CN107858302B

CN107858302B - Bacillus subtilis 7K and application thereof

Info

Publication number: CN107858302B
Application number: CN201711166108.8A
Authority: CN
Inventors: 秦启伟; 周胜; 黄晓红; 黄友华; 魏京广
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2017-11-21
Filing date: 2017-11-21
Publication date: 2020-07-28
Anticipated expiration: 2037-11-21
Also published as: CN107858302A

Abstract

The invention discloses Bacillus subtilis 7K and application thereof. The Bacillus subtilis 7K is preserved in Guangdong province microorganism strain preservation center, and the preservation number is GDMCC No: 60226 with preservation address of No. 59, No. 5, No. 100 college of Xieli Zhonglu, Guangzhou city and preservation date of 2017, 8 and 31. The Bacillus subtilis 7K has the characteristics of high temperature resistance at 100 ℃, low pH resistance, bile salt resistance, gastrointestinal digestive enzyme resistance and other extreme environments, and experiments prove that the screened new Bacillus subtilis 7K strain can promote the growth of aquatic animals and regulate the expression of various immune genes of the aquatic animals, can inhibit the growth of various pathogenic bacteria of the aquatic animals, further improves the pathogen infection resistance of the aquatic animals, and has good application prospect and market value.

Description

Bacillus subtilis 7K and application thereof

Technical Field

The invention belongs to the technical field of aquatics and microbiology, and particularly relates to a novel Bacillus subtilis 7K strain with disease-resistant probiotic effect, which can promote the growth of aquatic animals, regulate the expression of multiple immune genes of the aquatic animals, inhibit the growth of pathogenic bacteria of the aquatic animals, and further improve the disease infection resistance of the aquatic animals.

Background

According to the condition survey of the global aquaculture industry by the grain and agriculture organization of the united nations, 167,200,000 tons of fish were co-produced and produced a trade amount of nearly 1480 billion dollars in 2014 in the global fisheries. This also means that the consumption of water by humans reaches an unprecedented high value (Fisheres F A O. the state of world Fisheries and Aquaculture2016[ M)].2016.). At the same time, the prevalence and outbreaks of disease severely limit the development of the aquaculture industry, which almost annually entails nearly 300 billion dollars of losses in aquaculture (Subasinge R P, Bondad-Reantaso M G, McGladney S Eet al]Bangkok and FAO, NACA, 2012). Antibiotics have been widely used in the aquaculture industry to address the prevalence of disease among aquatic animals. A survey report of Thai shrimp breeders showed that 56 of 76 shrimp farmers who received the interview accepted the use of antibiotics, and many of them used antibiotics as a daily measure for preventing diseases every day, and the antibiotics used were also various and included gentamicin, chloramphenicol, tetracycline, quinolones, and trimethoprim (R) ((R))

K,

S,

A,et al.Antibiotic use in shrimp farming and implications forenvironmental impacts and human health[J].International journal of foodscience&technology,2003,38(3): 255-. In fact, antibiotics are not abused by breeders in asia alone, for example, oxytetracycline, florfenicol, sulfamethoxazole, sarafloxacin and enrofloxacin are also used in large quantities in shrimp farms in mexico (Roque a, Molina-Aja,

C,et al.In vitrosusceptibility to 15 antibiotics of vibrios isolated from penaeid shrimps inNorthwestern Mexico[J]an additional problem with large-scale antibiotic abuse, namely bacterial resistance (Elmahdi S, DaSilva L V, Parven S. antibiotic resistance of viral parahaemolyticus and Vibrio vulgaris in bacteria pools: A review [ J. sub.].Food microbiology,2016,57:128-134.)。

It is generally accepted that resistant bacteria are generated mainly from the antibiotic of the aquaculture industry and medical institutions using both pathways (Defoirdt, Boon N, Sorgelos P, et al, antibiotics to antibiotics bacterial infections: luminescent bacteria in aquatic as an example [ J ]. Trends in biological technology,2007,25(10): 472-. Drug-resistant bacteria generated in medical institutions can be carried out of hospitals by actions such as survival in intestinal tracts of patients, contact between patients or adsorption to medical instruments, and the like, and spread among other people. When the breeding industry uses antibiotics, the generated drug-resistant bacteria can be preserved in the meat of animals, when some cooking modes which cannot kill the bacteria well are adopted in the cooking process of consumers, the drug-resistant bacteria enter the intestinal tracts of diners, and the effective colonization of part of the drug-resistant bacteria enables the diners to become new infectious sources. Meanwhile, the manure containing drug-resistant bacteria often pollutes the irrigation water of crops, and the agricultural products irrigated by the polluted water become a reliable medium for transmitting the drug-resistant bacteria (centre for Disease Control and preservation (US). Perhaps we have difficulty assessing which of medical practice and food production is more responsible for the development of drug resistant bacteria, but many investigations have indicated that the amount of antibiotic abuse in the food industry is far beyond that of the medical institution.

From 1940, penicillin was introduced into clinical use, and it has been used for more than 70 years as a therapeutic against gram-positive bacterial infections such as streptococci and staphylococci (Chandra H, Bishmoi P, Yadav A, ethyl. antibacterial Resistance and the Alternative Resources with special antibiotics on Plants-Based antibiotics-A Review [ J ] Plants,2017,6(2): 16.). With the abuse of antibiotics, a large number of gram-positive bacteria appear to develop resistance to any available antibiotic, and people are faced with the difficult situation of returning again to the situation where no drug is available. Once a bacterium has acquired resistance to an antibiotic, there is no effective way to lose this property, and the spread of resistance among bacterial genomes is so rapid that it is predicted that the commonly used antibiotics will fail within the next five years (Bush K.antibacterial drug discovery in the 21 st center [ J ]. Clinical Microbiology and Infection,2004,10(s4): 10-17.). In the case of polymyxin, which has been discontinued soon after it has been placed into clinical use due to its great renal and neurological toxicity, it has recently been restored to clinical use due to its potent therapeutic effect against many drug-resistant gram-positive bacteria, whereas in 2015, an E.coli strain containing mcr1 was isolated and identified from a pig farm in the Shanghai of China (Crofs T S, Gasparrini A J, Dantas G.Next-generation a proacs to an understand and combatthe antigenic resistance [ J ]. Nature Reviews Microbiology, 2017.). Drug-resistant bacteria have been seriously detrimental to human health, and it has been reported by the U.S. centers for disease prevention and control that at least 2,000,000 people suffer from severe infections associated with drug-resistant bacteria annually and at least 23,000 people die directly from drug-resistant bacterial infections locally in the U.S. (Defoirdt, Boon N, Sorgelos P, et al. alternative to antibiotics in vitro infections: luminescence in a bacterial in vitro in an exo [ J ]. Trends in biotechnology,2007,25(10): 472-. More harmfully, infections with drug-resistant bacteria have been reported by the World Health organization to be more likely to occur in severe infections such as respiratory tract infections, meningitis, gonorrhea, diarrhea, syphilis and tuberculosis (World Health organization. antipicrobial resistance [ J ]. Retrievedonline August,2002,24: 2008.). This series of consequences also has led european authorities to choose to limit the use of antibiotics as growth promoters in animal farming; the guidelines of the U.S. Food and Drug administration also consider that the Use of antibiotics in Animal husbandry for the purpose of growth promotion is completely unnecessary and should strictly regulate the antibiotic usage behavior (Food and Drug administration. New Animal Drugs and New Animal Drug combination Products added in or on medical Feed or Drug administration Water for Food-production antibiotics: Recom. for Drug Sponsors for volumetric availability of antibiotic Products with GFI #209[ J ]. 2013.).

In this context, the use of probiotics in the aquaculture industry is increasingly becoming one of the methods of responding to disease epidemics. With the continuous and thorough understanding of the function of the intestinal flora, it is widely accepted that probiotics promote the healthy growth of animals in many ways: (i) inhibition of pathogenic bacteria by competitive growth; (ii) production of some trophic factors such as vitamins, or this promotes digestion by lytic enzymes; (iii) secreting a pathogen-inhibiting substance; (iv) (iv) enhancing host body immune expression, and (v) purifying water environment and the like (Defoirdt T, Boon N, Sorgelos P, et al].Trends in biotechnology,2007,25(10):472-479.；

-Atienza E,Gómez-Sala B,Araújo C,et al.Antimicrobial activity,antibiotic susceptibilityand virulence factors of lactic acid bacteria of aquatic origin intended foruse as probiotics in aquaculture[J].BMC microbiology,2013,13(1):15.)。

Many studies have demonstrated that probiotics have inhibitory activity against pathogenic bacteria, but outbreaks of viral infectious diseases have strikingly combated the development of the aquaculture industry. Similar to the situation of antibiotic abuse in the population, people in the aquatic field also lack effective weapons for dealing with viral infection, and it is difficult to distinguish viral infection from bacterial infection in time in the actual culture process, which also becomes an important reason for antibiotic abuse. Therefore, if the outbreak of the virus-derived diseases in aquatic animals cannot be controlled, the abuse of antibiotics and prohibited drugs by breeders is difficult to reduce.

Disclosure of Invention

The invention aims to: the Bacillus subtilis 7K new strain with disease-resistant probiotic effect can promote the growth of aquatic animals and regulate the expression of multiple immune genes of the aquatic animals, and can inhibit the growth of multiple pathogenic bacteria of the aquatic animals, so that the disease-resistant capability of the aquatic animals is improved, the Bacillus subtilis new strain can be used as a feed additive or a medicament for aquaculture, and the like, and the use of antibiotics and prohibited medicaments is reduced.

In order to achieve the aim, the invention provides Bacillus subtilis 7K, which is a new strain separated from the intestinal tract of healthy grouper by methods such as isolated culture, morphological observation, physiological and biochemical experiments, 16S rDNA sequence analysis and the like, and is preserved in Guangdong province microbial strain preservation center with the preservation number of GDMCCNo: 60226 with preservation address of No. 59, No. 5, No. 100 college of Xieli Zhonglu, Guangzhou city and preservation date of 2017, 8 and 31.

The 16S rDNA sequence of the Bacillus subtilis 7K is shown in SEQ ID NO. 1.

Experiments show that the Bacillus subtilis 7K has good spore heat resistance and gastrointestinal fluid resistance, the fermentation supernatant contains various antibacterial active substances, and has the function of broad-spectrum inhibition of pathogenic bacteria of common marine fishes. In addition, the Bacillus subtilis 7K can also improve the expression level of various immune genes in the grouper body. Therefore, the Bacillus subtilis 7K can be used for promoting the growth of aquatic animals, improving the expression level of immune genes of the aquatic animals, improving the immunity of the aquatic animals and preventing or treating diseases of the aquatic animals.

Preferably, the aquatic animal is a grouper or pomfret.

Preferably, the immune genes include I L-1 β, I L-8, TNF α, MX I, MX II, and ISG 15.

The invention also provides an aquatic animal feed additive which comprises an effective dose of Bacillus subtilis 7K as an active ingredient.

The invention also provides a microecological preparation for regulating the aquatic animal culture water environment, which comprises an effective dose of Bacillus subtilis 7K as an active ingredient.

The invention also provides a medicament for improving the immunity of aquatic animals, which comprises an effective dose of Bacillus subtilis 7K as an active ingredient.

The invention also provides a medicament for preventing or treating diseases of aquatic animals, which comprises an effective dose of Bacillus subtilis 7K as an active ingredient.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the invention separates and screens to obtain a new bacterial strain Bacillus subtilis 7K, which has the characteristics of high temperature resistance of 100 ℃, low pH resistance, bile salt resistance, gastrointestinal digestive enzyme resistance and other extreme environments.

(2) The Bacillus subtilis 7K disclosed by the invention is subjected to an extracellular bacteriostasis experiment, has a remarkable inhibition effect on various common marine fish pathogenic bacteria, and can be used for preparing a preparation for inhibiting aquatic animal related pathogenic bacteria and a microecological preparation for regulating aquatic animal culture water environment.

(3) The Bacillus subtilis 7K disclosed by the invention is subjected to a grouper growth influence experiment, and the weight of the grouper can be obviously improved, so that the Bacillus subtilis 7K disclosed by the invention has a function of promoting the growth of aquatic animals and can be used for preparing an aquatic animal feed additive.

(4) The Bacillus subtilis 7K disclosed by the invention is subjected to a virus infection experiment, and the result shows that the Bacillus subtilis 7K can be used for remarkably reducing the death rate of groupers infected with the Singapore grouper iridovirus and improving the expression level of multiple immune genes in spleen tissues of the groupers, so that the Bacillus subtilis 7K disclosed by the invention can be used for preparing medicines for improving the immunity of aquatic animals and medicines for preventing or treating diseases of the aquatic animals.

A Bacillus subtilis (Bacillus subtilis)7K is preserved in Guangdong province microorganism culture collection center, and the preservation number is GDMCC No: 60226 with preservation address of No. 59, No. 5, No. 100 college of Xieli Zhonglu, Guangzhou city and preservation date of 2017, 8 and 31.

Drawings

FIG. 1 is a phylogenetic tree constructed based on the 16S rDNA sequence of Bacillus subtilis 7K using the Neighbor-Joining method.

FIG. 2 shows the results of a heat resistance test of Bacillus subtilis 7K of the present invention.

FIG. 3 shows the results of gastric juice resistance test of Bacillus subtilis 7K of the present invention.

FIG. 4 shows the results of an intestinal juice resistance test of Bacillus subtilis 7K of the present invention.

FIG. 5 shows the inhibition zones formed by the supernatant after 7K fermentation culture of Bacillus subtilis according to the present invention on Aeromonas hydrophila (Aeromonas hydrophila), (b) Vibrio vulnificus (Vibrio vulgaris), (c) Vibrio harveyi (Vibrio harveyi), (d) Staphylococcus aureus (Staphylococcus aureus), (e) Micrococcus lysodeikticus, and (f) Vibrio alginolyticus (Vibrio algiryvein).

FIG. 6 shows the results of measuring the bacteriostatic substances in the supernatant after the fermentation culture of Bacillus subtilis 7K.

FIG. 7 is the cumulative mortality of giant grouper infected with SGIV for 10 days with the same letter superscript indicating no statistical difference where P < 0.05 is statistically different.

FIG. 8 shows spleen I L-1 β after infection of giant grouper with SGIV for 48h,I L-8, TNF α, MXI, MXII and ISG15mRNA relative expression levels, wherein "+" indicates P<0.05; ". indicates P<0.01, this figure shows only 10¹⁰cfu g^-1Statistical analysis of the group compared to other dose groups.

Detailed Description

In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are only for the purpose of illustrating the invention and are not to be construed as limiting the invention, and the parameters, proportions and the like of the embodiments may be suitably selected without materially affecting the results.

Unless otherwise specified, reagents and materials used in the following examples are commercially available.

Examples

Separation and identification of Bacillus subtilis 7K

1) Sampling: the cultured groupers are purchased from a culturing farm in Hainan; the marine bottom mud is derived from deep sea of mangrove forest, south sea and Indian ocean in Guangdong coast.

2) Culture medium:

liquid culture medium for enrichment (L B culture medium) comprises peptone 10g, yeast powder 5g, NaCl 10g, and distilled water 1000ml, and has pH of 7.0-7.2.

Separating culture medium L B adding agar with mass fraction of 1.5% -2%.

Salt tolerance test medium the amount of NaCl added to the L B medium was varied to produce media of different salinity (NaCl 2%, 5%, 7%, 10%).

Sugar fermentation test medium: (NH)₄)₂HPO₄1g，MgSO₄·7H₂0.2g of O, 0.2g of KCl, 0.2g of yeast extract, 10g of sugar or alcohol, 1000m L of distilled water and 1.2ml of 0.2% bromothymol blue solution, adjusting the pH value to 7.0-7.2, subpackaging test tubes, packaging 10m L of culture medium in each tube, and sterilizing at 115 ℃ for 20 min.

The gelatin culture medium comprises 5g of NaCl, 10g of peptone, 3g of beef extract, 120g of gelatin and 1000m of distilled water L, and the pH value is 7.2-7.4.

The starch culture medium comprises 5g of beef extract, 10g of peptone, 5g of sodium chloride, 2g of soluble starch and 20g of distilled water 1000m L pH7.0-7.2 agar.

And (3) preparing a milk plate, namely adding 5g of skimmed milk powder into 50m L distilled water (or 50m L skimmed milk), dissolving 1.5g of agar in 50m L distilled water, separately sterilizing the two solutions, cooling to 45-50 ℃, uniformly mixing the two solutions, and pouring the mixture into a plate to obtain the milk plate.

Citrate medium: 2g of sodium citrate, 5g of NaCl and MgSO₄·7H₂O 0.1g、(NH₄)₂HPO₄1g、K₂HPO₄·3H₂O1 g, 1% bromothymol blue aqueous solution 10m L, agar 20g, pH6.8-7.0.

Fermentation medium: 10g of soybean meal powder, 10g of glucose, 2.5g of yeast powder, (NH)₄)₂SO₄2.5g、K₂HPO₄·3H₂O0.6g、KH₂PO₄0.25g、MgSO₄·7H₂O 0.2g、CaCl₂0.05g、MnCl₂·4H₂O0.03g, distilled water 1000m L, and trace element solution 2m L, ferrous ion solution 2m L, and defoaming agent 1m L.

The above culture medium is sterilized in high pressure steam sterilizer at 110 deg.C for 20 min.

Spore production medium (DSM): 8g of nutrient broth, 10% (w/v) KCl 10ml, 1.2% (w/v) MgSO₄·7H₂O10ml, 1000ml of distilled water, pH adjusted to 7.6. Sterilizing at 121 deg.C for 20 min. After cooling, sterile 1MCa (NO) is added before use₃)₂1ml，0.01M MnCl₂1ml，1mM FeSO₄1ml。

Simulated gastric fluid: 4.8g NaCl, 1.56g NaHCO₃、2.2g KCl、0.22g CaCl₂1000ml of distilled water, adjusting the pH value to 1.5, sterilizing at 121 ℃ for 20min for later use, and adding a pepsin solution through a bacteria filter with the pore diameter of 0.22 mu m to ensure that the pepsin concentration in the final simulated gastric juice is 500 u/L before the spore gastric juice resistance test.

Simulating intestinal juice: mixing 5g of NaCl, 0.6g of KCl and 0.25g of CaCl₂8.5g of oxgall to 1L, 1M NaHCO₃In solution, adjust pH to 7.0Sterilizing at 121 deg.C for 20min, filtering with a filter with pore size of 0.22 μm, adding lipase solution with concentration of 20000U/L, amylase solution of 16000U/L and protease solution of 1200U/L, and making into simulated intestinal juice.

3) B, bacillus isolation: sterilizing the outer surface of the grouper with cotton stained with 75% alcohol, and dissecting on a super clean bench. The intestinal tract of the grouper is separated and the contents of the intestinal tract are squeezed out with forceps that have been sterilized.

The intestinal contents of grouper and about 1g of marine bottom mud are put into a triangular flask filled with 100ml of sterile normal saline to be fully mixed, and the mixture is subjected to water bath at 80 ℃ for 10-15 min to kill non-spore thalli. The supernatant was collected by centrifugation. Inoculating the supernatant into enrichment liquid culture medium, and shake culturing at 37 deg.C and 200r/min for 24 h. And taking 1ml of the enriched bacterial liquid for gradient dilution. Spreading on separation culture medium, and repeatedly streaking to obtain pure strain. 1 strain of the strain is isolated from the intestinal tract of the grouper and named as 7K. The colony is white and opaque, dry, irregular in edge and star-shaped in the middle. The purified colonies were picked from the isolation medium plate, inoculated in the enrichment liquid medium, cultured at 37 ℃ at 200r/min for 12 hours, and stored at 4 ℃ for further use. After gram staining, the cells were observed under a 1000-fold microscope. The two strains are gram-positive bacteria, clear spores in the bacteria can be seen, and the bacteria can be preliminarily identified as the genus of bacillus.

4) Sequencing to identify the isolated strains: total bacterial DNA was extracted using a bacterial DNA extraction kit (Tiangen Biochemical technology Co., Ltd.). Amplifying 16S rDNA by PCR, purifying PCR product and sequencing. The primers for the PCR reaction were: K1F is shown as SEQ ID NO. 2, and K2R is shown as SEQ ID NO. 3. Reaction system of PCR: 1 μ l of bacterial DNA; taq 0.2. mu.l; dNTP 1 u l; 10Xbuffer 2.5. mu.l; K1F 1 μ l; K2R 1 μ l; by dd H₂O to 25. mu.l, PCR conditions of 95 ℃ pre-denaturation for 3min, 94 ℃ denaturation for 1min, 55 ℃ annealing for 45S, 72 ℃ extension for 45S, 35 cycles, 72 ℃ extension for 5min, purification and sequencing of the PCR product were performed by Hiliefi Biotechnology Ltd, the determined sequence (as shown in SEQ ID NO: 1) was subjected to B L AST alignment analysis in NCBI and the 16S rDNA sequence of the obtained strain was compared with the reference Bacillus, Clostridium, Variosa, by means of software MEGA5.0,Comparing and analyzing the sequences of model strains in the Sporosarcina, the Sporolactobacillus, the Desulfoenterobacter and the Paenibacillus, and constructing a phylogenetic tree by using a Neighbor-Joining method.

The homology of B L AST of the obtained 7K 16S rDNA sequence with the logged-in Bacillus subtilis 16SrDNA was analyzed in NCBI, and the results showed that the homology of the 16S rDNA sequence of the strain 7K with the 16S rDNA sequence of Bacillus subtilis was 99%, and the separated strain was judged to be a new Bacillus subtilis strain according to Bergary' S Manual of systematic bacteriology, 2001 second edition, spring 2001, Springer-Verlag, Gerge M.Garrity, MattewWinters and Nenise B.Searles, and the phylogenetic tree obtained by constructing the 16S rDNA sequence of 7K by means of the Neighbor-Joining method with the software MEGA5.0 was shown in FIG. 1.

5) Physiological and biochemical characteristic test for identifying separated bacterial strain

Physiological and biochemical identification was performed with reference to "Manual of identification of common bacterial systems" (Dongxu bead, Chuia Miaoying. Manual of identification of common bacterial systems [ M ]. Beijing: scientific Press, 2001). The bacillus cereus biochemical identification tube (Guangdong Huanji microbial technology limited) is used for physiological and biochemical identification such as catalase, V-P reaction, nitrate reduction and the like. Casein hydrolysis, starch hydrolysis, gelatin liquefaction, citrate utilization test, sugar fermentation test and salt tolerance test were performed using a self-prepared milk plate, gelatin medium, starch medium, citrate medium, sugar fermentation medium and salt tolerance test medium, respectively.

The Bacillus subtilis 7K is subjected to biochemical identification and physiological and biochemical characteristic tests, and the experimental results are shown in Table 1. After comparing with the physiological and biochemical characteristics of the Bacillus subtilis in the handbook of identifying common bacteria systems, the physiological and biochemical characteristics of the Bacillus subtilis 7K are consistent with those of the Bacillus subtilis, and the separated strain is further shown to be a new Bacillus subtilis strain.

TABLE 1 Biochemical identification and test results of physiological and biochemical characteristics of Bacillus subtilis 7K

Note: "+" is positive and "-" is negative.

Experimental example 1 Bacillus subtilis 7K characteristic experiment

(1) Preparing spores, namely selecting 7K colonies of monoclonal Bacillus subtilis (Bacillus subtilis) to inoculate in 5ml of L B liquid culture medium, carrying out shake cultivation at 37 ℃ overnight, then diluting the Bacillus subtilis according to a ratio of 1:100, adding the Bacillus subtilis to a DSM culture medium, carrying out shake cultivation at 37 ℃ at 250r/min, taking 100 mu L culture solution smears for 0h, 12h and 24h of spore cultivation respectively, carrying out spore staining microscopy, observing spore generation, collecting and washing the spores, wherein the spore collection and washing method comprises the steps of centrifuging at 8000r/min for 15min, collecting thalli, removing supernatant, adding lysozyme to a final concentration of 4mg/m L, standing at room temperature for 15min, washing the spores with 1 mol/L NaCl, adding PMSF to a final solubility of 1 mu mol/L, fully mixing, centrifuging at 8000r/min for 15min, collecting thalli, washing the thalli with 1 mol/L KCl for 1 time, washing with distilled water for 2 times, dissolving the spores in a certain amount of distilled water, killing residual Bacillus subtilis propagulants at 68 ℃ for 1h, counting with distilled water, counting the number of 1 mu 25, and counting the number of the remaining Bacillus subtilis cells under a microscope at a temperature of 5920-20 ℃.

(2) The heat resistance test of the spores comprises the steps of selecting a vegetative mass, spores and escherichia coli of Bacillus subtilis 7K to carry out the heat resistance test in a 100 ℃ boiling water bath, placing the vegetative mass, the spores and the escherichia coli of the Bacillus subtilis 7K with the same initial concentration in the 100 ℃ boiling water bath, respectively sampling and coating the vegetative mass, the spores and the escherichia coli in L B plates when processing is carried out for 0s, 30s, 60s, 90s and 120s, carrying out colony counting after culturing is carried out for 24h at 37 ℃, carrying out three repetition on each sample, and drawing a survival curve by taking an average value.

The results of the thermotolerance test showed cell survival curves as shown in FIG. 2. After 2min of boiling water bath at 100 ℃, the spore of the Bacillus subtilis 7K is reduced slowly, and stronger heat resistance is embodied. Coli and 7k trophozoites were compared. The escherichia coli completely died after being subjected to boiling water bath for 30s, and the Bacillus subtilis 7K nutrient body completely died after being subjected to boiling water bath for 60 s.

(3) The Bacillus gastrointestinal fluid resistance test comprises the steps of selecting a Bacillus subtilis 7K vegetative mass, spores and escherichia coli to carry out a gastrointestinal fluid resistance test, placing the Bacillus subtilis 7K vegetative mass, the spores and the escherichia coli with the same initial concentration in simulated gastric fluid and intestinal fluid, treating the Bacillus subtilis 7K vegetative mass, the spores and the escherichia coli in the simulated gastric fluid for 0min, 15min, 30min, 45min and 60min, respectively sampling and coating the samples in the simulated intestinal fluid for 0h, 1h, 2h and 3h, carrying out colony counting after culturing at 37 ℃ for 24h, carrying out three repetition on each sample, and taking an average value to draw a survival curve.

The results of the gastric juice resistance test show that the cell survival curve is shown in figure 3, in simulated gastric juice, the survival number of the vegetative bodies of escherichia coli and Bacillus subtilis is sharply reduced within 15min until all the vegetative bodies die within 15min, while the death number of spores is lower than 0.03logCFU/m L after the Bacillus subtilis 7K is treated for 60min, and the Bacillus subtilis 7K spores have very strong resistance to the simulated gastric juice.

The intestinal fluid tolerance test shows that the cell survival curve is shown in FIG. 4. the Bacillus subtilis 7K shows higher tolerance and almost 100% survival rate at 3 hours of simulated intestinal fluid culture.the trophosome of the Bacillus subtilis 7K drops from 6.43log CFU/m L to 1.56log CFU/m L, showing high sensitivity, while the amount of E.coli in the simulated intestinal fluid is stable.

(4) An extracellular bacteriostatic test comprises culturing Bacillus subtilis 7K in L B culture medium at 37 deg.C for 24 hr, diluting at a ratio of 1:100, adding into a fermenter, fermenting at 28 deg.C in the culture mediumFermenting and culturing for 48h, centrifuging the fermentation medium at 4000rpm and 4 ℃ for 10min, taking the supernatant, passing the supernatant through a bacteria filter with the pore diameter of 0.22 mu m, storing the supernatant at 20 ℃ for later use, measuring 7K extracellular antibacterial activity of Bacillus subtilis by an Oxford cup method, taking common pathogenic bacteria of marine fishes such as staphylococcus aureus, aeromonas hydrophila, Micrococcus lyticus, Vibrio harveyi, Vibrio alginolyticus and Vibrio vulnificus as pathogens in the test, taking 150 mu L of the supernatant to be added into the inoculated 10 mu L⁵CFU/ml pathogens L B agar plate in the Oxford cup for each pathogen three replicates were made and the supernatant was initially assayed for chemical properties of antibacterial compounds using high performance liquid chromatography combined with high resolution mass spectrometry (HP L C-HRMS).

The extracellular bacteriostasis result of the Bacillus subtilis 7K is shown in a table 2, the result shows that the supernatant obtained after the fermentation culture of the strain 7K can form an obvious circular bacteriostasis area (shown in a figure 5) for six common marine fish pathogenic bacteria, and the circular bacteriostasis area indicates that the circular bacteriostasis area has extracellular bacteriostasis activity, the supernatant has more obvious bacteriostasis effect on vibrio harveyi, staphylococcus aureus, micrococcus lyticus and vibrio alginolyticus in the 6 selected common bacteria, and the supernatant is preliminarily determined to possibly contain various bacteriostasis related chemical substances according to a high performance liquid chromatography combined high resolution mass spectrometry (HP L C-HRMS) (shown in a figure 7).

TABLE 2 bacteriostatic test results of the supernatant from the fermentation of Bacillus subtilis 7K

Note: the diameter of the bacteriostatic area is 5mm-1mm, "+ +" is 1mm-15mm, and "+ + + +" is 15mm-20 mm.

Experimental example 2 probiotic action of Bacillus subtilis 7K on Epinephelus

1) Preparation of Bacillus subtilis 7K additive feed

Inoculating Bacillus subtilis 7K separated from intestine of Epinephelus macrorrhiza into L B liquid cultureCulturing at 37 deg.C for 24 hr, mixing the culture solution with spore-forming culture medium (soybean powder 10g/l, yeast powder 2.5 g/L, glucose 10g/L, (NH) at a ratio of 1:100₄)₂SO₄2.5 g/L, inorganic salts and trace elements) was cultured at 37 ℃ for 48h in a 5L automatic fermentor, the broth was then centrifuged at 8000rpm for 15min and the supernatant discarded all spores were transferred into lysozyme solution (4mg/ml) for 15min at room temperature to remove residual nutrients, the treated spores were rinsed with sterile water and centrifuged twice (8000rpm, 15min), the rinsed spores were placed in a 68 ℃ water bath for heat treatment for 1h and the treated spores were stored briefly at 4 ℃ for feed preparation.

Mixing the spores with sterilized commercial grouper feed (121 deg.C, 20min), granulating, drying at 70 deg.C, drying at room temperature, sealing, and storing. The 7K of the four feeds containing bacillus subtilis (Bacillus subtilis) used in the experiment is 0 and 10 respectively⁶,10⁸And 10¹⁰cfu g^-1. The bacterial content of the feed is determined by a flat plate bacterial colony counting method, and in order to ensure that the bacterial content of the feed is stable, the bacterial activity of each group of feed is measured at the beginning of an experiment, in the middle of the experiment and after the experiment is finished.

2) Grouper breeding

Healthy pearl giant grouper was purchased from a farm. Before the beginning of the formal experiment, the groupers are raised in a laboratory animal room water tank for two weeks to adapt to the environment, the water tank adopts circulating seawater, and the groupers are fed with disinfected and sterilized groupers feed in the period. In order to prevent bite between groupers due to excessive body size difference, groupers with weight of 7.5 g-10.0 g were selected and randomly divided into four feeding dose groups (7k content of 0,10 respectively)⁶,10⁸And 10¹⁰cfu g^-1) Each group comprised two 20L water jars, each containing 20 grouper.

3) Data analysis

And (3) encoding and sorting the recorded original data, inputting the encoded and sorted original data into a computer, establishing an Excel database, carrying out logic statistical analysis, and deleting records with abnormal values and missing values. The processing and analysis of the survey data are mainlyWhen the analysis of variance is used for comparison among groups, Student-Newman-Keulstest is used for analyzing differences among groups, t test is used for comparing relative expression of the immune genes, all results are expressed as mean value +/-SD, and p < 0.05 is significant difference, wherein the relative expression data of mRNA is obtained by 2-fold reaction between target gene and β -actin^△△ctThe method is expressed by adopting L ightCycler480 software for analysis, and the variance analysis is carried out by adopting SAS9.3 software.

4) Growth influence test

The growth impact test included 4 feeding dose groups: the 7K content of Bacillus subtilis in the feed is respectively 0 and 10⁶,10⁸And 10¹⁰cfu g^-1. Grouper was fed twice daily (9:00am,5:00pm) at 3% -5% of body weight. In order to prevent the mutual pollution of the groups of bacteria, an independent circulating system is adopted in each group of water jars, and the water jars are replaced by 70% of clean seawater every day. The test was run for 4 weeks, groups of grouper were weighed at the beginning of the test and the following week, and dead individuals were removed and recorded in time. Wherein the calculation formulas of the percentage growth body weight (PWG) and the Feeding Efficiency (FE) are respectively as follows:

percent gain weight ═ 100 × (final weight-initial weight) (initial weight)^-1]；

Efficiency of feeding [ (final weight-initial weight) (food intake)^-1]。

The data results of the experiment on the influence of the bacillus subtilis 7k on the growth of the grouper are shown in table 3. No significant differences (P < 0.05) were observed in the body weight of grouper groups before the start of the experiment. After 4 weeks of feeding, the feed had a 7k content of 10⁶,10⁸And 10¹⁰cfu g^-1The final body weight, weight gain, PWG and FE of the grouper are all significantly higher than the grouper group without 7k addition to the feed (P < 0.05). But with a bacterial content of 10⁶,10⁸And 10¹⁰cfu g^-1In the final body weight, body weightIn increments, the difference between PWG and FE was not statistically different (P < 0.05). In addition, no dead individuals were present in any of the four groups of groupers in the 4-week growth trial.

TABLE 3 influence of Bacillus subtilis 7K in feed on growth of grouper

Note: growth data values are expressed as mean ± standard deviation, with the same letter superscript indicating no statistical difference where P < 0.05 is statistically different.

EXAMPLE 3 viral infection assay

The virus strain used in the infection experiments, Singapore Grouper Iridovirus (SGIV), was stored in the laboratory. Before the start of the viral infection test, each group of groupers was fed with Bacillus subtilis 7K in an amount of 0,10, respectively⁶,10⁸And 10¹⁰cfu g^-1The feed of (2) for 35 days. Each group included 40 fish, 30 of which were housed in an average of 3 water jars as a source of data for statistical mortality, and 10 additional fish were used as a source of tissue for measuring immune gene expression levels. All grouper experimental groups were immersed in the solution containing SGIV 10 by soaking for contamination³cfu L^-1The cultivation is carried out in seawater for 2 days. At 48h the infection test was performed, 4 fish were randomly selected from each group as fish for measuring the expression level of the immune gene, spleen tissues were taken out and rapidly placed in liquid nitrogen for storage at-80 ℃. In the whole experiment, each group continues to feed the feed with corresponding bacteria content twice a day (9:00am,5:00pm), and independent circulating water is adopted among the groups without adding new seawater for updating. The dead individuals will be removed from the water tank in time and recorded for a total of 10 days throughout the test.

Three days before the grouper receives SGIV soaking infection, the bacteria content of the feeding is 10⁸And 10¹⁰cfu g^-1Some groupers in the two groups have the conditions of reduced appetite, abnormal swimming posture, even wound ulcer and the like, but no dead groupers exist. Viral infection over a period of ten daysTest, feeding 10⁸cfu g^-1The cumulative mortality rate (46.67 plus or minus 11.55 percent) of the groupers added with the feed of the Bacillus subtilis 7K is obviously higher than 10¹⁰cfu g^-1(30.00. + -. 0%). The cumulative mortality (73.33 +/-5.77%) of the groupers without the addition of Bacillus subtilis 7K is obviously higher than 10⁸cfu g^-1The grouper is added with feed by Bacillus subtilis 7K. But feed 10⁶cfu g^-1The difference between the cumulative mortality rates of the groupers fed with Bacillus subtilis 7K (60.00 + -10.00%) and the groupers without Bacillus subtilis 7K (P < 0.05) did not have a statistical difference. The results of the experiment are shown in FIG. 8.

EXAMPLE 4 Immunity Gene expression

EXAMPLES 3 expression levels of immune genes in spleen tissues of Epinephelus malabaricus in each experimental group were measured, including MX-1, MX-2, ISG, I L-1 β, I L-8, and TNF- α. Total RNA in spleen tissues was extracted using SV Total RNA Isolation Kit (Promega) according to the manual protocol the quality of extracted RNA was measured by electrophoresis on 1% agar gel and the extracted RNA was reverse transcribed into cDNA using ReverTraace Kit (TOYOBO, Japan). expression levels of immune genes were analyzed by Roche480 Real Time Detection System (Roche, German) using relatively quantitative PCR primers shown in Table 4.

TABLE 4 primer sequences for the fluorescent quantitative PCR detection of immune-related gene expression

After 48 hours of infection of the giant grouper by SGIV virus, the expression changes of 6 immune related genes are analyzed and detected, namely three proinflammatory cytokine genes (I L-1 β, I L-8 and TNF α) and three anti-virus related genes (MXI, MXII and ISG15), wherein 10 is added into the feed¹⁰cfu g^-1Grouper with Bacillus subtilis 7K compared with 10⁶And 0cfu g^-1The groupers in the group are all obviously up-regulated in three proinflammatory factors of I L-1 β, I L-8, TNF α and the like, and are fed with the feed 10⁶cfu g^-1The grouper I L-8 gene expression level of the group was also significantly higher than that of the control group without 7k addition for 10⁸And 10¹⁰cfu g^-1These two dose groups, 10¹⁰cfu g^-1The expression level of the group I L-1 β is obviously higher than 10⁸cfu g^-1Group, but significantly less than 10 levels of TNF α⁸cfu g^-1And (4) grouping.

The expression levels of the three antiviral genes showed a dose-dependent relationship (FIG. 8), which is 10¹⁰cfu g^-1The average expression level of three genes of MXI, MXII, ISG15 is obviously higher than 10⁶And 0cfu g^-1Gene expression level of group, and 10¹⁰cfu g^-1Groups MXI and ISG15 were also significantly higher than 10⁸cfu g^-1Group (d); and 10⁸cfu g^-1The average of the expression levels of three genes of MXI, MXII, ISG15 and the like is obviously higher than 10⁶And 0cfu g^-1Expression levels of the corresponding genes of the panel. In addition to this, feeding 10⁶cfu g^-1The grouper is also more than 0cfu g^-1The groups expressed higher levels of ISG 15.

In conclusion, the Bacillus subtilis 7K is determined to be the Bacillus subtilis 7K by separating, identifying and sequencing the Bacillus subtilis through 16S rDNA sequencing, performing B L AST comparison analysis on the obtained sequence in NCBI, and determining that the Bacillus subtilis 7K shows strong heat-resistant and gastrointestinal digestion resistant characteristics through biochemical identification and physiological and biochemical characteristic tests, so that the Bacillus subtilis 7K can survive in feed thermal processing and marine fish intestinal tracts better through heat-resistant and gastrointestinal fluid-resistant tests, and further play a probiotic role, and the extracellular bacteriostatic test shows that a supernatant obtained after fermentation culture of the Bacillus subtilis 7K can be used for developing staphylococcus aureus, aeromonas hydrophila, metolysate, vibrio harveyi, vibrio alginolyticus and wound and has obvious bacteriostatic effect on staphylococcus aureus, vibrio harveyi, staphylococcus harveyi Bacillus subtilis and wound, and has obvious bacteriostatic effect on staphylococcus aureus, vibrio harveyi, staphylococcus harveyi, vibrio alginolyticus and wound, and the primary bacteriostatic effect of Bacillus subtilis by combining with a Bacillus subtilis HP.

Respectively taking Bacillus subtilis (Bacillus subtilis)7K as 0 and 10⁶,10⁸And 10¹⁰cfu g^-1The four feeds are fed to the pearl giant grouper. The results show that the feed contains 0,10⁶,10⁸And 10¹⁰cfu g^-1The final weight, weight gain, percentage increase weight and feeding efficiency of the grouper with Bacillus subtilis 7K are obviously higher than those of a control group. In the SGIV infection test, feeding 10¹⁰，10⁸cfu g^-1The death rate of the grouper with the Bacillus subtilis 7K is obviously lower than 10⁶Group and control group. While the relative quantitative PCR results indicated that the feed contained 10⁸And 10¹⁰cfu g^-1The expression levels of six genes of I L-1 β, I L-8, TNF α, MXI, MXII, ISG15 and the like of grouper fed with 7K Bacillus subtilis (Bacillus subtilis) are obviously higher than 0 and 10⁶cfu g^-1And (4) grouping. Animal experiments show that the Bacillus subtilis 7K added into the feed can promote the growth of the giant grouper, and the resistance of the giant grouper to virus infection is increased through up-regulation of relevant immune gene expression, so that the death rate caused by virus infection is reduced. Therefore, the Bacillus subtilis 7K can obviously reduce the occurrence of aquatic animal diseases and is a probiotic strain with application value.

Appropriate changes and modifications to the embodiments described above will become apparent to those skilled in the art from the disclosure and teachings of the foregoing description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Sequence listing

<110> southern China university of agriculture

<120> bacillus subtilis 7K and application thereof

<160>17

<170>SIPOSequenceListing 1.0

<210>1

<211>1419

<212>DNA

<213> Bacillus subtilis 16SrDNA (Bacillus subtilis 7K)

<400>1

tacatgcaag tcgagcggac agatgggagc ttgctccctg atgttagcgg cggacgggtg 60

agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc ggggctaata 120

ccggatggtt gtttgaaccg catggttcag acataaaagg tggcttcggc taccacttac 180

agatggaccc gcggcgcatt agctagttgg tgaggtaacg gctcaccaag gcgacgatgc 240

gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac 300

gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt 360

gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa gtgccgttca 420

aatagggcgg caccttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag 480

ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa gggctcgcag 540

gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc attggaaact 600

ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga aatgcgtaga 660

gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac gctgaggagc 720

gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta aacgatgagt 780

gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa gcactccgcc 840

tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggt 900

ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg 960

acaatcctag agataggacg tccccttcgg gggcagagtg acaggtggtg catggttgtc 1020

gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgatctta 1080

gttgccagca ttcagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg 1140

gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggac 1200

agaacaaagg gcagcgaaac cgcgaggtta agccaatccc acaaatctgt tctcagttcg 1260

gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagcat 1320

gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgt 1380

aacacccgaa gtcggtgagg taacctttag gagccagcc 1419

<210>2

<211>24

<212>DNA

<213>K1F(Artificial Sequence)

<400>2

aactgaagag tttgatcctg gctc 24

<210>3

<211>22

<212>DNA

<213>K2R(Artificial Sequence)

<400>3

tacggttacc ttgttacgac tt 22

<210>4

<211>21

<212>DNA

<213>MXI-RT-F(Artificial Sequence)

<400>4

cgaaagtacc gtggacgaga a 21

<210>5

<211>23

<212>DNA

<213>MXI-RT-R(Artificial Sequence)

<400>5

tgtttgatct gctccttgac cat 23

<210>6

<211>25

<212>DNA

<213>MXII-RT-F(Artificial Sequence)

<400>6

gcttcatcaa ctacaagacc ttcga 25

<210>7

<211>26

<212>DNA

<213>MXII-RT-R(Artificial Sequence)

<400>7

cgccttccta acagtatctc ctattt 26

<210>8

<211>19

<212>DNA

<213>ISG15-RT-F(Artificial Sequence)

<400>8

gggtgtccct gctggtgat 19

<210>9

<211>21

<212>DNA

<213>ISG15-RT-R(Artificial Sequence)

<400>9

ctctctgccc tggtgaatga g 21

<210>10

<211>20

<212>DNA

<213>IL-1 β -RT-F(Artificial Sequence)

<400>10

aacctcatca tcgccacaca 20

<210>11

<211>21

<212>DNA

<213>IL-1 β -RT-R(Artificial Sequence)

<400>11

agttgcctca caaccgaaca c 21

<210>12

<211>22

<212>DNA

<213>IL-8-RT-F(Artificial Sequence)

<400>12

gccgtcagtg aagggagtct ag 22

<210>13

<211>19

<212>DNA

<213>IL-8-RT-R(Artificial Sequence)

<400>13

atcgcagtgg gagtttgca 19

<210>14

<211>22

<212>DNA

<213>TNFα-RT-F(Artificial Sequence)

<400>14

gtgtcctgct gtttgcttgg ta 22

<210>15

<211>24

<212>DNA

<213>TNFα-RT-R(Artificial Sequence)

<400>15

cagtgtccga cttgattagt gctt 24

<210>16

<211>20

<212>DNA

<213>Actin- RT-F(Artificial Sequence)

<400>16

tacgagctgc ctgacggaca 20

<210>17

<211>20

<212>DNA

<213>Actin- RT-R(Artificial Sequence)

<400>17

ggctgtgatc tccttctgca 20

Claims

1. A Bacillus subtilis (Bacillus subtilis)7K is preserved in Guangdong province microorganism culture collection center, and the preservation number is GDMCC No: 60226 with preservation address of No. 59, No. 5, No. 100 college of Xieli Zhonglu, Guangzhou city and preservation date of 2017, 8 and 31.

2. Use of Bacillus subtilis 7K according to claim 1 for the preparation of a medicament for promoting growth, enhancing immunity, enhancing expression level of immune genes of aquatic animals, and preventing or treating diseases of aquatic animals, wherein the aquatic animals are groupers.

3. An aquatic animal feed additive comprising an effective amount of Bacillus subtilis 7K according to claim 1 as an active ingredient.

4. A microecological formulation for use in regulating the aquatic environment of aquatic animal farming, comprising an effective amount of the Bacillus subtilis 7K of claim 1 as an active ingredient.

5. A medicament for enhancing immunity of grouper comprising an effective amount of Bacillus subtilis 7K of claim 1 as an active ingredient.

6. A medicament for preventing or treating grouper disease, comprising an effective amount of Bacillus subtilis 7K of claim 1 as an active ingredient.