Bacillus subtilis and application

Abstract

The invention discloses bacillus subtilis and application thereof. The bacillus subtilis LDH-Y3 is obtained by being separated from Guangzhou commercially available Rakang goat milk pasteurized milk, and is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 64442. The bacterial strain is high in growth speed and high in activity, the maximum viable count reaches 9.36 log CFU / mL, and the bacterial strain has good gastrointestinal tract digestion resistance. The bacillus subtilis is high in cellulase production capacity, CMCase (Carboxymethyl Cellulase) reaches 39.66 U / mL, beta-Gase (beta-Gase) reaches 2.36 U / mL, and Fpase (Fpasease) reaches 11.37 U / mL. The strain has the capability of inhibiting helminthosporium convex hilum and helminthosporium longibrachiatum, and after solid state fermentation of corn straw, the content of neutral detergent fibers in the corn straw can be reduced, and the digestibility of the straw is improved.

Description

Technical Field

[0001] The invention relates to Bacillus subtilis, in particular to Bacillus subtilis LDH-Y3 and application thereof, and belongs to the field of development and application of microecological preparations. Background Art

[0002] Bacillus subtilis is a Gram-positive bacterium. Its cells are straight rod-shaped, with a single cell of 0.7-0.8×2-3μm, uniform coloration, no capsule, flagella around it, and it can move. It can form endogenous stress-resistant spores, which are 0.6-0.9×1.0-1.5 microns and are elliptical to cylindrical. It grows and reproduces quickly, with wrinkles on the surface of the colony, which is dirty white or slightly yellow. When growing in liquid culture medium, it often forms wrinkles. It is an aerobic bacterium. Due to its ability to form spores under harsh conditions, Bacillus subtilis can adapt to a variety of environments well. It is widely present in the gastrointestinal tract of animals, the surface of the soil, and the water environment in nature. Bacillus subtilis has obtained the GRAS certification of the US FDA and is a food-grade microorganism. Due to the safety, antibacterial, and enzyme-producing characteristics of Bacillus subtilis, it has become an important strain of microecological agents and is active in food, feed, industry and other fields.

[0003] In terms of crops, in order to prevent crops from being attacked by fungi and reducing yields, chemical control, biological control and other means are usually used to inhibit the reproduction of pathogens. Although chemical control is effective, it will cause damage to the surrounding environment, and residual harmful substances are difficult to be degraded by the environment. The use of antibacterial secondary metabolites produced by Bacillus subtilis for control belongs to biological control. This method is green and pollution-free, and plays an important role in promoting sustainable development. The current role of Bacillus subtilis in crops is reflected in the following four aspects: Role 1, Bacillus subtilis can prevent and control root rot and wilt; Role 2, prevent crop diseases such as powdery mildew, gray mold, and ulcers; Role 3, the metabolites of Bacillus subtilis can take root and promote growth. Role 4, Bacillus subtilis is a beneficial bacterium that can kill harmful bacteria through biological oxygen deprivation and improve the soil environment.

[0004] At present, no Bacillus subtilis strain has been found that produces high cellulase activity and is suitable for the degradation of corn straw and the inhibition of corn leaf spot pathogens (Helminthosporium and Helminthosporium convexum). In addition, there are problems such as high enzyme production cost, poor enzyme activity, and a small applicable pH range. Therefore, there is an urgent need to discover Bacillus subtilis strains with better enzyme activity, greater functionality, and a wider range of action.

[0005] Chinese invention patent CN202310254957.8 discloses a strain of Bacillus subtilis and a biocontrol agent and their application. The Bacillus subtilis has an inhibitory effect on plant pathogens such as Arthomyces mulberryensis, Sangxin brown spot shell spore, Fusarium equisetifolia, Fusarium oxysporum, and Fusarium solani. Chinese invention patent application CN202311761064.9 discloses a strain of Bacillus subtilis, its microbial agent and its application in the prevention and control of sweet potato black spot disease. The Bacillus subtilis has an inhibitory effect on sweet potato black spot pathogens. However, neither of these two Bacillus subtilis was found to have an inhibitory effect on Helminthosporium longis and Helminthosporium convexum, nor was it found to have a degrading effect on corn stalks. Summary of the invention

[0006] The present invention aims to provide a strain of Bacillus subtilis LDH-Y3, whose Latin name is Bacillus subtilis, and the strain has a preservation number of GDMCC No: 64442, and is preserved in Guangdong Province Microbiological Culture Collection Center.

[0007] Another object of the present invention is to provide a Bacillus subtilis seed liquid prepared by the Bacillus subtilis LDH-Y3.

[0008] The present invention also provides the use of the Bacillus subtilis seed liquid in determining cellulase activity by Congo red staining, the use of Bacillus subtilis in testing the cellulase secretion ability, the use of Bacillus subtilis in degrading corn stalks, and the use of Bacillus subtilis in preparing drugs for inhibiting Helminthosporium and Helminthosporium.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A Bacillus subtilis LDH-Y3, characterized in that the deposit number is GDMCC No: 64442, and the strain is deposited in Guangdong Microbiological Culture Collection Center. The 16S-rDNA sequence of Bacillus subtilis LDH-Y3 is shown in Sequence Table 1.

[0011] A Bacillus subtilis seed liquid is obtained by inoculating frozen Bacillus subtilis into TSB culture medium for cultivation; the culture temperature is 32-42°C, the rotation speed is 120-160rpm, and the time is 20-28 hours; the inoculation is carried out by inoculating 100 μL of Bacillus subtilis into 20-40mL of TSB culture medium.

[0012] The invention provides the application of the Bacillus subtilis in determining the cellulase activity by Congo red staining method.

[0013] Preferably, the Bacillus subtilis seed solution is spotted on a CMC-Na agar plate, cultured at 30-37° C. for 1-3 days, stained with a Congo red solution for 1-2 hours, then washed with a NaCl solution, and the ratio of the transparent zone diameter to the colony diameter is calculated to determine the cellulase activity secreted by Bacillus subtilis using the Congo red staining method.

[0014] The invention provides the application of the Bacillus subtilis in the test of cellulase secretion ability.

[0015] Preferably, the Bacillus subtilis seed solution of claim 2 is inoculated on a sodium carboxymethyl cellulose liquid culture medium at a volume ratio of 3-4%, and after culturing at a temperature of 30-37° C. and a rotation speed of 120-160 rpm for 24-28 hours, the enzyme solution is centrifuged at 2-4° C. and 8000-10000 rpm to obtain the enzyme solution, and the carboxymethyl cellulase activity (CMCase), β-glucosidase activity (β-Gase) and filter paper enzyme activity (Fpase) are determined according to the cellulase preparation standard QB2583-2003 method to obtain the cellulase secretion ability of Bacillus subtilis;

[0016] Preferably, the sodium carboxymethyl cellulose liquid culture medium is obtained by adding 3-5 parts of CMC-Na, 1-3 parts of peptone, 0.3-0.5 parts of yeast extract, 1-2 parts of (NH4)2SO4, 2-4 parts of KH2PO4, and 0.3-0.5 parts of MgSO4·7H2O to every 1000 parts of water.

[0017] The invention provides the application of the Bacillus subtilis in degrading corn stalks.

[0018] Preferably, 40-50 wt % deionized water is added to the corn stalk powder, mixed, sterilized at 110-121° C. for 15-20 min, and then cooled to room temperature, and the Bacillus subtilis seed solution is inoculated into the corn stalk powder at an inoculation amount of 10-30 wt % and fermented for 6-7 days, with the powder being turned over 1-2 times every other day during the fermentation period.

[0019] The invention provides the use of the Bacillus subtilis in preparing a drug for inhibiting Helminthosporium and Helminthosporium umbilicus.

[0020] Preferably, the Bacillus subtilis seed solution is inoculated into PDB culture medium at a volume ratio of 3-4%, and cultured at 30-37°C and 120-160rpm for 20-24h. The obtained culture solution is centrifuged at 2-4°C and 10000-12000rpm for 1-2min, and the collected supernatant is filtered with a microporous filter membrane to obtain a drug for inhibiting Helminthosporium and Helminthosporium.

[0021] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0022] The Bacillus subtilis LDH-Y3 of the present invention is sensitive to the antibiotics vancomycin, erythromycin, chloramphenicol, gentamicin, streptomycin, tetracycline, kanamycin and clindamycin, and meets the requirements of the EFSA microbial characteristic guideline standard for use as feed additives or production organisms.

[0023] In the Bacillus subtilis LDH-Y3 genome of the present invention, two antibiotic genes, rifamycin and daptomycin, three virulence factor genes, capB, capC and capD, and gene clusters of antibacterial substances such as bacilysin, subtilosin A, bacilibactin, sublancin 168, fengycin and surfactin are found.

[0024] The Bacillus subtilis LDH-Y3 has a fast growth rate, enters the logarithmic growth phase in about 4 hours, and has a maximum viable bacterial count of 9.36 Log CFU / ml. It can grow in the pH range of 5 to 9.

[0025] The Bacillus subtilis LDH-Y3 of the present invention can withstand gastrointestinal digestion. After 15 hours of simulated gastrointestinal digestion at pH = 5, the number of viable bacteria decreased from 9.42 log CFU / mL to 8.08 log CFU / mL, and the Caco-2 cell adhesion was 186×10 5 CFU / mL.

[0026] The Bacillus subtilis LDH-Y3 of the invention has a significantly higher ability to produce carboxymethyl cellulase than other strains, can degrade neutral detergent fiber of corn stalks, and improves the digestibility of corn stalks by ruminants.

[0027] The Bacillus subtilis LDH-Y3 of the invention can inhibit the growth and reproduction of the pathogenic bacteria Helminthosporium convexum and Helminthosporium longifolium which cause corn leaf spot disease, and the inhibition rates are 25.45% and 41.23% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The colony morphology of the Bacillus subtilis LDH-Y3 strain of the present invention is shown in FIG.

[0029] Figure 2 The bacterial morphology of the Bacillus subtilis LDH-Y3 strain of the present invention is shown in FIG.

[0030] Figure 3 This is a gene cluster diagram of the secondary metabolites of Bacillus subtilis LDH-Y3 of the present invention;

[0031] Figure 4 is a growth characteristic curve diagram of the Bacillus subtilis LDH-Y3 strain of the present invention;

[0032] Figure 5 This is a graph showing the growth of Bacillus subtilis LDH-Y3 at different initial pH values ​​of the present invention;

[0033] Figure 6 This is a graph showing changes in the number of live bacteria of Bacillus subtilis LDH-Y3 in simulated gastrointestinal digestion of the present invention;

[0034] Figure 7 This is a graph showing changes in the number of live bacteria of Bacillus subtilis LDH-Y3 in simulated gastrointestinal digestion of the present invention;

[0035] Figure 8 This is a graph showing the results of the Congo red staining method for Bacillus subtilis LDH-Y3 in Example 8;

[0036] Fig. 9 This is a graph showing the results of the Congo red staining method for Bacillus subtilis LDH-Y3 in Example 9;

[0037] Fig.10 This is a graph showing the results of Bacillus subtilis LDH-Y3 inhibiting Helminthosporium and Helminthosporium in Example 14.

[0038] Fig.11 This is a graph showing the results of Bacillus subtilis LDH-Y3 inhibiting Helminthosporium and Helminthosporium in the present invention 15. DETAILED DESCRIPTION

[0039] In order to better understand the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the embodiments do not constitute any limitation on the protection scope of the present invention.

[0040] The Bacillus subtilis LDH-Y3 involved in the present invention is a strain extracted, isolated and identified by the inventor from pasteurized Ruikang goat milk sold in Guangzhou. The strain is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 64442 and a deposit date of March 21, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province. The 16S-rDNA sequence of Bacillus subtilis LDH-Y3 of the present invention is shown in Sequence Table 1.

[0041] The colony morphology of Bacillus subtilis LDH-Y3 is as follows Figure 1 As shown, the surface is rough and wrinkled, and the bacterial morphology is as follows Figure 2 The antibiotic resistance of the Bacillus subtilis meets the EFSA standard, has no potential antibiotic resistance genes and virulence genes, and has strong secondary metabolite secretion ability and gastrointestinal tolerance.

[0042] The Congo red staining method for determining cellulase activity is simple and convenient, and can intuitively distinguish whether the strain has the ability to decompose cellulose, but it cannot obtain the specific cellulase activity value. Determining the ability of Bacillus subtilis to secrete cellulase can obtain the specific enzyme activity of different types of cellulase, which is convenient for subsequent research on the degradation of cellulose-containing straw.

[0043] This strain has the following characteristics:

[0044] In terms of feed, mold or Bacillus is usually introduced to improve the digestibility of feed by farmed animals. Although mold has a strong ability to produce cellulase, its slow reproduction and metabolism hinders its application. Bacillus subtilis has the advantages of good safety and fast reproduction and metabolism, and can be added to feed as an animal probiotic.

[0045] The Bacillus subtilis of the present invention has passed the test, and the antibiotic sensitivity meets the EFSA guideline standard for microbial characteristics used as feed additives or production organisms, and does not carry vancomycin (VanA), erythromycin (ermA, ermB), chloramphenicol

[0046] The microorganism has 9 antibiotic resistance genes, including cat, gentamicin (aac(6′)-aph(2″)), streptomycin (strA), tetracycline (tetL, tetM), and kanamycin (aph(3′)-IIIa), and does not carry 8 virulence genes, including hemolytic enterotoxin (hblA, hblC, hblD), non-hemolytic enterotoxin (nehA, nheB, nehC), vomitoxin (cesB), and cytotoxin (cytK). It is safe for application and can be used as a feed microorganism.

[0047] It has strong gastrointestinal tolerance and still has a high number of live bacteria after digestion in artificial gastric juice and artificial intestinal juice, and can remain active after entering the animal body; it has strong Caco-2 cell adhesion ability and is able to adhere to the animal's gastrointestinal tract, increasing the number of microorganisms in the animal's gastrointestinal tract, thereby playing a role in feed digestion, straw degradation, etc.

[0048] The strain can produce extracellular cellulase and can significantly degrade neutral detergent fiber in the straw, thereby increasing the degradation rate of corn straw, facilitating the digestion of straw feed by ruminants, and realizing the application of Bacillus subtilis in ruminant feed.

[0049] This strain has a significant inhibitory effect on the pathogens of corn large and small leaf spot diseases, Helminthosporium and Helminthosporium. When used in corn planting, it can prevent corn from developing leaf spot diseases caused by Helminthosporium and Helminthosporium, thereby ensuring corn yield.

[0050] In the present invention, w / v refers to the mass-to-volume ratio, where the mass unit is g and the volume unit is cm 3 .

[0051] Unless otherwise specified, the Bacillus subtilis in the present invention refers to Bacillus subtilis LDH-Y3.

[0052] In the following examples, TSB medium was prepared by adding 17 g of casein pancreatic digest, 5 g of sodium chloride, 3 g of soy flour papain digest, 2.5 g of glucose, and 2.5 g of potassium dihydrogen phosphate to 1 L of deionized water, with a natural pH (1-2% (w / v) agar was added to the solid medium based on the liquid medium), and sterilized at 121° C. for 15 min.

[0053] Example 1: Whole genome

[0054] Whole genome analysis: 100 μl of Bacillus subtilis LDH-Y3 strain stored at -80°C was inoculated into 20 mL of sterilized TSB medium, cultured at 37°C for 24 h, subcultured twice, the cells were collected and quick-frozen with liquid nitrogen, genomic DNA was extracted using the CTAB method, DNA samples were library-built, and third-generation sequencing was performed using the long-read sequencing platform Oxford Nanopore PromethION. Second-generation sequencing was performed using paired-end technology (PE 150) on the Illumina Novaseq 6000 sequencer. Genome assembly was performed using third-generation sequencing data, and correction was performed using second-generation sequencing data. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was used to annotate the assembled genome.

[0055] The DNA sequence of Bacillus subtilis LDH-Y3 is SEQ.ID.NO1.

[0056] Analysis of drug resistance genes: The genome data of Bacillus subtilis LDH-Y3 was compared with the CARD and ARDB databases, and a similarity greater than 80% was used as a screening condition. Two antibiotic genes were annotated, rpoB and pqsA, and their resistance mechanisms were antibiotic target substitution and antibiotic target change, respectively. Neither of them is a major antibiotic gene, and they do not belong to the types of antibiotics determined in the EFSA guidelines for microbial characteristics used as feed additives or production organisms, and have good safety.

[0057] Virulence gene analysis: The Bacillus subtilis LDH-Y3 genome data was compared and analyzed with the VFDB database, and a similarity greater than 80% was used as the screening condition. Three virulence factors, capB, capC, and capD, were screened out, with similarities of 100%, 88%, and 80%, respectively. All of them are atypical virulence factors with good safety.

[0058] Secondary metabolite analysis: Using the antiSMASH database, it was annotated that Bacillus subtilis LDH-Y3 produced Bacilysin, Subtilosin A, Bacilibactin, Sublancin 168, Fengycin, and Surfactin. The results are as follows Figure 3 As shown, except for surfactin with a similarity of 82%, the similarity of the other products reached 100%, indicating that Bacillus subtilis LDH-Y3 can secrete a variety of secondary metabolites.

[0059] Example 3: Strain growth characteristics

[0060] (1) Preparation of culture medium: 100 μl of Bacillus subtilis LDH-Y3 stored at -80°C was added to 20 mL of sterile TSB medium for activation and cultured at 37°C for 24 h to obtain Bacillus subtilis seed liquid.

[0061] (2) Growth characteristics determination: 4% (v / v) seed solution was inoculated into TSB medium and cultured at 37°C. OD was measured every 2 h during 0-30 h. 600 nm The result is Figure 4 As shown, at 0-8h in culture, OD 600 nm The value increased rapidly from 0 to 1.01, then increased slowly, and stabilized at OD600 nm About 1.1, indicating that the strain is in the logarithmic growth phase at 2-8h, reaches the stable phase at 20h, has a fast growth rate, and the maximum number of viable bacteria is 9.36CFU / mL.

[0062] (3) Initial pH of culture: 4% (v / v) of the seed solution was inoculated into TSB medium with pH values ​​of 4, 5, 6, 7, 8, 9, and 10, respectively, and the OD was measured after culturing at 37°C for 24 h. 600 nm The result is Figure 5 As shown in Figure 2, the strain basically did not grow at pH 4 and 10, but could grow at pH 5-9, and grew best at pH 7-8. 600nm It reached the maximum, indicating that the strain grew better in a neutral environment.

[0063] Example 3: Antibiotic Sensitivity

[0064] (1) Preparation of bacterial suspension: 100 μl of the strain stored at -80°C was added to 20 mL of TSB medium for activation, and centrifuged at 4°C and 8000 rpm for 2 min. The supernatant was discarded and the precipitated cells were collected. 5 mL of sterile saline was added per gram of cells for washing and centrifugation. After repeating twice, the OD value of the bacterial suspension was adjusted with MH medium. 600 nm to 0.57 to obtain a Bacillus subtilis suspension.

[0065] (2) Preparation of antibiotic dilution solution: Use erythromycin, clindamycin, vancomycin, gentamicin, kanamycin, streptomycin, tetracycline, and chloramphenicol to prepare an antibiotic stock solution with a concentration of 5120 μg / mL, and dilute it to 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL before use.

[0066] (3) Add the gradient diluted antibiotic dilutions to the 96-well plate in sequence, and then add 100 μL of bacterial suspension, and set 200 μL of sterile broth MH medium as a negative control and 200 μL of Bacillus subtilis suspension as a positive control. Place the inoculated 96-well plate in a 37°C constant temperature incubator for 48 hours, and use a functional microplate reader to measure the OD 600 nm According to the EFSA guidance document on microbial characteristics used as feed additives or production organisms, the minimum inhibitory concentration range is determined, and the range is shown in Table 1. The test results are the no-growth critical values ​​in Table 1, which are all lower than the concentration specified in the EFSA document, indicating that the Bacillus subtilis is sensitive to antibiotics and meets the standards of the EFSA guidance document on microbial characteristics used as feed additives or production organisms.

[0067] Table 1 MIC values ​​of Bacillus subtilis LDH-Y3

[0068]

[0069] Example 4: Drug resistance gene determination

[0070] Table 2 Antibiotic gene primer information Reference Ji Xing et al. (Jixing, Li Jun, Wang Ran, et al. Research progress on drug resistance of common probiotics [J]. Journal of Jiangsu Agricultural Sciences, 2022, 38(06): 1722-1728.) Antibiotic resistance gene primer information, Bacillus subtilis LDH-Y3 was used to amplify 9 antibiotic genes according to the primers in Table 2, and 1.5% (w / v, where mass unit is g and volume unit is cm 3 ) PCR products were detected by agarose gel electrophoresis to determine whether there were specific target bands.

[0071] Strain activation: 100 μl of Bacillus subtilis LDH-Y3 strain stored at -80°C was inoculated into 20 mL of sterilized TSB medium and cultured at 37°C for 24 h. The extraction of Bacillus subtilis LDH-Y3 was consistent with that in Example 1. Nine antibiotic resistance genes were amplified using primers in Table 2. 1.5% (w / v, where mass is g and volume is cm 3 ) PCR products were detected by agarose gel electrophoresis to determine whether there were specific target bands. The results showed that there were no specific bands for vancomycin (VanA), erythromycin (ermA, ermB), chloramphenicol (cat), gentamicin (aac(6′)-aph(2″)), streptomycin (strA), tetracycline (tetL, tetM), and kanamycin (aph(3′)-IIIa).

[0072] Table 2 Antibiotic resistance gene primer information

[0073]

[0074] Example 5: Virulence gene assay

[0075] Table 3 Virulence gene primer information Refer to Liu Chun et al. (Liu Chun, Fang Ying, Long Zhu, et al. Safety analysis of commercial probiotic Bacillus [J]. China Animal Husbandry Journal, 2020, 56(08): 203-208.) Virulence gene primer information, Bacillus subtilis LDH-Y3 was used to specifically amplify 8 virulence genes according to the primers in Table 3, and 1.5% (w / v, where mass unit is g and volume unit is cm3 ) PCR products were detected by agarose gel electrophoresis to determine whether there were specific target bands.

[0076] Table 3 Virulence gene primer information

[0077]

[0078] Strain activation: 100 μl of Bacillus subtilis LDH-Y3 strain stored at -80°C was inoculated into 20 mL of sterilized TSB medium and cultured at 37°C for 24 h. After extracting Bacillus subtilis LDH-Y3 DNA, eight virulence genes were specifically amplified using primers in Table 3. The 1.5% (w / v, where mass is g and volume is cm 3 ) Agarose gel electrophoresis was used to detect the PCR products to determine whether there were specific target bands. The results showed that Bacillus subtilis did not carry the eight virulence genes of hemolytic enterotoxin (hblA, hblC, hblD), non-hemolytic enterotoxin (nehA, nheB, nehC), vomitoxin (cesB), and cytotoxin (cytK).

[0079] Example 6: Artificial Gastric Juice, Artificial Intestinal Juice Tolerance and Caco-2 Adhesion

[0080] (1) Preparation of culture medium: 100 μl of Bacillus subtilis LDH-Y3 strain stored at -80°C was added to 20 mL of sterile TSB medium for activation and cultured at 37°C for 24 h to obtain Bacillus subtilis seed liquid.

[0081] (2) Preparation of artificial gastric juice: Prepare 3 mg / mL pepsin solution using PBS (pH=5) buffer.

[0082] (3) Preparation of artificial intestinal fluid: Prepare 1 mg / mL pancreatic enzyme solution using PBS (pH=8) buffer.

[0083] (4) Gastrointestinal tolerance test: 1 mL of Bacillus subtilis seed solution was added to 9 mL of artificial gastric juice and incubated at 37°C for 3 h. Then 1 mL of artificial gastric juice containing the seed solution was added to 9 mL of artificial intestinal juice and incubated for 12 h. Samples were taken at 0, 1, 2, and 3 h after treatment with artificial gastric juice and at 4, 5, 6, 7, 10, and 15 h after treatment with artificial intestinal juice, and the number of viable bacteria was counted by the dilution plate method. The results were as follows: Figure 6As shown. Under the condition of pH = 5 simulated gastric fluid, the initial viable bacterial count was 9.42log CFU / mL, and the viable bacterial count of Bacillus subtilis was 8.08log CFU / mL after 15h of digestion, and its viable bacterial count only decreased by 1.34log CFU / mL, indicating that the strain has a high gastrointestinal tolerance.

[0084] (5) Caco-2 adhesion test: Caco-2 cells were cultured at 1.4×10 4 The cells were inoculated at a density of 10 cells / well in a 24-well plate and incubated at 37°C. The cell culture medium was replaced every other day. After Caco-2 formed a monolayer, the culture medium without antibiotics was replaced and cultured for 1 day. At the same time, the activated strains were centrifuged at low temperature to collect the cells, washed with PBS (pH = 7.4) to remove the culture medium, and centrifuged twice. The cells were finally diluted to a concentration of 10 with 20% (v / v) fetal bovine serum DMEM culture medium without antibiotics. 9 CFU / mL, after removing the Caco-2 culture medium from the 24-well plate, 1 mL of diluted bacterial solution was added and incubated at 37°C for 2 h. After that, the cell surface was washed twice with PBS buffer, and trypsin was added to lyse the cells to obtain a suspension of bacteria adhering to the surface of Caco-2 cells. The adherent bacteria were counted by the dilution plate method. The adhesion of Bacillus subtilis to Caco-2 cells was 186×10 5 CFU / mL, with good Caco-2 cell adhesion ability.

[0085] Example 7: Artificial Gastric Juice, Artificial Intestinal Juice Tolerance and Caco-2 Adhesion

[0086] (1) Preparation of culture medium: 100 μl of Bacillus subtilis LDH-Y3 stored at -80°C was added to 20 mL of sterile TSB medium for activation and cultured at 37°C for 24 h to obtain Bacillus subtilis seed liquid.

[0087] (2) Preparation of artificial gastric juice: Prepare 3 mg / mL pepsin solution using PBS (pH=3) buffer.

[0088] (3) Preparation of artificial intestinal fluid: Prepare 1 mg / mL pancreatic enzyme solution using PBS (pH=8) buffer.

[0089] (4) Gastrointestinal tolerance test: 1 mL of seed solution was added to 9 mL of artificial gastric juice and incubated at 37°C for 3 h. Then 1 mL of artificial gastric juice containing seed solution was added to 9 mL of artificial intestinal juice and incubated for 12 h. Samples were taken at 0, 1, 2, and 3 h after artificial gastric juice treatment and at 4, 5, 6, 7, 10, and 15 h after artificial intestinal juice treatment, and the number of viable bacteria was counted by the dilution plate method. The results were as follows: Figure 7 As shown in the figure, under the condition of pH=3 simulated gastric fluid, the initial viable bacterial count was 9.36log CFU / mL, and the viable bacterial count of Bacillus subtilis was 7.05log CFU / mL after 15h of digestion, and its viable bacterial count only decreased by 2.31log CFU / mL, indicating that the strain has a high gastrointestinal tolerance.

[0090] (5) Caco-2 adhesion test: Caco-2 cells were cultured at 1.4×10 4 The cells were inoculated at a density of 10 cells / well in a 24-well plate and incubated at 37°C. The cell culture medium was replaced every other day. After Caco-2 formed a monolayer, the culture medium without antibiotics was replaced and cultured for 1 day. At the same time, the activated strains were centrifuged at low temperature to collect the cells, washed with PBS (pH = 7.4) to remove the culture medium, and centrifuged twice. The cells were finally diluted to a concentration of 10 with 20% (v / v) fetal bovine serum DMEM culture medium without antibiotics. 9 CFU / mL, after removing the Caco-2 culture medium from the 24-well plate, 1.5 mL of diluted bacterial solution was added and incubated at 37°C for 2 h. Afterwards, the cell surface was washed twice with PBS buffer, and trypsin was added to lyse the cells to obtain a suspension of bacteria adhering to the surface of Caco-2 cells. The adherent bacteria were counted using the dilution plate method. The adhesion of Bacillus subtilis LDH-Y3 to Caco-2 cells was 215×10 5 CFU / mL, with good Caco-2 cell adhesion ability.

[0091] Example 8: Congo red staining method to determine cellulase activity

[0092] (1) Preparation of culture medium: 100 μL of Bacillus subtilis LDH-Y3 strain stored at -80°C was added to 20 mL of sterile TSB medium for activation and cultured at 37°C for 24 h to obtain Bacillus subtilis seed liquid.

[0093] (2) Preparation of sodium carboxymethyl cellulose agar medium: CMC-Na 10 g, (NH4)2SO4 4.0 g, peptone 1.0 g, MgSO 4·7H2O 0.5g, KH2PO4 1.0g, agar 20g, dissolved in 1000mL deionized water.

[0094] (3) Cultivation method: Use a sterile toothpick to dip Bacillus subtilis seed solution and spot it on a CMC-Na agar plate. After culturing at 37°C for 2 days, stain it with Congo red solution (1 mg / mL) for 1 hour, wash it with NaCl (1 mol / L) solution, and calculate the ratio of the transparent zone diameter D to the colony diameter d (the transparent zone diameter D is Figure 8 The diameter of the yellowish circle in the middle is the diameter of the colony. Figure 8 The diameter of the dark area in the center of the light yellow circle). The results of Congo red staining of Bacillus subtilis LDH-Y3 are shown in Figure 8 After Congo red staining and Nacl washing, an obvious transparent zone appeared around the strain growth, and its D / d value was 2.28±0.05, indicating that Bacillus subtilis secreted cellulase during growth and reproduction, degrading the carboxymethyl cellulose around the colony into cellobiose and glucose, which could not combine with Congo red to form a red complex, thus producing a transparent zone, with better cellulose degradation ability, and the larger the ratio of the transparent zone diameter to the colony diameter, the greater the cellulose degradation ability.

[0095] Example 9: Congo red staining method to determine cellulase activity

[0096] (1) Preparation of culture medium: 100 μL of Bacillus subtilis LDH-Y3 strain stored at -80°C was added to 20 mL of sterile TSB medium for activation and cultured at 37°C for 24 h to obtain Bacillus subtilis seed liquid.

[0097] (2) Preparation of sodium carboxymethyl cellulose agar medium: CMC-Na 10 g, (NH4)2SO4 4.0 g, peptone 1.0 g, MgSO 4· 7H2O 0.5g, KH2PO4 1.0g, agar 20g, dissolved in 1000mL deionized water.

[0098] (3) Cultivation method: Use a sterile toothpick to dip Bacillus subtilis seed solution and spot it on a CMC-Na agar plate. After culturing at 30°C for 1 day, stain it with Congo red solution (1 mg / mL) for 1 hour, wash it with NaCl (1 mol / L) solution, and calculate the ratio of the transparent zone diameter D to the colony diameter d (the transparent zone diameter D is Fig. 9 The diameter of the yellowish circle in the middle is the diameter of the colony. Fig. 9 The Congo red staining results of strain LDH-Y3 are shown in Fig. 9After Congo red staining and Nacl washing, an obvious transparent zone appeared around the strain growth, and its D / d value was 1.57±0.06, indicating that Bacillus subtilis secreted cellulase during growth and reproduction, degrading the carboxymethyl cellulose around the colony into cellobiose and glucose, which could not combine with Congo red to form a red complex, thus producing a transparent zone, and had good cellulose degradation ability.

[0099] Example 10: Determination of the ability of Bacillus subtilis LDH-Y3 to secrete cellulase

[0100] (1) Preparation of culture medium: 100 μl of Bacillus subtilis LDH-Y3 strain stored at -80°C was added to 20 mL of sterile TSB medium for activation and cultured at 37°C for 24 h to obtain Bacillus subtilis seed liquid.

[0101] (2) Preparation of sodium carboxymethylcellulose liquid culture medium: 5 g CMC-Na, 3 g peptone, 0.5 g yeast extract, 2 g (NH4)2SO4, 4 g KH2PO4, and 0.5 g MgSO4·7H2O were dissolved in 1000 ml deionized water.

[0102] (3) Enzyme solution preparation: Bacillus subtilis seed solution was inoculated into sodium carboxymethyl cellulose liquid culture medium at a ratio of 4% (v / v), cultured at 30°C and 120 rpm for 24 h, and then centrifuged at 4°C and 8000 rpm. The resulting supernatant was the enzyme solution.

[0103] (4) Plotting of glucose standard curve: Weigh 1 g of anhydrous glucose dried to constant weight at (103±2)°C, dissolve it in water and dilute to 100 ml to obtain a glucose standard stock solution. Pipette 0.00, 0.30, 0.60, 0.90, 1.20, 1.50, and 1.80 ml of glucose standard stock solution into a 10 ml volumetric flask, dilute to 10 ml with deionized water, and shake well for later use. Pipette 0.5 ml of glucose standard working solution, 2.5 ml of phosphate buffer, and 3 ml of DNS reagent into a 25 ml stoppered test tube. Place the standard tubes in a boiling water bath at the same time, react for 10 min, quickly cool to room temperature, dilute to 25 ml, and measure the absorbance at a spectrophotometric wavelength of 540 nm using a cuvette. Use the amount of glucose as the horizontal coordinate x and the absorbance as the vertical coordinate y to plot a standard curve to obtain the linear regression equation, y=0.3094x(R 2 =0.9968).

[0104] (5) Determination of carboxymethyl cellulose enzyme activity (CMCase): Take 2 g of CMC-Na, add 200 mL of PBS (pH = 7) buffer, and dissolve in a water bath at 80°C to obtain a sodium carboxymethyl cellulose solution. Add 0.5 mL of the supernatant and 2 mL of the CMC-Na solution to a 25 mL stoppered test tube, and add 3 mL of DNS buffer after a 50°C water bath for 30 min. After a boiling water bath for 10 min, the volume was adjusted to 25 mL. The average absorbance was measured at a wavelength of 540 nm using a spectrophotometer, and the absorbance value was substituted into the regression equation (y = 0.3094x (R 2 =0.9968)) The CMCase was calculated to be 39.66U / mL. The enzyme's function is to destroy β-1,4-glycosidic bonds to form short-chain oligosaccharides. The higher the enzyme activity, the stronger the ability to degrade β-1,4-glycosidic bonds.

[0105] (6) Filter paper enzyme activity (Fpase) assay: 0.5 mL of the supernatant obtained in step (3) and 50 mg of rapid qualitative filter paper were added to a 25 mL stoppered test tube, and the tube was incubated at 50°C for 60 min. Then 3 mL of DNS buffer was added and the volume was adjusted to 25 mL after a boiling water bath for 10 min. The average absorbance was measured at a wavelength of 540 nm using a spectrophotometer. The absorbance value was substituted into the regression equation (y = 0.3094x(R 2 =0.9968)) The Fpase was calculated to be 11.37 U / mL. The enzyme activity reflects the comprehensive cellulose degradation ability of multiple cellulases. The higher the enzyme activity, the stronger its cellulose degradation ability.

[0106] (7) β-glucosidase activity (β-Gase) assay: Take 1 mL of 0.5% (w / v, where mass is g and volume is cm 3 ) phosphate buffer solution (the concentration of the phosphate buffer solution is 0.05 mol / L, and the pH is 6.0) and 0.5 mL of the supernatant obtained in step (3) were added to a 25 mL stoppered test tube. After a 50° C. water bath for 30 min, 3 mL of DNS buffer solution was added. After a boiling water bath for 10 min, the volume was adjusted to 25 mL. The average absorbance was measured at a wavelength of 540 nm using a spectrophotometer. The absorbance value was substituted into the regression equation (y=0.3094x(R 2 =0.9968)), the β-Gase was calculated to be 2.36U / mL. The function of this enzyme is to hydrolyze short-chain oligosaccharides into glucose. The higher the enzyme activity, the stronger the ability to degrade short-chain oligosaccharides.

[0107] (8) The above enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μg of glucose per mL of enzyme solution per min, which is 1 enzyme activity unit (U / mL). After measurement, the enzyme activity of Bacillus subtilis LDH-Y3 CMCase was 39.66U / mL, β-Gase was 2.36U / mL, and Fpase was 11.37U / mL, which were significantly higher than the enzyme activity of Bacillus subtilis that can degrade cellulose disclosed in Chinese invention patent CN202211451060.6 (the carboxymethyl cellulase activity, β-glucosidase, and filter paper enzyme in Bacillus subtilis that can degrade cellulose disclosed in Chinese invention patent CN202211451060.6 were 4.06, 1.97, and 0.74U / mL, respectively). Chinese invention patent application CN202311296123.X discloses a Bacillus subtilis GJ231 and its application. The maximum value of the carboxymethyl cellulase activity of the Bacillus subtilis is only 22.16U / mL, which is also significantly lower than that of the present invention, indicating that Bacillus subtilis The cellulase produced by Bacillus subtilis LDH-Y3 has a higher ability to degrade β-1,4-glycosidic bonds and short-chain oligosaccharides, and has a stronger ability to degrade cellulose overall. This may be because Bacillus subtilis LDH Y3 can produce a richer or more active cellulase system, and its application in feed can help improve the digestibility of feed in farmed animals.

[0108] Example 11: Determination of the ability of Bacillus subtilis LDH-Y3 to secrete cellulase

[0109] (1) Preparation of culture medium: 100 μl of Bacillus subtilis LDH-Y3 strain stored at -80°C was added to 20 mL of sterile TSB medium for activation and cultured at 37°C for 24 h to obtain Bacillus subtilis seed liquid.

[0110] (2) Preparation of sodium carboxymethylcellulose liquid culture medium: 5 g CMC-Na, 3 g peptone, 0.5 g yeast extract, 2 g (NH4)2SO4, 4 g KH2PO4, and 0.5 g MgSO4·7H2O were dissolved in 1000 ml deionized water.

[0111] (3) Enzyme solution preparation: Bacillus subtilis seed solution was inoculated into sodium carboxymethyl cellulose liquid medium at a ratio of 4% (v / v), cultured at 37°C and 160 rpm for 28 h, and then centrifuged at 2°C and 10,000 rpm. The resulting supernatant was the enzyme solution.

[0112] (4) Plotting of glucose standard curve: Weigh 1 g of anhydrous glucose dried to constant weight at (103±2)°C, dissolve it in water and dilute to 100 ml to obtain a glucose standard stock solution. Pipette 0.00, 0.30, 0.60, 0.90, 1.20, 1.50, and 1.80 ml of glucose standard stock solution into a 10 ml volumetric flask, dilute to 10 ml with deionized water, and shake well for later use. Pipette 0.5 ml of glucose standard working solution, 2.5 ml of phosphate buffer, and 3 ml of DNS reagent into a 25 ml stoppered test tube. Place the standard tubes in a boiling water bath at the same time, react for 10 min, quickly cool to room temperature, dilute to 25 ml, and measure the absorbance at a spectrophotometric wavelength of 540 nm using a cuvette. Use the amount of glucose as the horizontal coordinate x and the absorbance as the vertical coordinate y to plot a standard curve to obtain the linear regression equation, y=0.3094x(R 2 =0.9968).

[0113] (5) Determination of carboxymethyl cellulose enzyme activity (CMCase): Take 2 g of CMC-Na, add 200 mL of PBS (pH = 7) buffer, and dissolve in a water bath at 80°C to obtain a sodium carboxymethyl cellulose solution. Add 0.5 mL of the supernatant and 2 mL of the CMC-Na solution to a 25 mL stoppered test tube, and add 3 mL of DNS buffer after a 50°C water bath for 30 min. After a boiling water bath for 10 min, the volume was adjusted to 25 mL. The average absorbance was measured at a wavelength of 540 nm using a spectrophotometer, and the absorbance value was substituted into the regression equation (y = 0.3094x (R 2 =0.9968)) The CMCase was calculated to be 35.26U / mL. The enzyme's function is to destroy β-1,4-glycosidic bonds to form short-chain oligosaccharides. The higher the enzyme activity, the stronger the ability to degrade β-1,4-glycosidic bonds.

[0114] (6) Filter paper enzyme activity (Fpase) assay: 0.5 mL of the supernatant obtained in step (3) and 50 mg of rapid qualitative filter paper were added to a 25 mL stoppered test tube, and the tube was incubated at 50°C for 60 min. Then 3 mL of DNS buffer was added and the volume was adjusted to 25 mL after a boiling water bath for 10 min. The average absorbance was measured at a wavelength of 540 nm using a spectrophotometer. The absorbance value was substituted into the regression equation (y = 0.3094x(R 2 =0.9968)), and the Fpase was 8.21U / mL. The enzyme activity reflects the comprehensive cellulose degradation ability of multiple cellulases. The higher the enzyme activity, the stronger its cellulose degradation ability.

[0115] (7) β-glucosidase activity (β-Gase) assay: Take 1 mL of 0.5% (w / v, where mass is g and volume is cm 3) was added to a 25 mL stoppered test tube with 0.05 mol / L salicin prepared in a phosphate buffer solution with a pH of 6.0 and 0.5 mL of the supernatant. After a 50°C water bath for 30 min, 3 mL of DNS buffer was added. After a boiling water bath for 10 min, the volume was adjusted to 25 mL. The average absorbance was measured at a wavelength of 540 nm using a spectrophotometer. The absorbance value was substituted into the regression equation (y = 0.3094x (R 2 =0.9968)), the obtained β-Gase was 1.96U / mL. The function of this enzyme is to hydrolyze short-chain oligosaccharides into glucose. The higher the enzyme activity, the stronger the ability to degrade short-chain oligosaccharides.

[0116] (8) The enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μg of glucose per mL of enzyme solution per minute, which is 1 unit of enzyme activity (U / mL). After measurement, the CMCase of Bacillus subtilis LDH-Y3 was 35.26 U / mL, β-Gase was 1.96 U / mL, and Fpase was 8.21 U / mL, indicating that Bacillus subtilis LDH-Y3 also has a strong ability to degrade β-1,4-glycosidic bonds, short-chain oligosaccharides and cellulose under this condition.

[0117] Example 12: Corn straw degradation test

[0118] (1) Preparation of culture medium: 100 μL of Bacillus subtilis LDH-Y3 strain stored at -80°C was added to 20 mL of sterile TSB medium for activation and cultured at 37°C for 24 h to obtain Bacillus subtilis seed liquid.

[0119] (2) Corn stalk solid fermentation: The test group added corn stalk powder into a 1000 mL conical flask, added deionized water according to the mass ratio of corn stalk powder to water of 2:1, and mixed well. After sterilization at 121°C for 15 min, the mixture was cooled to room temperature. The activated seed liquid was inoculated into the corn stalk powder according to the inoculation amount of 20% (w / w, the mass of the seed liquid relative to the mass of the corn stalk powder), and fermented for 7 days. The samples were turned over every other day during the fermentation period. After the fermentation was completed, the samples were sent to Hangzhou Aike Testing Technology Co., Ltd., and the physical and chemical indicators of the fermented corn stalk powder were analyzed by near infrared detection method.

[0120] The blank control group was different from the test group in that the activated seeds were not inoculated in the corn stalk powder at an inoculation amount of 20% (w / w, the mass of the seed liquid was relative to the mass of the corn stalk powder), but 20% (w / w, the mass of the seed liquid was relative to the mass of the corn stalk powder) TSB culture medium was directly added, and other aspects were the same.

[0121] After testing, the acid detergent fiber content of the blank control group was 29.61% (w / w, the mass of acid detergent fiber relative to the mass of corn straw after fermentation), and the neutral detergent fiber content was 37.72% (w / w, the mass of neutral detergent fiber relative to the mass of corn straw after fermentation); the acid detergent fiber content of the test group after fermentation was 25.16% (w / w, the mass of acid detergent fiber relative to the mass of corn straw after fermentation), and the neutral detergent fiber content was 26.18% (w / w, the mass of neutral detergent fiber relative to the mass of corn straw after fermentation).

[0122] According to the test, acidic detergent fiber contains cellulose and lignin, while neutral detergent fiber contains hemicellulose, cellulose and lignin, which are important indicators for evaluating the degradation effect of corn straw. Among them, the acidic detergent fiber and neutral detergent fiber of corn straw were significantly reduced after fermentation with Bacillus subtilis LDH-Y3, and the neutral detergent fiber content was reduced more, indicating that it has a significant corn straw degradation effect, which helps ruminants digest straw feed and realizes the application of Bacillus subtilis in ruminant feed.

[0123] Example 13: Corn straw degradation test

[0124] (1) Preparation of culture medium: 100 μL of Bacillus subtilis LDH-Y3 strain stored at -80°C was added to 20 mL of sterile TSB medium for activation and cultured at 30°C for 24 h to obtain Bacillus subtilis seed liquid.

[0125] (2) Corn stalk solid fermentation: The test group added corn stalk powder into a 1000 mL conical flask, added deionized water according to the mass ratio of corn stalk powder to water of 2:1, and mixed well. After sterilization at 121°C for 15 min, the mixture was cooled to room temperature. The activated seed liquid was inoculated into the corn stalk powder according to the inoculation amount of 20% (w / w, the mass of the seed liquid relative to the mass of the corn stalk powder), and fermented for 6 days. The samples were turned over every other day during the fermentation period. After the fermentation was completed, the samples were sent to Hangzhou Aike Testing Technology Co., Ltd., and the physical and chemical indicators of the fermented corn stalk powder were analyzed by near infrared detection method.

[0126] The blank control group was different from the test group in that the activated seed liquid was not inoculated into the corn stalk powder at an inoculation rate of 20% (w / w, the mass of the seed liquid was relative to the mass of the corn stalk powder), but 20% (w / w, the mass of the seed liquid was relative to the mass of the corn stalk powder) of TSB culture medium was directly added, and other aspects were the same.

[0127] After testing, the content of acidic detergent fiber in the blank control group was 29.61% (w / w, the mass of acidic detergent fiber relative to the mass of corn stalk after fermentation), and the content of neutral detergent fiber was 37.72% (w / w, the mass of neutral detergent fiber relative to the mass of corn stalk after fermentation); the content of acidic detergent fiber in the test group after fermentation was 27.53% (w / w, the mass of acidic detergent fiber relative to the mass of corn stalk after fermentation), and the content of neutral detergent fiber was 30.37% (w / w, the mass of neutral detergent fiber relative to the mass of corn stalk after fermentation). It can be seen that the Bacillus subtilis seed solution cultured at 30°C can also significantly improve the degradation rate of corn stalk.

[0128] Example 14: Antibacterial test of Helminthosporium and Helminthosporium

[0129] (1) Preparation of culture solution: 100 μL of Bacillus subtilis LDH-Y3 strain stored at -80°C was inoculated into 20 mL of NB medium, and cultured at 37°C, 120 rpm for 24 hours to obtain seed solution. The seed solution was inoculated into 100 mL of PDB medium at a ratio of 4% (v / v), and cultured at 37°C, 120 rpm for 24 hours. The obtained culture solution was centrifuged at 4°C, 10000 rpm for 2 minutes, and the supernatant was collected and filtered with a 0.22 um microporous filter membrane to obtain a drug for inhibiting Helminthosporium and Helminthosporium, which was stored at 4°C for future use.

[0130] (2) Determination of inhibition rate: The antibacterial activity was determined using the mycelium growth rate method and Bacillus subtilis culture medium. For the test group, 1 mL of the microporous filtered drug and 19 mL of PDA medium were placed in a culture dish and mixed. For the blank control group, 1 mL of PDB medium and 19 mL of PDA medium were mixed and placed in a culture dish. After the culture medium solidified, Helminthosporium convexum and Helminthosporium longum were inoculated in the center of the culture dish using an inoculation loop, and the inhibition rate at 37°C for 7 days was calculated. The inhibitory effect is Fig.10 After 7 days of culture, the diameters of Helminthosporium and Helminthosporium convexum were significantly reduced when the culture filtrate of Bacillus subtilis was added to the culture medium. The inhibition rate of Bacillus subtilis LDH-Y3 on Helminthosporium was 41.23%, and the inhibition rate on Helminthosporium convexum was 25.45%.

[0131] The inhibitory effect of Bacillus subtilis LDH-Y3 on Helminthosporium and Helminthosporium may be related to the antifungal active substances surfactant (surfactin) and fengycin (fengycin) secreted by it as found in Example 4. These two substances inhibit the germination of mold spores by destroying the cell structure, thereby reducing the growth rate of the mold. When used in corn planting, it can prevent the occurrence of corn large and small leaf spot diseases.

[0132] Example 15: Antibacterial test of Helminthosporium and Helminthosporium

[0133] (1) Preparation of culture fluid: 100 μL of Bacillus subtilis LDH-Y3 strain stored at -80°C was inoculated into 20 mL of NB medium, and cultured at 37°C, 120 rpm for 24 hours to obtain seed fluid. The seed fluid was inoculated into 100 mL of PDB medium at a ratio of 3% (v / v), and cultured at 30°C, 160 rpm for 20 hours. The obtained culture fluid was centrifuged at 2°C, 12000 rpm for 1 min, and the supernatant was collected and filtered with a 0.22 um microporous filter membrane to obtain a drug for inhibiting Helminthosporium and Helminthosporium, which was stored at 4°C for future use.

[0134] (2) Determination of inhibition rate: The mycelium growth rate method was used to determine the antibacterial activity using Bacillus subtilis culture medium. For the test group, 1 mL of the microporous filtered drug and 19 mL of PDA culture medium were placed in a culture dish and mixed. For the blank control group, 1 mL of PDB culture medium and 19 mL of PDA culture medium were mixed and placed in a culture dish. After the culture medium solidified, the mold was inoculated in the center of the culture dish with an inoculation loop, and the inhibition rate at 37°C for 7 days was calculated. The inhibitory effect is shown in Fig.11 After 7 days of cultivation, the diameters of Helminthosporium and Helminthosporium in the culture medium added with Bacillus subtilis culture filtrate were significantly reduced. The inhibition rate of Bacillus subtilis LDH-Y3 on Helminthosporium was 40.37%, and the inhibition rate on Helminthosporium was 23.64%. Therefore, the filtrate obtained under 30℃ culture conditions also has an inhibitory effect on Helminthosporium and Helminthosporium.

[0135] .

Claims

1. A Bacillus subtilis LDH-Y3, characterized in that: The deposit number is GDMCCNo:64442.

2. A Bacillus subtilis seed solution, characterized in that: The method is obtained by inoculating the frozen Bacillus subtilis of claim 1 into TSB medium for cultivation; the cultivation temperature is 32-42°C, the rotation speed is 120-160rpm, and the time is 20-28 hours; the inoculation is performed by inoculating 100 μL of Bacillus subtilis into 20-40 mL of TSB medium.

3. Use of the Bacillus subtilis described in claim 1 in determining cellulase activity by Congo red staining.

4. The use of Bacillus subtilis in determining cellulase activity by Congo red staining according to claim 3, characterized in that: The Bacillus subtilis seed liquid of claim 2 is spotted on a CMC-Na agar plate, cultured at 30-37° C. for 1-3 days, stained with a Congo red solution for 1-2 hours, then washed with a NaCl solution, and the ratio of the transparent zone diameter to the colony diameter is calculated to determine the cellulase activity secreted by Bacillus subtilis by the Congo red staining method.

5. Use of the Bacillus subtilis according to claim 1 in testing the ability to secrete cellulase.

6. The use of Bacillus subtilis in the cellulase secretion ability test according to claim 5, characterized in that: Inoculate the Bacillus subtilis seed liquid of claim 2 on a sodium carboxymethyl cellulose liquid culture medium at a volume ratio of 3-4%, culture at a temperature of 30-37° C. and a rotation speed of 120-160 rpm for 24-28 hours, centrifuge at 2-4° C. and 8000-10000 rpm to obtain an enzyme solution, and determine the carboxymethyl cellulase activity, β-glucosidase activity and filter paper enzyme activity according to the cellulase preparation standard QB2583-2003 method to obtain the cellulase secretion ability of Bacillus subtilis; The sodium carboxymethyl cellulose liquid culture medium is obtained by adding 3-5 parts of CMC-Na, 1-3 parts of peptone, 0.3-0.5 parts of yeast extract, 1-2 parts of (NH4)2SO4, 2-4 parts of KH2PO4 and 0.3-0.5 parts of MgSO4·7H2O to every 1000 parts of water.

7. Use of the Bacillus subtilis according to claim 1 in degrading corn stalks.

8. The use of Bacillus subtilis in degrading corn stalks according to claim 7, characterized in that: Add 40-50wt% deionized water into the corn stalk powder, mix well, sterilize at 110-121°C for 15-20min, and then cool to room temperature. Inoculate the Bacillus subtilis seed solution of claim 2 in an inoculation amount of 10-30wt% into the corn stalk powder and ferment for 6-7 days, turning the corn stalk powder 1-2 times every day during the fermentation.

9. Use of the Bacillus subtilis according to claim 1 in preparing drugs for inhibiting Helminthosporium and Helminthosporium.

10. The use of Bacillus subtilis in the preparation of a drug for inhibiting Helminthosporium and Helminthosporium according to claim 9, characterized in that: The Bacillus subtilis seed solution of claim 2 is inoculated into PDB culture medium at a volume ratio of 3-4%, and cultured at 30-37°C and 120-160rpm for 20-24h. The obtained culture solution is centrifuged at 2-4°C and 10000-12000rpm for 1-2min, and the collected supernatant is filtered through a microporous filter membrane to obtain a drug for inhibiting Helminthosporium and Helminthosporium.

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