Bovine-derived cellulase-producing bacillus capable of inhibiting gastrointestinal pathogenic bacteria and application of bovine-derived cellulase-producing bacillus

Abstract

The preparation method comprises the following steps: carrying out sterile operation: taking 1.0 g of a fresh excrement sample, putting the fresh excrement sample into 9 mL of sterile normal saline, oscillating at 37 DEG C at 200 r / min for 30 min to fully and uniformly mix the excrement sample, carrying out water bath in a water bath kettle at 75 DEG C for 15 min to kill non-spore thalli, and then centrifuging at 900 r / min for 10 min to precipitate impurities in the excrement sample; the method comprises the following steps: respectively carrying out gradient dilution on the treated excrement sample stock solution by using sterile water until 10 <-1 >-10 <-6 >, aseptically absorbing 200 mu L of 10 <-4 >, 10 <-5 > and 10 <-6 > supernate, uniformly coating on a sodium carboxymethyl cellulose solid culture medium, culturing at 37 DEG C for 48 hours, adding Congo red dye liquor (1g / L) on a flat plate to submerge bacterial colonies, and discarding the dye liquor at normal temperature for 1 hour. The strain separated from the feces of the bred animals comes from the animals, can better adapt to the gastrointestinal tract environment in the animals, is easy to colonize, has a remarkable inhibition effect on gastrointestinal tract pathogenic bacteria, and has the effects of promoting digestion and improving the growth performance of the feed.

Description

Technical Field

[0001] The invention relates to the technical field of bovine cellulase-producing bacillus, in particular to a bovine cellulase-producing bacillus capable of inhibiting gastrointestinal pathogens and application thereof. Background Art

[0002] In recent years, due to the rapid development of my country's animal husbandry industry, the abuse of antibiotics has become increasingly obvious. Antibiotics, as growth promoters for livestock and poultry, have played a huge role in the growth, disease prevention and treatment of livestock and poultry. However, with the abuse of antibiotics, problems such as drug residues, bacterial resistance, and endogenous infections in animals have emerged, seriously endangering food safety and human life. Since January 2020, the addition of antibiotics to feed in my country has been completely banned. With the advent of the era of reducing and replacing antibiotics in the breeding industry, exploring the role of microbial feed additives in reducing and replacing the use of antibiotics, improving the balance of intestinal flora in farmed animals, and enhancing the digestive system function of farmed animals has gradually become a new research hotspot.

[0003] As a type of probiotic with outstanding resistance-reducing properties, Bacillus can increase the activity of digestive enzymes, maintain the normal physiological state of animals, and promote animal growth. In commercial application and promotion, it has the characteristics of strong heat resistance and a long shelf life. It has strong stress resistance. Some excellent strains can also produce enzymes such as protease, amylase, lipase, cellulase and other metabolites and various antibacterial substances, such as surfactin, iturin, fentaminocin, bacillus, and polymyxin. It has a significant regulatory effect on the intestinal microecology of farmed animals, and at the same time has a significant inhibitory effect on conditional pathogens.

[0004] Therefore, we propose a bovine cellulase-producing Bacillus that inhibits gastrointestinal pathogens and its application to solve the above-mentioned problems. Summary of the invention

[0005] The purpose of the present invention is to provide a bovine cellulase-producing Bacillus and its application for inhibiting gastrointestinal pathogens, so as to solve the above-mentioned research problems on the role of microbial feed additives in reducing and replacing the use of antibiotics, improving the balance of intestinal flora of farmed animals, and enhancing the digestive system function of farmed animals.

[0006] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a bovine cellulase-producing Bacillus for inhibiting gastrointestinal pathogens and its application, wherein the bovine cellulase-producing Bacillus for inhibiting gastrointestinal pathogens comprises the following materials for isolating the strain: fresh cow dung sample, sterile physiological saline, sterile water, sodium carboxymethyl cellulose solid culture medium, Congo red dye, NaCl solution, and LB solid culture medium.

[0007] The application of bovine cellulase-producing Bacillus for inhibiting gastrointestinal pathogens in antibiotic-free breeding of livestock and poultry comprises the following steps:

[0008] 1. Methods

[0009] (1) Cow dung samples and treatment

[0010] Aseptically take 1.0 g of fresh fecal sample, place it in 9 mL of sterile saline, shake it at 37°C and 200 r / min for 30 min to fully mix it, then place it in a 75°C water bath for 15 min to kill non-spore-forming bacteria, and then centrifuge it at 900 r / min for 10 min to precipitate impurities in the fecal sample;

[0011] (2) Initial screening of cellulose-producing Bacillus

[0012] The processed fecal sample stock solution was diluted with sterile water to 10 -1 ~10 -6 , aseptically pipette 10 -4 , 10 -5 , 10 -6 200 μL of the supernatant was evenly spread on a sodium carboxymethyl cellulose solid culture medium and cultured at 37°C for 48 h. Congo red dye (1 g / L) was added to the plate to cover the colonies. After 1 h at room temperature, the dye was discarded and NaCl solution (0.06%, w / v) was added. The plate was rinsed 3 to 5 times. A sufficient amount of NaCl solution was added to the plate for 1 h. The diameter of the transparent zone (H) was measured and the H / D value was calculated.

[0013] The strains with larger hydrolysis zone diameter / pore diameter (H / D) ratio obtained through preliminary screening were inoculated into LB solid medium and streaked and purified three times to obtain purified strains;

[0014] (3) Identification of cellulose-producing bacteria

[0015] S1. Morphological identification

[0016] The purified strain was streaked on LB solid medium to grow a single colony, the morphology and size of the strain were observed, and a few colonies were selected for Gram staining and microscopic examination;

[0017] S2. Physiological and biochemical identification

[0018] By starch hydrolysis, gelatin liquefaction, 7% sodium chloride tolerance, VP test, propionate utilization, citrate utilization, catalase test, methyl red test, casein hydrolysis, L-arabinose fermentation, D-xylose fermentation, D-mannitol fermentation;

[0019] S3, 16S rDNA identification

[0020] The strains that can form hydrolysis circles in the initial screening were inoculated into LB solid culture medium, cultured at 28°C and 200r / min until OD = 0.8, 1mL of bacterial solution was taken respectively, centrifuged at 12000r / min for 2min, the supernatant was discarded, and the total DNA was extracted by chelex-100 method, and stored at -20°C for later use. The total genomic DNA of the strain was used as a template, and the gene sequence was amplified with the universal primers 27F / 1492F for bacterial genes. The PCR amplification system was 50.0μL: 2×Taq MasterMix 25.0μL, 1.0μL of upstream and downstream primers (10μmol / L), 1.0μL of DNA template, 22.0μL of ddH2O, and the amplification program was 95°C pre-denaturation for 4min; 95°C denaturation for 1min, 55°C annealing for 1min, and 72°C extension for 2min, for a total of 32 cycles, and the PCR products were sequenced, and the sequences were compared by EzBioCloud for homology analysis;

[0021] (4) Cellulase activity assay

[0022] S1. Preparation of crude enzyme solution

[0023] The strains to be tested were inoculated into 50 mL of seed culture medium respectively, and then inoculated into fermentation medium after culturing for 18 h. The culture was shaken at 37 °C and 180 r / min for 48 h. 5 mL of fermentation liquid was centrifuged at 5000 r / min for 10 min. The supernatant was the crude enzyme solution.

[0024] S2. Drawing of glucose standard curve

[0025] 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mL of 1 mg / mL glucose standard solution were respectively taken and placed in a colorimetric tube, and distilled water was added to 1.5 mL, and then 2 mL of DNS reagent was added. After thorough mixing, the mixture was boiled in boiling water for 5 min, and the volume was cooled and fixed to 15 mL. The OD value was measured at 540 nm with an enzyme marker, and the glucose content (mg) was used as the horizontal axis and the OD value was used as the vertical axis to draw a glucose standard curve, and a regression equation was fitted;

[0026] S3. Determination of cellulase activity of strains

[0027] Filter paper enzyme activity assay: Take 4 15mL test tubes, add 1.5mL acetic acid-sodium acetate buffer and 0.5mL crude enzyme solution to the test tubes, preheat the 4 test tubes at 50℃ for 10min, cut Xinhua quantitative filter paper into 50mg strips as substrate, roll them into small rolls and place them in three of the 15mL test tubes, and leave the remaining one without filter paper as a blank control, and react at this temperature for 60min. After taking out the samples, immediately add 2.0mL DNS reagent to each sample, place them in a 100℃ water bath for 5min, cool them down to 15mL after taking them out, measure the absorbance of each sample with an enzyme marker, compare the measured OD value with the drawn glucose standard curve, and convert it into glucose concentration;

[0028] S4. Calculation of enzyme activity

[0029] Definition of enzyme activity: At 50°C, the amount of enzyme required for 1 mL of crude enzyme solution to hydrolyze the substrate to produce 1 μg of glucose within 1 min is 1 cellulase activity unit (U / mL);

[0030] (5) Optimization of enzyme production conditions

[0031] Single-factor experiments were conducted on strains with strong enzyme production ability, and their enzyme activity was measured only when the culture time, initial pH of the culture medium, and inoculation amount changed; the initial pH of the fermentation medium was set to 6, the inoculation amount was 1%, and the effect of the fermentation time on the enzyme production of the strain was measured; the initial pH of the fermentation medium was set to 6, the inoculation amount was 1%, 2%, 3%, 4%, and 5%, and the optimal fermentation time was set, and the effect of the inoculation amount on the enzyme production of the strain was measured; the initial pH of the fermentation medium was set to 5, 6, 7, 8, and 9, the optimal inoculation amount, and the optimal fermentation time, and the effect of the initial pH of the fermentation medium on the enzyme production of the strain was measured;

[0032] (6) Drug sensitivity test

[0033] Eight antimicrobial drugs were selected to test the drug sensitivity of the selected probiotics by the disc method to understand the resistance of the probiotics to the current human and veterinary antimicrobial drugs. The drug sensitivity discs were selected and the drug sensitivity tests of the relevant pathogens were carried out according to the operation steps of the drug sensitivity disc diffusion method, including chloramphenicol, neomycin, gentamicin, amoxicillin, erythromycin, streptomycin, and rifampicin.

[0034] (7) Strain stress resistance

[0035] S1. Artificial gastric juice tolerance

[0036] The three strains of Bacillus were inoculated into NB medium respectively, and shaken for fermentation for 24 hours. Then, 10% of the inoculum was inoculated into 10 ml of artificial gastric juice with pH values ​​of 6.0, 7.0, and 8.0, respectively. After shaking for 3 hours, samples were taken for gradient dilution. Three gradients were taken for each sample, and three replicates were taken for each gradient. Plates with 30-300 colonies were selected for viable bacterial counts, and the counts at 0 hours were used as controls to calculate the survival rate.

[0037] S2. Artificial intestinal fluid tolerance

[0038] The three strains of Bacillus were inoculated into NB medium respectively, and shake-flask fermented for 24 hours. 1 mL of bacterial fermentation liquid was added into 9 mL of artificial intestinal fluid containing 0.1%, 0.2%, and 0.3% bile salts and shake-flask culture was performed. The count at 0 h was used as a control. After 3 hours, samples were taken for gradient dilution. Three gradients were taken for each sample, and three replicates were taken for each gradient. Plates with 50-300 colonies were selected for live bacteria count and survival rate was calculated.

[0039] (8) Antibacterial experiment

[0040] Agar diffusion method (punch diameter 9mm) was used, with Escherichia coli, Mycobacterium tuberculosis, and Staphylococcus aureus as indicator bacteria. 100μL of diluted solution was applied and then punched. Then 100μL of fermentation liquid, supernatant, and bacterial resuspension of the strain to be tested were added to the wells, and the wells were placed in a 30℃ constant temperature incubator for 24h. This was repeated 3 times, and the diameter of the inhibition zone was measured.

[0041] (9) Hemolysis test

[0042] The strain to be tested was streaked onto Columbia blood agar medium, with Staphylococcus aureus as the indicator strain, and observed after 48 hours of culture to see whether a hemolytic zone was produced;

[0043] (10) Data Statistical Analysis

[0044] The experimental data were statistically analyzed using SPSS21.0 software, and Excel software was used to draw graphs;

[0045] 2. Results and Analysis

[0046] (1) Screening of cellulose-degrading bacteria

[0047] S1. Initial screening results

[0048] Bacteria of different morphologies were picked from the separation plate, and 15 strains were finally selected based on the size, morphology and color of the single colony. A strain that could produce a transparent hydrolysis zone was obtained by Congo red staining, and was named 13068-2. The diameter of its transparent zone (D) was 23.20 mm, the strain diameter (d) was 16.17 mm, and the EI value was 2.53.

[0049] (2) Strain identification results

[0050] S1. Morphological characteristics of strains

[0051] The surface of 13068-1 colony is wrinkled, the colony is grayish white and frosted, the colony edge is irregular, and the surface is dry. Gram staining and spore staining show that the bacteria are Gram-positive bacteria under microscope, blue-purple, short rod-shaped, with oval spores;

[0052] S2. Physiological and biochemical identification

[0053] The results of physiological and biochemical tests of strain 13068-2 are shown in Table 1. Starch hydrolysis, gelatin liquefaction, 7% NaCl tolerance, VP test, casein hydrolysis, L-arabinose fermentation, D-xylose fermentation, D-mannose fermentation, and propionate utilization of strain 13068-2 were all positive, while citrate utilization, methyl red test, and catalase test were all negative. "+" means positive; "-" means negative.

[0054] Table 1 Physiological and biochemical characteristics of strain 13068-2

[0055]

[0056] (3) Sequencing analysis of strain 16S rDNA

[0057] The 16S rDNA sequencing results of the target strain 13068-2 were uploaded to EzBioCloud for comparison analysis, and the results showed that the sequence similarity between strain 13068-2 and Bacillus tequilensis was 99.87%;

[0058] (3) Optimization results of enzyme fermentation conditions of strains

[0059] When the initial pH of the fermentation medium was 6 and the inoculation amount was 1%, the fermentation time had an effect on the enzyme production capacity of 13068-2. When the fermentation time was 48 h, the enzyme production capacity of the strain reached the maximum, and the enzyme activity of 13068-2 was 92.01 U / mL.

[0060] When the initial pH of the fermentation medium was 6 and the fermentation time was 48 h, the effect of the inoculation amount on the enzyme production capacity of the strain was that when the inoculation amount was 4%, the enzyme production capacity of the strain reached the maximum, and the enzyme activity of strain 13068-2 was 103.62 U / mL;

[0061] When the inoculation amount was 4% and the fermentation time was 48h, the effect of the initial pH of the fermentation medium on the enzyme production capacity of the strain was that when the pH was 6, the enzyme activity of 13068-2 reached a maximum of 103.906U / mL;

[0062] The optimal fermentation conditions for strain 13068-2 were pH 6, inoculum size 4, and fermentation time 48 h.

[0063] (4) Results of drug sensitivity test

[0064] According to the test requirements and standards of the National Committee for Clinical Standards (NCCLS) as shown in Table 2, "S" means sensitive, "I" means intermediate, and "R" means resistant. The sensitivity of 13068-2 to 8 drugs is analyzed as shown in Table 3. 13068-2 is sensitive (S) to chloramphenicol, neomycin, gentamicin, and erythromycin, moderately sensitive (I) to neomycin, streptomycin, and amoxicillin, and resistant to rifampicin;

[0065] Table 2 Drug sensitivity judgment criteria

[0066]

[0067] Table 313068-2 Drug sensitivity test results for 8 drugs

[0068]

[0069] (5) Stress resistance analysis

[0070] S1, resistance to artificial gastric juice

[0071] The results of the artificial gastric juice resistance test of strain 13068-2 showed that the survival rate of strain 13068-2 after being treated with artificial gastric juice of pH 6, pH 7, and pH 8 for 3 hours was above 70%, indicating that it had good tolerance to artificial gastric juice, and the survival rate tended to increase with the increase of gastric juice pH.

[0072] S2, resistance to artificial intestinal fluid

[0073] The results of the artificial intestinal fluid resistance test of strain 13068-2 showed that after being treated with intestinal fluid with bile salt concentrations of 0.1%, 0.2%, and 0.3% for 3 hours, the survival rate showed a downward trend as the bile salt concentration increased, but was above 60%. This indicates that 13068-2 has a good tolerance to bile salts and can tolerate the bile salts secreted by the animal intestines to play a probiotic role.

[0074] (6) Antibacterial test results

[0075] The results of the in vitro antibacterial test of 13068-2 are shown in Table 4. The fermentation broth, supernatant and bacteria of 13068-2 all have antibacterial activity against Mycobacterium tuberculosis, the fermentation broth has antibacterial activity against Staphylococcus aureus, and 13068-2 has no antibacterial activity against Escherichia coli;

[0076] Table 413068-2 Antibacterial activity against different pathogenic indicator bacteria

[0077]

[0078] (7) Hemolytic analysis

[0079] Staphylococcus aureus showed a β-hemolysis zone on Columbia blood agar, but 13068-2 showed no hemolysis zone, indicating that 13068-2 had no hemolytic activity;

[0080] 3. Use

[0081] The isolated and selected bovine cellulase-producing Bacillus is used as a feed probiotic product to feed livestock, thereby inhibiting livestock gastrointestinal pathogens and promoting animal digestion and absorption.

[0082] Compared with the prior art, the present invention has the following beneficial effects:

[0083] (1) The bovine cellulase-producing Bacillus and its application for inhibiting gastrointestinal pathogens. The strain isolated from the feces of farmed animals comes from the animals themselves, can better adapt to the gastrointestinal environment of the animals, and is easy to colonize, providing new ideas for the development and application of probiotic products for feed. This study combines the characteristics of the local breeding environment in Guangxi Zhuang Autonomous Region, uses local materials, and screens out the Bacillus strain 13068-2 that is resistant to gastric juice, bile salts, high heat, and high enzyme activity. It also has a significant inhibitory effect on gastrointestinal pathogens. This strain has good cellulase production ability and is particularly suitable for use in tropical and subtropical habitats in the Beibu Gulf region. It has the effect of promoting digestion and improving feed growth performance;

[0084] (2) The bovine cellulase-producing Bacillus strain that inhibits gastrointestinal pathogens and its application, through antibacterial experiments, found that the strain has obvious antibacterial effects on Staphylococcus aureus and Mycobacterium tuberculosis; it has a certain therapeutic effect on diseases caused by Staphylococcus aureus and Mycobacterium tuberculosis infection; at the same time, it has a good tolerance to the gastrointestinal colonization environment, is sensitive to most commonly used antibiotics and has no hemolytic activity; at the same time, the strain has simple nutritional conditions, reproduces quickly, and has strong resistance to contamination by foreign bacteria, which is very suitable for use in the front line of aquaculture, conforms to the concept of reducing resistance in the aquaculture industry, and provides an excellent candidate strain for the further development of microbial preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 The bacterial strain of the present invention is point-connected with a hydrolysis transparent circle;

[0086] Figure 2 The colony morphology, Gram staining and spore staining microscopic morphology of the strain 13068-2 of the present invention are shown;

[0087] Figure 3 The effect of fermentation time on the enzyme production capacity of strain 13068-2 of the present invention;

[0088] Figure 4The effect of the inoculation amount of the present invention on the enzyme production capacity of strain 13068-2;

[0089] Figure 5 The effect of the initial pH on the enzyme production capacity of strain 13068-2 of the present invention;

[0090] Figure 6 The results of the drug sensitivity test of the strain 13068-2 of the present invention are as follows;

[0091] Figure 7 is the survival rate of the strain 13068-2 of the present invention in artificial gastric juice;

[0092] Figure 8 is the survival rate of the strain 13068-2 of the present invention in artificial intestinal fluid;

[0093] Fig. 9 This is the hemolytic test of the present invention. DETAILED DESCRIPTION

[0094] 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.

[0095] The present invention provides a bovine cellulase-producing bacillus for inhibiting gastrointestinal pathogens and application thereof. The method is introduced in detail below.

[0096] A bovine cellulase-producing bacillus for inhibiting gastrointestinal pathogens comprises the following materials for isolating the strain: fresh cow dung sample, sterile physiological saline, sterile water, sodium carboxymethyl cellulose solid culture medium, Congo red dye, NaCl solution, LB solid culture medium;

[0097] Application of bovine cellulase-producing Bacillus for inhibiting gastrointestinal pathogens in antibiotic-free breeding of livestock and poultry comprises the following steps:

[0098] 1. Methods

[0099] (1) Cow dung samples and treatment

[0100] Aseptically take 1.0 g of fresh fecal sample, place it in 9 mL of sterile saline, shake it at 37°C and 200 r / min for 30 min to fully mix it, then place it in a 75°C water bath for 15 min to kill non-spore-forming bacteria, and then centrifuge it at 900 r / min for 10 min to precipitate impurities in the fecal sample;

[0101] (2) Initial screening of cellulose-producing Bacillus

[0102] The processed fecal sample stock solution was diluted with sterile water to 10 -1 ~10 -6 , aseptically pipette 10 -4 , 10 -5 , 10 -6 200 μL of the supernatant was evenly spread on a sodium carboxymethyl cellulose solid culture medium and cultured at 37°C for 48 h. Congo red dye (1 g / L) was added to the plate to cover the colonies. After 1 h at room temperature, the dye was discarded and NaCl solution (0.06%, w / v) was added. The plate was rinsed 3 to 5 times. A sufficient amount of NaCl solution was added to the plate for 1 h. The diameter of the transparent zone (H) was measured and the H / D value was calculated.

[0103] The strains with larger hydrolysis zone diameter / pore diameter (H / D) ratio obtained through preliminary screening were inoculated into LB solid medium and streaked and purified three times to obtain purified strains;

[0104] (3) Identification of cellulose-producing bacteria

[0105] S1. Morphological identification

[0106] The purified strain was streaked on LB solid medium to grow a single colony, the morphology and size of the strain were observed, and a few colonies were selected for Gram staining and microscopic examination;

[0107] S2. Physiological and biochemical identification

[0108] By starch hydrolysis, gelatin liquefaction, 7% sodium chloride tolerance, VP test, propionate utilization, citrate utilization, catalase test, methyl red test, casein hydrolysis, L-arabinose fermentation, D-xylose fermentation, D-mannitol fermentation;

[0109] S3, 16S rDNA identification

[0110] The strains that can form hydrolysis circles in the initial screening were inoculated into LB solid culture medium, cultured at 28°C and 200r / min until OD = 0.8, 1mL of bacterial solution was taken respectively, centrifuged at 12000r / min for 2min, the supernatant was discarded, and the total DNA was extracted by chelex-100 method, and stored at -20°C for later use. The total genomic DNA of the strain was used as a template, and the gene sequence was amplified with the universal primers 27F / 1492F for bacterial genes. The PCR amplification system was 50.0μL: 2×Taq MasterMix 25.0μL, 1.0μL of upstream and downstream primers (10μmol / L), 1.0μL of DNA template, 22.0μL of ddH2O, and the amplification program was 95°C pre-denaturation for 4min; 95°C denaturation for 1min, 55°C annealing for 1min, and 72°C extension for 2min, for a total of 32 cycles, and the PCR products were sequenced, and the sequences were compared by EzBioCloud for homology analysis;

[0111] (4) Cellulase activity assay

[0112] S1. Preparation of crude enzyme solution

[0113] The strains to be tested were inoculated into 50 mL of seed culture medium respectively, and then inoculated into fermentation medium after culturing for 18 h. The culture was shaken at 37 °C and 180 r / min for 48 h. 5 mL of fermentation liquid was centrifuged at 5000 r / min for 10 min. The supernatant was the crude enzyme solution.

[0114] S2. Drawing of glucose standard curve

[0115] 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mL of 1 mg / mL glucose standard solution were respectively taken and placed in a colorimetric tube, and distilled water was added to 1.5 mL, and then 2 mL of DNS reagent was added. After thorough mixing, the mixture was boiled in boiling water for 5 min, and the volume was cooled and fixed to 15 mL. The OD value was measured at 540 nm with an enzyme marker, and the glucose content (mg) was used as the horizontal axis and the OD value was used as the vertical axis to draw a glucose standard curve, and a regression equation was fitted;

[0116] S3. Determination of cellulase activity of strains

[0117] Filter paper enzyme activity assay: Take 4 15mL test tubes, add 1.5mL acetic acid-sodium acetate buffer and 0.5mL crude enzyme solution to the test tubes, preheat the 4 test tubes at 50℃ for 10min, cut Xinhua quantitative filter paper into 50mg strips as substrate, roll them into small rolls and place them in three of the 15mL test tubes, and leave the remaining one without filter paper as a blank control, and react at this temperature for 60min. After taking out the samples, immediately add 2.0mL DNS reagent to each sample, place them in a 100℃ water bath for 5min, cool them down to 15mL after taking them out, measure the absorbance of each sample with an enzyme marker, compare the measured OD value with the drawn glucose standard curve, and convert it into glucose concentration;

[0118] S4. Calculation of enzyme activity

[0119] Definition of enzyme activity: At 50°C, the amount of enzyme required for 1 mL of crude enzyme solution to hydrolyze the substrate to produce 1 μg of glucose within 1 min is 1 cellulase activity unit (U / mL);

[0120] (5) Optimization of enzyme production conditions

[0121] Single-factor experiments were conducted on strains with strong enzyme production ability, and their enzyme activity was measured only when the culture time, initial pH of the culture medium, and inoculation amount changed; the initial pH of the fermentation medium was set to 6, the inoculation amount was 1%, and the effect of the fermentation time on the enzyme production of the strain was measured; the initial pH of the fermentation medium was set to 6, the inoculation amount was 1%, 2%, 3%, 4%, and 5%, and the optimal fermentation time was set, and the effect of the inoculation amount on the enzyme production of the strain was measured; the initial pH of the fermentation medium was set to 5, 6, 7, 8, and 9, the optimal inoculation amount, and the optimal fermentation time, and the effect of the initial pH of the fermentation medium on the enzyme production of the strain was measured;

[0122] (6) Drug sensitivity test

[0123] Eight antimicrobial drugs were selected to test the drug sensitivity of the selected probiotics by the disc method to understand the resistance of the probiotics to the current human and veterinary antimicrobial drugs. The drug sensitivity discs were selected and the drug sensitivity tests of the relevant pathogens were carried out according to the operation steps of the drug sensitivity disc diffusion method, including chloramphenicol, neomycin, gentamicin, amoxicillin, erythromycin, streptomycin, and rifampicin.

[0124] (7) Strain stress resistance

[0125] S1. Artificial gastric juice tolerance

[0126] The three strains of Bacillus were inoculated into NB medium respectively, and shaken for fermentation for 24 hours. Then, 10% of the inoculum was inoculated into 10 ml of artificial gastric juice with pH values ​​of 6.0, 7.0, and 8.0, respectively. After shaking for 3 hours, samples were taken for gradient dilution. Three gradients were taken for each sample, and three replicates were taken for each gradient. Plates with 30-300 colonies were selected for viable bacterial counts, and the counts at 0 hours were used as controls to calculate the survival rate.

[0127] S2. Artificial intestinal fluid tolerance

[0128] The three strains of Bacillus were inoculated into NB medium respectively, and shake-flask fermented for 24 hours. 1 mL of bacterial fermentation liquid was added into 9 mL of artificial intestinal fluid containing 0.1%, 0.2%, and 0.3% bile salts and shake-flask culture was performed. The count at 0 h was used as a control. After 3 hours, samples were taken for gradient dilution. Three gradients were taken for each sample, and three replicates were taken for each gradient. Plates with 50-300 colonies were selected for live bacteria count and survival rate was calculated.

[0129] (8) Antibacterial experiment

[0130] Agar diffusion method (punch diameter 9mm) was used, with Escherichia coli, Mycobacterium tuberculosis, and Staphylococcus aureus as indicator bacteria. 100μL of diluted solution was applied and then punched. Then 100μL of fermentation liquid, supernatant, and bacterial resuspension of the strain to be tested were added to the wells, and the wells were placed in a 30℃ constant temperature incubator for 24h. This was repeated 3 times, and the diameter of the inhibition zone was measured.

[0131] (9) Hemolysis test

[0132] The strain to be tested was streaked onto Columbia blood agar medium, with Staphylococcus aureus as the indicator strain, and observed after 48 hours of culture to see whether a hemolytic zone was produced;

[0133] (10) Data Statistical Analysis

[0134] The experimental data were statistically analyzed using SPSS21.0 software, and Excel software was used to draw graphs;

[0135] 2. Results and Analysis

[0136] (1) Screening of cellulose-degrading bacteria

[0137] S1. Initial screening results

[0138] Bacteria of different forms were picked from the separation plate, and 15 strains were finally selected based on the size, form and color of the single colony. The strains were initially screened by Congo red staining to obtain a strain that could produce a transparent hydrolysis zone. Figure 1 , named it 13068-2, and measured its transparent zone diameter (D) to be 23.20 mm, strain diameter (d) to be 16.17 mm, and EI value to be 2.53;

[0139] (2) Strain identification results

[0140] S1. Morphological characteristics of strains

[0141] The morphological characteristics and Gram staining and spore staining microscopy of strain 13068-2 are as follows Figure 2 As shown, the surface of the colony of 13068-1 is wrinkled, the colony is grayish white and frosted, the colony edge is irregular, and the surface is dry. Gram staining and spore staining show that the bacteria are Gram-positive bacteria under a microscope, blue-purple, short rod-shaped, and have oval spores.

[0142] S2. Physiological and biochemical identification

[0143] The results of physiological and biochemical tests of strain 13068-2 are shown in Table 1. Starch hydrolysis, gelatin liquefaction, 7% NaCl tolerance, VP test, casein hydrolysis, L-arabinose fermentation, D-xylose fermentation, D-mannose fermentation, and propionate utilization of strain 13068-2 were all positive, while citrate utilization, methyl red test, and catalase test were all negative. "+" means positive; "-" means negative.

[0144] Table 1 Physiological and biochemical characteristics of strain 13068-2

[0145]

[0146] (3) Sequencing analysis of strain 16S rDNA

[0147] The 16S rDNA sequencing results of the target strain 13068-2 were uploaded to EzBioCloud for comparison analysis, and the results showed that the sequence similarity between strain 13068-2 and Bacillus tequilensis was 99.87%;

[0148] (3) Optimization results of enzyme fermentation conditions of strains

[0149] When the initial pH of the fermentation medium was 6 and the inoculation amount was 1%, the effect of fermentation time on the enzyme production capacity of 13068-2 was as follows: Figure 3 ,When the fermentation time was 48h, the enzyme production capacity of the strain reached the maximum, and the enzyme activity of 13068-2 was 92.01U / mL;

[0150] When the initial pH of the fermentation medium was 6 and the fermentation time was 48 h, the effect of inoculation amount on the enzyme production capacity of the strain was as follows: Figure 4 , when the inoculation amount was 4%, the enzyme production capacity of the strain reached the maximum, and the enzyme activity of strain 13068-2 was 103.62U / mL;

[0151] When the inoculation amount was 4% and the fermentation time was 48 h, the effect of the initial pH of the fermentation medium on the enzyme production capacity of the strain was as follows: Figure 5 , when pH was 6, the enzyme activity of 13068-2 reached a maximum of 103.906U / mL;

[0152] The optimal fermentation conditions for strain 13068-2 were pH 6, inoculum size 4, and fermentation time 48 h.

[0153] (4) Results of drug sensitivity test

[0154] According to the test requirements and standards of the National Committee for Clinical Standards (NCCLS) as shown in Table 2, "S" means sensitive, "I" means intermediate, and "R" means resistant. The sensitivity of 13068-2 to 8 drugs is analyzed as shown in Table 3. 13068-2 is sensitive (S) to chloramphenicol, neomycin, gentamicin, and erythromycin, moderately sensitive (I) to neomycin, streptomycin, and amoxicillin, and resistant to rifampicin;

[0155] Table 2 Drug sensitivity judgment criteria

[0156]

[0157] Table 313068-2 Drug sensitivity test results for 8 drugs

[0158]

[0159] (5) Stress resistance analysis

[0160] S1, resistance to artificial gastric juice

[0161] The results of the artificial gastric fluid resistance test of strain 13068-2 are as follows Figure 7 As shown, the survival rate of 13068-2 treated with artificial gastric juice of pH 6, pH 7, and pH 8 for 3 hours was above 70%, and it had good tolerance to artificial gastric juice. With the increase of gastric juice pH, the survival rate showed an upward trend.

[0162] S2, resistance to artificial intestinal fluid

[0163] The results of the artificial intestinal fluid resistance test of strain 13068-2 are as follows Figure 8 As shown, after 3 hours of treatment with intestinal fluid at 0.1%, 0.2%, and 0.3% bile salt concentrations, there was a downward trend with the increase of bile salt concentration, but the survival rate was above 60%, which shows that 13068-2 has a good tolerance to bile salts and can tolerate the bile salts secreted by the animal intestines to play a probiotic role;

[0164] (6) Antibacterial test results

[0165] The results of the in vitro antibacterial test of 13068-2 are shown in Table 4. The fermentation broth, supernatant and bacteria of 13068-2 all have antibacterial activity against Mycobacterium tuberculosis, the fermentation broth has antibacterial activity against Staphylococcus aureus, and 13068-2 has no antibacterial activity against Escherichia coli;

[0166] Table 413068-2 Antibacterial activity against different pathogenic indicator bacteria

[0167]

[0168] (7) Hemolytic analysis

[0169] The test results are as follows Fig. 9 As shown, Staphylococcus aureus showed a β-hemolysis zone on Columbia blood agar medium ( Fig. 9 Left culture medium), 13068-2 without hemolysis zone ( Fig. 9 The right culture medium), indicating that 13068-2 has no hemolytic activity;

[0170] 3. Use

[0171] The isolated and selected bovine cellulase-producing Bacillus is used as a feed probiotic product to feed livestock, thereby inhibiting livestock gastrointestinal pathogens and promoting animal digestion and absorption.

[0172] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A bovine cellulase-producing Bacillus for inhibiting gastrointestinal pathogens, characterized in that: The materials for isolating the strains include the following: fresh cow dung samples, sterile physiological saline, sterile water, sodium carboxymethyl cellulose solid culture medium, Congo red dye solution, NaCl solution, and LB solid culture medium.

2. Use of the bovine cellulase-producing Bacillus for inhibiting gastrointestinal pathogens as claimed in claim 1 in antibiotic-free breeding of livestock and poultry.

3. The use of a bovine cellulase-producing Bacillus for inhibiting gastrointestinal pathogens in antibiotic-free breeding of livestock and poultry as claimed in claim 2, characterized in that: The steps include:

1. Methods (1) Cow dung samples and treatment Aseptically take 1.0 g of fresh fecal sample, place it in 9 mL of sterile saline, shake it at 37°C and 200 r / min for 30 min to fully mix it, then place it in a 75°C water bath for 15 min to kill non-spore-forming bacteria, and then centrifuge it at 900 r / min for 10 min to precipitate impurities in the fecal sample; (2) Initial screening of cellulose-producing Bacillus The processed fecal sample stock solution was diluted with sterile water to 10 -1 ~10 -6 , aseptically pipette 10 -4 , 10 -5 , 10 -6 200 μL of the supernatant was evenly spread on a sodium carboxymethyl cellulose solid culture medium and cultured at 37°C for 48 h. Congo red dye (1 g / L) was added to the plate to cover the colonies. After 1 h at room temperature, the dye was discarded and NaCl solution (0.06%, w / v) was added. The plate was rinsed 3 to 5 times. A sufficient amount of NaCl solution was added to the plate for 1 h. The diameter of the transparent zone (H) was measured and the H / D value was calculated. The strains with larger hydrolysis zone diameter / pore diameter (H / D) ratio obtained through preliminary screening were inoculated into LB solid medium and streaked and purified three times to obtain purified strains; (3) Identification of cellulose-producing bacteria S1. Morphological identification The purified strain was streaked on LB solid medium to grow a single colony, the morphology and size of the strain were observed, and a few colonies were selected for Gram staining and microscopic examination; S2. Physiological and biochemical identification By starch hydrolysis, gelatin liquefaction, 7% sodium chloride tolerance, VP test, propionate utilization, citrate utilization, catalase test, methyl red test, casein hydrolysis, L-arabinose fermentation, D-xylose fermentation, D-mannitol fermentation; S3, 16S rDNA identification The strains that can form hydrolysis circles in the initial screening were inoculated into LB solid culture medium, cultured at 28°C and 200r / min until OD = 0.8, 1mL of bacterial solution was taken respectively, centrifuged at 12000r / min for 2min, the supernatant was discarded, and the total DNA was extracted by chelex-100 method, and stored at -20°C for later use. The total genomic DNA of the strain was used as a template, and the gene sequence was amplified with the universal primers 27F / 1492F for bacterial genes. The PCR amplification system was 50.0μL: 2×Taq MasterMix 25.0μL, 1.0μL of upstream and downstream primers (10μmol / L), 1.0μL of DNA template, 22.0μL of ddH2O, and the amplification program was 95°C pre-denaturation for 4min; 95°C denaturation for 1min, 55°C annealing for 1min, and 72°C extension for 2min, for a total of 32 cycles, and the PCR products were sequenced, and the sequences were compared by EzBioCloud for homology analysis; (4) Cellulase activity assay S1. Preparation of crude enzyme solution The strains to be tested were inoculated into 50 mL of seed culture medium respectively, and then inoculated into fermentation medium after culturing for 18 h. The culture was shaken at 37 °C and 180 r / min for 48 h. 5 mL of fermentation liquid was centrifuged at 5000 r / min for 10 min. The supernatant was the crude enzyme solution. S2. Drawing of glucose standard curve 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mL of 1 mg / mL glucose standard solution were respectively taken and placed in a colorimetric tube, and distilled water was added to 1.5 mL, and then 2 mL of DNS reagent was added. After thorough mixing, the mixture was boiled in boiling water for 5 min, and the volume was cooled and fixed to 15 mL. The OD value was measured at 540 nm with an enzyme marker, and the glucose content (mg) was used as the horizontal axis and the OD value was used as the vertical axis to draw a glucose standard curve, and a regression equation was fitted; S3. Determination of cellulase activity of strains Filter paper enzyme activity assay: Take 4 15mL test tubes, add 1.5mL acetic acid-sodium acetate buffer and 0.5mL crude enzyme solution to the test tubes, preheat the 4 test tubes at 50℃ for 10min, cut Xinhua quantitative filter paper into 50mg strips as substrate, roll them into small rolls and place them in three of the 15mL test tubes, and leave the remaining one without filter paper as a blank control, and react at this temperature for 60min. After taking out the samples, immediately add 2.0mL DNS reagent to each sample, place them in a 100℃ water bath for 5min, cool them down to 15mL after taking them out, measure the absorbance of each sample with an enzyme marker, compare the measured OD value with the drawn glucose standard curve, and convert it into glucose concentration; S4. Calculation of enzyme activity Definition of enzyme activity: At 50°C, the amount of enzyme required for 1 mL of crude enzyme solution to hydrolyze the substrate to produce 1 μg of glucose within 1 min is 1 cellulase activity unit (U / mL); (5) Optimization of enzyme production conditions Single-factor experiments were conducted on strains with strong enzyme production ability, and their enzyme activity was measured only when the culture time, initial pH of the culture medium, and inoculation amount changed; the initial pH of the fermentation medium was set to 6, the inoculation amount was 1%, and the effect of the fermentation time on the enzyme production of the strain was measured; the initial pH of the fermentation medium was set to 6, the inoculation amount was 1%, 2%, 3%, 4%, and 5%, and the optimal fermentation time was set, and the effect of the inoculation amount on the enzyme production of the strain was measured; the initial pH of the fermentation medium was set to 5, 6, 7, 8, and 9, the optimal inoculation amount, and the optimal fermentation time, and the effect of the initial pH of the fermentation medium on the enzyme production of the strain was measured; (6) Drug sensitivity test Eight antimicrobial drugs were selected to test the drug sensitivity of the selected probiotics by the disc method to understand the resistance of the probiotics to the current human and veterinary antimicrobial drugs. The drug sensitivity discs were selected and the drug sensitivity tests of the relevant pathogens were carried out according to the operation steps of the drug sensitivity disc diffusion method, including chloramphenicol, neomycin, gentamicin, amoxicillin, erythromycin, streptomycin, and rifampicin. (7) Strain stress resistance S1. Artificial gastric juice tolerance The three strains of Bacillus were inoculated into NB medium respectively, and shaken for fermentation for 24 hours. Then, 10% of the inoculum was inoculated into 10 ml of artificial gastric juice with pH values ​​of 6.0, 7.0, and 8.0, respectively. After shaking for 3 hours, samples were taken for gradient dilution. Three gradients were taken for each sample, and three replicates were taken for each gradient. Plates with 30-300 colonies were selected for viable bacterial counts, and the counts at 0 hours were used as controls to calculate the survival rate. S2. Artificial intestinal fluid tolerance The three strains of Bacillus were inoculated into NB medium respectively, and shake-flask fermented for 24 hours. 1 mL of bacterial fermentation liquid was added into 9 mL of artificial intestinal fluid containing 0.1%, 0.2%, and 0.3% bile salts and shake-flask culture was performed. The count at 0 h was used as a control. After 3 hours, samples were taken for gradient dilution. Three gradients were taken for each sample, and three replicates were taken for each gradient. Plates with 50-300 colonies were selected for live bacteria count and survival rate was calculated. (8) Antibacterial experiment Agar diffusion method (punch diameter 9mm) was used, with Escherichia coli, Mycobacterium tuberculosis, and Staphylococcus aureus as indicator bacteria. 100μL of diluted solution was applied and then punched. Then 100μL of fermentation liquid, supernatant, and bacterial resuspension of the strain to be tested were added to the wells, and the wells were placed in a 30℃ constant temperature incubator for 24h. This was repeated 3 times, and the diameter of the inhibition zone was measured. (9) Hemolysis test The strain to be tested was streaked onto Columbia blood agar medium, with Staphylococcus aureus as the indicator strain, and observed after 48 hours of culture to see whether a hemolytic zone was produced; (10) Data Statistical Analysis The experimental data were statistically analyzed using SPSS21.0 software, and Excel software was used to draw graphs; 2. Results and Analysis (1) Screening of cellulose-degrading bacteria S1. Initial screening results Bacteria of different morphologies were picked from the separation plate, and 15 strains were finally selected based on the size, morphology and color of the single colony. A strain that could produce a transparent hydrolysis zone was obtained by Congo red staining, and was named 13068-2. The diameter of its transparent zone (D) was 23.20 mm, the strain diameter (d) was 16.17 mm, and the EI value was 2.

53. (2) Strain identification results S1. Morphological characteristics of strains The surface of 13068-1 colony is wrinkled, the colony is grayish white and frosted, the colony edge is irregular, and the surface is dry. Gram staining and spore staining show that the bacteria are Gram-positive bacteria under microscope, blue-purple, short rod-shaped, with oval spores; S2. Physiological and biochemical identification The results of physiological and biochemical tests of strain 13068-2 are shown in Table 1. Starch hydrolysis, gelatin liquefaction, 7% NaCl tolerance, VP test, casein hydrolysis, L-arabinose fermentation, D-xylose fermentation, D-mannose fermentation, and propionate utilization of strain 13068-2 were all positive, while citrate utilization, methyl red test, and catalase test were all negative. "+" indicates positive; "-" indicates negative. Table 1 Physiological and biochemical characteristics of strain 13068-2 (3) Sequencing analysis of strain 16S rDNA The 16S rDNA sequencing results of the target strain 13068-2 were uploaded to EzBioCloud for comparison analysis, and the results showed that the sequence similarity between strain 13068-2 and Bacillus tequilensis was 99.87%; (3) Optimization results of enzyme fermentation conditions of strains When the initial pH of the fermentation medium was 6 and the inoculation amount was 1%, the fermentation time had an effect on the enzyme production capacity of 13068-2. When the fermentation time was 48 h, the enzyme production capacity of the strain reached the maximum, and the enzyme activity of 13068-2 was 92.01 U / mL. When the initial pH of the fermentation medium was 6 and the fermentation time was 48 h, the effect of the inoculation amount on the enzyme production capacity of the strain was that when the inoculation amount was 4%, the enzyme production capacity of the strain reached the maximum, and the enzyme activity of strain 13068-2 was 103.62 U / mL; When the inoculation amount was 4% and the fermentation time was 48h, the effect of the initial pH of the fermentation medium on the enzyme production capacity of the strain was that when the pH was 6, the enzyme activity of 13068-2 reached a maximum of 103.906U / mL; The optimal fermentation conditions for strain 13068-2 were pH 6, inoculum size 4, and fermentation time 48 h. (4) Results of drug sensitivity test According to the test requirements and standards of the National Committee for Clinical Standards (NCCLS) as shown in Table 2, "S" is sensitive, "I" is intermediate, and "R" is resistant. The sensitivity analysis of 13068-2 to 8 drugs is shown in Table 3. 13068-2 is sensitive (S) to chloramphenicol, neomycin, gentamicin, and erythromycin, moderately sensitive (I) to neomycin, streptomycin, and amoxicillin, and resistant to rifampicin; Table 2 Drug sensitivity judgment criteria Table 313068-2 Drug sensitivity test results for 8 drugs (5) Stress resistance analysis S1, resistance to artificial gastric juice The results of the artificial gastric juice resistance test of strain 13068-2 showed that the survival rate of strain 13068-2 after being treated with artificial gastric juice of pH 6, pH 7, and pH 8 for 3 hours was above 70%, indicating that it had good tolerance to artificial gastric juice, and the survival rate tended to increase with the increase of gastric juice pH. S2, resistance to artificial intestinal fluid The results of the artificial intestinal fluid resistance test of strain 13068-2 showed that after being treated with intestinal fluid with bile salt concentrations of 0.1%, 0.2%, and 0.3% for 3 hours, the survival rate showed a downward trend as the bile salt concentration increased, but was above 60%. This indicates that 13068-2 has a good tolerance to bile salts and can tolerate the bile salts secreted by the animal intestines to play a probiotic role. (6) Antibacterial test results The results of the in vitro antibacterial test of 13068-2 are shown in Table 4. The fermentation broth, supernatant and bacteria of 13068-2 all have antibacterial activity against Mycobacterium tuberculosis, the fermentation broth has antibacterial activity against Staphylococcus aureus, and 13068-2 has no antibacterial activity against Escherichia coli; Table 413068-2 Antibacterial activity against different pathogenic indicator bacteria (7) Hemolytic analysis Staphylococcus aureus showed a β-hemolysis zone on Columbia blood agar, but 13068-2 showed no hemolysis zone, indicating that 13068-2 had no hemolytic activity; 3. Use The isolated and selected bovine cellulase-producing Bacillus is used as a feed probiotic product to feed livestock, thereby inhibiting livestock gastrointestinal pathogens and promoting animal digestion and absorption.

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