are as high as 2902.72 U / mL, 11026.33 U / mL and 2983.04 U / mL respectively, far exceeding those of similar engineering bacteria. At the same time, this strain can maintain a high survival rate in a high-temperature environment.

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

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

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

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

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

[0020] In addition, the present invention also discloses the use of fermenting a plant-derived raw material containing plant fibers with the above-mentioned Bacillus subtilis.

[0021] Preferably, the raw materials are one or a combination of forage, palm meal, soybean meal, corn meal, peanut meal, fruit peels, straws, and tree branches and leaves.

[0022] In addition, the present invention also discloses the use of the Bacillus subtilis as described above for preparing a feed additive.

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

[0024] In the above-mentioned feed, the raw materials are one or a combination of forage, palm meal, soybean meal, corn meal, peanut meal, fruit peels, straws, and tree branches and leaves.

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

[0026] In some application scenarios of the present invention, the fermentation inoculant may further contain other strains for fermentation purposes, 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 for adding to the feeds of growing pigs and sows. During the feeding process of growing pigs and sows, the addition amount of the Bacillus subtilis in the feed is 10^8 - 10^9 cfu / kg feed.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] The Bacillus subtilis Yirun2024 provided by the present invention is isolated from healthy Tibetan pigs, grows rapidly, and has high cellulase production. The activities of its endoglucanase, exoglucanase, and β-glucosidase in the shake flask fermentation for 12 h are respectively as high as 2902.72 U / mL, 11026.33 U / mL, and 2983.04 U / mL, far exceeding those of similar engineered bacteria, and can significantly improve the nutritional components 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. Description of the Drawings

[0032] Figure 1 is the glucose standard curve;

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

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

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

[0036] Figure 5 Growth curve of Bacillus subtilis Yirun2024;

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

[0038] Figure 7 Photo of the primary screening culture result of high cellulose - enzyme - producing strains. Detailed implementation manners

[0039] Next, in combination with the accompanying drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall 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 Solarbio Science & Technology Co., Ltd.

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

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

[0044] Gram staining solution: Qingdao Hi - tech Industrial Park Haibo Biotechnology Co., Ltd.

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

[0046] 1. Culture media and reagents

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

[0048] Nutrient Agar (NA): Peptone 10 g / L, Beef Extract Powder 3.0 g / L, NaCl 5.0 g / L, Agar 15.0 g / L, pH 7.3 ± 0.1, autoclaved at 121 °C for 15 min.

[0049] Cellulase Enrichment Medium: CMC-Na 10 g / L, MgSO 4 0.5 g / L, NaNO 3 3.0 g / L, KCl 0.5 g / L, K 2 HPO 4 0.4 g / L, FeSO 4 0.01 g / L, autoclaved at 121 °C for 15 min.

[0050] Isolation Medium: CMC-Na 10 g / L, MgSO 4 0.5 g / L, NaNO 3 3.0 g / L, KCl 0.5 g / L, K 2 HPO 4 0.4 g / L, FeSO 4 0.01 g / L, Agar 15 g / L, autoclaved 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 amounts, then weigh 5.0 g of the mixed fecal samples and place them in a 250 mL conical flask containing 100.0 mL of cellulase enrichment medium. Add glass beads and vortex thoroughly. Incubate at 37 °C, 200 r / min for 24 h. Take 1.0 mL of the bacterial liquid and inoculate it into 100 mL of cellulase enrichment medium. Continue to incubate at 37 °C, 200 r / min for 24 h, and repeat 2 times. Take 1.0 mL of the bacterial liquid and add it to 9.0 mL of physiological saline. At this time, the dilution concentration is 10 -1 , and then dilute it to 10 -9 respectively at a ratio of 1:10. Take 100 μL of the bacterial suspension of each dilution gradient and spread it on the isolation medium. Incubate at 37 °C for 24 - 72 h until most colonies grow. Pick the colonies and streak them on the NA medium until a single colony is purified. Subsequently, pick the purified strain and inoculate it into the NB medium for amplification for 24 h, and store it at 4 °C for later use.

[0053] 3. Screening of High-Cellulase-Producing Strains

[0054] 3.1. Primary Screening

[0055] The activated strain was inoculated onto the isolation medium by the three-point inoculation method and cultured in an inverted position at 37°C for 48 h. After the strain culture was completed, 10 mL of 0.5% congo red solution was slowly poured from the edge of the petri dish, and gently shaken to make the congo red solution cover the entire plate. After standing for 10 min, the liquid in the petri dish was poured out, and the plate was rinsed once with 1 mol / L NaCl solution. Then, 10 mL of 1 mol / L NaCl solution was added to the petri dish, and after standing for "decolorization" for 15 min, the liquid in the petri dish was poured out. The diameter (D) of the clear zone and the diameter (d) of the colony on the culture medium plate were observed and measured, and the ratio of the diameter (D) of the clear zone to the diameter (d) of the colony was calculated. Strains with a ratio > 4 were subjected to a rescreening for cellulase determination.

[0056] Five strains with obvious hydrolysis zones were isolated from the feces of Tibetan pigs. Among them, the diameter ratio of 3-1 and 1-1 was greater than 4 (Table 1, the hydrolysis zone of strain 3-1 is visible Figure 7 ), and cellulase activity determination was carried out.

[0057] Table 1 Hydrolysis zone diameters of each strain

[0058] Strain number Diameter D of hydrolysis zone (mm) Diameter d of strain (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. Determination of cellulase enzyme activity

[0060] 3.2.1. Preparation of glucose standard curve

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

[0062] Table 2 Preparation table of glucose standard curve solution

[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 solution (mL) 10 9 8 7 6 5 4 3

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

[0065] The glucose standard curve is as Figure 1 shown. With the glucose content (mg) as the abscissa and OD540nm as the ordinate, the equation of the drawn glucose standard curve is: y = 0.1494x + 0.0260, R2 = 0.9974, meeting the requirements of the standard curve.

[0066] 3.2.2. Determination of cellulase activity

[0067] After activating the strains obtained from the primary screening, inoculate them into a 250 mL conical flask containing 100 mL of cellulase enrichment medium at an inoculation amount of 3% (v / v). Conduct 3 parallel experiments. After culturing at 37 °C and 200 r / min for 12 h, centrifuge at 10000 rpm and 4 °C for 10 min. The supernatant is the crude enzyme solution.

[0068] Determination of endo - cellulase activity (carboxymethyl cellulase): After appropriately diluting the crude enzyme solution, take 0.5 mL and add 1 mL of a buffer solution containing 1% sodium carboxymethyl cellulose (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 carry out a boiling water bath for 10 min. After cooling, add 6.5 mL of distilled water to make up the volume to 10 mL. Measure the absorbance value at 540 nm, and calculate the content of reducing sugar produced by the reaction through the glucose standard curve.

[0069] Determination of exo - cellulase activity (microcrystalline cellulase): After appropriately diluting the crude enzyme solution, take 0.5 mL and add 1 mL of a buffer solution containing 1% microcrystalline cellulose (pH 4.8, 0.2 mol / L acetic acid - sodium acetate buffer). React at 50 °C for 30 min, then add 2 mL of DNS reagent to terminate the reaction, and carry out a boiling water bath for 10 min. After cooling, add 6.5 mL of distilled water to make up the volume to 10 mL. Measure the absorbance value at 540 nm.

[0070] Determination of β - glucosidase activity: Add 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 to a centrifuge tube in sequence, mix well. After reacting at 50 °C for 30 min, add 2 mL of DNS reagent to terminate the reaction, carry out a boiling water bath for 10 min. After cooling, add 6.5 mL of distilled water to make up the volume to 10 mL. Measure the absorbance value at 540 nm.

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

[0072]

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

[0074] N is the dilution factor

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

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

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

[0078] Table 3 Cellulase Activity of Strains

[0079]

[0080] 4. Strain Identification

[0081] The genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit produced by Beijing Solarbio Science & Technology Co., Ltd. The extracted DNA sample was appropriately diluted and used as a PCR template, and the universal primers for bacterial 16S rDNA were amplified with Tsingke 1×TSE101 Gold Mix.

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

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

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

[0085] Table 4 Amplification System Formulation

[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 specifically:

[0088] Table 5 Amplification Program Table

[0089]

[0090]

[0091] After PCR amplification, it was identified by 2% agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue, 12 minutes at 300 V voltage), and then sent to Tsingke Biotechnology Co., Ltd. in Beijing for sequencing. The complete genome sequence of this strain is shown as SEQ ID No.1; The sequence results were compared in the NCBI database, and the results showed that strain 3-1 is Bacillus subtilis ( Figure 2)。This strain is named Bacillus subtilis Yirun2024, which was deposited at 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, China.

[0092] Example 2 Biological Characteristics of Bacillus subtilis Yirun2024

[0093] 1. Observation of Colony Morphology

[0094] The strain was inoculated on NA medium by the streak plate method and incubated in an inverted position at 37 °C for 16 - 18 h, then the colony morphology was observed, and the colonies were picked for Gram staining.

[0095] The colonies of Bacillus subtilis Yirun2024 in NA culture were semi - transparent irregular circles, with irregular edges, dry and wrinkled surfaces ( Figure 3 ). Further Gram staining showed positive results, and the bacterial cells were long rod - shaped ( Figure 4 ).

[0096] 2. Growth Curve

[0097] After activating the strain, the bacterial liquid was adjusted to 1.0×10^8 cfu / mL, inoculated into 100 mL of NB medium at an inoculation amount of 3% (v / v), cultured in a shaker at 30 °C and 200 r / min, and three parallel experiments were carried out. Samples were taken every 2 hours, and the absorbance value was measured at 600 nm until there was no obvious growth. The growth curve was plotted with the culture time as the abscissa and OD600 as the ordinate.

[0098] The growth curve of Bacillus subtilis Yirun2024 is as Figure 5 shown. Bacillus subtilis Yirun2024 had a lag phase during 0 - 2 h of fermentation culture, entered the logarithmic growth phase after 2 h, and entered the stationary phase at 16 h.

[0099] 3. High - Temperature Tolerance Test

[0100] The activated strain was adjusted to a bacterial liquid of 1.0×10^8 cfu / mL, inoculated into 1 mL of NB medium at an inoculation amount of 3% (v / v), and placed in a water bath at 30 °C, 60 °C, and 80 °C for 10 min respectively. Three parallel experiments were carried out, and then dilution coating and counting were performed. Taking 30 °C 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] As Figure 6 can be seen, the survival rate of Bacillus subtilis Yirun2024 reached 89.76% at 60 °C and still reached 85.34% at 80 °C.

[0102] Example 3 Improvement of Palm Kernel Meal Quality by Fermentation of Bacillus subtilis Yirun2024

[0103] 1. Preparation of Seed Liquid

[0104] After activating the strain, the bacterial liquid was adjusted to 1.0×10^8 cfu / mL and inoculated into NB medium at an inoculation amount of 3% (v / v). It was cultured at a constant temperature of 37°C and 200 r / min for 12 h to obtain the seed liquid.

[0105] 2. Fermentation of Palm Kernel Meal

[0106] The palm kernels were crushed and sieved. Exactly 500.0 g was weighed and the seed liquid was added at ratios of 1%, 3%, 5% and 7% (v / w). The ratio of material to water was 1:1. It was put into a fermentation bag with a valve, mixed evenly, and fermented at 30°C for 1 d, 2 d, and 3 d respectively, with 3 parallel tests carried out.

[0107] 3. Detection of Nutritional Components of Palm Kernel Meal before and after Fermentation

[0108] 100.0 g of unfermented and fermented palm kernel meal were dried at 105°C and sieved through a 60-mesh sieve, and the material passing through the sieve was collected. The crude protein and crude fiber of palm kernel meal before and after fermentation were determined with reference to GB / T 6432 - 2018 and GB / T 6434 - 2022 respectively. The data were analyzed for significance using independent-sample t-test in SPSS 26.0. P < 0.05 indicates a significant difference.

[0109] As can be seen from Table 4, the crude fiber content of palm kernel meal after fermentation decreased compared with that before fermentation. Among them, the crude fiber content decreased the most when fermented for 2 d at an inoculation amount of 3%, decreasing by 32.67% compared with that before fermentation.

[0110] Table 4 Crude Fiber Content in Palm Kernel Meal before and after Fermentation (Dry Matter Basis)

[0111]

[0112] Through the above experiments, it can be found that:

[0113] 1. As a strain naturally isolated from Tibetan pig feces, Bacillus subtilis Yirun2024 of the present invention has a fast growth rate, and the cellulase in its fermentation products has an enzyme activity exceeding that of cellulases produced by other Bacillus subtilis and their genetically engineered strains;

[0114] It should be noted that in the literature in this field, the enzyme activity performance recorded in some literatures exceeds the above records. For example, in CN101643708B, a constitutive acid endocellulase high-yielding strain, the detected endocellulase is purified 542.98 times. For the purified enzyme, its activity can be increased exponentially. In the relevant technical records in this field, no enzyme expressed by a 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 very excellent high-temperature resistance, can adapt to different harsh fermentation environments, and reduces the difficulty of fermentation process control.

[0116] 3. Fermenting palm meal with the Bacillus subtilis Yirun2024 of the present invention can significantly reduce the crude fiber content.

[0117] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claimed claim.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis )Yirun2024, characterized in that, The deposit number is CCTCCNO:M 20241752, the deposit date is August 7, 2024, the depositor is China Center for Type Culture Collection, and the deposit 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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