CN110904013A - Bacillus subtilis strain and application thereof - Google Patents

Abstract

The invention provides a bacillus subtilis strain and application thereof. The preservation number of the Bacillus subtilis (Bacillus paralicheniformis) strain is CGMCC No. 17375. The application range is wide, and the preparation method comprises the preparation of livestock and poultry feed and antibacterial drugs. The fermentation product is added into the feed, and has good effects on the aspects of laying hen production performance, weaned piglet growth, animal immunity improvement and the like.

Description

Bacillus subtilis strain and application thereof

Technical Field

The invention relates to the field of microbiology, in particular to a bacillus subtilis strain and application thereof.

Background

China is the biggest antibiotic producing country and the largest using country in the world, and is also a serious disaster area of antibiotic abuse and bacterial drug resistance. In the fields of food, medicine, livestock, poultry, crops and the like, the problems of antibiotic abuse, overproof, antibiotic residue and the like are frequent. Especially in the field of animal husbandry, antibiotics have the functions of preventing and treating diseases, promoting growth, saving nutrients and the like. The farmers commonly use antibiotics in the links of preventive medication, epidemic disease treatment, feed addition and the like. At present, about half of antibiotics in China are generally used in livestock and poultry breeding as feed additives every year, and due to the reasons that a supervision system is not perfect enough and is driven by benefits and the like, manufacturers and farmers have a series of problems of blindly adding and abusing antibiotics in the use process of the antibiotics, so that the problems of increase of bacterial drug resistance, drug residues, environmental pollution, harm to human health and the like are caused. These problems are challenging the health bottom line of the nation. The search for safe, healthy and high-quality foods, medicines and the like becomes an urgent and highly important problem for people at present.

As one of the beneficial bacteria of human microorganisms, the probiotic is an important flora of intestinal tract and reproductive system in human body, can generate health effect for providing effective function for human body, and is a general term of flora organisms for maintaining human body health. The probiotic is the only compound probiotic capable of taking products as main microorganisms in the world at present, and plays an important role in the development processes of biological development engineering, industry, agriculture and food. With the intensive study of probiotics by scholars at home and abroad, the probiotics are widely used in the fields of food, medical treatment, health and the like at present.

With the comprehensive disablement of pharmaceutical additives such as colistin sulfate and olaquindox in animal feed, the feed industry has raised the need for replacing the hot tide of antibiotic-free products. The probiotics are considered as the best choice for replacing antibiotics due to the advantages of greenness, high efficiency, definite effect, no drug resistance, no residue and the like. Lactic acid bacteria, bifidobacteria, enterococcus, yeast, etc. are widely used in food. Lactic acid bacteria and bacillus are commonly used in the feed industry. After entering the gastrointestinal tract, the lactobacillus has strong adhesion and colonization capacity on intestinal mucosa, and L-lactic acid generated by metabolism can reduce the pH value of the intestinal tract, inhibit the growth of alkalophilic pathogenic bacteria, optimize the healthy environment of the intestinal tract and have obvious effect on reducing the incidence rate of enteritis and diarrhea. Compared with lactic acid bacteria, the bacillus can form spores with strong stress resistance and good stability, has strong tolerance to strong acid and high temperature in the stomach, and has wider application range.

Bacillus genus, a type of gram-positive (G)⁺) Bacteria, aerobic or facultative anaerobic, produce spores, mostly non-capsulated, move with a peritrichous flagellum. Cells are in the shape of straight rods, often arranged in pairs or chains. Because of their spore-forming properties, they are resistant to various extreme environments such as high temperature, extreme acids, extreme salts, and are resistant to various fungicides, and thus, can be isolated in various natural environments. The microorganisms of the genus bacillus have common characteristics, are capable of forming biofilms with spores, and are similar in metabolites, and are capable of producing cyclopeptide, antibiotics, acids (polyglutamic acid, polyaspartic acid), polysaccharides, and the like.

Currently used bacillus as probiotics include bacillus licheniformis, bacillus coagulans, bacillus subtilis, bacillus amyloliquefaciens, bacillus firmus, bacillus pumilus, bacillus megaterium, bacillus circulans, bacillus cereus, bacillus clausii, and the like. The probiotic bacillus can promote the growth and development of immune organs of animals, increase the number of T, B lymphocytes, and enhance the effects of cellular immunity and humoral immunity, thereby improving the immune function of the animals. Research results of Liu Kelin and the like show that the probiotic bacillus can promote the growth and development of immune organs of carps such as thymus and spleen, and T, B lymphocytes in the immune organs of a test group are mature faster and have more quantity than those of a control group. The influence of the probiotic bacillus on the nonspecific immunity of organisms is mainly reflected in that the bacillus enhances the mucosal barrier function of animals, activates nonspecific immune cells and promotes the generation of nonspecific bactericidal substances. Research shows that the bacillus preparation added into the daily ration of the broiler chicken can increase thymus index, spleen index and bursal index to different degrees. Severson et al reported that Bacillus subtilis could promote the development of intestinal tract-associated lymphoid tissue in rabbits and enhance the formation of B-lymphoid follicles. At present, probiotics bacillus has less research on erythrocyte immune function, and the like proves that the bacillus can obviously improve the rosette rate of the broiler erythrocyte C3b receptor and the rosette rate of the erythrocyte immune complex and promote erythrocyte immunity. Zhang Fu et al added the bacillus preparation in the chicken daily ration inoculated with Newcastle Disease (ND) vaccine, the result was not obvious to the red blood cell immune effect, the analysis reason is that besides animal species, age, physiological status, feeding way and dosage, and other factors, also probably because inoculated ND vaccine formed Immune Complex (IC) occupy red blood cell C3b receptor vacancy, thus showed slight compensation red blood cell immune deficiency, indirectly reduced the probiotic bacillus effect.

Bacillus subtilis (Bacillus paralicheniformis) is a relatively rare resource in the genus Bacillus. At present, relatively few researches on Bacillus paralicheniformis are carried out at home and abroad, and the number of related reports is limited. In 2015, Dunlap CA et al isolated a strain from fermented soybean sauce for the first time, and performed phylogenetic tree analysis to find that the strain has close homology of 99.4% and 99.5% with Bacillus licheniformis and Bacillus sojae respectively, and named as Bacillus paralicheniformis. In 2016, a strain of Bacillus paralicheniformis APC 1576 was isolated from the intestinal tract of mackerel by Collins FWJ et al, and lantibiotics with broad-spectrum bacteriostatic action were purified from the fermentation supernatant, suggesting that they have potential therapeutic value. In 2017, Wang Y and the like separate a strain of Bacillus paralicheniformis MDJK30 from the root zone of peony, and find that the strain has a certain inhibiting effect on pathogenic bacteria of the root rot of the peony and can promote the growth of plants. In 2018, Liu QT and the like take Bacillus paralicheniformis ATCC 9945a as raw materials to prepare the iron-and oxygen-containing stable acid urease,can remove urea and carcinogenic substance Ethyl Carbamate (EC) in yellow wine, and has great potential in food industry; the Bacillus paraffinformis KMS 80 is separated from rice root tissues by Annapurna K and the like, and is found to have biological nitrogen fixation and plant growth promotion capability; khan MM et al isolated a strain of Bacillus paralicheniformis CBS3 from korean traditional food (kimchi) and found that it produced a novel extracellular heat-resistant alkaline cohesive galacturonase (BPN 3). A strain of Bacillus paralicheniformis is separated from stratum produced water by Maxin and the like, and is found to have the effects of degrading guanidine gum and producing mannanase and can be applied to the aspect of microbial repairing of fracturing fluid. In 2019, the potential that Bacillus parafiniformis CBMAI1303 separated from Brazil rape has the capability of controlling the growth of plant pathogenic microorganisms rhizoctonia solani and anthrax is discovered by Pylro VS and the like; xiao X et al have studied that the selenium-rich Bacillus paralicheniformis SR14 can reduce H through MAPK pathway₂O₂Induced oxidative damage to porcine jejunal epithelial cells; khan MM and the like separate a strain of alkaline polygalacturonase PN32 producing strain (Bacillus paraffinformis CBS32) from Korean traditional fermented food pickle, and the results of enzymatic depolymerization and degumming experiments on ramie fibers show that the depolymerization rate of PN32 is 28% higher than that of commercial pectinase and has very high industrial application potential; muccee F et al found that Bacillus paralicheniformis KJ-16 has tolerance to toluene and the ability to degrade toluene. From the current research progress, the Bacillus subtilis (Bacillus paracoccidentaformis) is undoubtedly a novel Bacillus with great application potential.

Disclosure of Invention

The invention provides a Bacillus subtilis (Bacillus Paralicheniformis) strain and application thereof.

The technical scheme of the invention is realized as follows:

a Bacillus subtilis (Bacillus Paralicheniformis) strain with preservation number of CGMCC No. 17375.

The 16S rDNA sequence of the strain is shown in SEQ ID No. 1; the gyrB sequence is shown in SEQ ID No. 2.

Culturing the bacillus subtilis to obtain a fermentation product.

A preparation method of fermentation product comprises slant culture step and solid fermentation step.

Preferably, the slant culture step comprises a primary slant culture and a secondary slant culture.

Preferably, the primary slant culture step comprises: inoculating the Bacillus subtilis strain to a primary inclined plane for culture, and culturing for 2-4 days at 37 ℃ to obtain primary inclined plane seeds;

the secondary slant culture step comprises: inoculating the first-stage slant seeds to the slant of a second-stage eggplant-shaped culture bottle in batches, culturing for 2-4 days at 37 ℃, and then refrigerating for 24 hours at 4 ℃ to obtain second-stage slant seeds;

the solid fermentation step comprises: preparing the secondary bevel seeds into a bacterium suspension according to the sterile water amount of 20-30mL per bottle;

inoculating 10-15 bottles of the bacterial suspension into a solid culture medium per ton of solid material; the initial water content of the solid culture medium is controlled to be 50-60%, and the pH range is 7.0-7.2; the relative humidity of the koji chamber is kept above 52%, the material temperature is controlled at 35-40 ℃, and the culture is carried out for 2-4 days.

After the solid fermentation is finished, the spore rate of the Bacillus subtilis strain can reach more than 85 percent, and the spore number reaches 3.8 multiplied by 10¹⁰CFU/g。

The culture medium can be selected from:

the formula of the culture medium of the first-level inclined plane is as follows: 0.5-1% of beef extract, 1-2% of peptone, 0.5-1% of sodium chloride and 1.2-1.6% of agar powder, and adjusting the pH value to 7.0-7.2.

The culture medium formula of the secondary inclined plane is as follows: 0.5-1% of beef extract, 1-2% of peptone, 0.5-1% of sodium chloride and 1.6-1.8% of agar powder, and adjusting the pH value to 7.0-7.2.

The solid culture medium is prepared according to the following proportion: 70-80% of bran, 1-6% of corn flour, 1-6% of corn cob powder, 10-20% of soybean meal, 0.5-2% of calcium carbonate, 0.5-1% of ammonium chloride, 0.2-1% of sodium hydroxide, 0.2-1% of dipotassium phosphate, 0.1-0.5% of magnesium sulfate, 0.02-0.03% of manganese sulfate, 0.01-0.02% of ferric sulfate and 7.0-7.2% of pH.

A livestock and poultry feed comprises the fermentation product.

A preparation method of livestock and poultry feed comprises the step of culturing the bacillus subtilis (Bacillus Paralicheniformis) strain.

The fermentation product is applied to bacteriostatic drugs.

Advantageous effects

(1) The solid fermentation process of the strain is different from the existing liquid fermentation process, the solid fermentation can ensure that the strain keeps the original natural state for growth, and the metabolite is richer and more diverse.

(2) The spore rate of the fermentation product of the invention can reach more than 85 percent, and the spore number can reach 3.8 multiplied by 10¹⁰CFU/g。

(3) The Bacillus subtilis Bacillus paracasei CGMCC No. 17375 can be propagated in a nutrient-poor culture medium, the production cost can be greatly reduced by adopting industrial leftover materials, and simultaneously, higher spores are obtained.

(4) The fermentation product has antibacterial effect on various pathogenic bacteria, and has obvious antibacterial effect and wide antibacterial range.

(5) The fermentation product is added into the feed, and has good effects on the aspects of laying hen production performance, weaned piglet growth, animal immunity improvement and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive exercise.

FIG. 1 is a graph of the bacteriostatic results produced by the solid fermentation of example 2; wherein a-Clostridium perfringens (Clostridium perfringens GIM1.307), b-Bacillus subtilis (Bacillus subtilis test # 01), c-Staphylococcus aureus (Staphylococcus aureus D48), D-Listeria monocytogenes (Listeria monocytogenes LM1), e-Escherichia coli (Escherichia coli DH5), f-Salmonella enteritidis subspecies (Salmonella enterica GIM 1.114); the left part of the sample hole of the graph is CGMCC No. 17375 solid fermentation product, and the right part is blank control (sterile water).

FIG. 2 is the effect of the solid fermentation product of example 3 on egg color; wherein (a) is the color of the eggshell of the control group, and (b) is the color of the eggshell of the test group.

FIG. 3 is a photograph of a plate of Bacillus subtilis according to the present invention after culturing for 24 hours.

FIG. 4 is a gram chromosome of Bacillus subtilis according to the present invention.

FIG. 5 shows a phylogenetic tree (16S rDNA) in the Bacillus subtilis molecular characterization test of the present invention.

FIG. 6 shows a phylogenetic tree (gyrB r gene) in the molecular characterization test of Bacillus subtilis according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

And (3) biological preservation:

the Bacillus subtilis (Bacillus Paralicheniformis) strain is preserved in China general microbiological culture Collection center (CGMCC for short; address: China academy of sciences microorganism research institute No. 3, national institute of sciences No. 1, North Cheng West Lu, No. 1, Beijing, the rising district, the postal code: 100101) in 20 days 3 months and 3 days in 2019, and the preservation number is as follows: CGMCC No. 17375.

And (3) strain identification:

1. morphological identification

NA medium, incubated at 37 ℃ for 24 hours.

The diameter of a bacterial colony of the strain on a nutrient medium is 2.0mm-3.0mm, the shape of the bacterial colony is nearly circular, the edge is not neat, the bacterial colony is flat, and the bacterial colony is grey white and opaque. The cells are observed under a microscope to be rod-shaped, the size of the thallus is (0.5-0.7) mum multiplied by (1.2-2.4) mum, the thallus is arranged singly or in pairs, the spores are oval, the mesogen or the proximal end is grown, and the cysts are expanded. The strain Bacillus paraheteroformis can produce spores under aerobic conditions, has flagella and can move. Can grow at a pH of 6.0-11.0 and a temperature of 15-60 ℃, and can tolerate NaCl up to 10% (w/v).

2. Physiological and biochemical characteristics

The symbols indicate "+", positive: "-", negative.

3. Molecular characterization

The phylogenetic tree is constructed by using MEGA5.0 software as shown in FIGS. 5 and 6. The results show that the relationship with Bacillus paralicheniformis is recent.

Example 1

A strain of Bacillus subtilis CGMCC No. 17375 which is a new strain is screened out by Hangzhou Baoankang biotechnology limited.

The fermentation process comprises the following steps:

(1) first-grade slant culture of Bacillus subtilis CGMCC No. 17375

Inoculating the screened original strain to a first-stage test tube slant for culture, and culturing at 37 ℃ for 2-4d, wherein the culture medium formula is as follows: 0.5-1% of beef extract, 1-2% of peptone, 0.5-1% of sodium chloride and 1.2-1.6% of agar powder, and adjusting the pH value to 7.0-7.2.

(2) Secondary slant culture of Bacillus subtilis CGMCC No. 17375

Inoculating the first-stage test tube slant seeds obtained in the step (1) to a second-stage eggplant-shaped culture bottle slant in batches, culturing at 37 ℃ for 2-4d, and refrigerating at 4 ℃ for 24 h. The formula of the culture medium is as follows: 0.5-1% of beef extract, 1-2% of peptone, 0.5-1% of sodium chloride and 1.6-1.8% of agar powder, and adjusting the pH value to 7.0-7.2.

(3) sub-Bacillus subtilis and Bacillus paralicheniformis CGMCC No. 17375.

Preparing the two-stage eggplant-shaped culture flask slant seeds in the step (2) into a strain suspension according to the sterile water amount of 20-30 mL/bottle, inoculating 10-15 bottles of strain suspension into a solid culture medium according to each ton of solid material, controlling the initial water content of the culture medium to be 50-60%, and controlling the pH value to be 7.0-7.2. The relative humidity of the koji chamber is kept above 52 percent, the material temperature is controlled between 35 ℃ and 40 ℃, the culture is carried out for 2 to 4d, the spore rate of Bacillus paralicheniformis CGMCC No. 17375 can reach above 85 percent after the fermentation is finished, and the spore number can reach 3.8 multiplied by 10¹⁰CFU/g。

The solid fermentation medium is prepared according to the following proportion: 70-80% of bran, 1-6% of corn flour, 1-6% of corn cob powder, 10-20% of soybean meal, 0.5-2% of calcium carbonate, 0.5-1% of ammonium chloride, 0.2-1% of sodium hydroxide, 0.2-1% of dipotassium phosphate, 0.1-0.5% of magnesium sulfate, 0.02-0.03% of manganese sulfate, 0.01-0.02% of ferric sulfate and 7.0-7.2% of pH.

Example 2

The solid fermentation product prepared in the step (3) of the example 1 is subjected to in vitro bacteriostatic activity detection:

the method comprises the following steps:

dissolving the obtained fermentation product with sterile water for dilution, magnetically stirring the fermentation product on a magnetic stirrer at 160rpm for 30min, centrifuging the fermentation product at 3000rpm for 5min, and performing in-vitro antibacterial test on the Bacillus paralicheniformis CGMCC No. 17375 solid fermentation product by adopting an oxford cup method in an agar diffusion method. The detection indicator bacteria include Clostridium (Clostridium perfringens GIM1.307), Staphylococcus (Staphylococcus aureus Staphylococcus D48, Staphylococcus citreus LC5), Listeria (Listeria monocytogenes LM1), and Enterobacter (Escherichia coli DH5), Salmonella (Salmonella enterica subsp. enterica GIM1.114, Salmonella paratyphoid A GIM1.235), Bacillus (Bacillus subtilis 01 #). The indicator bacteria are cultured by using LB culture medium except clostridium perfringens enrichment culture medium, the culture temperature is 37 ℃, and the culture time is 12 h.

And (3) detection results:

the Bacillus subtilis Bacillus subtilis CGMCC No. 17375 solid fermentation product has better inhibiting effect on gram-positive bacteria, wherein the solid fermentation product comprises Clostridium perfringens (Clostridium perfringens GIM1.307), Bacillus subtilis (Bacillus subtilis test No. 01), staphylococcus aureus (Staphylococcus aureus D48) and Listeria monocytogenes (Listeria monocytogenes LM1), and also has certain inhibiting effect on gram-negative bacteria, including Escherichia coli DH5 and Salmonella enteritidis subsp.coli GIM 1.114. The diameter of the bacteriostatic circle of the Bacillus paralicheniformis CGMCC No. 17375 for the detected indicator bacterium can reach 13.95mm to 14.28mm at most.

The second method comprises the following steps:

dissolving the obtained fermentation product with sterile water for dilution, magnetically stirring the fermentation product on a magnetic stirrer at 160rpm for 30min, centrifuging the fermentation product at 3000rpm for 5min, and performing in-vitro antibacterial test on the solid fermentation product of Bacillus paralicheniformis CGMCC No. 17375 by adopting a plate perforation method in an agar diffusion method. The detection indicator bacteria include Clostridium (Clostridium perfringens GIM1.307), Staphylococcus (Staphylococcus aureus Staphylococcus D48, Staphylococcus citreus LC5), Listeria (Listeria monocytogenes LM1), and Enterobacter (Escherichia coli DH5), Salmonella (Salmonella enterica subsp. enterica GIM1.114, Salmonella paratyphoid A GIM1.235), Bacillus (Bacillus subtilis 01 #). The indicator bacteria are cultured by using LB culture medium except clostridium perfringens enrichment culture medium, the culture temperature is 37 ℃, and the culture time is 12 h.

The third method comprises the following steps:

performing in vitro bacteriostatic test on a Bacillus subtilis paracerini CGMCC No. 17375 solid fermentation product by a broth dilution method, dissolving the obtained fermentation product with sterile water for dilution, magnetically stirring the fermentation product on a magnetic stirrer at 160rpm for 30min, centrifuging the fermentation product at 3000rpm for 5min, adding the fermentation product into a liquid culture medium containing indicator bacteria according to a certain volume, culturing the fermentation product at 37 ℃ and 200rpm/min for 12h, and measuring OD (optical density) of each test tube under an ultraviolet spectrophotometer₆₀₀The value is obtained. The detection indicator bacteria include Clostridium (Clostridium perfringens GIM1.307), Staphylococcus (Staphylococcus aureus D48, Staphylococcus citreus LC5), Listeria (Listeria monocytogenes LM1), and Enterobacter (Escherichia coli DH5), Salmonella (Salmonella enterica GIM1.114, Salmonella paratyphoid A GIM1.235), Bacillus (Bacillus subtilis 01 #). The indicator bacteria are cultured by using LB culture medium except that clostridium perfringens enrichment culture medium is used for clostridium perfringens.

Example 3

The solid fermentation product obtained in step (3) of example 1 was subjected to animal test analysis:

in the test, 100 laying hens which are healthy and have the weight close to 24 weeks are randomly selected, 50 laying hens are randomly allocated to a control group and a test group, 150g/t times of Bacillus subtilis parac CGMCC No. 17375 solid fermentation products are added into the daily ration of the laying hens in the test group, and the control group feeds the laying hens normally. The whole experimental feeding period is 42 d.

The experimental results are as follows:

(1) the addition of the Bacillus subtilis CGMCC No. 17375 solid fermentation product in the daily ration can deepen the color of the eggshell, improve the smoothness of the eggshell, enhance the strength of the eggshell and reduce the egg breaking rate.

(2) Increase laying rate and reduce feed-egg ratio.

TABLE 1 influence of Bacillus paralicheniformis CGMCC No. 17375 solid fermentation product on egg laying performance of layer chicken

Example 4

The effect of the solid fermentation product prepared in step (3) of example 1 on the growth performance of weaned pigs was as follows:

in a certain large-scale feed company test field in Guangxi, 28-day-old weaned pigs with similar body conditions are selected, the number of test heads is 100, the weaned pigs are divided into two groups, each group has 50 heads, the test period is 30d, and the test result is as follows, the result shows that the addition of 200g/t times of Bacillus subtilis paraceriniformis CGMCC No. 17375 solid fermentation product into the daily ration of the weaned pigs can improve the daily weight gain and the material weight ratio of the weaned pigs, and effectively prevent the diarrhea of the weaned pigs.

TABLE 2 influence of Bacillus paralicheniformis CGMCC No. 17375 solid fermentation product on growth performance of weaned pig

Example 5

The Bacillus subtilis Bacillus parahalinformis CGMCC No. 17375 has the following fermentation process:

(1) first order slant culture

Inoculating the screened original strain to an LB culture medium test tube slant for culture, and culturing at 37 ℃ for 2-4d, wherein the culture medium formula is as follows: tryptone 1%, yeast extract powder 0.5%, sodium chloride 1%, agar powder 1.4-1.6%, and adjusting pH to 6.8-7.2.

(2) Two stage slant culture

Inoculating the first-stage test tube slant seeds obtained in the step (1) to a second-stage eggplant-shaped culture bottle slant in batches, culturing at 37 ℃ for 2-4d, and refrigerating at 4 ℃ for 24 h. The formula of the culture medium is as follows: tryptone 1%, yeast extract powder 0.5%, sodium chloride 1%, agar powder 1.4-1.6%, and adjusting pH to 6.8-7.2.

(3) Solid fermentation

Preparing the second grade eggplant type culture flask slant seeds in the step (2) into bacterial suspension according to the sterile water amount of 20-30 mL/bottle, inoculating the bacterial suspension into a solid culture medium according to the seed amount of 10-15 bottles of second grade eggplant type culture flasks per ton of solid material, controlling the initial water content of the culture medium to be 50-60%, and controlling the pH range to be 7.0-7.2. The relative humidity of the koji chamber is kept above 52%, the material temperature is controlled at 35-40 ℃, and the culture is carried out for 2-4 d.

The solid fermentation medium is prepared according to the following proportion: 70-80% of bran, 1-6% of corn flour, 1-6% of corn cob powder, 10-20% of soybean meal, 0.5-2% of calcium carbonate, 0.5-1% of ammonium chloride, 0.2-1% of sodium hydroxide, 0.2-1% of dipotassium phosphate, 0.1-0.5% of magnesium sulfate, 0.02-0.03% of manganese sulfate, 0.01-0.02% of ferric sulfate and 7.0-7.2% of pH.

Example 6

The Bacillus subtilis Bacillus parahalinformis CGMCC No. 17375 has the following fermentation process:

(1) first order slant culture

Inoculating the screened original strain to a TSA culture medium test tube slant for culture, and culturing at 37 ℃ for 2-4d, wherein the culture medium formula is as follows: tryptone 1.5%, soybean papain hydrolysate 0.5%, sodium chloride 0.5%, and agar 1.4-1.6%, and adjusting pH to 6.8-7.2.

(2) Two stage slant culture

Inoculating the first-stage test tube slant seeds obtained in the step (1) to a second-stage eggplant-shaped culture bottle slant in batches, culturing at 37 ℃ for 2-4d, and refrigerating at 4 ℃ for 24 h. The formula of the culture medium is as follows: tryptone 1.5%, soybean papain hydrolysate 0.5%, sodium chloride 0.5%, and agar 1.4-1.6%, and adjusting pH to 6.8-7.2.

(3) Solid fermentation

Preparing the second grade eggplant type culture flask slant seeds in the step (2) into bacterial suspension according to the sterile water amount of 20-30 mL/bottle, inoculating the bacterial suspension into a solid culture medium according to the seed amount of 10-15 bottles of second grade eggplant type culture flasks per ton of solid material, controlling the initial water content of the culture medium to be 50-60%, and controlling the pH range to be 7.0-7.2. The relative humidity of the koji chamber is kept above 52%, the material temperature is controlled at 35-40 ℃, and the culture is carried out for 2-4 d.

The solid fermentation medium is prepared according to the following proportion: 70-80% of bran, 1-6% of corn flour, 1-6% of corn cob powder, 10-20% of soybean meal, 0.5-2% of calcium carbonate, 0.5-1% of ammonium chloride, 0.2-1% of sodium hydroxide, 0.2-1% of dipotassium phosphate, 0.1-0.5% of magnesium sulfate, 0.02-0.03% of manganese sulfate, 0.01-0.02% of ferric sulfate and 7.0-7.2% of pH.

Example 7

The Bacillus subtilis Bacillus parahalinformis CGMCC No. 17375 has the following fermentation process:

(1) first order slant culture

Inoculating the screened original strain to a first-stage test tube slant for culture, and culturing at 37 ℃ for 2-4d, wherein the culture medium formula is as follows: 0.5-1% of beef extract, 1-2% of peptone, 0.5-1% of sodium chloride and 1.2-1.6% of agar powder, and adjusting the pH value to 7.0-7.2.

(2) Two stage slant culture

Inoculating the first-stage test tube slant seeds obtained in the step (1) to a second-stage eggplant-shaped culture bottle slant in batches, culturing at 37 ℃ for 2-4d, and refrigerating at 4 ℃ for 24 h. The formula of the culture medium is as follows: 0.5-1% of beef extract, 1-2% of peptone, 0.5-1% of sodium chloride and 1.6-1.8% of agar powder, and adjusting the pH value to 7.0-7.2.

(3) Solid fermentation

Preparing the second grade eggplant type culture flask slant seeds in the step (2) into bacterial suspension according to the sterile water amount of 20-30 mL/bottle, inoculating the bacterial suspension into a solid culture medium according to the seed amount of 10-15 bottles of second grade eggplant type culture flasks per ton of solid material, controlling the initial water content of the culture medium to be 50-60%, and controlling the pH range to be 7.0-7.2. The relative humidity of the koji chamber is kept above 52%, the material temperature is controlled at 35-40 ℃, and the culture is carried out for 2-4 d.

The solid fermentation medium is prepared according to the following proportion: the solid fermentation medium is prepared according to the following proportion: 40-50% of bran, 1-6% of corn flour, 1-6% of corn cob powder, 20-30% of soybean meal, 0.5-2% of calcium carbonate, 0.2-1% of sodium hydroxide, 0.2-1% of dipotassium phosphate, 0.1-0.5% of magnesium sulfate, 0.02-0.03% of manganese sulfate, 0.01-0.02% of ferric sulfate and pH 7.0-7.2.

Example 8

The Bacillus subtilis Bacillus parahalinformis CGMCC No. 17375 has the following fermentation process:

(1) first order slant culture

Inoculating the screened original strain to a first-stage test tube slant for culture, and culturing at 37 ℃ for 2-4d, wherein the culture medium formula is as follows: 0.5-1% of beef extract, 1-2% of peptone, 0.5-1% of sodium chloride and 1.2-1.6% of agar powder, and adjusting the pH value to 7.0-7.2.

(2) Two stage slant culture

Inoculating the first-stage test tube slant seeds obtained in the step (1) to a second-stage eggplant-shaped culture bottle slant in batches, culturing at 37 ℃ for 2-4d, and refrigerating at 4 ℃ for 24 h. The formula of the culture medium is as follows: 0.5-1% of beef extract, 1-2% of peptone, 0.5-1% of sodium chloride and 1.6-1.8% of agar powder, and adjusting the pH value to 7.0-7.2.

(3) Solid fermentation

Preparing the second grade eggplant type culture flask slant seeds in the step (2) into bacterial suspension according to the sterile water amount of 20-30 mL/bottle, inoculating the bacterial suspension into a solid culture medium according to the seed amount of 10-15 bottles of second grade eggplant type culture flasks per ton of solid material, controlling the initial water content of the culture medium to be 50-60%, and controlling the pH range to be 7.0-7.2. The relative humidity of the koji chamber is kept above 52%, the material temperature is controlled at 35-40 ℃, and the culture is carried out for 2-4 d.

The solid fermentation medium is prepared according to the following proportion: 40-50% of bran, 2-8% of corn flour, 20-30% of soybean meal, 0.5-1% of glucose, 2-5% of sodium chloride, 0.5-2% of calcium carbonate, 0.2-1% of sodium hydroxide, 0.5-2% of dipotassium phosphate, 0.1-0.5% of magnesium sulfate, 0.02-0.03% of manganese sulfate and pH 7.0-7.2.

Example 9

The Bacillus subtilis Bacillus parahalinformis CGMCC No. 17375 has the following fermentation process:

(1) first order slant culture

Inoculating the original strain to the first-stage slant culture medium, and culturing at 37 deg.C for 2-4 days. The formula of the culture medium is as follows: 0.5-1% of beef extract, 1-2% of peptone, 0.5-1% of sodium chloride and 1.2-1.6% of agar powder, and adjusting the pH value to 7.0-7.2.

(2) Secondary liquid seed culture

Inoculating 17375 the first-level Bacillus subtilis CGMCC No. 17375 in the step 1 into a second-level culture solution, and culturing for 2-4 days at 37 ℃ and 160-.

The secondary liquid culture medium is a mixed solution prepared according to the following proportion: 0.5-1% of beef extract, 1-2% of peptone, 0.5-1% of sodium chloride and pH 7.0-7.2.

(3) Three-stage amplification culture

Inoculating the second-level Bacillus subtilis Paralicheniformis CGMCC No. 17375 seed solution obtained in the step 2 into a third-level culture solution, wherein the inoculation amount is 1-5% by volume percent, and culturing for 2-4d under the conditions of 37 ℃ and 160-level and 200rpm/min to obtain the third-level Bacillus subtilis Paralicheniformis CGMCC No. 17375 seed solution.

The secondary liquid culture medium is a mixed solution prepared according to the following proportion: 0.5-1% of beef extract, 1-2% of peptone, 0.5-1% of sodium chloride and pH 7.0-7.2.

(4) Solid fermentation culture

Inoculating the third-level Bacillus subtilis Bacillus parahalifaciens CGMCC No. 17375 seed solution into a solid fermentation culture medium according to the weight ratio of 1-10%, wherein the initial moisture of the culture medium is controlled at 50-60%, and the pH range is 7.0-7.2. The relative humidity of the koji chamber is kept above 52%, the material temperature is controlled at 35-40 ℃, and the culture is carried out for 2-4 d.

The solid fermentation medium is prepared according to the following proportion: 70-80% of bran, 1-6% of corn flour, 1-6% of corn cob powder, 10-20% of soybean meal, 0.5-2% of calcium carbonate, 0.5-1% of ammonium chloride, 0.2-1% of sodium hydroxide, 0.2-1% of dipotassium phosphate, 0.1-0.5% of magnesium sulfate, 0.02-0.03% of manganese sulfate, 0.01-0.02% of ferric sulfate and 7.0-7.2% of pH.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Hangzhou Bao' ankang biotechnologies Limited

<120> bacillus subtilis strain and application thereof

<141>2019-12-19

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>1389

<212>DNA

<213> Bacillus subtilis (Bacillus paralicheniformis)

<400>1

ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa 60

cctgcctgta agactgggat aactccggga aaccggggct aataccggat gcttgattga 120

accgcatggt tcaattataa aaggtggctt ttagctacca cttacagatg gacccgcggc 180

gcattagcta gttggtgagg taacggctca ccaaggcaac gatgcgtagc cgacctgaga 240

gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag gcagcagtag 300

ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg atgaaggttt 360

tcggatcgta aaactctgtt gttagggaag aacaagtacc gttcgaatag ggcggtacct 420

tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 480

ggtggcaagc gttgtccgga attattgggc gtaaagcgcg cgcaggcggt ttcttaagtc 540

tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga acttgagtgc 600

agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt ggaggaacac 660

cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggcgcgaaag cgtggggagc 720

gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa gtgttagagg 780

gtttccgccc tttagtgctg cagcaaacgc attaagcact ccgcctgggg agtacggtcg 840

caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 900

attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaac cctagagata 960

gggcttcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 1020

gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc cagcattcag 1080

ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat gacgtcaaat 1140

catcatgccc cttatgacct gggctacaca cgtgctacaa tgggcagaac aaagggcagc 1200

gaagccgcga ggctaagcca atcccacaaa tctgttctca gttcggatcg cagtctgcaa 1260

ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg 1320

ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac ccgaagtcgg 1380

tgaggtaac 1389

<210>2

<211>1053

<212>DNA

<213> Bacillus subtilis (Bacillus paralicheniformis)

<400>2

gttaacgccc tttcaaccga gctcgatgta acggtttaca gagatggaaa agtccattat 60

caggaatttg aacggggcgt tccgaaagct gatttgaaag tcatcggaga gacggaagtg 120

acgggaacga ccactcactt caagcctgat ccggaaatat tcacggaaac gactgaatac 180

gactatgata cgctcgccac tcgtgtccgg gagctcgctt tcttgacaaa aggcgtcaaa 240

atcacgattg aagacaagcg agatggaaaa gaacgcaaga atgaatactg ctatgaaggc 300

ggtattaaaa gctatgttga acacttgaac cgttcgcggg aagttgttca tgaagagccg 360

gtctacattg aaggatccaa agacggcatt accgtcgagg tggcgctcca atacaacgac 420

agctatacga gcaacattta ttcgtttgcc aacaacattc atacgtatga aggcggaacg 480

catgaagccg gctttaagac aggtttgaca cgggttatca atgattacgc gagaaggaac 540

ggcgtattca aagaaagcga tccaaactta agcggggaag atgtccggga agggttgaca 600

gcgatcattt caatcaagca cccggatcct caatttgaag gacagacgaa aacaaagctt 660

ggcaactcag aagcgcgtac gataacagat gcgctatttt cagaagcgct cgaaaagttt 720

ctgcttgaaa acccggattc agcgaaaaaa atcgttgaaa aaggggttat ggctgccaga 780

gcacgaatgg cggcaaagaa agcgcgcgaa ttgacgcgca gaaaaagcgc ccttgaagtg 840

tcaaatctgc cagggaaact ggctgactgt tcttctaaag atccgacgat ttccgaactt 900

tacatcgttg agggtgactc tgcgggcgga tcggcaaaac agggccgcga tcgtcatttc 960

caagccattt tgcctttgag agggaaaatc ttgaacgtcg aaaaagcacg cctggacaaa 1020

attttgtcca acaatgaggt tcgttctatg atc 1053

Claims (9)

1. A Bacillus subtilis (Bacillus Paralicheniformis) strain with preservation number of CGMCC No. 17375.

2. The strain according to claim 1, wherein the 16S rDNA sequence is shown in SEQ ID No. 1; the gyrB sequence is shown in SEQ ID No. 2.

3. A fermentation product obtained by culturing the Bacillus subtilis (Bacillus Paralicheniformis) strain according to claim 1.

4. A process for producing a fermentation product according to claim 3, which comprises a slant culture step and a solid fermentation step.

5. The method of claim 4, wherein the step of slant culture comprises primary slant culture and secondary slant culture.

6. The method of claim 5, wherein the primary slant culture step comprises: inoculating the bacillus subtilis strain to a primary inclined plane for culture, and culturing for 2-4 days at 37 ℃ to obtain primary inclined plane seeds;

the secondary slant culture step comprises: inoculating the first-stage slant seeds to the slant of a second-stage eggplant-shaped culture bottle in batches, culturing for 2-4 days at 37 ℃, and then refrigerating for 24 hours at 4 ℃ to obtain second-stage slant seeds;

the solid fermentation step comprises: preparing the secondary bevel seeds into a bacterium suspension according to the sterile water amount of 20-30mL per bottle;

inoculating 10-15 bottles of the bacterial suspension into a solid culture medium per ton of solid material; the initial water content of the solid culture medium is controlled to be 50-60%, and the pH range is 7.0-7.2; the relative humidity of the koji chamber is kept above 52%, the material temperature is controlled at 35-40 ℃, and the culture is carried out for 2-4 days.

7. A livestock feed comprising the fermentation product of claim 3.

8. The preparation method of the livestock and poultry feed is characterized by comprising the step of culturing the bacillus subtilis strain.

9. Use of a fermentation product according to claim 3 in a bacteriostatic medicament.

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