CN111004741B - Deep-sea-derived campylobacter R29-2, microecological preparation and application thereof - Google Patents
Deep-sea-derived campylobacter R29-2, microecological preparation and application thereof Download PDFInfo
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention relates to a campylobacter strain, which has a preservation name: bacillus flexusBacillus flexusR29-2, depository: china general microbiological culture Collection center (CGMCC) with the collection number of CGMCC No. 18731. The strain is derived from the Western Pacific ocean Carolina mountains and presents a round beige, smooth and opaque colony in an LB agar culture medium; under the microscope, the sample is in a short rod shape, and gram staining is positive. The bacteria have good antibacterial effect on gram-positive bacteria such as staphylococcus aureus, streptococcus agalactiae, enterococcus faecalis, bacillus cereus and the like, and can be used as a feed additive in poultry cultivation and aquaculture by being prepared into a microecological preparation, so that the survival rate of poultry can be effectively improved, the feed conversion ratio is reduced, and the disease resistance of aquaculture animals is improved.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to deep-sea-derived Bacillus flexus R29-2 which can effectively inhibit gram-positive bacteria such as staphylococcus aureus, streptococcus agalactiae, enterococcus faecalis, bacillus cereus and the like, can obviously improve the survival rate of poultry, reduce the feed-meat ratio and simultaneously effectively improve the disease resistance of aquaculture animals.
Background
In recent years, the misuse and abuse of antibiotics to people and animals greatly accelerates the formation of microbial drug resistance, and as the time limit for comprehensive resistance banning of the breeding industry is gradually approached in 2020, the search for safe and efficient alternative products is urgent. A large number of microbial flora exist in animal intestinal tracts, and when the antibacterial agent is used for killing harmful bacteria in animal bodies, normal flora in the intestinal tracts can be killed, so that the intestinal flora of the animals is unbalanced, the immunity is reduced, and the difficulty in preventing and treating the animal epidemic diseases is increased. The use of probiotics to replace antibiotic additives in breeding feed has become increasingly accepted. By selecting a proper microecological preparation, the secreted antibacterial active substances not only can specifically kill pathogenic bacteria in animal intestinal tracts and inhibit the reproduction of harmful bacteria, but also can promote the growth of healthy flora in other intestinal tracts, so that the intestinal tracts are healthier, the animals can develop larger growth potential, and stronger immunity can be obtained to resist other diseases.
Most of the probiotics commonly used at present are strains from terrestrial organisms or terrestrial environments, and the application of the probiotics derived from the ocean in the culture is less reported. The special extreme conditions of high salt, high pressure, low temperature, oxygen deficiency, low light and the like in the deep sea environment endow marine microorganisms with a metabolic system and a defense system different from those of terrestrial microorganisms, generate metabolic products with various biological activities and have unique effect on the aspect of antibiosis.
The campylobacter is a gram-positive bacterium, and has good application in the aspects of degrading organic nitrogen and catechol, promoting methane fermentation, producing beta-amylase and the like in the prior literature report, but has no report related to antibacterial activity of the campylobacter.
Disclosure of Invention
Based on the urgent need of currently searching for a substitute for an antibiotic product, the invention screens and provides a strain of campylobacter with the activity of inhibiting partial gram-positive bacteria such as staphylococcus aureus, streptococcus agalactiae, enterococcus faecalis and the like from the western Pacific ocean Carolinia mountains, the campylobacter is used as an antibiotic additive for substituting for the growth promotion of the gram-positive bacteria, the strain is prepared into a microecological preparation, the survival rate of poultry can be effectively improved, the feed conversion ratio is reduced, and the disease resistance of aquaculture animals is improved.
In order to realize the purpose, the invention adopts the following technical scheme:
the invention provides a bacillus flexus R29-2, which is characterized in that: the Bacillus flexus strain is classified and named as Bacillus flexus, and is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the following addresses: western road No.1, north school of republic of beijing, west road No. 3, institute of microbiology, china academy of sciences; the preservation date is as follows: 24 days 10 and 2019; the preservation number is CGMCC No. 18731.
Further, the curvulus R29-2 was beige opaque on LB plates, smooth-edged, moist and flat; gram-positive bacteria, which are short rod-shaped under an optical microscope.
The invention provides application of Bacillus curvatus R29-2 in preparations for inhibiting Staphylococcus aureus (Staphylococcus aureus), Streptococcus agalactiae (Streptococcus agalactiae), Bacillus cereus (Bacillus cereus) and Enterococcus faecalis (Enterococcus faecalis).
The invention provides a microecological preparation containing Bacillus curvatus R29-2.
Further, the microecological preparation is a liquid microecological preparation or a solid microecological preparation, and the preparation method of the liquid microecological preparation comprises the steps of taking Bacillus flexus R29-2 to culture and activate in a culture medium to obtain a culture solution, inoculating the culture solution into a fermentation tank according to the inoculation amount of 1-2% of the total volume of the culture medium, stirring and fermenting at the rotation speed of 180-200rpm and the ventilation rate of 10-20L/min, adding a proper amount of a sterilizing antifoaming agent, and culturing at the temperature of 30-37 ℃ for 24h to obtain a fermentation liquid, namely the liquid microecological preparation.
Furthermore, the thallus density in the liquid microecological preparation is more than or equal to 108CFU/ml。
The preparation method of the solid microecological preparation comprises the steps of culturing the curvibacillus curvatus R29-2 in a liquid culture medium to obtain a seed solution, inoculating the seed solution into a fermentation tank according to the inoculation amount of 1-2% of the total volume of the culture medium, stirring and fermenting under the conditions of the rotation speed of 180-200rpm and the ventilation of 10-20L/min, adding a proper amount of a sterilizing antifoaming agent, culturing at 30-37 ℃ for 24h to obtain a fermentation liquid, adding a protective agent, and then spraying to dry to obtain the thallus and metabolic products of the curvibacillus curvatus R29-2.
Furthermore, the thallus density of thallus and metabolite in the solid microecological preparation is more than or equal to 1010CFU/g。
The invention provides application of a microecological preparation in increasing the survival rate of poultry, increasing the average slaughter weight of the poultry and reducing the feed-meat ratio.
Furthermore, in the application of the microecological preparation in improving the survival rate of poultry, improving the average slaughter weight of poultry and reducing the feed-meat ratio, the addition amount of the microecological preparation in the poultry breeding water line accounts for 0.2-0.5% by mass.
The invention provides application of a microecological preparation in improving the disease resistance of aquaculture animals.
Furthermore, in the application of the microecological preparation in improving the disease resistance of the aquaculture animals, the addition amount of the microecological preparation in each gram of the basic feed accounts for 0.1-0.3% by mass.
The invention has the advantages that:
the invention provides a Bacillus flexus (Bacillus flexus) R29-2 strain with gram-positive bacteria resistance activity for the first time. Bacillus flexus R29-2 was isolated from the Western Pacific ocean Callin sea mountain and no reports have been made before on the antibacterial activity of this species of bacteria. The bacillus curvatus R29-2 microecological preparation has antibacterial activity on a plurality of gram-positive bacteria such as staphylococcus aureus, enterococcus faecalis, streptococcus agalactiae and bacillus cereus, can be used as a substitute for antibiotics by adding drinking water, can effectively improve the survival rate of poultry and reduce the feed conversion ratio, and can improve the disease resistance of aquaculture animals by adding the bacillus curvatus R29-2 microecological preparation into daily basic feed.
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FIG. 1 is a photomicrograph (X1000) of Bacillus curvatus R29-2;
FIG. 2 is a graph of the antimicrobial activity of fermentation supernatants during different periods of fermentation;
FIG. 3 shows the effect of Bacillus curvatus R29-2 solid microecological formulation on disease resistance of tilapia.
Detailed Description
The invention is further illustrated by the following examples, which are intended to illustrate the invention but are not intended to limit the scope thereof. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 identification of the Strain R29-2
(1) Morphological identification
Colonies were beige opaque on LB plates, smooth-edged, moist and flat; after gram staining, the cells were observed as purple short rods under an optical microscope (see FIG. 1).
(2) Hemolytic property
A colony of the campylobacter R29-2 is picked, spotted on a blood agar plate, cultured for 48 hours at 37 ℃, observed and free of hemolysis.
(3) Identification of strains
16S rRNA gene amplification and sequencing: r29-2 was inoculated into LB broth and cultured overnight at 37 ℃ to amplify the 16S rRNA gene sequence of the bacterium using the bacterial suspension as a template and the bacterial universal primers 27F and 1492R. The PCR amplification system is 50 μ L, 1 μ L of bacterial liquid as template, 1 μ L of upstream and downstream primers, 2 Xmix 25 μ L, ddH2O22. mu.L. PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 55 deg.C for 30s and stretching at 72 deg.C for 90s, 34 times of cycles, and stretching at 72 deg.C for 10 min. And (3) sequencing the unpurified PCR product by using a biological engineering (Shanghai) limited company, wherein the sequence is shown as SEQ ID No: 1, and the sequence was aligned in the NCBI database, and the final strain R29-2 was identified as Bacillus flexus.
The same result is obtained by performing whole genome sequencing on the strain based on the sample evolution analysis of housekeeping genes, and the strain is Bacillus flexus.
Example 2 determination of antibacterial Activity of Bacillus curvatus R29-2
For the determination of the bacteriostatic activity of the curvulus curvatus R29-2, the gram-negative indicator bacteria are selected from Escherichia coli K88, Aeromonas hydrophila T2W-1 and Klebsiella pneumoniae THM 1; the gram-positive indicator bacteria are Staphylococcus aureus JP, streptococcus agalactiae Staphylococcus agalactiae Y405, Enterococcus faecalis faecalium 2# XB and Bacillus cereus 3.
The single colony of R29-2 is picked up and put on an LBH plate, and cultured for 24h at 28 ℃, 100 mul of the indicator bacterium liquid is taken and put in 10ml of LB or MRS culture medium (containing 0.8 percent of agar), and the indicator bacterium liquid is poured on the cultured single colony of R29-2 after being mixed evenly, and the diameter of the inhibition zone is observed after the culture is carried out for 12h at 37 ℃.
The bacteriostatic effect is shown in the following table 1: the strain has antagonistic action on various gram-positive bacteria including staphylococcus aureus, streptococcus agalactiae, enterococcus faecalis and bacillus cereus, and has no inhibiting effect on gram-negative bacteria such as escherichia coli, aeromonas hydrophila, and bacillus pneumoniae.
TABLE 1 bacteriostatic Activity of Strain R29-2
Example 3 groping of liquid submerged aerobic fermentation conditions of Bacillus curvatus R29-2
(1) Preparing a seed solution: picking single colony from a test tube slant LBH agar culture medium of the campylobacter R29-2, inoculating the single colony in a test tube of the LBH liquid culture medium, and culturing at 32 ℃ and 120rpm for 16-20h to obtain a first-stage seed solution; inoculating the first-stage seed solution into LBH liquid culture medium at 2%, culturing at 32 deg.C and 120rpm to logarithmic phase to obtain 10% seed solution7-108cfu/ml of secondary seed liquid.
(2) Liquid submerged fermentation: the preparation culture is carried out in a 50L fermentation tank with the inoculation amount of 1-2%, separately prepared glucose solution is sterilized, and glucose is supplemented as a carbon source in the fermentation process. The dissolved oxygen condition in the fermentation tank is adjusted by setting the rotating speed and the ventilation quantity, and the fermentation liquid OD is sampled and monitored in the fermentation process600Residual sugar content, thallus density and antibacterial activity (sample is diluted by double dilution method until 10 μ l sample still has the minimum gradient of antibacterial activity by overlap or spot-on-lawn method detection, and the antibacterial activity of 10 μ l sample under the dilution is defined as 1Au) (see table 2 and figure 2), and the OD is waited600The value tends to be stable, namely the fermentation tank is stopped to obtain the fermentation liquor.
The optimal conditions are as follows: the thallus density and the bacteriostatic activity of the fermentation liquid obtained by fermenting for 24 hours are optimal under the conditions that the rotating speed is 180 plus 200rpm and the ventilation is 10L/min.
TABLE 2 examination of the respective indices under different conditions
Example 4 fermentation of Bacillus curvatus R29-2 to prepare a probiotic
Preparing a liquid microecological preparation, activating Bacillus curvatus R29-2 in an LBH liquid culture medium, carrying out shake culture for 18-24h at 30-37 ℃ and 100-150rpm, and inoculating the strain accounting for 2% of the total volume of the culture medium into a 250ml triangular flask for culture for 18h to serve as a secondary seed solution. Inoculating the seed solution into a 50L fermentation tank at an inoculation amount of 1% of the total volume of the culture medium, rotating at a speed of 180-8CFU/ml。
Preparing a solid microecological preparation, culturing and activating Bacillus flexus R29-2 in a culture medium to obtain a culture solution, inoculating the culture solution into a fermentation tank at an inoculum size of 1% of the total volume of the culture medium at a rotation speed of 180-200R/min and an aeration rate of 10L/min, adding a proper amount of a sterilizing antifoaming agent, culturing at 30-37 ℃ for 24h to obtain a fermentation broth, adding a protective agent, and spraying to dry to obtain the thallus and metabolite of the Bacillus flexus R29-2. The thallus density in the solid microecological preparation is more than or equal to 1010CFU/g。
Example 5 influence of Bacillus curvatus R29-2 on broiler survival rate and feed-to-meat ratio
Taking white feather broilers in a certain farm in Shandong as experimental objects, selecting one henhouse to carry out the following experiments: two experimental groups are set, 2000 chickens with consistent size and health condition are selected from each group, and the daily water intake of 0.2% in the first group of drinking water line is 3.7 multiplied by 108CFU/ml R29-2 liquid microecological preparation, and the water content of the second group of drinking water line is 0.5% of daily drinking water content, and the added concentration is 3.7 × 108CFU/ml R29-2 liquid microecological preparation, and blank CK as the third group. The liquid microecological preparation is used for 3 days at intervals of 4 days, and 35 days are used as an experimental period. The average body weight, survival rate, feed meat ratio of the chicken flocks of the two experimental groups and the blank control group at the time of slaughtering are shown in table 3:
TABLE 3 influence of Bacillus curvatus R29-2 on broiler survival rate and feed-to-meat ratio
Example 6 Effect of Bacillus curvatus R29-2 on disease resistance of Tilapia mossambica
The method comprises the steps of taking tilapia as an experimental object, selecting 30 tilapia with uniform size and good growth state as one group, setting two experimental groups, adding 0.1% of R29-2 solid microecological preparation into basic feed every day for the first group, adding 0.3% of R29-2 solid microecological preparation into basic feed every day for the second group, additionally setting a blank control group CK, not adding the solid microecological preparation into the basic feed, and setting three times for each group, wherein the tilapia is fed three times every day and fed for 7 days continuously. Then, the tilapia in the experimental group and the blank control group is subjected to challenge test, the tilapia is anesthetized firstly, and then 0.1 ml of tilapia with the concentration of 1 × 10 is injected into the intraperitoneal cavity of the tilapia7CFU/ml of Streptococcus agalactiae liquid, and a toxicity attacking control group is additionally arranged: equal amounts of phosphate buffer were injected, feeding was stopped within one day after challenge of fish, and then all tilapia were fed control feed.After continuously monitoring for 21 days, tilapia death was observed, and the results are shown in fig. 3, in which all tilapia injected with an equal amount of phosphate buffer solution survived.
Sequence listing
<110> Zaozhuang City Jieno Bio-enzyme Co., Ltd
<120> deep-sea-derived campylobacter R29-2, microecological preparation and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1404
<212> DNA
<213> Bacillus flexus (Bacillus flexus)
<400> 1
cgaactgatt agaagcttgc ttctatgacg ttagcggcgg acgggtgagt aacacgtggg 60
caacctgcct gtaagactgg gataactccg ggaaaccgga gctaataccg gataacattt 120
tctcttgcat aagagaaaat tgaaagatgg tttcggctat cacttacaga tgggcccgcg 180
gtgcattagc tagttggtga ggtaacggct caccaaggca acgatgcata gccgacctga 240
gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 300
agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag tgatgaaggc 360
tttcgggtcg taaaactctg ttgttaggga agaacaagta caagagtaac tgcttgtacc 420
ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt 480
aggtggcaag cgttatccgg aattattggg cgtaaagcgc gcgcaggcgg tttcttaagt 540
ctgatgtgaa agcccacggc tcaaccgtgg agggtcattg gaaactgggg aacttgagtg 600
cagaagagaa aagcggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca 660
ccagtggcga aggcggcttt ttggtctgta actgacgctg aggcgcgaaa gcgtggggag 720
caaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttagag 780
ggtttccgcc ctttagtgct gcagctaacg cattaagcac tccgcctggg gagtacggtc 840
gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 900
aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa ctctagagat 960
agagcgttcc ccttcggggg acagagtgac aggtggtgca tggttgtcgt cagctcgtgt 1020
cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgatcttagt tgccagcatt 1080
tagttgggca ctctaaggtg actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1140
aatcatcatg ccccttatga cctgggctac acacgtgcta caatggatgg tacaaagggc 1200
tgcaagaccg cgaggtcaag ccaatcccat aaaaccattc tcagttcgga ttgtaggctg 1260
caactcgcct acatgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat 1320
acgttcccgg gccttgtaca caccgcccgt cacaccacga gagtttgtaa cacccgaagt 1380
cggtggggta acctttatgg agcc 1404
Claims (9)
1. A strain of Bacillus flexus R29-2 is characterized in that: the Bacillus flexus strain is classified and named as Bacillus flexus, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation number of CGMCC No.18731, and is characterized in that the Bacillus flexus R29-2 is beige and opaque on an LB flat plate, has smooth edges, is moist and flat; gram-positive bacteria, which are short rod-shaped under an optical microscope.
2. Use of Bacillus curvatus R29-2 according to claim 1 for the preparation of a preparation for inhibiting Staphylococcus aureus, Streptococcus agalactiae, enterococcus faecalis, Bacillus cereus.
3. A microecological formulation comprising Bacillus curvatus R29-2 of claim 1.
4. The microecological formulation according to claim 3, wherein: the microecological preparation is a liquid microecological preparation or a solid microecological preparation;
the preparation method of the liquid microecological preparation comprises the steps of taking Bacillus flexus R29-2 to culture and activate in a culture medium to obtain a seed solution, then inoculating the seed solution into a fermentation tank according to the inoculation amount which is 1-2% of the total volume of the culture medium, stirring and fermenting under the conditions that the rotation speed is 180-200rpm and the ventilation is 10-20L/min, adding a proper amount of a sterilizing antifoaming agent, and culturing at 30-37 ℃ for 24h to obtain a fermentation broth, namely the liquid microecological preparation;
the preparation method of the solid microecological preparation comprises the steps of culturing the curvibacillus curvatus R29-2 in a liquid culture medium to obtain a seed solution, inoculating the seed solution into a fermentation tank according to the inoculum size of 1-2% of the total volume of the culture medium, stirring and fermenting under the conditions of the rotation speed of 180-200rpm and the ventilation of 10-20L/min, adding a proper amount of a sterilizing antifoaming agent, culturing at 30-37 ℃ for 24h to obtain a fermentation liquid, adding a protective agent, and then spraying to dry to obtain the thallus and metabolic products of the curvibacillus curvatus R29-2.
5. The microecological formulation according to claim 4, wherein: the thallus density of the liquid microecological preparation is more than or equal to 108CFU/ml; the thallus density of the solid microecological preparation is more than or equal to 1010CFU/g。
6. The microecological preparation of claim 3, wherein the microecological preparation is capable of increasing the survival rate of white feather broilers, increasing the average slaughter weight of the white feather broilers and reducing the feed-meat ratio.
7. The microecological formulation according to claim 6, wherein: the method for improving the survival rate of the white feather broilers, improving the average slaughter weight of the white feather broilers and reducing the feed-meat ratio is characterized in that the addition amount of the microecologics in the white feather broilers breeding drinking water line is controlled to be 0.2-0.5% by mass ratio.
8. The microecological formulation according to claim 3, wherein the microecological formulation is capable of improving disease resistance in tilapia.
9. The microecological preparation according to claim 8, wherein the disease resistance of tilapia is controlled by controlling the addition amount of the microecological preparation in per gram of basal feed to be 0.1-0.3% by mass.
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