CN105524855A - Bacillus coagulans having aquatic pathogen antagonistic characteristic and application of bacillus coagulans - Google Patents
Bacillus coagulans having aquatic pathogen antagonistic characteristic and application of bacillus coagulans Download PDFInfo
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Abstract
The invention discloses bacillus coagulans having an aquatic pathogen antagonistic characteristic and application of the bacillus coagulans, and belongs to the technical field of microorganisms. The strain is classified and named as bacillus coagulans JSSW-LA-07-1 and is preserved in China General Microbiological Culture Collection Center with preservation number of CGMCC No. 10182. The invention also discloses a preparation method of a strain powder preparation, wherein the method comprises the following steps: activating and fermentation-cultivating the bacillus coagulans JSSW-LA-07-1 which serves as an initial strain, so that a fermentation broth is obtained, centrifuging the fermentation broth at a high speed and collecting a wet strain; mixing the wet strain with dry starch in the mass ratio of 1 to (2-10); drying at 30-50 DEG C for 20-24h; grinding by virtue of a grinder; and filtering by virtue of a 0.9mm sieve, so that the strain powder preparation is obtained; in the strain powder preparation, bacterium concentration is no less than 5.0 to the power of 8 (CFU)/g; the strain powder preparation, when used as an additive of fish culture feed, is added by 1.0-5.0mg/kg to feed basal diet; and the strain powder preparation, when used as a water quality improver for aquaculture, is sprayed to a full tank by the dosage of 50-200g/mu.m (mu is a unit of area, equal to 0.0667 hectares).
Description
Technical field
The present invention relates to a kind of Bacillus coagulans and the application thereof with aquatic pathogenic bacterium antagonistic properties, belong to microbial technology field.
Background technology
In recent years, China's culture fishery is flourish, has greatly enriched the material life of people.But along with high-density intensive culture scale expanding day, aquaculture water ecotope goes from bad to worse, and substantially increases the chance of cross infections between aquatic animal, causes all kinds of fish disease multiple, the even large-area death of aquatic animal.For the control of fish disease, traditional method uses microbiotic, and short-term may play good effect, but life-time service easily causes the resistance of pathogenic bacteria more and more stronger, hydrocoles intestinal flora is unbalance, and long-term enrichment affects the drawbacks such as human health by food chain in vivo.The control carrying out aquatic animal disease with probiotics is a kind of novel method that developed recently gets up, effectively can avoid taking resistance that microbiotic causes and superinfection etc., it relies on function of unique regulating intestinal canal flora and enjoys and catch people's attention.
Lactic acid bacteria class probiotic bacterium is that the quantity of the government permission use both at home and abroad such as the Ministry of Agriculture of current China, U.S. food and FAD (FDA) is maximum, the good class safe microorganisms bacterial classification of effect.It has following characteristics: acidproof, still can grow when pH is 3.0-4.5, is adapted to the sour environment of stomach and intestine; Milk-acid bacteria is the normal microflora of animal intestinal, and in the field planting of intestines system, in vivo by biological antagonist, antagonism gram negative pathogenic bacteria, maintains the normal eubiosis in enteron aisle; Can gut pH be reduced, stop and suppress objectionable impurities, strengthening anti-infection ability; Containing higher superoxide-dismutase (SOD) in endobacillary meta-bolites alive, humoral immunization and the cellular immunization of animal can be increased, the immunoregulatory activity of enhancing body, growth promoting effects.But ordinary lactic acid bacteria is in process of growth, do not form gemma, resistance is poor, and easy in inactivation is difficult to ensure Tibetan, so the application in fodder industry is subject to a definite limitation.The Bacillus coagulans that the present invention applies just in time makes up this shortcoming.Bacillus coagulans (
bacilluscoagulans), be that a class can sporiferous milk-acid bacteria, except the feature with milk-acid bacteria, also there is strong stress resistance, high temperature resistant, easy preservation, survival rate advantages of higher.
The present invention relates to the wide spectrum antagonistic action of Bacillus coagulans JSSW-LA-07-1 to the common germ of multiple aquatic products, this bacterial strain also has the feature of the growth of Aquaculture Environmental Control function and promotion hydrocoles concurrently simultaneously, is a kind of novel multifunctional microbial.There is no Bacillus coagulans patent report in this respect at present.
Apply in aquaculture probiotics prepared by this bacterial strain can high efficiency regulatory water quality, disease preventing and treating, the growth of promotion hydrocoles, meet aquaculture and control and prevention of disease, water ecological setting administered and the height requirement of healthy aquaculture.In the cultivation of fresh water environmental health is applied; compare with disease prevention techniques with traditional regulating and controlling water quality based on drug use; probiotics has initiatively prevention; nontoxic, noresidue; free of contamination advantage, to forming good breeding ecological environment, reduces the discharge of aquaculture wastewater; eliminate the residual of fishery products Chinese traditional medicine, protection of the environment is significant.Can effectively promote aquatic animal healthy growth as feeding additive aquatic animal simultaneously, drive the raising of aquaculture economic benefit, promote the increasing both production and income of aquaculture family.
Summary of the invention
The object of this invention is to provide a kind of to aquaculture encountered pathogenic bacteria have antagonistic action, can purifying aquatic water, promote hydrocoles growth coagulating bacillus strain.
For reaching above object, technical scheme of the present invention is: provide a strain to have the coagulating bacillus strain of aquatic pathogenic bacterium antagonistic properties, be separated from fish intestines, and the Classification And Nomenclature of this bacterial strain is: Bacillus coagulans (
bacilluscoagulans) JSSW-LA-07-1, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number: CGMCCNo.10182.Coagulating bacillus strain JSSW-LA-07-1 of the present invention, gram-positive microorganism, microscopy is shaft-like, and cell dia < 1 μm, gemma expands.On MRS flat board, colony diameter is 2.0-3.0mm, and form is irregular, surface folding, opaque, non-pigment.LB substratum can obtain circle, surface folding, opaque, white bacterium colony in a large number.
Preparation section of the present invention is as follows:
(1) actication of culture:
The freeze-drying preservation of bacteria strain of aseptic unlatching Bacillus coagulans JSSW-LA-07-1, streak inoculation, in wheat bran nutrient agar medium test tube slant, cultivates 24-48h in 30-37 DEG C, and then line is transferred in wheat bran nutrient agar medium eggplant bottle inclined-plane, cultivates 24-48h for 30-37 DEG C.Microscopy, when more than 90% thalline forms gemma, is maturation.Repeatedly activate 2-3 time, seed bacteria suspension is equipped with in the triangular flask of fermention medium with the access of volume ratio 1%-10% inoculum size, and triangular flask liquid amount is volume ratio 10%-20%, and rotating speed is 100-180rpm, 30-37 DEG C of constant-temperature shaking culture 20-24h;
Slant medium composition is in g/L: peptone 10, extractum carnis 3, NaCl5, agar 15-20, distilled water 1L, pH7.0-7.2.
Fermention medium composition is in g/L: wheat bran 5-20, yeast extract paste 5-10, bean cake powder 5-10, K
2hPO
43, NaCl5, MnSO
4h
2o0.3, distilled water 1L, pH7.0.
(2) biocidal property measures:
Aseptic filter paper sheet is about 10 in concentration
71.0h is soaked in CFU/mL Bacillus coagulans JSSW-LA-07-1 fresh medium.Get concentration about 5 × 10
6the liquid medium 0.1mL of common pathogen Aeromonas hydrophila, Aeromonas sobria, Aeromonas veronii, Aeromonas caviae, Listonella anguillarum, tarda, Vibrio parahemolyticus, intestinal bacteria, vibrio alginolyticus in the aquaculture of CFU/mL, coat on LB agar culture plate respectively, then the filter paper soaking bacterium liquid is affixed on culture dish, each plate pastes 3, and each plate does 3 repetitions.Plate is placed in 25-30 DEG C of incubator 24,48h, measures inhibition zone size.
(3) preparations and applicatio of Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation:
The fermented liquid high speed centrifugation obtained through fermentation culture after bacterial classification JSSW-LA-07-1 activates collects wet thallus, wet thallus mixes with mass ratio 1:2-10 with dry starch, dry 20-24h at 30-50 DEG C, pulverizer is pulverized, cross 0.9mm sieve, the Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation obtained, concentration is not less than 5.0 × 10
8individual (CFU)/gram.
The application of the Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation prepared by described method, as the additive of fishery cultivating feed, adds 1.0-5.0mgkg feeding in basal diet
-1bacillus coagulans JSSW-LA-07-1 bacterium powder preparation; As aquaculture water quality improving agent, by the consumption full pool spilling head Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation of 50-200g/ mu rice.
Beneficial effect of the present invention:
Bacillus coagulans JSSW-LA-07-1 effectively can suppress the growth of Aeromonas hydrophila, Aeromonas sobria, Aeromonas veronii, Aeromonas caviae, Listonella anguillarum, tarda, Vibrio parahemolyticus, is wherein better than other kind pathogenic bacterium to the inhibition of Aeromonas sobria, Aeromonas caviae.Therefore, in aquaculture, use Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation can effective caused by pathogenic bacteria aquatic animal disease of prevention and control, protection hydrocoles healthy growth.
Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation can significantly improve the rate of body weight gain of hydrocoles, specific rate of increase as fishery cultivating fodder additives, significantly reduces feed coefficient.
Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation effectively can reduce the pollutant load in water body as improver of water quality used for aquiculture, improve cultivation water.
Biological material specimens preservation: Bacillus coagulans (
bacilluscoagulans) JSSW-LA-07-1, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center the sixth of the twelve Earthly Branches, be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation date on December 15th, 2014, preserving number is: CGMCCNo.10182.
Accompanying drawing explanation
The phylogenetic tree of Fig. 1 JSSW-LA-07-1.
Embodiment
Embodiment 1: bacteria selection be separated
Gather hybridized prussian carp sample at Wuxi E Hu cultivating pool, fish body solution is taken enteron aisle, adds physiological saline, homogenate is made in grinding, and 1.0mL drawn by each sample, 80 DEG C of heating 10min, get liquid-transfering gun and draw 0.1mL on YPD solid medium, coating, 37 DEG C of cultivations.Choose well-grown bacterium colony according to bacterium colony size and repeatedly inoculate screening, until obtain uniform single bacterium colony, called after JSSW-LA-07-1.
Embodiment 2: strain identification
(1) morphological specificity
Bacterial strain JSSW-LA-07-1, gramstaining is positive, and microscopy is shaft-like, and cell dia < 1 μm, can form gemma.On MRS flat board, colony diameter is 2.0-3.0mm, and form is irregular, surface folding, opaque, non-pigment.LB substratum can obtain circle, surface folding, opaque, white bacterium colony in a large number.
(2) biochemical characteristic
Bacterial strain JSSW-LA-07-1, gramstaining is positive, and catalase, beta-galactosidase enzymes, VP test, methyl red test are positive, can grow under 50 DEG C of environment.The test of oxydase, urase, arginine dihydrolase, ornithine decarboxylase, lysine decarboxylase, can not hydrolyzed starch, gelatin, casein for negative, edwardsiella hoshinae and H
2s, can not grow on citric acid.
Growth experiment result shows this bacterial strain can utilize D-fiber two pool, alpha-D-glucose, D-MANNOSE, D-Fructose, D-trehalose, dextrin, D-melibiose, N-acetyl-D-grape amine, glycerine, produces Pfansteihl, Pyruvic Acid Methyl ester.Denitrification test, for positive, illustrates the nitrogenous compound that this bacterial strain can be degraded in water body.
Chemical-sensitive experimental result shows, this bacterial strain is to lincomycin, MINOCYCLINE HCL, fusidic acid, Guanidinium hydrochloride, lithium chloride, sodium tetradecyl sulfate, D-Ser, vancomycin, troleomycin, Rifamycin Sodium, Sodium propanecarboxylate, sodium bromate, 4%NaCl, 8%NaCl sensitivity, insensitive to 1% Sodium.alpha.-hydroxypropionate, Nalidixic Acid, potassium tellurite, 1%NaCl, aztreonam, can grow under pH5.0, pH6.0 environment.
(3) structure of 16SrRNA sequential analysis and phylogenetic tree
Utilize bacterial 16 S rRNA universal primer:
Forward primer 27F:5'-AGAGTTTGATCCTGGCTCAG-3',
Reverse primer 1492r:5'-GGTTACCTTGTTACGACTT-3' amplifying target genes.Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..With the genomic dna of strain isolated JSSW-LA-07-1 for template, PCR reaction conditions is: 94 DEG C of denaturation 4min; 94 DEG C of sex change 45s, 55 DEG C of 45s, 72 DEG C of 1min, 30 circulations; Repair for 72 DEG C and extend 10min.Get 5 μ L products electrophoresis on 1% sepharose containing goldview staining agent, 150V, 100mA, 20min.After electrophoresis terminates, gel is put into gel imaging system, observations.Pcr amplification product transfers to Sangon Biotech (Shanghai) Co., Ltd. to carry out sequencing.The 16SrRNA gene order of bacterial strain JSSW-LA-07-1 is SEQIDNO:1.
The 16SrRNA gene order of bacterial strain JSSW-LA-07-1 and nucleotide sequence known from GenBank are carried out Blast analysis, select the higher sequence of homology and complete sequence alignment at ClusterX software, comparison terminates rear use MEGA4.1 software building phylogenetic tree.
Bacterial strain JSSW-LA-07-1 gene order sequencing result 16SrRNA mrna length is 1466bp, the 16SrRNA gene order increased by bacterial strain carries out homology search at NCBI by Blast, result retrieval goes out the 16SrRNA gene order for bacillus, adopt adjacent method to build bacterial strain molecule and grow tree, isolated strains on phylogenetic tree with Bacillus coagulans (
bacilluscoagulans) (accession number: KM096994.1) belong to same, homology reaches more than 99% (see figure 1).Combining form and physiological and biochemical property institute's isolated strains is accredited as Bacillus coagulans (
bacilluscoagulans).
Embodiment 3: biocidal property measures
Be 1.0 × 10 by aseptic filter paper sheet in concentration
71.0h is soaked in CFU/mL Bacillus coagulans JSSW-LA-07-1 fresh medium.Getting concentration is 5.0 × 10
6the liquid medium 0.1mL of common pathogen Aeromonas hydrophila, Aeromonas sobria, Aeromonas veronii, Aeromonas caviae, Listonella anguillarum, tarda, Vibrio parahemolyticus, intestinal bacteria, vibrio alginolyticus in the aquaculture of CFU/mL, coat on LB agar culture plate respectively, then the filter paper soaking bacterium liquid is affixed on culture dish, each plate pastes 3, and each plate does 3 repetitions.Plate is placed in 28 DEG C of incubators 24,48h, measures inhibition zone size.Measurement result is as shown in table 1:
Table 1 Bacillus coagulans JSSW-LA-07-1 biocidal property measurement result (antibacterial circle diameter: mm)
Note: "-" indicates without inhibition zone, scraps of paper diameter is 7.00mm.
As shown in Table 1, Bacillus coagulans JSSW-LA-07-1 all has certain antagonistic action to Aeromonas hydrophila, Aeromonas sobria, Aeromonas veronii, Aeromonas caviae, Listonella anguillarum, tarda, Vibrio parahemolyticus, wherein the most remarkable to the inhibition of Aeromonas sobria, Aeromonas caviae.
Embodiment 4: the preparation of Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation
The fermented liquid that bacterial strain JSSW-LA-07-1 obtains through fermentation culture after activating, wet thallus is collected through 10000rpm high speed centrifugation fermented liquid, wet thallus mixes with mass ratio 1:2 ~ 10 with dry starch, cryodrying 20 ~ 24h at 30 ~ 50 DEG C, pulverizer is pulverized, cross 0.9mm sieve, obtain Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation concentration and be not less than 5.0 × 10
8individual (CFU)/gram.
Embodiment 5: the cultivation application of Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation
Test Juvenile Gibel Carp adopts cycling stream water temperature controlling system to cultivate.The hybridized prussian carp that test and Selection health, specification, weight are basically identical, original body mass is that the hybridized prussian carp of (17.7 ± 0.07) g is divided into 3 groups at random, and often group 3 is parallel, each parallel 30 tail fishes.Control group fed basal diet is crucian commodity material, is provided (table 2) by Tong Wei company.Based on test daily ration, daily ration adds 1.0 ~ 5.0mgkg respectively
-1bacillus coagulans JSSW-LA-07-1 bacterium powder preparation (preparing in embodiment 4).
Feeding and management: before test, carries out official test after first raising and train 2 weeks with basal feed.Duration of test, throw something and feed every day 2 times (8:00-8:30,15:30-16:00), and till apparent being satiated with food, daily ration of feeding is the 3%-4% of fish body weight, and appropriately adjust according to situation about growing and ingest.Every day, timing measured water temperature, pH, ammonia nitrogen, dissolved oxygen, and during cultivation, water temperature is about 26.5 DEG C, pH7.6 ~ 7.8, and dissolved oxygen is greater than 5mg/L, and ammonia nitrogen is less than 0.01mg/L.Weigh in time throwing something and feeding and test daily ration 45d.
Table 2 hybridized prussian carp basal diet
Table 3 is fed the impact (n=3) of Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation on Juvenile Gibel Carp growth performance
Note: with have in data line different letter representation significant difference (
p<0.05).
45d cultivates result and shows: compared with control group, add in basal diet Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation can significantly improve the rate of body weight gain of fish body, specific growth rate (
p<0.05), and can significantly reduce feed coefficient (
p<0.05), survival rate, liver body ratio, coefficient of condition without significant difference (
p>0.05), the result Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation that shows to ingest can significantly improve the growth of hybridized prussian carp.
Embodiment 6: Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation purifies water the application of aspect
Random selecting cultivation 6, black carp pond, 3 is test group, and 3 is control group.Test group is for pressing
The consumption full pool spilling head Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation of 50g/ mu rice, makes to enter that Bacillus coagulans starter bacteria in water body is dense is about 10
2-10
3cFU/mL, splashes one week continuously.Control group is not for splashing Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation.Test period is 1 week, tests forward and backwardly to sample respectively, detects every water-quality guideline.
Table 4 Bacillus coagulans is on the impact of cultivation black carp pond water quality
Note: with have in data line different letter representation significant difference (
p<0.05).
As can be seen from Table 4, the every water-quality guideline of test group all significantly reduction before comparatively testing (
p<0.05), and be starkly lower than control group (
p<0.05).Therefore, can illustrate that Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation effectively can reduce the pollutant load in water body, improve cultivation water, aquaculture can be applied to as water quality cleansing agent.
<210>SEQIDNO:1
<211>1446
<212>DNA
The 16SrRNA gene order of <213> bacterial strain JSSW-LA-07-1
<400>1
tatacatgcagtcgtgcggaccttttaaaagcttgcttttaaaaggttagcggcggacgg60
gtgagtaacacgtgggcaacctgcctgtaagatcgggataacgccgggaaaccggggcta120
ataccggatagttttttcctccgcatggaggaaaaaggaaagacggcttcggctgtcact180
tacagatgggcccgcggcgcattagctagttggtggggtaatggctcaccaaggcaacga240
tgcgtagccgacctgagagggtgatcggccacattgggactgagacacggcccaaactcc300
tacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccg360
cgtgagtgaagaaggccttcgggtcgtaaaactctgttgccggggaagaacaagtgccgt420
tcgaacagggcggcgccttgacggtacccggccagaaagccacggctaactacgtgccag480
cagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagcgcgcg540
caggcggcttcttaagtctgatgtgaaatcttgcggctcaaccgcaagcggtcattggaa600
actgggaggcttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgt660
agagatgtggaggaacaccagtggcgaaggcggctctctggtctgtaactgacgctgagg720
cgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatg780
agtgctaagtgttagagggtttccgccctttagtgctgcagctaacgcattaagcactcc840
gcctggggagtacggccgcaaggctgaaactcaaaggaattgacgggggcccgcacaagc900
ggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcct960
ctgacctccctggagacagggccttccccttcgggggacagagtgacaggtggtgcatgg1020
ttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttga1080
ccttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggagga1140
aggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaa1200
tggatggtacaaagggctgcgagaccgcgaggttaagccaatcccagaaaaccattccca1260
gttcggattgcaggctgcaacccgcctgcatgaagccggaatcgctagtaatcgcggatc1320
agcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagag1380
tttgtaacacccgaagtcggtgaggtaacctttacggagccagccgccgaaggtgggaca1440
gatgattggggtgaagtcgtaacaag1466
Claims (3)
1. a strain has the Bacillus coagulans of aquatic pathogenic bacterium antagonistic properties, its Classification And Nomenclature be Bacillus coagulans (
bacilluscoagulans) JSSW-LA-07-1, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is: CGMCCNo.10182.
2. the preparation method of a Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation, it is characterized in that with Bacillus coagulans JSSW-LA-07-1 for starting strain, the fermented liquid high speed centrifugation obtained through fermentation culture after bacterial strain JSSW-LA-07-1 activates collects wet thallus, wet thallus mixes with mass ratio 1:2-10 with dry starch, dry 20-24h at 30-50 DEG C, pulverizer is pulverized, and crosses 0.9mm sieve, the Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation obtained, concentration is not less than 5.0 × 10
8cFU/ gram.
3. the application of the Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation prepared by method described in claim 2, is characterized in that: as the additive of fishery cultivating feed, adds 1.0-5.0mgkg feeding in basal diet
-1bacillus coagulans JSSW-LA-07-1 bacterium powder preparation; As aquaculture water quality improving agent, by the consumption full pool spilling head Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation of 50-200g/ mu rice.
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| CN105524855B (en) | 2019-03-12 |
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