CN111187730B

CN111187730B - Bacillus subtilis and application thereof

Info

Publication number: CN111187730B
Application number: CN201911194242.8A
Authority: CN
Inventors: 李有全; 贾丹; 王锦明; 关贵全; 孟林明; 李贺海; 王佳慧; 刘军龙; 刘爱红; 殷宏; 罗建勋; 刘志杰; 白小明; 独军政; 高闪电; 郝建刚; 刘光远; 任巧云; 杨吉飞; 李东平
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2019-11-28
Filing date: 2019-11-28
Publication date: 2021-04-20
Anticipated expiration: 2039-11-28
Also published as: CN111187730A

Abstract

The invention discloses a bacillus subtilis BSC16a and application thereof. The bacillus subtilis is preserved in the general microbiological culture collection center of China Committee for culture Collection of microorganisms in 2019, 9 and 6, and the preservation number is CGMCC NO: 18473. The strain has good in-vitro probiotic characteristics. The strain provided by the invention plays a good probiotic role in the aspects of improving the growth performance of the fattening pigs, improving the body immunity and reducing the intestinal pathogenic bacteria. In addition, the bacillus subtilis BSC16a is used as a probiotic preparation to feed diarrhea young animals, and the strain has good prevention and treatment effects on diarrhea livestock. The strain can also be used as a feed additive to be applied to the prevention and treatment of diarrhea of piglets, lambs and calves.

Description

Bacillus subtilis and application thereof

Technical Field

The invention relates to bacillus subtilis and application thereof.

Background

Antibiotics have brought great benefits to human medicine and livestock breeding since their discovery. However, with the long-term use of antibiotics, the problems of the increase of the drug resistance of pathogenic bacteria, the antibiotic residue in livestock and poultry products, the pollution of the environment caused by excessive excretion and the like bring new challenges to the health of human beings. Subsequently, various countries have successively issued prohibitions that limit and prohibit the use of antibiotics in livestock farming. In 1986, sweden became the first country that regulated antibiotics in livestock and poultry feeds, and subsequently the european union, korea, usa, etc. successively proposed that antibiotics be banned for use as feed additives. The agricultural rural area of China declares that the drug feed additive will quit in 2019 in 2020. However, with the expansion of the breeding industry, the development of the livestock breeding industry is not limited to the production performance alone, and the quality and safety of livestock products are more and more valued by human beings. Therefore, it is imperative to find alternatives to antibiotics.

Disclosure of Invention

The invention provides a bacterium which can be used for livestock products.

The bacterium is a bacillus subtilis BSC16a which has been preserved in Beijing China general microbiological culture Collection center (CCTCC) in 2019, 9 and 6 days, and is classified as bacillus subtilisBacillus subtilis The preservation number is CGMCC NO: 18473.

The bacillus subtilis BSC16a can be applied to feeds of pigs, piglets, cows, calves, sheep and lambs, can also be applied to feeds, and can also be used for preparing veterinary drugs for treating diarrhea of the pigs, the cows and the sheep.

Furthermore, the bacillus subtilis of the invention can be used for preparing probiotic preparations.

The bacillus subtilis BSC16a is separated from fresh excrement of healthy pigs.

The strain provided by the invention has good tolerance to acid, cholate and gastrointestinal tract, and can inhibit the growth of various pathogenic bacteria, such as pathogenic escherichia coli, listeria monocytogenes, proteus vulgaris, clostridium perfringens and other pathogenic bacteria.

The strain provided by the invention has extremely strong capability of producing protease and amylase, and has strong capability of adhering to Caco-2 cells.

The strain provided by the invention has the effects of improving the growth performance and the organism immunity of the fattening pigs and reducing the number of escherichia coli in intestinal tracts. Experiments show that 30 healthy fattening pigs with similar age in days and weight are randomly divided into a test group and a control group, and 15 fattening pigs are selected. The test group feed is basic daily ration added with the bacillus subtilis with the dosage of 2 multiplied by 10⁸CFU/mL/head/day, control group fed normal basal diet, test period 60 d. After the test is finished, the average body weight and the average daily gain weight of the test group are higher than those of the control group; the test groups have IgG, IgM, IFN-gamma and IL-2 secretion levels higher than those of the test groupsA control group; the content of lactobacillus in the feces of the test group is higher than that of the control group, the content of bacillus is slightly higher than that of the control group, and the content of escherichia coli is lower than that of the control group.

The bacillus subtilis and other probiotic strains provided by the invention are combined into a composite microecological preparation, and the composite microecological preparation is applied to diarrhea piglets, has the effects of relieving and curing diarrhea diseases, and has an obvious effect.

Probiotics have received extensive attention and research as a new green, safe, residue-free antibiotic alternative. The American FDA and American society for feed control officials published a 42 microbial strain list capable of being directly fed to animals in 1989, microbial strains are supplemented to 46 in 2009, and 34 probiotics capable of being used for feed addition are proposed in 2013 by the Ministry of agriculture in China, wherein bacillus subtilis is contained, and the strains are generally considered to be safe. Bacillus subtilis belongs to unicellular prokaryotes, generally has no capsule, is peritrichogenous, and is motile. The bacillus subtilis is a gram-positive bacterium with strong stress resistance, and can form dormant spores to protect the bacteria under a severe environment.

After entering the intestinal tract of animals, the bacillus subtilis can play a probiotic role in the following modes: (1) oxygen is consumed, the oxygen concentration and the oxidation-reduction potential of the intestinal tract are reduced, the growth of anaerobic probiotics such as lactic acid bacteria is kept, and the growth of animals is promoted; (2) the growth of pathogenic microorganisms and the like is inhibited in a mode of secreting bacteriostat or generating acid substances to reduce the pH value of the intestinal tract; (3) the bacillus subtilis has the capability of degrading xylan, pectin and cellulose, decomposing sulfide and the like; (4) the adhesive is adhered and planted in the intestinal tract, so that the invasion of pathogenic bacteria to the intestinal mucosa is prevented, and the protective and repair effects on the intestinal mucosa barrier are exerted; (5) generating various nutrients such as vitamin B group, vitamin C, amino acid, growth promoting factor, etc., activating immunocyte activity and promoting animal growth; (6) accelerating the development of immune organs, stimulating the immune system of the organism and improving the immune function of the organism.

In the livestock breeding industry, Diarrhea (Diarrhea Disease) is a factor which seriously affects the healthy development of livestock breeding in China. Diarrhea can cause the function of the intestinal tract of animals to be declined, and the digestion and absorption of the intestinal tract are influenced, so that malnutrition is caused, and the body health is influenced. The factors causing diarrhea can be divided into two types of infectious factors and non-infectious factors, wherein the infectious factors mainly comprise bacterial diarrhea, viral diarrhea and parasitic diarrhea; non-infectious factors include environmental factors, improper feeding management, animal factors and the like. Animal diarrhea occurs mostly in young animals, and can cause death of young animals when severe. Research shows that normal microbial flora in gastrointestinal tract can be established as soon as possible by supplementing probiotics in the stage that intestinal flora is not established in the newborn animals, and beneficial microbial flora in young animals can be increased to inhibit pathogenic microbial attack so as to reduce diarrhea. The probiotic preparation can effectively prevent piglets from diarrhea and prevent the piglets from dying due to the diarrhea. In addition, the growth performance and the feed utilization rate of the bred animals are another important factor influencing the economic development. The probiotics can promote the digestion and absorption of various nutrient components in the feed by animal organisms, promote the growth and development of the animal organisms and reduce the feed cost. Therefore, the selection of proper probiotics as the feed additive to enhance the body resistance of the young livestock, reduce the diarrhea rate and improve the growth performance of the animals has important significance.

Drawings

FIG. 1, morphology of a single colony of Bacillus subtilis BSC16 a. The strain BSC16a was white, flat, round or irregular on NA plates, rough in surface and opaque.

FIG. 2, PCR amplification plot of Bacillus subtilis BSC16 a. The first lane is a DNAmarker with a DNA molecular mass of 2000. 1. Lane 2 shows the fragment length of the strain GHZ10a, which is about 1500 bp.

FIG. 3, a graph of Bacillus subtilis BSC16a inhibiting large intestine ATCC 43888.

FIG. 4, Bacillus subtilis BSC16a shows inhibition of Staphylococcus aureus ATCC 6538.

FIG. 5, growth graph of Bacillus subtilis BSC16 a.

Fig. 6, a proteolytic diagram of bacillus subtilis BSC16 a.

Detailed Description

The following is a detailed description of aspects of the invention with reference to the drawings.

EXAMPLE isolation and characterization of Bacillus subtilis

1.1 isolation of the Strain

The Bacillus subtilis BSC16a (Bacillus subtilis BSC16a) is used for separating and screening fresh excrement of healthy pigs in Sichuan province, and the total amount of the samples is 86 parts. The specific separation and purification method comprises the following steps: weighing 10g of a feces sample, diluting and uniformly mixing the feces sample with 90mL of sterile PBS, filtering the feces sample, taking 1mL of filtrate, centrifuging the filtrate, and removing supernatant; resuspending with 1mL nutrient broth culture solution, shake culturing at 37 deg.C for 48h, heating at 80 deg.C for 10min, centrifuging, and removing supernatant; adding 1mL of nutrient broth culture solution again, and performing shaking culture at 37 ℃ for 24h after vortexing; the mixture was diluted to a suitable amount, spread on Nutrient Agar (NA) plates and incubated at 37 ℃ for 10 h.

The formulation of the sterile PBS used in the invention is as follows:

weighing 8.0g of NaCl, 0.2g of KCl and Na₂HPO₄1.42 g，KH₂PO₄Adding 0.27 g of the mixture into 800mL of deionized water, fully stirring and dissolving, adding deionized water to a constant volume of 1L, dropwise adding hydrochloric acid to adjust the pH value to 7.4, carrying out autoclaving at 121 ℃ for 20min, and storing at room temperature for later use.

The nutrient broth culture medium of the invention comprises the following components:

nutrient broth culture medium: weighing 10.0g of peptone, 3.0g of beef extract and 5.0g of NaCl, adding into deionized water, adjusting the pH to 7.3 +/-0.2 after the volume is 1L, carrying out autoclaving at 121 ℃ for 20min, and storing at 4 ℃.

Nutrient Agar (NA) medium: 15g of agar powder is added on the basis of the nutrient broth culture medium.

Picking the suspected bacillus subtilis strain 952 from the NA plate, numbering the suspected bacillus subtilis strain 952, and respectively scribing and purifying on the NA plate; after each isolate is purified, selecting a single colony to be cultured in a nutrient broth culture medium at 37 ℃ for 18h by oscillation, and then preserving the bacterial liquid for later use.

1.2 morphological Observation of the Strain

Strain BSC16a was white, flat, round or irregular on NA plates, rough and opaque (fig. 1). Gram-positive is observed under an oil microscope after gram staining of the strain BSC16a, the thallus is in a bluish purple rod shape, and the size of a single cell is 0.7 mu m multiplied by 2 mu m to 3 mu m.

1.3 molecular biological characterization of the strains

Extracting the genome of the isolate by using a DNA extraction kit, identifying 16S rDNA universal primers 27F and 1492R by using bacteria, amplifying specific primers gyrA, gyrB, groEL and the like, and carrying out PCR amplification procedures as follows: 95 ℃ for 5 min; 95 ℃ for 45 s; 45s at 55 ℃; 72 ℃ for 1 min; repeat 35 cycles; 72 ℃ for 8 min; the amplified product was subjected to 1% agarose gel electrophoresis to identify a target fragment of about 1500bp in length (FIG. 2). The PCR product was sequenced by Western Onck department, Zessen biology, Inc., and the sequence was submitted to NCBI for BLAST analysis, among the 952 new isolates, 673 were Bacillus subtilis, the rest were Bacillus licheniformis, Bacillus cereus, Bacillus amyloliquefaciens, etc. Wherein the sequence homology of the isolated strain BSC16a and Bacillus subtilis is 100%, and the strain is identified as Bacillus subtilis by combining the identification result of the specific primer and the homology analysis of a phylogenetic tree. The sequence of Bacillus subtilis BSC16a gene is submitted to NCBI database, and the accession number MK208493 is obtained. The strain is submitted in 2019, 9 and 6 days and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO: 18473.

Example screening of Bacillus subtilis

2.1 screening of tolerant Bacillus subtilis

Inoculating 1mL of fresh culture solution of Bacillus subtilis into sterile PBS (pH 2.5) and containing 0.3% pig bile salt, respectively, culturing at 37 deg.C for 3 hr, sampling, and continuously diluting to 10%^-4、10^-50.1mL of diluent is absorbed and coated on an NA flat plate, the viable count is counted and determined after 12 hours of culture at 37 ℃, and the survival rate is calculated by taking the viable count of 0 hour as a control.

The tolerance test result shows that only 18 percent of tested samples in the tested bacillus subtilis are extremely sensitive to acid and bile salt, and the survival rate is lower than 30 percent; the rest can simultaneously tolerate the environment with pH 2.5 and 0.3 percent of bile salt, and the survival rate is higher than 80 percent.

2.2 screening of bacteriostatic Bacillus subtilis

Centrifuging the activated bacterial liquid at 4 deg.C and 6000r/min for 10min, filtering the supernatant with 0.22 μm microporous membrane, and storing at 4 deg.C; selecting 10 common pathogenic bacteria such as Escherichia coli and Staphylococcus aureus as indicator bacteria, and adjusting the activated pathogenic bacteria to 10% concentration with sterile PBS⁶CFU/mL bacterial suspension, using a sterile cotton swab to dip, and then evenly spread on a solid plate.

The antibacterial activity of the strain is determined by an Oxford cup method. Adding 150 μ L of the above sterile supernatant into Oxford cup, pre-diffusing at room temperature for 2h, culturing in an incubator at 37 deg.C for 18h, measuring the size of the zone of inhibition with a vernier caliper, and evaluating the effect of inhibition. The results show that only 34% of the tested samples have no growth inhibition effect on any pathogenic bacteria, and the rest tested samples have different degrees of inhibition effects on 10 common pathogenic bacteria. The bacteriostatic effect of the bacillus subtilis BSC16a is shown in table 1, the strain can inhibit the growth of various pathogenic bacteria such as escherichia coli (figure 3), listeria monocytogenes, proteus vulgaris and the like, has extremely strong inhibiting effect on clostridium perfringens, the diameter of an inhibition zone reaches 26.25 mm, the inhibiting effect on staphylococcus aureus ATCC6538 is shown in figure 4, and the BSC16a does not inhibit the growth of salmonella BSC.

TABLE 1 Bacillus subtilis BSC16a determination of bacteriostatic ability (mm)

2.3 screening of highly adhesive Bacillus subtilis

Fluorescence labeling of bacillus subtilis: centrifuging the activated bacillus subtilis liquid at 4 ℃ and 6000r/min and collecting thalli; washing with sterile PBS for 3 times, and adding into Fluorescein Isothiocyanate (FITC) labeling solution with working concentration of 500 μ g/mL; after vortex mixing, dark incubation is carried out for 2h at 37 ℃; bacteria centrifugally marked at 6000r/minThe solution was washed 3 times with PBS to remove unbound FITC; resuspending the cells in RPMI-1640 cell culture medium, adjusting the cell concentration to 2X 10⁸CFU/mL; and (3) sucking 100 mu L of the fluorescence-labeled bacterial liquid into a 96-well plate, and measuring the relative fluorescence intensity of the bacteria in the conditions that the absorption light wavelength is 485nm and the emission light wavelength is 530nm by using an enzyme-labeling instrument, namely the initial relative fluorescence value.

Preparation (1mL) of the Fluorescein Isothiocyanate (FITC) labeling solution of the present invention: taking out FITC stored at 4 ℃ and placing for 30min at room temperature, weighing 0.5g FITC and dissolving in 1mL dimethyl sulfoxide (DMSO) to obtain 500mg/mL solution; pipette 1. mu.L of the above solution into 999. mu.L of sterile PBS, a fluorescent labeling solution with a working concentration of 500. mu.g/mL.

Culture of Caco-2 cells: taking out 1 frozen Caco-2 cell from a liquid nitrogen tank, and rapidly thawing in a water bath at 37 ℃; sucking the cells in the cryopreserved tube into a 15mL centrifuge tube, adding 5mL of cell culture solution, and uniformly mixing; horizontally centrifuging for 15min under the condition of 1000 r/min; centrifuging, discarding supernatant, resuspending cells in 6mL cell culture medium supplemented with 10% fetal calf serum, 100U/mL penicillin, and 0.1mg/mL streptomycin, and standing at 37 deg.C with 5% CO₂Culturing in an incubator; after the cells are cultured for 24 hours, liquid in the bottle is discarded, cells growing adherent to the walls are rinsed by sterile PBS and cell culture solution is replenished; continuously culturing for 48h, and removing liquid in the bottle; after rinsing the cells with sterile PBS, 0.6mL of pancreatin was aspirated to digest the cells for 2min at 37 ℃; repeatedly blowing and beating the cells by using a pipette until the cells are in a single dispersed state; continuously culturing the cells after the cells are subcultured according to the ratio of 1: 6; and (4) carrying out cell passage for a plurality of times, and when the cell state is good, subpackaging the freshly-passaged cells into 12-hole culture plates for culture.

Adhesion test: 0.5mL of FITC-labeled Bacillus subtilis was added to each of 12-well plates cultured to give monolayer cells, and the mixture was incubated at 37 ℃ and 5% CO₂After the culture box is sealed and incubated for 2h, rinsing the culture box for 3-5 times by using sterile PBS to remove non-adhered bacteria; and transferring 300 mu L of pancreatin digested cells for 5min, and adding a cell culture solution to stop the reaction when the cells scatter like snowflakes under a microscope. 100 μ L of cell suspension was pipetted into a 96-well plate and the fluorescence intensity was measured and recorded as the relative fluorescence after adhesion.The adhesion rate of the strain is calculated by the formula:

the adhesion rate (relative fluorescence value after adhesion/initial relative fluorescence value) × 100%

The adhesion test result shows that in the 100 bacillus subtilis strains, the adhesion rate of 91 percent of the bacillus subtilis strains to Caco-2 cells is lower than 10 percent, the lowest adhesion rate is only 1.08 percent, the adhesion rate of only 9 bacillus subtilis strains to Caco-2 cells is higher than 10 percent, and the adhesion rate of the bacillus subtilis BSC16a is the highest and is 10.92 percent.

TABLE 2 Bacillus subtilis BSC16a adhesion rates

Bacterial strains	Adhesion Rate (%)
		Bacillus subtilis BSC16a	10.92±0.71

Therefore, bacillus subtilis BSC16a was selected as probiotic.

Example biological Properties Studies of Bacillus subtilis BSC16a

3.1 activation of Bacillus subtilis BSC16a and seed liquid preparation

The frozen Bacillus subtilis BSC16a was removed from the-20 ℃ freezer, rapidly thawed in a 37 ℃ water bath and streaked onto NA plates, cultured for 10h at 37 ℃ and then single colonies were picked up and inoculated into 10mL of nutrient broth medium, and shake-cultured for 18h at 37 ℃ and 220 rpm/min. After two successive passages, liquid is prepared for standby.

3.2 determination of the growth Curve of Bacillus subtilis BSC16a

The bacillus subtilis BSC16a strain of 3.1 is inoculated into a nutrient broth with the inoculation amount of 2 percent, and is cultured for 24 hours at 37 ℃ by shaking. By using non-inoculated bacteriaThe liquid culture medium is used as a control, and bacterial liquid OD is determined by sampling every 2h after inoculation₆₀₀The value is obtained. As shown in FIG. 5, the Bacillus subtilis BSC16a entered the logarithmic phase 2 hours after inoculation, and the cells entered the stationary phase 10 hours after inoculation, at which time the cell concentration was the highest and the activity was the best, and the OD was the highest₆₀₀Was 3.55.

3.3 Bromoidic acid and bile salt tolerance assay

Adding 1mL of the new isolate into 9mL of PBS with different pH values (1.0, 1.5, 2.0, 2.5, 3.0) or different concentrations of porcine bile salt (0.0%, 0.1%, 0.2%, 0.3%, 0.6%), culturing at 37 deg.C for 0, 2, 4 hr, sampling, and diluting to 10%^-4、 10^-5And spread on NA plates, incubated at 37 ℃ for 10 hours, and counted. The survival rate of the strain BSC16a in acid and bile salt environments is shown in Table 3, the survival rate of the strain BSC16a is higher than 75.00% after the strain BSC16a is respectively endured for 2h and 4h under the condition of pH being 3.0 and above, and the survival rate of the strain BSC16a is lower in the environment of pH being 2.5 and below. The bacillus subtilis BSC16a can grow under different bile salt conditions, but has poor tolerance to the environment with 0.6% bile salt concentration.

TABLE 3 survival rate of Bacillus subtilis BSC16a in acid and bile salt environments

Gradient of pH	pH＝2.0	pH＝2.5	pH＝3.0	pH＝3.5	pH＝4.0
						2h	20.14％	57.19％	89.04％	98.94％	100.00％
4h	1.05％	38.23％	75.00％	79.48％	84.78％
						Concentration of bile salts	0.0％	0.1％	0.2％	0.3％	0.6％
2h	96.16％	90.57％	86.32％	77.13％	55.00％
						4h	90.16％	87.14％	70.15％	58.12％	30.14％

3.4 tolerance assay of Bacillus subtilis BSC16a in simulated gastrointestinal solutions

Simulated gastric fluid: pepsin (1:15000) was weighed and dissolved in sterilized normal saline (0.9% w/v, pH adjusted to 3.0) to prepare a solution with a concentration of 3 g/L. After filtration through a 0.22 μm sterile filter membrane, the cells were stored at 4 ℃.

Simulating intestinal juice: trypsin (1:250) was weighed out and dissolved in sterilized normal saline (0.9% w/v, pH adjusted to 8.0) to prepare a solution with a concentration of 1g/L, and 0.3% bile salt was added. After filtration through a 0.22 μm sterile filter membrane, the cells were stored at 4 ℃.

After overnight incubation of the strain BSC16a, it was centrifuged and resuspended in sterile PBS at a concentration of 1X 10⁹CFU/mL. Adding 1mL of the bacterial liquid into 9mL of simulated gastric juice, culturing at 37 ℃ for 1, 2 and 3 hours, sampling and counting respectively. After 3h, 1mL of the above solution was added to the simulated intestinal fluid, and after incubation at 37 ℃ for 2, 4, and 6h, samples were taken and counted, respectively. The results of the BSC16a tolerance are shown in Table 4, after 3h of simulated gastric juice treatment and 6h of simulated intestinal juice treatment, the survival rate of the strain BSC16a is still over 88%, and the strain is known to have good tolerance to the gastrointestinal environment.

TABLE 4 detection of tolerance of Bacillus subtilis BSC16a in gastrointestinal solutions [ (X. + -. s). times.10⁷CFU/mL]

3.5 determination of protease and amylase producing ability of Bacillus subtilis BSC16a

Proteolytic assay agar plates: weighing 50g of skimmed milk powder and 15g of agar, adding into 1L of water, and sterilizing at 113-115 ℃ for 20 min.

Starch hydrolysis test agar plate: weighing 5g of beef extract, 10g of peptone, 2g of soluble starch and 15g of agar, adding the beef extract, the peptone, the soluble starch and the agar into 1L of water, adjusting the pH value to 7.2-7.6, and sterilizing at 113-115 ℃ for 20 min.

Centrifuging the activated BSC16a bacterial solution at 4 ℃ and 6000r/min for 10min, filtering by a 0.22-micron microporous filter membrane, and storing the supernatant at 4 ℃ for later use; uniformly punching holes on the two agar plates by using an oxford cup; 150 μ LBSC16a cell-free fermentation supernatant was pipetted into the wells, incubated at 37 ℃ and the diameter of the hydrolysis loop was measured at 3h, 6h, 9h, 12h and 24h, respectively. As shown in Table 5, the BSC16a has extremely high protease and amylase producing capability, and has the effects of decomposing nutrients and promoting nutrient absorption when being applied to feed additives to a certain extent. The proteolytic capacity of the strain BSC16a is shown in fig. 6.

TABLE 5 Bacillus subtilis BSC16a results of hydrolysis Ring diameter variation with time (mm)

	3h	6h	9h	12h	24h
						Proteolytic loop	13.65	14.53	16.32	18.44	20.04
Starch hydrolysis ring	10.33	12.57	13.29	15.87	18.63

Example application of bacillus subtilis BSC16a in improving growth performance and body immunity of fattening pigs

4.1 animal feeding management

Selecting 30 healthy fattening pigs with similar age in days and similar weight, randomly dividing the pigs into a test group and a control group, and numbering, wherein 15 fattening pigs are selected in each group. The control group is fed with normal feed, and the feed of the test group is added with Bacillus subtilis BSC16a (2 × 10) based on the control group⁸CFU/mL/head/day). The test period was 60 days.

4.2 study of growth enhancement

The weight of each pig was weighed and recorded at 0d, 30d and 60d of the feeding trial, respectively. As can be seen from Table 6, in the initial stage of the test, the initial body weights of the test group and the control group are not obviously different, and after the test group and the control group are fed for 30d and 60d, the average body weight and the average daily gain weight of the test group are higher than those of the control group, which shows that the strain BSC16a has the effect of improving the growth performance of the fattening pigs.

TABLE 6 influence of Bacillus subtilis BSC16a on weight of fattening pigs

	Test group	Control group
			0d body weight (kg)	36.41±2.03	35.77±1.18
30d body weight (kg)	59.81±1.69	57.37±1.17
			Average daily gain (kg) of 30 days	0.78±0.16	0.72±0.06
60d body weight (kg)	82.61±1.99	77.17±2.25
			Average daily gain (kg) over 60 days	0.77±0.06	0.69±0.15

4.3 study of improving immune function

Collecting blood sample of each pig after feeding experiment, standing at room temperature for 2 hr, centrifuging at 3500r/min for 15min, collecting supernatant, and storing serum sample at-20 deg.C. Detecting the content of IgA, IgM, IgG, IFN-gamma and IL-2 in serum by using an ELASA enzyme linked immunosorbent assay kit. The influence of the bacillus subtilis BSC16a on the immune function of the fattening pigs is shown in Table 7, and in three detected immune globulins and two immune cell factors, compared with a control group, the four immune indexes of a test group are obviously changed except IgA, which shows that the bacillus subtilis can improve the secretion levels of IgG, IgM, IFN-gamma and IL-2 in serum.

TABLE 7 influence of Bacillus subtilis BSC16a on the immune function of fattening pigs

4.4 Effect on gut microbiology

After the test, one pig was randomly selected from the two groups of test pigs, and fresh fecal samples were collected and stored separately using sterile spoons. Weighing 1g of the feces, mixing with 9mL of sterile PBS, uniformly mixing by vortex, and performing gradient dilution. Heating 1mL of the diluted solution at 80 deg.C for 10min, and diluting to 10%^-3-10^-5Coating the diluted solution with the concentration on an NA flat plate, culturing at 37 ℃ for 12h, and counting the number of the bacillus; coating the diluent on an MRS solid plate, carrying out anaerobic culture at 37 ℃ for 24h, and counting the number of lactic acid bacteria; and coating the diluent on a MacconKa solid plate, culturing at 37 ℃ for 18h, and counting the number of escherichia coli in the feces.

The MRS solid flat plate used in the invention has the following formula:

10.0g of peptone, 10.0g of beef extract powder, 5.0g of yeast extract powder, 20.0g of glucose, 801.0 mL of Tween and K₂HPO₄·3H₂O2.0 g, anhydrous sodium acetate 5.0g, triammonium citrate 2.0g, MgSO₄·7H2O 0.29g，MnSO₄·H₂0.058g of O and 15g of agar. Adding the above components into distilled water, diluting to 1L, and autoclaving at 121 deg.C for 20 min.

The recipe of the mcnkai solid flat plate used in the invention is as follows:

20.0g of peptone, 10.0g of lactose, 5.0g of bile salt, 5.0g of sodium chloride, 0.075g of neutral red and 15.0g of agar, adding the components into distilled water, fixing the volume to 1L, adjusting the pH to 7.2 +/-0.2, and autoclaving at 121 ℃ for 20 min.

The effect on microorganisms in feces after feeding bacillus subtilis BSC16a is shown in Table 8, the content of bacillus in feces of the test group is slightly higher than that of the control group, the content of lactic acid bacteria is increased, and the content of escherichia coli is decreased.

TABLE 8 Effect of Bacillus subtilis BSC16a feeding on microorganisms in pig manure (lg CFU/g)

	Bacillus	Lactic acid bacteria	Escherichia coli
				Control group	4.97	6.05	7.05
Test group	5.18	6.42	6.49

Example application of five-bacillus subtilis BSC16a in prevention and treatment of diarrhea of young livestock

The bacillus subtilis BSC16a is mixed with laboratory self-separated lactobacillus johnsonii GHZ10a and lactobacillus salivarius ZLp4b according to a certain proportion to form a composite microecological preparation which is primarily applied to prevention and treatment of piglet diarrhea. The results are shown in table 9, 1511 head diarrhea piglets in 5 farms of gansu yongjing, Pingyang, folk music, Shanxi Yangling and Henan Minggu are fed respectively, the diarrhea symptoms are obviously relieved in the next day of feeding, 80% of sick animals do not have the diarrhea symptoms after 3 days of feeding, the sick animals are basically cured after 3-5 days of continuous feeding, no animal death occurs in the Gansu Pingyang farms during feeding, the cure rate reaches 100%, individual sick animals in the rest farms die due to severe diarrhea and other factors, and 5 head pigs in the Gansu Mingyu farms die due to severe dehydration. Except that the cure rate of diarrhea of piglets in the Yangtze rabdosia Shaanxi farm is 87.60%, the cure rate of diarrhea cases in other farms is higher than 90.00%. In the feeding test, the probiotic preparation has obvious effect of curing diarrhea of piglets and has no phenomena of repeated disease conditions and the like. In addition, the composite microecological preparation is primarily applied to diarrhea lambs and calves, and has the effects of relieving and curing diarrhea diseases. Therefore, the bacillus subtilis BSC16a can be used as a compound microbial ecological agent compound strain to be applied to prevention and treatment of diarrhea of young animals.

TABLE 92019 statistical table of control results of diarrhea cases of piglets

Region of land

Animal(s) production

Cultivation scale

Number of onset of disease

Number of rehabilitation

Number of deaths

Cure rate

Mortality rate

Gansu yongjing

Piglet

2000

326

302

20

92.63％

6.67％

Gansu calm and cool

Piglet

600

56

0

100.00％

0.00％

Gansu folk music

Piglet

8000

532

496

36

93.23％

7.67％

Shaanxi Yangling

Piglet

4000

121

106+14*

3

87.60％

2.47％

Right of south river

Piglet

6000

476

452

24

94.96％

5.04％

Remarking: wherein 14 of the above drugs are drenched for 3 days to find no obvious improvement, and then the drugs are treated with antibiotics, wherein 11 of the drugs are recovered, and 3 of the drugs die. But in statistics, it was not calculated according to the number of convalescent pigs.

Claims

1. Bacillus subtilis (B.subtilis)Bacillus subtilis) The BSC16a is preserved in China general microbiological culture Collection center (CGMCC) at 2019, 9 and 6, with the preservation number of CGMCC NO: 18473.

2. The use of the bacillus subtilis BSC16a of claim 1 in the preparation of piglet, lamb, calf feed.

3. The use of the bacillus subtilis BSC16a of claim 1 in the preparation of veterinary drugs for the treatment of diarrhea in piglets, lambs, and calves.

4. Use of the bacillus subtilis according to claim 1 for the preparation of a probiotic formulation.