CN111411053B

CN111411053B - Bacillus subtilis JCL16 capable of synergistically producing three antibacterial metabolites and screening and application thereof

Info

Publication number: CN111411053B
Application number: CN202010193730.3A
Authority: CN
Inventors: 罗楚平; 管明; 强晓刚; 陈树桥; 王小花; 仲海静; 刘佳琛; 杨雪婷; 游庆红; 尹秀莲; 李相前
Original assignee: Huaian Fisheries Technical Guidance Station; Huaiyin Institute of Technology
Current assignee: Huaian Fisheries Technical Guidance Station; Huaiyin Institute of Technology
Priority date: 2020-03-17
Filing date: 2020-03-17
Publication date: 2023-02-17
Anticipated expiration: 2040-03-17
Also published as: CN111411053A

Abstract

The invention discloses a Bacillus subtilis JCL16 capable of synergistically producing three antibacterial metabolites and screening and application thereof, wherein the Bacillus subtilis JCL16 capable of synergistically producing the three antibacterial metabolites is identified as Bacillus subtilis, and is preserved in China center for type microorganism preservation, the preservation time is 2018, 06, 01 days, and the preservation number is M2018336. The bacillus subtilis JCL16 can generate three antibacterial metabolites which are respectively: surfactin, bacilysin, and subtilisina; the compound preparation has excellent control effect on the disease of the Micropterus salmoides, and particularly, the Micropterus salmoides disease caused by Nocardia seriolae can be used for preparing related biological preparations and feeds.

Description

Bacillus subtilis JCL16 capable of synergistically producing three antibacterial metabolites and screening and application thereof

Technical Field

The invention belongs to disease control of aquaculture, relates to a bacillus subtilis JCL16 capable of synergistically producing three antibacterial metabolites and screening and application thereof, and also relates to a control effect of a bacterial strain JCL16 for producing surfactin, lysobactin and subtilisina on nocardiosis of seria quinqueradiata and application thereof in feed.

Background

The Lateolabrax japonicas is named as Micropterus salmoides (Lacep e 1802) and belongs to Perciformes, sunglatidae and Perolabrax, and is originally produced in the water system of Mississippi river of California. The micropterus salmoides mainly inhabit water areas with low turbidity and aquatic plant distribution, and are greatly expanded in the culture area of China due to fresh and delicious meat, rapid growth and easy fishing, so that the micropterus salmoides become important economic fishes.

The nocardia seriolae is widely distributed in water, soil and activated sludge, and both human and animals can be infected with diseases under certain conditions. Nocardia seriolae is usually not obvious in characterization after initial infection of juvenile fish and is difficult to find in time. Infection and pathogenesis are lengthy, with typical symptoms and significant harm often present during adult fish. Nocardiosis in seriolae has a very slow growth and reproduction speed in fish body, so the disease course is very long, and especially a long proliferation development period and a long disease course are needed before symptoms appear or the fish dies. The cultured fish population infected with Nocardia seriolae can cause great reduction of yield, very low feed conversion, high mortality, and poor body condition of the fish harvested at the end of summer, which is very weak. The mode of transmission of the disease varies, and the initial stage of small fish may be infected by iced bait or nocardia seriolae with the transmission of infection by diseased fish, and feeding together with diseased or infected fish is also one of the causes of infection.

The bacillus is a microorganism with important use value and economic value, is rich in variety and wide in distribution, and produces various bioactive substances. Bacillus is a gram-positive bacterium that produces resting spores to spend harsh environments. Because the spores have very strong stress resistance and are not easy to inactivate in the transportation or storage process, the bacilli are ideal biological pesticide. And secondly, the bacillus has simple nutritional requirement and high growth speed, so that the cost of production and fermentation is low, and the bacillus is favorable for developing a biocontrol microbial inoculum. Most importantly, a large number of bacilli are also capable of producing a variety of secondary metabolites, which are important sources of antimicrobially active substances. The bacillus has good ecological adaptability, can compete with various microorganisms, is beneficial to controlling pathogenic bacteria, has lasting effect and strong struggle, and shows good application prospect. If bacillus with antagonistic effect on micropterus salmoides can be separated from soil such as lakes, ponds and the like, the bacillus has important significance on preventing and treating nocardia seriolae.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a strain JCL16 which can synergistically produce three antibacterial metabolites and is identified as Bacillus subtilis, and the Bacillus subtilis JCL16 can produce three antibacterial metabolites which are respectively as follows: surfactin, bacilysin, and subtilisina; has excellent prevention and treatment effect on Micropterus salmoides nocardioides.

The invention also provides screening and application of the strain JCL16.

The technical scheme is as follows: in order to achieve the above object, the strain JCL16, which is a strain that synergistically produces three antibacterial metabolites and is identified as Bacillus subtilis, is deposited in the chinese type culture collection, with the following storage addresses: wuhan, wuhan university, zip code: 430072, preservation time of 2018, 06 months and 01 days, preservation number of CCTCC NO: M2018336. The strain JCL16 is derived from the soil of an aquaculture institute in Huaian city, the form of a single colony on a culture medium is observed through aseptic operation, and the bacillus subtilis JCL16 is circular on an LB culture medium, dark white, opaque and neat in edge.

Wherein the three antibacterial metabolites are: surfactin, bacilysin, and subtilisnA.

The screening method of the strain JCL16 capable of synergistically producing three antibacterial metabolites comprises the following steps:

fully soaking the soil sample in sterile water, diluting 1mL of soil suspension by 10 times of the sterile water, dropwise adding 100uL of diluted test solution on an LB solid culture medium, uniformly coating, and inverting for overnight culture; and observing the morphology of a single colony on the culture medium through aseptic operation, wherein the morphology comprises the color, the size, the colony edge and the surface roughness of the colony, and obtaining the primary screening strain of the bacillus 86 strain. And (3) selecting the primarily screened strains obtained by separation and screening as test bacteria, selecting the Nocardia seriolae as an indicator bacteria, and screening the Bacillus subtilis JCL16 with the best antagonistic effect on the Nocardia seriolae by an Oxford cup method.

The invention discloses application of a strain JCL16 capable of synergistically producing three antibacterial metabolites in production of antibacterial metabolites including Surfactin, lysobactin Bacilysin and subtilisina.

The three antibacterial metabolites of the invention have obvious synergistic effect after being mixed in different proportions.

Preferably, the strain JCL16 is inoculated into a culture medium for fermentation, and the antibacterial metabolite is separated from a fermentation broth.

Preferably, the strain JCL16 is inoculated into a culture medium for fermentation under the conditions that a shaking table is used for culturing at 37 ℃ and the constant temperature of 160rpm, 100mL of the culture medium is contained in a 250mL triangular flask, and the fermentation time is 60 hours.

Wherein the culture medium is a fermentation culture medium and comprises the following components in percentage by mass: soybean cake powder 2-2.5%, millet flour 2-3%, glucose 2-3%, (NH) ₄ ) ₂ SO ₄ 0.8-1％、KCl 0.3-0.5％，MgSO ₄ 0.01 percent, and the balance of water.

The application of the strain JCL16 which synergistically produces three antibacterial metabolites in preventing and treating nocardiosis of seriolala quinqueradiata, in particular to the application of the strain JCL16 in preventing and treating the nocardiosis of the seriolala quinqueradiata.

The invention relates to application of a strain JCL16 which synergistically produces three antibacterial metabolites in preparation of a biocontrol preparation or feed for preventing and treating Micropterus salmoides diseases, in particular to diseases caused by Nocardia seriolae.

The biocontrol agent or the feed contains thalli, spores or fermentation liquor of the bacillus subtilis JCL16.

The biocontrol preparation or the feed is obtained by adding 180mL of fermentation liquor of the strain JCL16 into 1kg of basic feed (42.2% of crude protein, 11.9% of crude fat and 2.5mm of particle size which are purchased from the aquatic product market in Huaian city and expanded feed).

Preferably, the contents of three antibacterial metabolites, surfactant, bacilysin and subtilisina, in the fermentation broth are 2.4g/L, 1.8g/L and 1.3g/L, respectively.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention finds a bacillus subtilis JCL16, in particular to a strain which synergistically produces three antibacterial metabolites and the prevention and treatment effects of the three antibacterial metabolites on nocardia seriolana of weever, wherein the three antibacterial metabolites are respectively as follows: surfactin, bacilysin, subtilisin. Meanwhile, the invention further researches a fermentation culture medium for high yield of three antibacterial metabolites, which comprises 2-2.5% of soybean cake powder, 2-3% of millet flour, 2-3% of glucose and (NH) ₄ ) ₂ SO ₄ 0.8-1％、KCl 0.3-0.5％，MgSO ₄ 0.01% of water as the rest; three anti-bacterial strains generated by strain JCL16The bacterial metabolite has obvious inhibition effect on Nocardia seriolae, and the three antibacterial metabolites have synergistic effect.

According to the invention, the basal feed and the basal feed added with the Bacillus subtilis JCL16 are respectively fed to the Micropterus salmoides injected with the Nocardia seriolae (180 mL of Bacillus subtilis JCL16 fermentation liquor is added to each 1kg of the basal feed), and the result shows that the survival rate of the Micropterus salmoides infected with the Nocardia seriolae is far higher than that of the Micropterus salmoides fed with the common feed by adding the Bacillus subtilis JCL16 fermentation liquor to the fed feed, so that the Bacillus subtilis JCL16 has a good control effect on the Nocardia salmoides, and can be effectively applied to the preparation or the feed for controlling the Micropterus salmoides disease.

In conclusion, the bacillus subtilis JCL16 has good prevention and treatment effects on relevant diseases of micropterus salmoides, and can be used for preparing relevant biological preparations and feeds.

Drawings

FIG. 1 is a morphology chart of Bacillus subtilis JCL16, wherein the left picture is a colony photograph cultured in LB medium at 28 ℃ for 24h, and the right picture is a colony morphology chart;

FIG. 2 is a graph showing the measurement of the bacteriostatic activity of Bacillus subtilis JCL16 against Nocardia seriolae by the Oxford cup method;

FIG. 3 is a liquid chromatogram of surfactant in a fermentation product of Bacillus subtilis JCL 16;

FIG. 4 is a mass spectrum of surfactant in a fermentation product of Bacillus subtilis JCL 16;

FIG. 5 is a liquid chromatogram of lysobactin in a fermentation product of Bacillus subtilis JCL 16;

FIG. 6 is a mass spectrum of lysobactin in a fermentation product of Bacillus subtilis JCL 16;

FIG. 7 is a liquid chromatogram of subtilosinA in a Bacillus subtilis JCL16 fermentation product;

FIG. 8 is a graph showing the bacteriostatic activity of surfactin, lysobactin and subtilisina against Nocardia seriolae by the Oxford cup method.

Detailed Description

The invention is further illustrated by the following figures and examples.

Materials, reagents and the like used in examples of the present invention are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer. The Nocardia seriolae used in the method is provided by a biomass conversion process and an integrated laboratory of Huaiyin institute of Industrial science, and other wild Nocardia seriolae can also be used.

Example 1

Separation and screening of the strain JCL16 and determination of the antibacterial activity:

the fertilizer is obtained by separating soil from aquaculture institute in Huaian city, and the separation process is as follows: selecting 10g of soil with the depth of 10-20 cm, and adding 100ml of normal saline to mix evenly. Water bath is carried out for 1h at the temperature of 80 ℃, then 4g of NaCl is added, and water bath is carried out for 2h at the temperature of 37 ℃. Fully soaking the soil in sterile water, diluting 1mL of soil suspension by 10 times of the sterile water, dropwise adding 100uL of diluted test solution on an LB solid culture medium, uniformly coating, and inverting at 37 ℃ for overnight culture. And observing the morphology of a single colony on the culture medium through aseptic operation, wherein the morphology comprises the color, the size, the colony edge and the surface roughness of the colony, and obtaining the primary screening strain of the bacillus 86 strain. The cultured primary screened strain is used as a test strain, nocardia seriolae is used as an indicator strain, a strain with good antagonistic effect on the Nocardia seriolae is screened out by an oxford cup method, and the strain is named as JCL16. The morphology of a single colony on the culture medium is observed through aseptic operation, and the strain JCL16 is round, dark white, opaque and neat in edge on an LB culture medium, and is shown in figure 1.

The method for measuring the bacteriostatic activity by adopting the Oxford cup method comprises the following specific steps of: taking a single bacterial colony of the strain to be cultured in an LB liquid culture medium for 24 hours at the temperature of 28 ℃, trisecting a plate with the Nocardia seriolae as an indicator bacterium, clamping 3 Oxford cups in an aseptic operation, vertically placing the Oxford cups on the plate, respectively taking 0.2mL of bacterial liquid to be injected into the Oxford cups, carrying out experimental comparison, culturing for 24 hours at the temperature of 28 ℃, observing the zone around the Oxford cups, wherein the diameter/mm of the zone = the total diameter/mm-the diameter/mm of the Oxford cups, and obtaining the result as shown in figure 2, wherein the strain JCL16 has a good inhibition effect on the Nocardia seriolae, and the diameter of the zone is 17.8 +/-0.3 mm.

Identification of strain JCL 16:

culturing the selected strain JCL16 to logarithmic growth phase, and extracting the genome DNA of the strain. Amplifying a 16srDNA fragment thereof by using bacterial universal primers SEQ ID NO.1F27 (AGAGAGTTTGATCCTGGCTCAG) and SEQ ID NO.2R1492 (TACGGCTACCTTGTTACGACTT) for sequencing, wherein the total sequence length is 1455bp, and the sequence is shown in a sequence table SEQ ID NO.3; through sequence comparison, the complete sequence of the strain JCL16 is compared with sequences of Bacillus subtilis BS916 and the like registered by NCBI by adopting a BLAST analysis method, the homology is 99%, the strain JCL16 is identified as Bacillus subtilis by combining the results of physiological and biochemical experiments of the strain JCL16, the strain is named as Bacillus subtilis JCL16, the strain is preserved in China center for type microorganism preservation, the preservation time is 2018, 06 months and 01 days, and the preservation number is CCTCC NO: M2018336.

Example 2

Bacillus subtilis JCL16 fermentation culture by using industrial fermentation culture medium

(1) Seed culture: taking a loop of bacillus subtilis JCL16 stored on a slant, streaking and inoculating the bacillus subtilis JCL16 in a plate of an LB culture medium (tryptone 1%, yeast powder 0.5%, naCl1%, agar 1.5%, the balance water and pH 7.0), after overnight culture, taking a single colony to inoculate in an LB test tube culture medium, culturing at 37 ℃ for 180r/min for 24 hours, and preparing a seed solution.

(2) Fermentation culture: inoculating the seed solution into 1L of industrial fermentation culture medium according to the volume ratio of 3%, wherein the seed solution comprises the following components in percentage by mass: 2% of soybean cake powder, 2% of millet flour, 2% of glucose and (NH) ₄ ) ₂ SO ₄ 0.8％、KCl 0.3％、MgSO ₄ 0.01 percent and the balance of water, the pH is natural, and the sterilization is carried out for 15 minutes at 115 ℃. Fermentation culture conditions: culturing at 37 deg.C and 160rpm for 60h.

(3) Preparing a crude extract: performing acid precipitation and methanol extraction, centrifuging the fermentation liquor at 7000rpm/min for 8min, adjusting the fermentation liquor to pH 2.0,4 ℃ by using HCL, standing overnight, centrifuging at 7000rpm for 8min, collecting the precipitate, adding methanol (the amount of the methanol is 1mL per 100mL of the fermentation liquor) into the precipitate, adding NAOH to adjust the pH to 7.0,4 ℃, magnetically stirring for 5h, centrifuging at 12000rpm for 10min, collecting the supernatant, and filtering by using a 0.22 mu m filter membrane to obtain the clear liquid, namely the crude extract).

(4) Analyzing by a liquid phase analysis system to obtain a fermentation product with the surfactant content of 2.4g/L and the lysobactin content of 1.8g/L, and detecting conditions of the surfactant and the lysobactin: a chromatographic column: agilent zorbax sb-Aq C18 (specification: 4.6X 250mm, particle size: 5 μm); the column temperature is 29 ℃; the sample injection amount is 20 mu L; mobile phase: acetonitrile: water (20; the flow rate is 0.5ml/min; the detection wavelength was 230nm. The content of the subtilisnA is 1.3g/L, and the detection condition of the subtilisnA is as follows: the chromatographic column is C18 (specification: 4.6X 250mm, particle size: 5 μm); the column temperature is 29 ℃; the sample injection amount is 20uL; the mobile phase A is water containing 1 per mill, and the mobile phase B is acetonitrile containing 1 per mill; the flow rate is 0.6ml/min; the detection wavelength was 230nm. The amount of Bacillus subtilis JCL16 in the fermentation medium was 5X 10 by turbidity method ¹⁰ cfu/mL。

The liquid phase diagrams of the surfactant, the lysobactin and the subtilisina are shown in fig. 3, fig. 5 and fig. 7, which show that the fermentation liquor obtained by fermenting and culturing the bacillus subtilis JCL16 disclosed by the invention contains abundant surfactant, lysobactin and subtilisina, and the fermentation liquor has higher purity, and the HPLC purity is achieved.

Example 3

(2) Fermentation culture: inoculating the seed liquid into 1L of industrial fermentation culture medium according to the volume ratio of 3%, wherein the seed liquid comprises the following components: soybean cake powder 2.5%, millet flour 3%, glucose 3%, (NH) ₄ ) ₂ SO ₄ 1％、KCl 0.5％、MgSO ₄ 0.01 percent and the balance of water, the pH is natural, and the sterilization is carried out for 15 minutes at 115 ℃. Fermentation culture conditions: culturing at 37 deg.C and 160rpmCulturing for 60h.

(3) Crude extract preparation same as example 2

(4) The fermentation product was analyzed by a liquid phase analysis system (detection conditions were the same as in example 2) to have a surfactant content of 2.2g/L, a lysobactin content of 1.7g/L, and a subtilisin content of 1.2g/L. The amount of Bacillus subtilis JCL16 in the fermentation medium was 4X 10 as determined by turbidity method ¹⁰ cfu/mL。

Example 4

(1) Seed culture: taking a loop of bacillus subtilis JCL16 stored on a slant, streaking and inoculating the bacillus subtilis JCL16 in a plate of an LB culture medium (tryptone 1%, yeast powder 0.5%, naCl1%, agar 1.5%, the balance of water and pH 7.0), culturing at 37 ℃ at 180r/min for 24h, and preparing a seed solution.

(2) Fermentation culture: inoculating the seed liquid into 1L of industrial fermentation culture medium according to the volume ratio of 3%, wherein the seed liquid comprises the following components: soybean cake powder 2.5%, millet flour 2%, glucose 3%, (NH) ₄ ) ₂ SO ₄ 1％、KCl 0.3％、MgSO ₄ 0.01 percent and the balance of water, the pH is natural, and the sterilization is carried out for 15 minutes at 115 ℃. Fermentation culture conditions: the shaking table is used for culturing at the constant temperature of 37 ℃ and 160rpm, and the fermentation time is 60h.

(3) Crude extract preparation same as example 2

(4) The fermentation product obtained by the analysis by a liquid phase analysis system (the detection conditions were the same as in example 2) had a surfactant content of 2.1g/L, a lysobactin content of 1.6g/L, and a subtilisina content of 1.3g/L. The amount of Bacillus subtilis JCL16 in the fermentation medium was 5X 10 by turbidity method ¹⁰ cfu/mL。

Example 5

(1) Seed culture: taking a loop of bacillus subtilis JCL16 stored on a slant, streaking and inoculating the bacillus subtilis JCL16 in a plate of an LB culture medium (tryptone 1%, yeast powder 0.5%, naCl1%, agar 1.5%, the balance water and pH 7.0), culturing at 37 ℃ and 180r/min for 24h, and preparing a seed solution.

(2) Fermentation culture: inoculating the seed liquid into 1L of industrial fermentation culture medium according to the volume ratio of 3%, wherein the seed liquid comprises the following components: soybean cake powder 2%, millet flour 3%, glucose 3%, (NH) ₄ ) ₂ SO ₄ 1％、KCl 0.5％、MgSO ₄ 0.01 percent and the balance of water, the pH is natural, and the sterilization is carried out for 15 minutes at 115 ℃. Fermentation culture conditions: culturing at 37 deg.C and 160rpm for 60h.

(3) Crude extract preparation same as example 2

(4) The fermentation product was analyzed by a liquid phase analysis system (detection conditions were the same as in example 2) to have a surfactant content of 2.2g/L, a lysobactin content of 1.6g/L, and a subtilisin content of 1.1g/L. The bacterial amount of the bacillus subtilis JCL16 in the fermentation medium is 4.5 multiplied by 10 measured by a turbidity method ¹⁰ cfu/ml。

Comparative example 1

Bacillus subtilis JCL16 fermentation culture by utilizing LB fermentation culture medium

(2) Fermentation culture: inoculating the seed liquid into 1L of LB fermentation medium according to the volume ratio of 3%, wherein the fermentation culture conditions are as follows: culturing at 37 deg.C and 160rpm for 60h.

(3) Crude extract preparation same as example 2

(4) The fermentation product obtained by the analysis was analyzed by a liquid phase analysis system (detection conditions were the same as in example 2) to have a surfactant content of 1.3g/L, a lysobactin content of 0.9g/L, and a subtilisina content of 0.6g/L. The bacillus subtilis JCL16 bacterial amount in the fermentation medium is 4 multiplied by 10 measured by a turbidity method ⁹ cfu/ml。

Compared with comparative example 1, example 2, example 3, example 4 and example 5, the industrial culture medium provided by the invention can greatly improve the bacterial content and three antibacterial generations of bacillus subtilis JCL16Production of metabolite, wherein when the composition of the culture medium is: soybean cake powder 2%, millet flour 2%, glucose 2%, (NH 4) 2SO4.8%, KCl 0.3%, mgSO 2 ₄ 0.01 percent, the balance being water, natural pH, and 15 minutes of sterilization at 115 ℃, the yield of the antibacterial metabolite of the bacillus subtilis JCL16 is the highest, wherein the content of the surfactant is 2.4g/L, the content of the lysobactin is 1.8g/L, and the content of the subtilisina is 1.3g/L.

Example 6

Inhibition of Nocardia by three antibacterial metabolites

The effluent corresponding to the peak of the substance separated in the liquid phase in example 2 was taken and purified by mass spectrometry (crude extraction was determined by mass spectrometry under conditions of capillary voltage 32V, spray voltage 20kV, capillary temperature 320 ℃ and detection mode of positive ion, saturated alpha-cyano-4-hydroxycinnamic acid, 0.1% trifluoroacetic acid, acetonitrile and water (volume ratio 3: 1) diluted 1 000 times and directly injected) to prepare pure products of tensioacetin, lysobactin and subtilisina at concentrations of 2.4g/L, 1.8g/L and 1.3g/L, respectively, and mass spectrograms of tensinogen and lysobactin are shown in FIGS. 4 and 6. The inhibition effect of pure tenside, lysobactin and subtilisina on nocardia seriolae was investigated according to the oxford cup method of example 1, and the results are shown in fig. 8: A. b, C is respectively the bacteriostatic activity diagrams of surfactin, lysobactin and subtilisina for Nocardia seriolae, and the diameters of the bacteriostatic circle are respectively 14.1 + -0.3 mm, 14.7 + -0.4 mm and 15.3 + -0.3 mm.

Example 7

Synergistic effect of three antibacterial metabolites

(1) Preparation of inoculum: single colony of Nocardia seriolae is picked by an inoculating loop, inoculated in 5ml hydrolyzed casein MH broth (2 g/L beef powder, 1.5g/L soluble starch, 17.5g/L hydrolyzed casein, the balance being water, pH 7.4), and incubated for 6h at 35 ℃. The logarithmic phase bacterial liquid after enrichment is corrected to 0.5 McLeod turbidity standard by normal saline or MH broth, and the concentration is about 1-2 × 10 ⁸ CFU/ml, diluted 100-fold with MH broth.

(2) Antibacterial metabolite minimal inhibitory concentration experiments: taking a sterile test tube (13X 100 mm) 13, arranging in a row, adding 1ml of MH broth into each tube except 2ml of MH broth into the 1 st tube, adding 512ug of lysobactin into the 1 st tube, uniformly mixing, sucking 1ml into the 2 nd tube, sucking 1ml into the 3 rd tube after uniform mixing, continuously diluting to the 11 th tube in a multiple ratio, sucking 1ml from the 11 th tube, and discarding, wherein the 12 th tube is a growth control without antibacterial metabolites. In this case, the lysobactin concentration in each tube was 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25. Mu.g/ml in this order. Then 1ml of the prepared inoculum was added to each tube to give a final bacterial liquid concentration of about 5X 10 ⁵ CFU/ml. The drug concentrations of the 1 st tube to the 11 th tube are respectively 128, 64, 32, 16, 8, 4, 2, 1, 05, 0.25 and 0.125 mu g/ml, and the mixture is cultured in an incubator at 35 ℃ for 24 hours. Experiments of the minimum inhibitory concentrations of surfactant and subtilisina were performed in the same manner.

(3) As a result, it was judged that before the MIC of each antibacterial metabolite was read, the growth of the bacteria in the growth control tube should be checked for good growth, and the subculture of the inoculum should be checked to determine whether it was contaminated. The lowest concentration tube of the drug is observed by naked eyes, and the tube without bacteria growth is the MIC of the tested bacteria. The results show that: the minimum inhibitory concentrations of lysobactin, surfactin and subtilisina were 128ug/mL, 64ug/mL and 32ug/mL, respectively.

(4) Synergistic effect of three antibacterial metabolites: mixing lysobactin, surfactant and subtilisina in different proportions in pairs, mixing three kinds of mixture at the same time, measuring the MIC value of the mixture, actually expressing the MIC value, and calculating the theoretical MIC value of the mixture by the following formula:

coefficient of Synergy (SR) = actual MIC of mixture/theoretical MIC of mixture

SR > 1.5 is synergistic effect; SR is more than or equal to 0.5 and less than or equal to 1.5, which is the additive effect; SR < 0.5 is antagonistic.

The results are shown in table 1: except for lysobactin-surfactant 1:1, surfactant-subtilisina 1:1, surfactant-subtilisina 1:2 as additive, the remaining proportions of the mixture are synergistic, with the original mixing ratio of anti-bacterial metabolites in bacillus subtilis JCL16, lysobactin-surfactant-subtilisina 1.8, having an SR maximum of 3.9063.

TABLE 1 Combined Effect of three antibacterial metabolites on Nocardia seriolae

Example 8

Three micropterus salmoides are randomly selected, dissected under aseptic operation, and the tissue parts are taken and plated to determine that the micropterus salmoides are free from bacterial infection. The experimental fish were divided into three groups, ten in each group: one infection group, one experimental group, one blank control group. In the infected group and experimental group, nocardia seriolae strain was cultured in BHI medium and diluted to 2.5X 10 ⁷ cfu/mL bacterial suspension, adopting intraperitoneal injection, each piece of micropterus salmoides is injected with 0.2mL. The blank control group was injected intraperitoneally with the same amount of sterile water. The infected group was fed with a basal diet (expanded diet, 42.2% crude protein, 11.9% crude fat, 2.5mm particle size), and the experimental group added the bacillus subtilis JCL16 fermentation broth prepared in example 2 to the basal diet (180 mL broth per 1kg basal diet). The experiment is raised for 7 days at the water temperature of 28 ℃, and the infection and death conditions are recorded at any time.

TABLE 2 prevention and treatment of Nocardia disease by Bacillus subtilis JCL16

The in-vivo prevention and treatment experiment proves that the strain JCL16 has good prevention and treatment effects on the Micropterus salmoides diseases caused by the Nocardia seriolae, the morbidity of an experimental group is far slower than that of an infected group, when all Micropterus salmoides in the infected group die, the survival rate of the experimental group added with the bacillus subtilis is 70%, and the bacillus subtilis JCL16 has good prospects as Micropterus salmoides probiotics and can be effectively applied to preparation of biocontrol preparations or feeds for preventing and treating the Micropterus salmoides diseases.

Sequence listing

<110> Huaiyin institute of Industrial and research

<120> strain JCL16 with antibacterial activity and capable of producing three antibacterial metabolites and separation, screening and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agagtttgat cctggctcag 20

<210> 2

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tacggctacc ttgttacgac tt 22

<210> 3

<211> 1455

<212> DNA

<213> Bacillus subtilis

<400> 3

acattcttgt caccttcggc ggctggctcc taaaaggtta cctcaccgac ttcgggtgtt 60

acaaactctc gtggtgtgac gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc 120

atgctgatcc gcgattacta gcgattccag cttcacgcag tcgagttgca gactgcgatc 180

cgaactgaga acagatttgt gggattggct taacctcgcg gtttcgctgc cctttgttct 240

gtccattgta gcacgtgtgt agcccaggtc ataaggggca tgatgatttg acgtcatccc 300

caccttcctc cggtttgtca ccggcagtca ccttagagtg cccaactgaa tgctggcaac 360

taagatcaag ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 420

gacaaccatg caccacctgt cactctgccc ccgaagggga cgtcctatct ctaggattgt 480

cagaggatgt caagacctgg taaggttctt cgcgttgctt cgaattaaac cacatgctcc 540

accgcttgtg cgggcccccg tcaattcctt tgagtttcag tcttgcgacc gtactcccca 600

ggcggagtgc ttaatgcgtt agctgcagca ctaaggggcg gaaaccccct aacacttagc 660

actcatcgtt tacggcgtgg actaccaggg tatctaatcc tgttcgctcc ccacgctttc 720

gctcctcagc gtcagttaca gaccagagag tcgccttcgc cactggtgtt cctccacatc 780

tctacgcatt tcaccgctac acgtggaatt ccactctcct cttctgcact caagttcccc 840

agtttccaat gaccctcccc ggttgagccg ggggctttca catcagactt aagaaaccgc 900

ctgcgagccc tttacgccca ataattccgg acaacgcttg ccacctacgt attaccgcgg 960

ctgctggcac gtagttagcc gtggctttct ggttaggtac cgtcaaggta ccgccctatt 1020

cgaacggtac ttgttcttcc ctaacaacag agctttacga tccgaaaacc ttcatcactc 1080

acgcggcgtt gctccgtcag actttcgtcc attgcggaag attccctact gctgcctccc 1140

gtaggagtct gggccgtgtc tcagtcccag tgtggccgat caccctctca ggtcggctac 1200

gcatcgttgc cttggtgagc cgttacctca ccaactagct aatgcgccgc gggtccatct 1260

gtaagtggta gccgaagcca ccttttatgt ttgaaccatg cggttcaaac aaccatccgg 1320

tattagcccc ggtttcccgg agttatccca gtcttacagg caggttaccc acgtgttact 1380

cacccgtccg ccgctaacat cagggagcaa gctcccatct gtccgctcga cttgcatgta 1440

tagcacccgc aggcc 1455

Claims

1. A strain JCL16 for synergistically producing three antibacterial metabolites and identificationIs Bacillus subtilis (B.subtilis)Bacillus subtilis) The microbial culture medium has been preserved in China center for type microbial Collection with the preservation time of 2018, 06 and 01 months and the preservation number of CCTCC NO: M2018336.

2. The strain JCL16 synergistically producing three antibacterial metabolites according to claim 1, wherein the three antibacterial metabolites are respectively: surfactin, lysobactin, and subtilisina.

3. Use of the strain JCL16 synergistically producing three antibacterial metabolites according to claim 1 for the production of antibacterial metabolites surfactant, lysobactin, and subtilisina.

4. The use according to claim 3, wherein the strain JCL16 is inoculated into a culture medium for fermentation and the antibacterial metabolite is isolated from the fermentation broth.

5. The use according to claim 4, wherein the strain JCL16 is inoculated into a culture medium for fermentation, and the culture medium is a fermentation medium and comprises the following components in percentage by mass: 2-2.5% of soybean cake powder, 2-3% of millet flour, 2-3% of glucose and (NH) ₄ ） ₂ SO ₄ 0.8-1%、KCl 0.3-0.5%，MgSO ₄ 0.01 percent, and the balance of water.

6. The application of the strain JCL16 which synergistically produces three antibacterial metabolites according to claim 1 in preparing a biocontrol agent or feed for preventing and treating nocardia seriolae diseases of weever.

7. A feed for preventing and treating Nocardia disease of Larges quinqueradiata, which is characterized in that the feed contains thallus, spores or fermentation liquor of the bacillus subtilis JCL16 in claim 1.

8. The feed of claim 7, wherein the feed is obtained by adding fermentation broth of strain JCL16 to a basal feed.