CN111635878A - Bacillus amyloliquefaciens and application thereof in pomfret culture - Google Patents

Abstract

The invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZM506 strain which is preserved in a China general microbiological culture collection center (CGMCC) in 2019, 10 and 21 months, and has the number of CGMCC NO: 18714; the address is No. 3 Xilu-1 Hospital, Beijing, Chaoyang, North Chen. The bacillus amyloliquefaciens ZM506 strain provided by the invention is used for preparing a probiotic product for pomfret cultivation; the product is a soft capsule and comprises a wall material and a core material part. The bacillus amyloliquefaciens provided by the invention can enhance the natural barrier of the intestinal tract of a host, improve the composition of the intestinal flora of pomfret, reduce the relative abundance of vibrio, reduce the risk of flatulence and vibriosis and improve the activity of digestive enzyme of the host. The feeding mode of the prepared pomfret oral soft capsule can delay the medicine from entering a circulatory system, and ensure that effective ingredients of probiotics cannot escape in water in a large amount.

Description

Bacillus amyloliquefaciens and application thereof in pomfret culture

Technical Field

The invention belongs to the technical field of microorganism screening, and particularly relates to bacillus amyloliquefaciens and application thereof in pomfret culture.

Background

Silvery pomfret (Pampus argenteus) belongs to Perciformes (Perciformes), Pomfrataceae (Stromateidae) and Pomfret (Pampus), has delicious taste, rich nutrition and wide distribution, and is an important seawater economic fish in China. In recent years, under the dual requirements of protecting fishery resources and meeting market demands, artificial breeding of pomfret is carried out, and industrialization is gradually realized. However, the problems of diseases exposed in the culture process, particularly in the juvenile fish stage, are not inconstant, and on one hand, the problems of flatulence caused by the increase of aerogenic bacteria in intestinal tracts and the outbreak of vibriosis at high temperature in summer can cause great loss.

In aquaculture, EM bacterial liquid or lactic acid bacteria are used for improving water quality, regulating intestinal flora of pomfret and preventing the pomfret from suffering from flatulence and vibriosis. However, in the pomfret culture process, the daily water change amount is more than 100%, the probiotics splashing effect is very little, a stable flora cannot be formed, and the probiotics need to be infused into the pomfret body through ingestion. On one hand, the existing probiotics cannot be well adapted to the environment in the intestinal tract of the pomfret due to the difference of separation sources. On the other hand, as the feed of the pomfret is artificially prepared viscous feed, the dissolving feed rate is higher than that of mature pellet feed, and the content of the effective components of the pomfret is difficult to control only by adding probiotics into the feed. The existing aquaculture technology cannot provide a reasonable solution for the problems, and the problem of probiotic screening and feeding of pomfret must be solved by innovative technology.

In order to solve the problems, the isolation of the pomfret intestine-derived probiotics is urgently needed, the rejection phenomenon after ingestion is avoided, the digestion and absorption are promoted, and the composition of the pomfret intestinal flora is optimized.

Disclosure of Invention

The invention aims to provide bacillus amyloliquefaciens and application thereof in pomfret culture, thereby making up the defects of the prior art.

The invention firstly provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZM506 strain which is preserved in China general microbiological culture collection center (CGMCC) in 2019, 10 and 21 months, and the number of the strain is CGMCC NO: 18714; the address is No. 3 Xilu-1 Hospital, Beijing, Chaoyang, North Chen.

The bacillus amyloliquefaciens ZM506 strain provided by the invention is used for preparing a probiotic product for pomfret cultivation;

the product is a soft capsule and comprises a wall material and a core material part; the wall material comprises gelatin, a plasticizer, water and a phagostimulant; wherein the phagostimulant is jellyfish homogenate;

the jellyfish homogenate is obtained by homogenizing jellyfish with the diameter of 20-50cm, and filtering the homogenate;

the plasticizer is preferably glycerol;

the preparation method of the wall material comprises the steps of adding gelatin into distilled water, heating while stirring to dissolve the gelatin, adding jellyfish homogenate, and uniformly stirring; adding glycerol and stirring to complete the preparation of the wall material;

preferably, the mass part ratio of the gelatin to the water to the glycerin to the phagostimulant is 2: 2: 1: 1.

the bacillus amyloliquefaciens provided by the invention can enhance the natural barrier of the intestinal tract of a host, improve the composition of the intestinal flora of pomfret, reduce the relative abundance of vibrio, reduce the risk of flatulence and vibriosis and improve the activity of digestive enzyme of the host. The feeding mode of the prepared pomfret oral soft capsule can delay the medicine from entering a circulatory system, and ensure that effective ingredients of probiotics cannot escape in water in a large amount.

Drawings

FIG. 1: a colony morphology of bacillus amyloliquefaciens;

FIG. 2: TOP10 dominant bacterial map;

FIG. 3: trypsin activity detection graphs;

FIG. 4: lipase activity detection profiles;

FIG. 5: amylase activity assay graph.

Detailed Description

The isolation and purification medium used in the examples of the present invention is preferably LB (Luria Broth) medium: tryptone 10.0g, yeast extract 5.0g, NaCl 10.0g, fully dissolved in 1000mL double distilled water, 121 ℃ high pressure sterilization for 15 minutes, pH value to adjust 7.0, adding 15.0g agar, at 4 ℃ for standby.

The pathogenicity detection medium used was as follows:

(1) mannitol yolk polymyxin agar base (MYP) medium (Haibo HB 0248-1): weighing MYP culture medium 46.0g, heating to dissolve in 1000ml distilled water, subpackaging 100ml per bottle, autoclaving at 121 deg.C for 15 min, cooling to 50 deg.C, adding 50% egg yolk emulsion 5ml and polymyxin B10000IU per bottle, shaking, and pouring into sterile culture dish.

(2) Sheep blood TSA medium: weighing 40 g of commercial TSA culture medium powder, supplementing 5g of NaCl, dissolving in 1000ml of distilled water, autoclaving at 121 ℃ for 15 minutes, cooling to 40-50 ℃, adding a certain proportion of sheep whole blood (6-8ml of sheep whole blood/100 ml of TSA culture medium), mixing uniformly, and pouring into a sterile culture dish for later use.

The preferable trace biochemical identification tube is selected for the physicochemical property detection, and comprises a 7% NaCl experiment, a nitrate reduction experiment, oxidase, glycerol utilization, gelatin liquefaction, starch hydrolysis, arabinose fermentation, mannitol utilization, an acetyl methyl methanol (V-P) experiment, a methyl red experiment and a hydrogen sulfide experiment.

The present invention will be described in detail with reference to examples.

Example 1: isolation of Bacillus amyloliquefaciens

(1) Experimental materials:

experimental cultured Pomfret is obtained from aquatic offspring seed Co Ltd in Bay of Xiangshan county, Ningbo, Zhejiang province. Is a healthy young fish which is not infected with diseases during the outbreak period of flatulence disease and vibriosis in the culture process. The initial body weight is 70.02 + -10.94 g, the body length is 15.59 + -2.16 cm, the water temperature is 18 + -1 deg.C, the pH is 8.21 + -0.3, the salinity is 20.3 + -0.8, and the day and night alternate for 12 h. Randomly taking 6 silvery pomfret in the culture pond, and taking 3 silvery pomfret as a mixed sample.

(2) Collecting samples:

the silvery pomfret is anesthetized with anesthetic, then dissected rapidly, the intestinal tract is separated, and fat, blood and the like on the outer wall of the intestinal tract are washed clean with 1 x Phosphate Buffer Saline (PBS). Gently squeezing out intestinal contents with forceps, slowly squeezing the gastrointestinal contents into a 10mL centrifuge tube containing PBS buffer, then cutting the gastrointestinal tract longitudinally with a sterilized ophthalmic scissors, washing with PBS buffer several times, removing residual intestinal contents, slowly scraping off the mucosa on the inner wall of the intestinal tract with a rubber scraper, mixing the separated contents and mucosa together, and uniformly mixing.

(3) Heating treatment:

in the early research on healthy pomfret, the pathogenic bacteria are mostly separated at normal temperature, and strains with certain heat resistance need to be screened in order to remove the pathogenic bacteria and not influence the screening of probiotics and meet the requirement of the later soft capsule preparation process.

Treating intestinal dissolved substance by heat treatment: heating in 80 deg.C water bath for 20 min, spreading 100 μ L on LB solid culture medium, and culturing in electrothermal constant temperature incubator at 30 deg.C for 48 h. According to colony morphology, the method comprises the following steps: and (5) carrying out primary screening on the characteristics of color, whether the edge is regular, whether the surface is neat and the like. And selecting a single colony to be subjected to amplification culture in an LB liquid culture medium, wherein one part is used for molecular identification, and the other part is used for strain preservation.

The heat-treated bacillus was isolated and purified to obtain a single purified colony, designated ZM 506. As shown in FIG. 1, the colony on LB culture medium is small, round, regular in edge, smooth in surface, convex in center, yellowish in color, and turbid in LB liquid culture medium. The gram stain shows positive, the microscopic observation shows that the sample is in a short rod shape, the two ends are blunt and round, the sample is different in length, and secondary terminal spores larger than thalli are formed.

(4) And (3) molecular identification:

extraction of genomic DNA

The total DNA of the bacterial liquid was extracted using a bacterial DNA kit, and the specific procedure was followed according to the instructions. Bacterial cell walls were digested with lysozyme followed by lysis of the cells with proteinase K. And (3) passing through a DNA adsorption column after cracking, quickly washing the DNA by using a washing solution prepared in the kit to remove trace salt and protein pollutant impurities, and finally eluting the DNA by using a low-ionic-strength washing solution. The purified DNA can be directly used for subsequent reactions without further purification. The DNA concentration was determined using a NanoDrop 1000, with a ratio of A260/A280 between 1.7 and 1.9. The extracted DNA product was monitored on a 1% agarose gel. The DNA was diluted to 1ng/L with sterile water according to concentration. The resulting DNA was stored at-20 ℃ and used for PCR amplification of the bacterial 16S rRNA gene.

② PCR amplification

The V3 region of 16S rRNA was PCR amplified using bacterial universal primers 27F (5-: AGAGAGTTTGATCCTGGCTCAG-3) and 1492R (5-TACGGCTACCTTGTTACGACTT-3).

Reaction system:

reaction conditions are as follows:

the PCR products were detected by electrophoresis on a 1% agarose gel and observed under an ultraviolet lamp having a wavelength of 253 nm. The PCR products were sent to Yokoukang Biotechnology Inc. of Hangzhou for sequencing, and the sequencing results were compared with the homologous sequence by the BLAST search system of NCBI (http:// www.ncbi.nlm.nih.gov/BLAST /) and the public database of EzTaxon (http:// www.eztaxon.org /).

The screened strain ZM506 has the 16S rRNA total length of 1379bp (SEQ ID NO:1) and has the highest similarity with the strain with the number MK310270.1 in NCBI, so the strain is classified into a Bacillus amyloliquefaciens strain, is named as the Bacillus amyloliquefaciens ZM506 strain, is stored in the China general microbiological culture Collection center (CGMCC) and has the preservation number of CGMCC NO: 18714.

example 2: safety detection of bacillus amyloliquefaciens

(1) Selective medium screening:

screening was performed with a special medium, MYP and sheep blood TSA medium. The purified bacteria are inoculated on a sheep blood TSA culture medium, cultured for 6-12h at 28 ℃, and the used single colony is inoculated on the sheep blood TSA culture medium by a puncture method to compare the size of a hemolytic cycle. Hemolysis was observed and the behavior was recorded (total hemolysis (. beta. -hemolysis); partial hemolysis (. alpha. -hemolysis); non-hemolysis (. gamma. -hemolysis)). Yellow colonies on MYP medium do not produce a precipitating ring, indicating no egg-laying phospholipase. The blood shows gamma hemolysis on a blood plate, has no hemolytic ring, and is preliminarily identified as non-pathogenic bacteria.

(2) Animal safety detection

Activation of strains: transferring the strain preserved at-80 ℃ to an LB slant culture medium; after culturing at 30 ℃ for 24 hours, a single colony was picked up, inoculated into a flask containing 100mL of LB liquid medium, and cultured overnight at 30 ℃ with shaking at 120 rpm. Diluting overnight-cultured bacterial liquid by 10 times or 100 times, counting with a blood counting chamber, and collecting10 preparation of bacterial liquid⁶、10⁷、10⁸、10⁹Gradient concentration of CFU/mL.

Selecting 100 tail silvery pomfret with similar specification, and the total length is 5.8 + -0.6 cm. The test solution is divided into a blank control group and 4 treatment groups, wherein 20 animals are kept for 7 d. ZM506 bacillus amyloliquefaciens is selected as potential probiotics to carry out animal safety detection experiments, and 10 is used⁶、10⁷、10⁸、10⁹And (5) carrying out intraperitoneal injection on experimental silvery pomfret by using bacterial liquid with the concentration of CFU/mL and equivalent physiological saline, temporarily culturing for 7d, observing the condition of the pomfret, and counting the death rate. The results show that the bacillus amyloliquefaciens with different concentration gradients can not cause death of experimental fishes, the body surfaces of the fishes are not damaged, the fishes can normally swim, the fishes can be fed again after the experiment is finished, no ingestion blockage is caused, and the screened bacillus amyloliquefaciens can be inferred to be non-toxic and harmless.

Example 3: detection of physicochemical properties of Bacillus amyloliquefaciens

The strain is identified by a trace biochemical identification tube, and the result shows that the strain is positive in catalase, positive in nitrate reduction reaction, negative in hydrolyzed starch, liquefied gelatin and indole experiment, negative in V-P reaction, capable of fermenting by utilizing mannose and arabinose, negative in glycerol and methyl red reaction, free of hydrogen sulfide production and capable of growing in 7% NaCl.

Example 4: effect verification of bacillus amyloliquefaciens

(1) Experiment grouping

The experiment was divided into three groups, blank control group (group C), purchased bacillus amyloliquefaciens group NO: 1.857 (group 1), and the self-screened bacillus amyloliquefaciens group NO: 18714(2 groups), 100 fish per group. The addition amount of the probiotics is 10⁸CFU/mL, making probiotic into enteric-coated soft capsule, feeding, and feeding control group with soft capsule wrapped with normal salineFeeding the feed three times a day, and carrying out the experiment for 28 days totally, wherein the daily water change amount is 150%. During the period, samples were taken every 7 days for 5 times, each sample was a mixed sample of 3 fish intestines, 3 replicates each time. Samples of 1d and 28d were used for 16S rRNA microbial sequencing.

(2) Preparing the soft capsule:

manufacturing a wall material part:

melting glue: selecting high-quality gelatin, wherein the mass percentage of the gelatin is 1: 1 into distilled water, and heating and stirring the mixture for 10 minutes in a water bath at 40 ℃ until the glue is completely dissolved.

Preparing a phagostimulant: weighing iced fresh jellyfish with diameter of 20-50cm, thawing with fresh water in advance, removing fascia and impurities, and pulverizing with pulverizer at high speed to obtain homogenate. And (3) screening the jellyfish slurry by a 80-mesh screen, removing impurities, adding the jellyfish slurry into the prepared gelatin solution, and uniformly stirring for later use.

(iii) plasticizer: and (3) the plasticizer is preferably glycerol, the glycerol with the same mass as that of the jellyfish is weighed and added into the solution for three times, the glycerol is fully stirred for 3-5 minutes every time, and the rest glycerol is added after the glycerol is uniformly mixed. The ratio of the four components is gelatin: water: glycerol: phagostimulant equal to 2: 2: 1: 1.

preparation of core material part:

the core materials are respectively freeze-dried substances of bacillus amyloliquefaciens purchased from CGMCC and numbered as NO: 1.857, respectively; and the bacillus amyloliquefaciens ZM506 strain screened by the method is subjected to amplification culture by using an LB liquid culture medium, counted by a blood counting plate, and the concentration of a bacterial liquid is 10⁸CFU/ml；

Preparing the capsule wall material and the liquid medicine, pressing and forming, and drying, washing pills, evaporating the washing solvent, picking pills and the like to obtain the silvery pomfret oral soft capsule; or adopting other preparation methods commonly used in the soft capsule field.

(2)16S rRNA microbial sequencing

Total DNA in intestinal samples was extracted using the bacterial OMEGA bacterial DNA extraction kit, following the instructions, using a NanoDrop 1000 to determine the DNA concentration, with a ratio of A260/A280 between 1.7 and 1.9. The extracted DNA product was monitored on a 1% agarose gel. Root of herbaceous plantAccording to the concentration, the DNA was diluted to 1ng/L with sterile water. Barcode sequence amplification was performed on the V3-V4 hypervariable region of 16S rRNA using the universal primers 515F (5-GTGCCAGCMGCCGCGG-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3). PCR products were mixed and purified using the Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany). By Tru

DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, Calif., USA) according to the manufacturer' S instructions, adding the index code, according to the amplified 16S region characteristics, construction of small fragment library, generating sequencing library. Library quality was assessed using the Qubit @2.0 fluorometer (Thermo Scientific) and Agilent bioanalyzer 2100 system. Finally, the library was subjected to double-end sequencing on the Illumina High-Seqence 2500 platform to obtain a 250bp double-ended sequence. Through Reads splicing filtration, OTUs (operational Taxonomic units) clustering, species annotation and abundance analysis are carried out.

The results show that: the treatment group with probiotic addition significantly reduced the relative abundance of vibrio, P <0.05, compared to the control group (group C). Reduce the infection risk of vibriosis and avoid loss: the vibrio of the group 1 is reduced from 24.19% to 6.98%, the vibrio of the group 2 is reduced from 37.30 to 3.35%, and the relative abundance of the vibrio is not significantly changed and is 22.68% to 22.91% when the control group is fed with 28 d. The effect of reducing Vibrio bacteria was better in group 2 than in group 1 (see FIG. 2).

(3) Determination of digestive enzyme Activity

Preparation of crude enzyme solution:

intestinal tissue samples were thawed at 4 ℃, weighed, added with 10 volumes of pre-cooled normal saline (0.9% NaCl solution), and rapidly homogenized on ice for 1min with a homogenizer. Centrifuging the tissue homogenate with a 4 deg.C pre-cooled refrigerated centrifuge at 3000r/min for 10min, transferring the supernatant to a new 1.5ml sterilized centrifuge tube with a pipette, storing at 4 deg.C, and measuring within 24 h.

Measurement of digestive enzyme activity:

the determination of the digestive enzyme activity is carried out according to the instruction of the kit: a protein quantitative test kit (A045-4, BCA microplate method, Nanjing construction), a lipase test kit (A054, Nanjing construction), a trypsin test kit (A080-2, Nanjing construction) and an amylase test kit (C016, Nanjing construction). One blank, three replicates, was assayed for each sample.

The results show that when the probiotics are fed for 7 days, the enzyme activities of the three digestive enzymes are obviously changed, the enzyme activities of the three digestive enzymes are obviously inhibited by the group 1, P is less than 0.05, and the enzyme activities of the three digestive enzymes are obviously promoted by the group 2, P is less than 0.05 (see figures 3-5). The effect of the bacillus amyloliquefaciens ZM506 strain derived from the pomfret is better than that of the bacillus amyloliquefaciens from other sources.

Sequence listing

<110> Ningbo university

<120> bacillus amyloliquefaciens and application thereof in pomfret culture

<160>1

<170>SIPOSequenceListing 1.0

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<211>1379

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atgggagctt gctccctgat gttagcggcg gacgggtgag taacacgtgg gtaacctgcc 60

tgtaagactg ggataactcc gggaaaccgg ggctaatacc ggatggttgt ctgaaccgca 120

tggttcagac ataaaaggtg gcttcggcta ccacttacag atggacccgc ggcgcattag 180

ctagttggtg aggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgat 240

cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct 300

tccgcaatgg acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc 360

gtaaagctct gttgttaggg aagaacaagt gccgttcaaa tagggcggca ccttgacggt 420

acctaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca 480

agcgttgtcc ggaattattg ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg 540

aaagcccccg gctcaaccgg ggagggtcat tggaaactgg ggaacttgag tgcagaagag 600

gagagtggaa ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc 660

gaaggcgact ctctggtctg taactgacgc tgaggagcga aagcgtgggg agcgaacagg 720

attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag ggggtttccg 780

ccccttagtg ctgcagctaa cgcattaagc actccgcctg gggagtacgg tcgcaagact 840

gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa 900

gcaacgcgaa gaaccttacc aggtcttgac atcctctgac aatcctagag ataggacgtc 960

cccttcgggg gcagagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt 1020

tgggttaagt cccgcaacga gcgcaaccct tgatcttagt tgccagcatt cagttgggca 1080

ctctaaggtg actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg 1140

ccccttatga cctgggctac acacgtgcta caatggacag aacaaagggc agcgaaaccg 1200

cgaggttaag ccaatcccac aaatctgttc tcagttcgga tcgcagtctg caactcgact 1260

gcgtgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1320

gccttgtaca caccgcccgt cacaccacga gagtttgtaa cacccgatgt cggtgaggt 1379

Claims (10)

1. The bacillus amyloliquefaciens is characterized in that the preservation number of the bacillus amyloliquefaciens is CGMCC NO: 18714.

2. use of bacillus amyloliquefaciens according to claim 1 for preparing a probiotic preparation for pomfret culture.

3. The use of claim 2, wherein the product is a softgel formulation.

4. A probiotic soft capsule, which is characterized by comprising a wall material and a core material, wherein the core material used comprises the bacillus amyloliquefaciens of claim 1.

5. The probiotic soft capsule of claim 4, wherein the wall material comprises gelatin, a plasticizer, water and a phagostimulant.

6. The probiotic soft capsule of claim 5, wherein the phagostimulant is a jellyfish homogenate.

7. The probiotic soft capsule according to claim 6, characterized in that the jellyfish homogenate is obtained by homogenizing jellyfish having an umbrella diameter of 20 to 50cm, and filtering the homogenate.

8. The probiotic soft capsule of claim 5, wherein the plasticizer is glycerol.

9. The probiotic soft capsule according to claim 5, wherein the wall material is prepared by adding gelatin into distilled water, heating while stirring to dissolve the gelatin, adding jellyfish homogenate, and stirring uniformly; and adding glycerol and stirring to complete the preparation of the wall material.

10. The probiotic soft capsule according to any one of claims 4 to 9, characterized in that the ratio of the gelatin, water, glycerol and the phagostimulant in the wall material is 2: 2: 1: 1.

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