CN113862196B

CN113862196B - Bacillus subtilis SD-KC-001 and application thereof

Info

Publication number: CN113862196B
Application number: CN202111295487.7A
Authority: CN
Inventors: 张秉胜; 李锦�; 刘吉华; 王梅雪; 程英杰; 张永立
Original assignee: Zhonggu Biotechnology Dalian Co ltd
Current assignee: Zhonggu Biotechnology Dalian Co ltd
Priority date: 2021-11-03
Filing date: 2021-11-03
Publication date: 2023-09-08
Anticipated expiration: 2041-11-03
Also published as: CN113862196A

Abstract

The invention belongs to the technical field of feed additives, and discloses bacillus subtilis SD-KC-001 and application thereof. The invention provides bacillus subtilis which is separated from soil, is obtained through 16S rRNA molecular biology identification, NCBI sequence BLAST comparison and phylogenetic tree construction, and is named as bacillus subtilis SD-KC-001. The bacillus subtilis SD-KC-001 has strong acid resistance, salt resistance and heat resistance, has strong growth capacity, can produce enzymes such as amylase and protease at high yield, and has good antibacterial capacity. Through the discovery of feeding animals, the strain can improve the weight gain, reduce the drug cost, reduce the death rate and the like after being added into the feed, and can be used as a feed additive for livestock and poultry farming.

Description

Bacillus subtilis SD-KC-001 and application thereof

Technical Field

The invention belongs to the technical field of feed additives, and relates to bacillus subtilis SD-KC-001 and application thereof.

Background

Feed additives (Feed additives) play an important role in the field of animal husbandry production, and research and application of Feed additives have been a major research focus of animal nutritionists for a long time. Antibiotics (antibiotics) play an important role in animal feeding as feed additives, and can effectively reduce the morbidity of livestock and poultry and improve the production performance of animals. However, after the 40 s of the 20 th century, along with the wide application of antibiotics in the feeding field, people gradually realize the disadvantages of using antibiotics in large quantities, such as causing the problems of dysbacteriosis of the gastrointestinal tract of animals, reducing the immune function level, and drug residues, thereby affecting human health. Thus, most countries in the world have established relevant laws that limit and prohibit the use of antibiotics as feed additives. The search for new safe, effective additives that can be used in place of antibiotics has become a hotspot for additive research.

Microbial feed additives (DFMs) are important development directions for developing safe feed additives and replacing antibiotics in recent years. The research shows that the microbial feed additive for raising livestock and poultry has the characteristics of no pollution, no public hazard and the like, and has the advantages of adjusting the balance of intestinal flora, improving the immunity of animals, improving the digestion and absorption functions and the like. In particular, bacillus is a microorganism feed additive with wide application, has high temperature resistance and high pressure resistance compared with other microorganism feed additives, can still keep good activity through the processes of feed pelletization and the like, and is more suitable for being added and applied in the feed processing process. However, the existing problems are that bacillus products are mainly focused on the application of good enzyme production characteristics, and the bacteriostasis and the immunity improvement of the bacillus products are inferior to those of lactobacillus or cannot withstand gastric juice, bile, intestinal juice and other environments. Most of the lactobacillus has the characteristics of producing antibacterial substances and improving immunity, but is sensitive to temperature, and even if the lactobacillus is subjected to coating stabilization treatment, the heat resistance and bile acid resistance of the lactobacillus are still poor, so that the lactobacillus is influenced to be added and applied in the feed processing process.

Disclosure of Invention

In order to solve the problems, the bacillus subtilis (Bacillus subtilis) strain with excellent efficacy and characteristics has the advantages of enzyme production capacity, bacteriostasis capacity, growth capacity, gastric acid resistance, intestinal juice survival capacity, bile salt resistance, heat resistance and the like, is used for producing microbial feed additives, and has outstanding efficacy when applied to livestock and poultry cultivation.

The invention provides bacillus subtilis (Bacillus subtilis SD-KC-001), which is separated from soil, and is obtained by BLAST comparison of NCBI sequences through 16S rRNA molecular biology identification, and is named as bacillus subtilis SD-KC-001 (Bacillus subtilis SD-KC-001).

Based on the above findings, the present invention provides a Bacillus subtilis SD-KC-001 (Bacillus subtilis SD-KC-001) with a deposit number of: CGMCC No.23135.

Specific strain deposit information is as follows:

preservation unit name: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

Preservation address: beijing city, the North Chen Xili No.1, 3 national academy of sciences of China for microbiology

Preservation date: 2021, 8 and 10 days

Preservation number: CGMCC No.23135

Further, the bacillus subtilis SD-KC-001 16S rRNA gene sequence is shown in SEQ.NO. 1.

The biological characteristics of the bacillus subtilis SD-KC-001 are as follows: single cells (0.7-0.8) mu m x (2.0-3.0), gram positive, no capsule, periphyton and capable of moving, can form endogenous stress-resistant spores, are elliptic to columnar, are positioned in the center of the thalli or slightly deviated, do not expand after the spores are formed, and have the spore (0.6-0.9) mu m x (1.0-1.5) mu m.

The invention also provides a screening method of the bacillus subtilis SD-KC-001, which comprises the following steps:

step S1: preparation 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Soil dilutions of different dilution factors;

step S2: coating the soil diluents with different dilution factors in the step S1 on a flat-plate culture medium;

step S3: step S2, performing primary screening after the plate culture;

step S4: step S3, performing primary screening of extracellular enzyme activity by a plate method, determination of extracellular enzyme activity by a fermentation broth method, determination of bacteriostasis capacity, determination of growth capacity, determination of tolerance to artificial gastric juice and intestinal juice, determination of tolerance to bile salts and determination of heat resistance to high temperature on the strain subjected to primary screening, and verifying the characteristics of the strain;

step S5: and (3) carrying out morphological, physiological and biochemical analysis and identification on the strains screened in the step S4.

The invention also provides a fermentation method of the bacillus subtilis SD-KC-001, which comprises the following steps:

step S1: preparing seeds; activating the strain, preserving the strain at the temperature of-80 ℃ for later use in a glycerol making tube, inoculating the strain into a corresponding culture medium for expansion culture before fermentation production, wherein a container is an eggplant bottle or a triangular bottle, and the culture medium consists of the following components in percentage by mass: beef extract 0.20-0.50%, sodium chloride 0.30-0.80%, peptone 0.80-1.20%, agar 1.50-2.00%, and water in balance, pH 7.0-7.5, wrapping cotton plug part with oilpaper after bacteria grow sufficiently, and transferring to a refrigerator at 2-8deg.C for preservation as primary strain;

step S2: weighing and preparing a culture medium; during fermentation production, the raw materials of each component of the culture medium of the seed tank and the main fermentation tank are accurately weighed strictly according to the production formula of the culture medium, the volume of the seed fermentation tank is 30L, and the filling amount of the culture medium is 20L; the main fermentation tank has a volume of 2000L and a culture medium loading amount of 1400L, the seed tank and the main fermentation tank have the same culture medium production formula, and the culture medium consists of the following components in percentage by mass: 0.20 to 0.70 percent of sucrose, 0.10 to 0.40 percent of urea, 0.40 to 0.80 percent of dipotassium hydrogen phosphate, 0.20 to 0.40 percent of monopotassium phosphate, 0.02 to 0.06 percent of yeast extract, 0.01 to 0.04 percent of ferric chloride, 0.01 to 0.04 percent of calcium carbonate, 0.05 to 0.20 percent of magnesium sulfate, 0.01 to 0.04 percent of manganese sulfate, 0.30 to 0.80 percent of starch, 1.00 to 1.50 percent of bean pulp, 0.80 to 1.20 percent of soybean, 0.01 to 0.02 percent of dichlord, the balance of water and pH value of 6.0 to 7.0;

step S3: sterilizing for the first time; and (3) introducing high-temperature steam into all pipelines, seed tanks, material supplementing tanks and fermentation tanks of the fermentation equipment for the first time to sterilize before fermentation culture, wherein the sterilization temperature is 121 ℃ and the time is 20min.

Step S4: feeding materials; after the temperature of the fermentation tank is reduced to room temperature, respectively putting the raw materials of each component of the culture medium which are weighed and prepared into a seed tank and the fermentation tank;

step S5: sterilizing for the second time; introducing high-temperature steam to perform secondary sterilization and disinfection, wherein the sterilization temperature is 121 ℃ and the sterilization time is 20min;

step S6: inoculating a seed tank; the fermentation conditions are as follows: fermenting and culturing for 8-14 h at 35-38 ℃ under the conditions that the tank pressure is 0.02-0.06 Mpa, the aeration ratio is 1:0.5-1:1 and the stirring revolution is 150 rpm-200 rpm, and inoculating the first-level strain into a seed tank for secondary strain culture;

step S7: inoculating the main fermentation tank; after the secondary strain is spread and cultivated, the secondary strain is directly inoculated into a fermentation tank for fermentation production through a system control pipeline, and the fermentation process conditions are the same as those of the seed tank in the step S6.

Step S8: judging the fermentation end point, carrying out sampling microscopic examination on fermentation liquor at regular time in the fermentation process, and stopping fermentation when the spore rate reaches more than 90%;

step S9: centrifuging and accumulating bacteria, conveying fermented bacterial liquid to a high-speed tubular centrifuge through a pipeline, centrifuging and collecting bacterial mud, wherein the inner diameter of a centrifuge drum is 142mm, the rotating speed of the drum is 14000r/min, and the throughput capacity is 2500L/h, so that the fermentation liquid is ensured to be treated within 60 min;

step S10: preparing a bacterial agent, adding a certain amount of adsorbent into centrifugally collected bacterial mud according to the proportion of 1:1-3, drying at low temperature, crushing, screening, packaging and the like to prepare a high-concentration bacterial agent, and sealing and preserving for later use;

step S11: and (3) detecting the number of beneficial bacteria, sampling the high-concentration microbial inoculum, and then conveying the high-concentration microbial inoculum to a laboratory for detecting the number of living bacteria so as to accurately calculate the dosage in the process of producing the microbial feed additive.

The invention also provides application of the bacillus subtilis SD-KC-001 or fermentation liquor thereof in preparing feed additives for livestock and poultry raising industry, which is mainly used for animals such as pigs, chickens and cattle, and the bacillus subtilis SD-KC-001 or fermentation liquor is added into compound feed for uniform mixing and feeding during use.

The invention also provides a production method of the bacillus subtilis SD-KC-001 feed additive, which comprises the following steps:

step S1: weighing, namely accurately weighing a high-concentration microbial inoculum, a carrier and a diluent according to the following feed additive formula, wherein the feed additive formula comprises the following components in percentage by mass: 15-30 parts of bacillus subtilis SD-KC-001 high-concentration microbial inoculum, 5-10 parts of maltodextrin, 20-35 parts of montmorillonite and 25-60 parts of soluble starch;

step S2: feeding, namely feeding the raw materials weighed in the step S1 into a lifting machine or a vacuum feeding machine through a feeding port, and conveying the raw materials to a mixer;

step S3: after mixing, determining the optimal mixing time to be 8-10 min according to the determined mixing uniformity to ensure that the variation coefficient is not more than 5%, and determining the optimal mixing time to be 8min by using a V-shaped mixer or a biconical mixer;

step S4: packaging, wherein after the mixing time is reached, the mixer is automatically stopped, weighing is carried out according to preset packaging specifications through an automatic quantitative packaging scale, the metering deviation is not more than 3 per mill, and after quantitative weighing, an automatic bag sewing machine is used for sewing;

step S5: and (5) checking, warehousing, sampling and detecting that the number of beneficial bacteria reaches the product standard requirement during packaging, confirming that the product is qualified, and warehousing and preserving.

Further, when the bacillus subtilis SD-KC-001 feed additive is used for processing the feed of the cultured animals, the addition proportion of the bacillus subtilis SD-KC-001 feed additive in the compound feed is as follows: 0.1-0.3%, and the specific dosage is adjusted according to different animal varieties or different physiological stages of the same animal.

Compared with the prior art, the invention has the beneficial effects that:

the bacillus subtilis SD-KC-001 has strong acid resistance, salt resistance and heat resistance, has strong growth capacity, can produce enzymes such as amylase and protease at high yield, and has good antibacterial capacity. Through the discovery of feeding animals, the strain can improve the weight gain, reduce the drug cost, reduce the death rate and the like after being added into the feed, and can be used as a feed additive for livestock and poultry farming.

Drawings

The invention will be further described with reference to the drawings and examples.

FIG. 1 shows the quantitative measurement results of enzyme production by three bacillus subtilis strains;

FIG. 2 is a graph showing the growth of three strains of Bacillus subtilis;

FIG. 3 shows the survival rate of three strains of Bacillus subtilis in artificial gastric juice;

FIG. 4 shows the survival rate of three strains of Bacillus subtilis in artificial intestinal juice;

FIG. 5 shows the survival rate of three strains of Bacillus subtilis in 0.30% bile salts;

FIG. 6 shows the survival rate of three Bacillus subtilis strains at 45 ℃;

FIG. 7 shows the survival rate of three Bacillus subtilis strains at 65 ℃;

FIG. 8 shows the survival rate of three Bacillus subtilis strains at 85 ℃.

Detailed Description

The present invention is described in detail below by way of specific examples, but the scope of the present invention is not limited thereto. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and all experimental equipment, materials, reagents, etc. used can be obtained from commercial sources.

EXAMPLE 1 screening of Bacillus subtilis SD-KC-001

1. Primary screen

(1) Preparation of Medium plates

Preparing culture medium and sterile water, sterilizing at 121deg.C for 20min, pouring into a plate, and placing on an ultra-clean workbench for use.

(2) Preparation of soil dilution

The bacillus subtilis strain in the natural environment is purposefully screened. And (3) at a predetermined sampling place, 50-100 g of the sample is regularly sampled by using a sterile appliance, the sample is put into a sterile plastic bag to be pricked, and the sample is sent to a laboratory for drying as soon as possible and is screened by a 60-mesh analysis screen for standby. At the position ofAdding 9mL of sterile water into a centrifuge tube, weighing 1g of soil sample, adding into the centrifuge tube, and mixing the sample with water to obtain 10 ^-1 The diluted soil solution is placed in a water bath at 80 ℃ for 20min. Sequentially taking 1mL of the solution, adding the solution into a centrifuge tube added with 9mL of sterile water, and sequentially preparing 10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Soil solutions of different dilution factors.

(3) Plate coating

Plates of different dilutions were labeled, from 10 with sterile pipettes ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 The centrifuge tubes with different dilution factors are respectively used for sucking 0.1mL of diluent, and carefully dropping the diluent at the central position of the corresponding plate culture medium.

(4) Culturing

The flat plate is placed in an incubator at 37 ℃ in an inverted mode for culturing for 24-72 h.

(5) Primary screen

And (3) according to the colony morphology of the bacillus subtilis, observing that the colony surface is rough and opaque and white or yellowish, inoculating a small number of cells to a culture medium, placing the culture medium in a 37 ℃ incubator for culturing, and repeating the steps (3), (4) and (5) if the mixed bacteria are found, separating and purifying again until the strain with the form similar to that of the bacillus subtilis is screened out, and observing the strain under a microscope through gram staining, and comparing the strain with a standard bacillus subtilis strain to determine the bacillus subtilis.

2. Double screen

And the bacillus subtilis SD-KC-001 is continuously passaged on a solid culture medium for 40-50 times by adopting a plate streaking method, the shape and growth rate of the strain are measured, no obvious difference exists between the strain and the primary strain, and the genetic stability is good.

3. Comparison of Strain Properties

In order to facilitate the comparison of the characteristics of the strains, besides the initially screened bacillus subtilis strain SD-KC-001, two bacillus subtilis product strains KC-JR and KC-HB which are separated and preserved in a laboratory and are derived from better domestic market sales are simultaneously cultured and compared, so that the comparison advantages of the SD-KC-001 in the aspects of enzyme production capacity, bacteriostasis capacity, growth capacity, gastric acid resistance, intestinal juice survival capacity, bile salt resistance, heat resistance and the like are determined.

KC-JR is a Bacillus subtilis strain isolated from the Japanese imported Bacillus subtilis microbial feed additive product. The strain has the main characteristics of strong enzyme production capability, and high enzyme activities of amylase, protease and cellulase, but poor tolerance in intestinal juice.

KC-HB is a bacillus subtilis strain separated from domestic bacillus subtilis microbial feed additive products, and the bacillus subtilis strain has the main characteristics of strong growth capacity, high viable count, strong enzyme production capacity, high enzyme activity of amylase, protease and cellulase, good tolerance to gastric juice, intestinal juice and bile, but almost no antibacterial capacity.

The following determination is carried out by respectively adopting three strains of bacillus subtilis SD-KC-001, KC-JR and KC-HB, and each strain is selected from 3 strains of bacillus subtilis.

(1) Plate method extracellular enzyme activity assay

3 strains of bacillus subtilis single colony are selected to be inoculated on a cellulase screening culture medium, an amylase screening culture medium and a protease screening culture medium, and are cultured for a certain time at 37 ℃ and observed, and if transparent circles exist, the bacteria produce enzymes.

(2) Fermentation broth method extracellular enzyme activity determination

3 bacillus single colonies are selected and cultured for 24 hours at 37 ℃ and 160r/min in beef extract peptone liquid culture medium. Centrifuging the bacterial liquid at 6000r/min for 10min, and taking the supernatant as crude enzyme liquid for enzyme activity determination.

Adding 0.4mL of 3 strains of bacillus subtilis crude enzyme solution into a test tube respectively, adding 2mL of 1% casein solution as a substrate, accurately reacting for 30min in a water bath at 40 ℃, stopping the reaction by using 2mL of 10% trichloroacetic acid, centrifuging for 10min at 12000r/min, colorizing at 275nm wavelength, and determining the optical density value OD _275nm . Neutral protease activity was measured in 3 replicates per group.

3 strains of bacillus subtilis crude enzyme liquid are respectively added into a test tube, the temperature is kept for 10 minutes in a constant-temperature water bath at 40 ℃, 1.0mL of 1% starch solution is added, accurate reaction is carried out for 5 minutes at 40 ℃, 2.0mL of DNS reagent is added, the mixture is shaken uniformly and placed into a boiling water bath for 5 minutes, and the mixture is taken out, cooled and distilled water is added to 20mL. Shaking, colorimetry at 540nm wavelength, and OD value were measured. Amylase activity was measured in 3 replicates per group.

3 strains of crude bacillus subtilis enzyme solution (1.0 mL) are respectively added into a test tube, 1mL of 1% sodium carboxymethyl cellulose is added, and the mixture is placed into a water bath kettle at 40 ℃ for reaction for 30min. Then immediately adding 1.5mL of DNS to terminate the reaction, fully mixing, then boiling the mixture for 10min, immediately cooling the mixture to room temperature by cold water, fixing the volume to 10mL, and measuring the OD value, wherein 3 parallel measurement are arranged for each group.

After screening by using cellulase, amylase and protease screening culture medium, bacillus subtilis SD-KC-001 only produces amylase and protease, and other two strains of bacteria can produce amylase, protease and cellulase. As shown by the determination of extracellular enzyme activity by a fermentation broth method, the amylase produced by bacillus subtilis SD-KC-001 is 65.9U/mL, and the protease activity is 44.7U/mL. As shown in figure 1, the bacillus has stronger protease and amylase activities compared with other two strains in enzyme production activity, but the difference of enzyme production activities among different strains is larger. The three bacillus subtilis strains can produce protease and amylase, wherein the KC-JR produces the highest amylase, the SD-KC-001 is centered, the KC-HB is less, the SD-KC-001 is produced highest in the protease yield, and the KC-JR and KC-HB are similar in enzyme yield.

(3) Antibacterial ability measurement

The antibacterial activity of each strain was measured by oxford cup method. 3 bacillus single colonies are selected and cultured for 24 hours at 37 ℃ and 160r/min in beef extract peptone liquid culture medium. Inoculating the indicator bacteria into a specific culture medium for activation culture, mixing 1mL of the indicator bacteria into 100mL of the specific solid culture medium, placing sterile oxford cups on the mixture, enabling the sterile oxford cups to be clung to the culture medium, and adding 200 mu L of bacillus supernatant into each oxford cup. The plates were incubated at 37℃for 24 hours and then observed.

Antibacterial analysis was performed with staphylococcus aureus 834 (Staphylococcus aureus), staphylococcus aureus ATCC 12600 (Staphylococcus aureus ATCC 12600), escherichia coli, salmonella, pseudomonas aeruginosa and micrococcus luteus as indicator bacteria, and the results are shown in table 1.

Table 1 determination of bacteriostatic ability

Note that: "+" indicates antibacterial effect, and "-" indicates no antibacterial effect

From the bacteriostasis capability, bacillus realizes the inhibition effect on pathogenic bacteria through two ways of generating metabolic products such as bacteriocin and competing nutrition with pathogenic bacteria. The bacillus subtilis SD-KC-001 shows good antibacterial property, can inhibit staphylococcus aureus 834, staphylococcus aureus 12600, salmonella and micrococcus luteus, KC-JR can inhibit staphylococcus aureus 12600, escherichia coli, salmonella, pseudomonas aeruginosa and micrococcus luteus, and KC-HB has no antibacterial capability on indicator bacteria.

(4) Growth ability assay

3 strains of bacillus subtilis are activated on a solid LB plate and cultured overnight at 37 ℃, and the activated single colony is selected and cultured in an LB liquid medium for 24 hours at 37 ℃ and 160 r/min. The OD value was measured by colorimetry at 600nm wavelength at intervals of 2 hours. The growth capacities of three strains of Bacillus subtilis were compared.

The bacillus subtilis can enter a logarithmic growth phase in a short time due to the stabilization of nutrient substances and growth conditions in the growth process, and the difference exists due to the different growth capacities of strains. As can be seen from fig. 2: 3 strains of bacillus subtilis are in logarithmic growth phase for 2-12 h, which indicates that they can be rapidly propagated in a short time. After 12 hours, the number of 3 strains of bacteria reaches the maximum value, and the logarithmic growth phase is short, so that nutrient substances are supplemented to increase the concentration of the bacteria in the later culture. From the growth trend, the growth trend of 3 strains was approximately the same. SD-KC-001 and KC-JR have better growth capacity and similar growth capacity.

(5) Determination of tolerance to Artificial gastric juice and intestinal juice

3 bacillus subtilis strains are picked up and cultured in LB liquid medium at 37 ℃ and 160r/min for 72 hours. When the spore yield is more than 80% after microscopic examination, centrifuging at 5000rpm for 10min, and discarding the supernatant. Adding an equal volume of physiological saline for redissolution, and standing in a water bath at 80 ℃ for 20min to obtain three bacillus subtilis spores.

The artificial gastric juice is taken and split into 100mL triangular bottles, and each bottle is 30mL. Inoculating 3 strains of bacillus subtilis spores which are collected respectively into artificial gastric juice according to 10 percent of inoculum size, culturing in a shaking table at 37 ℃, and measuring the bacterial count by a dilution mixing dish method at 0, 0.5, 1, 1.5 and 2 hours respectively; the artificial intestinal juice is taken and split-packed in 100mL triangular flasks, 30mL per flask. The collected 3 bacillus subtilis spores are inoculated into artificial intestinal juice with an inoculation amount of 10 percent, cultured in a shaking table at 37 ℃, and the bacterial count is measured by a dilution mixing dish method at 0, 2, 4, 6 and 8 hours respectively, and data are recorded.

The results of measuring the bacterial count of bacillus in different times of artificial gastric juice are shown in figure 3, and the results of measuring the bacterial count of bacillus in different times show that SD-KC-001, KC-JR and KC-HB have good tolerance in the artificial gastric juice through figure 3, and the survival rate of the bacillus reaches more than 70%.

The small intestine is an important place where various probiotic preparations function. Bacillus only has the effect of tolerating intestinal juice and can play a probiotic role. In this test, the results of the bacterial count of 3 strains were measured at different time points after the three strains were subjected to the action of the artificial intestinal juice, and the results are shown in FIG. 4, and from FIG. 4, it is clear that SD-KC-001 and KC-HB are well tolerated in the intestinal juice, the survival rate is above 50%, and the survival rate of KC-JR after 6h in the artificial intestinal juice is 0%, and are not tolerated in the artificial intestinal juice.

(6) Determination of resistance to bile salts

Preparing 0.30% bile salt solution, packaging into small triangular bottles of 100mL, 30mL each bottle, and sterilizing at 121deg.C for 30min. The collected 3 bacillus subtilis spores were inoculated into a bile salt solution at an inoculum size of 10%, cultured in a shaker at 37 ℃, and the bacterial count was measured by a dilution mixing dish method at 0, 2, 4, 6 and 8 hours, respectively.

Cholate has antibacterial properties and can decompose fat in bacterial cell walls to inactivate bacteria. It is generally believed that the greater the anti-bile salt capacity of the probiotic, the greater the chance of survival in the intestine, the greater the content of human and animal duodenal bile salts is between 0.03% and 0.30%. The results of the bacterial count of three bacteria at different time points under the condition of 0.30% of bile salt concentration are shown in FIG. 5, and the results of FIG. 5 show that SD-KC-001 and KC-JR have strong tolerance to 0.30% of bile salt, the survival rate is more than 70%, and the survival rate of KC-HB at 0.30% of bile salt is about 50%.

(7) Heat resistance to high temperatures

3 bacillus subtilis strains are picked up and cultured in beef extract peptone liquid medium continuously at 37 ℃ and 160 r/min. When the spore yield is about 50% after microscopic examination, respectively treating the bacterial liquid at 45 ℃,65 ℃ and 85 ℃ for 10min,30min,2h,4h and 8h, and measuring the bacterial count by a dilution mixing dish method.

As is clear from FIGS. 6, 7 and 8, the three strains of Bacillus subtilis have similar tolerance at 45℃and 65℃and 85℃and the three strains of Bacillus subtilis have higher bacterial count at 30min of treatment and lower bacterial count at 2h of treatment at 45℃and the survival rate of the three strains of Bacillus subtilis is stabilized at about 70%. The bacterial count of three strains is obviously reduced at the temperature of 65 ℃ for 2 hours, the survival rate of SD-KC-001 and KC-JR is stabilized at about 40% and the survival rate of KC-HB is about 20% at the temperature of 65 ℃. At 85 ℃, the three strains tolerance gradually decreased with time, and at 8h, the three strains tolerance was 0. The temperature of the granulating step in the feed production process is generally controlled to be 65-90 ℃, the tempering time of the material contacted with steam is not more than 60s, and the SD-KC-001 still has more than 50% of survival rate in the environment of 10min at 85 ℃ and can resist short-time high temperature in the production process.

Through the analysis and comparison of the biological characteristics of 3 bacillus subtilis strains, SD-KC-001 has relatively good biological characteristics compared with other two bacillus subtilis strains, and has good growth capacity, enzyme production capacity, bacteriostasis capacity of gastrointestinal fluid and bile salt tolerance and high temperature tolerance, and other two bacillus subtilis strains have certain functional defects to a certain extent. Therefore, the strain SD-KC-001 has great application potential in the aspect of research of microbial feed additives.

Example 2 application of Bacillus subtilis SD-KC-001 as feed additive in pigs

A large amount of application data show that the bacillus subtilis feed additive can be added into pig feed to regulate microecological balance of digestive tracts and prevent intestinal diseases such as diarrhea and constipation; various digestive enzymes are generated, so that the absorption of nutrient substances is promoted, and the utilization rate of the feed is improved; stimulating the immune system of the organism and enhancing the immunity. Thereby improving the health of pigs and reducing disease loss; the cultivation cost is reduced; improving the quality of animal products; improving the environment of the livestock house.

The bacillus subtilis feed additive is added into weaned pig feed, so that weaning stress and nutritional diarrhea can be reduced, mortality can be reduced, and daily gain can be improved.

The whole process of growing and fattening pigs uses bacillus subtilis SD-KC-001 feed additive according to the proportion of 0.1%, the daily gain is improved by about 7%, and the growing and fattening pigs are put out of the fence 8-10 days in advance; the feed conversion ratio is reduced by about 5 percent.

The bacillus subtilis SD-KC-001 feed additive is added into pregnant and lactating sow feed, after continuous use for 4 weeks, the colibacillus in the sow feces is reduced by about 90%, and the quantity of lactobacillus and bifidobacterium is increased by 160 times. After the sow is used for 1 month before delivery, the delivery time is shortened to be within 3 hours, constipation is rarely caused, the feed intake and the lactation amount are improved, and the milk fat and the milk protein content are also improved. The birth weight of the piglets is uniform, the weaning survival rate is high, and the number of dead fetus and weak piglets is small.

The bacillus subtilis SD-KC-001 can generate amino oxidase and enzymes for decomposing sulfides in animal intestinal tracts, so that the concentration of harmful substances such as ammonia, indole and the like in blood and feces is reduced; the method can also supplement beneficial flora in the digestive tract to increase endogenous enzymes in the organism, thereby directly participating in metabolism of nitrogenous substances, and has remarkable effects of improving feed conversion rate and nitrogen utilization rate and reducing nitrogenous substances in feces.

The pig house environment is monitored, and the bacillus subtilis SD-KC-001 feed additive can reduce the ammonia emission by 40% -52% and improve the livestock house environment after long-term use.

TABLE 2 influence of Bacillus subtilis SD-KC-001 on Ammonia gas emission in pig house

Ammonia concentration unit: ppm of

Note that: the control group was not added with the bacillus subtilis SD-KC-001 feed additive, and the test group 1 and the test group 2 were added with the bacillus subtilis SD-KC-001 feed additive in a ratio of 0.1% and 0.2%, respectively.

EXAMPLE 3 use of Bacillus subtilis SD-KC-001 as a feed additive in poultry

A large amount of application data show that the bacillus subtilis feed additive is applied to poultry feed, so that the intestinal health can be improved, the production performance is improved, the product quality is improved, the immunity is enhanced, the survival rate is improved, the feed reward is improved, and the manure odor is reduced. The bacillus subtilis is added into the feed for the breeding hens, so that the laying time of the breeding hens can be prolonged, the laying rate, the egg weight and the hatching rate of the eggs are improved, weak chicks are reduced, the feed utilization rate is improved, and the cultivation income is increased.

Through the feeding test of bacillus subtilis SD-KC-001 feed additive according to the proportion of 0.2%, the laying rate of the laying hen in the egg laying peak period is increased by 5.21% compared with that of a control group, the egg laying peak period is prolonged by 2 weeks, the total death rate in the egg laying period is reduced by 23%, the egg breaking rate is reduced by 28%, and the egg weight is increased by 2.19%.

TABLE 3 test data of influence of Bacillus subtilis SD-KC-001 on the productivity of laying hens

Note that: table 3 is test statistics for 12000 layers continuously monitored for 80 weeks.

EXAMPLE 4 use of Bacillus subtilis SD-KC-001 as a feed additive in ruminants

The bacillus subtilis feed additive can be used as a functional feed additive product for eliminating stress of cattle and sheep and improving daily gain. Can effectively prevent the diseases of fragile nervous system, metabolic dysfunction, abnormal immune system and the like caused by the environmental change of cattle and sheep. Effectively reduces the occurrence of body weight loss and diseases of respiratory tract and digestive system in the stress reaction process. Can effectively relieve acidosis, reduce abdominal fat and improve the weight of the slaughtered house by 0.5-2%. The bacillus subtilis is added into the concentrate supplement for dairy cows, so that the milk yield of the dairy cows is improved, the milk fat and milk protein content is also improved, and the incidence rate of mastitis can be reduced, thereby reducing the somatic cell number in the milk.

The bacillus subtilis SD-KC-001 feed additive feeding test is carried out on the cows in the proportion of 0.2%, after continuous use for 5 weeks, the milk protein, the milk fat and the somatic cell number change obviously, and the daily milk yield is increased by 0.49%; milk protein and milk fat are respectively increased by 2.05 percent and 2.25 percent; the somatic cell number in milk is reduced by 17.97%. Thus, the bacillus subtilis SD-KC-001 feed additive can be used for a long time in a farm to obtain good benefits.

TABLE 4 comparison of various indicators of Bacillus subtilis SD-KC-001 milk

Note that: table 4 is experimental statistics for 200 cows continuously monitored for 6 weeks.

The above-described embodiments are only preferred embodiments of the invention, and not all embodiments of the invention are possible. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.

Claims

1. Bacillus subtilisBacillus subtilisSD-KC-001, which is characterized in that the bacillus subtilis SD-KC-001 is deposited in China general microbiological culture Collection center, and the deposit number is: CGMCC No.23135.

2. The fermentation method of bacillus subtilis SD-KC-001 according to claim 1, comprising the steps of:

step S3: sterilizing for the first time; before fermentation culture, introducing high-temperature steam into all pipelines, seed tanks, material supplementing tanks and fermentation tanks of the fermentation equipment for first sterilization and disinfection, wherein the sterilization temperature is 121 ℃ and the time is 20min;

step S6: inoculating a seed tank; the fermentation conditions are as follows: fermenting and culturing for 8-14 h at the temperature of 35-38 ℃ under the pressure of 0.02-0.06 Mpa with the aeration ratio of 1:0.5-1:1 and the stirring revolution of 150-200 rpm, and inoculating the primary strain into a seed tank for secondary strain culture;

step S7: inoculating the main fermentation tank; after the secondary strain is spread and cultivated, the secondary strain is directly inoculated into a fermentation tank for fermentation production through a system control by a pipeline, and the fermentation process conditions are the same as those of the seed tank in the step S6;

step S9: centrifugally accumulating bacteria, conveying fermented bacterial liquid to a high-speed tubular centrifuge through a pipeline, centrifugally collecting bacterial mud, wherein the inner diameter of a centrifuge drum is 142 and mm, the rotating speed of the drum is 14000 and r/min, and the throughput is 2500 and L/h, so that the fermentation liquid is ensured to be treated within 60 min;

step S10: preparing a bacterial agent, adding a certain amount of adsorbent into centrifugally collected bacterial mud according to the proportion of 1:1-3, drying at a low temperature, crushing, screening, packaging and the like to prepare a high-concentration bacterial agent, and sealing and preserving for later use;

3. The use of the fermentation broth of bacillus subtilis SD-KC-001 according to claim 1, wherein the feed additive for preparing livestock and poultry farming is mainly used for animals such as pigs, chickens and cattle, and the feed additive is added into compound feed for uniform mixing and feeding during use.

4. The use of a fermentation broth of bacillus subtilis SD-KC-001 according to claim 3, wherein the production method of the feed additive comprises the steps of: