CN103740624B - Bacillus pumilus and application thereof to preparation of exopolysaccharides - Google Patents
Bacillus pumilus and application thereof to preparation of exopolysaccharides Download PDFInfo
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- CN103740624B CN103740624B CN201410022110.8A CN201410022110A CN103740624B CN 103740624 B CN103740624 B CN 103740624B CN 201410022110 A CN201410022110 A CN 201410022110A CN 103740624 B CN103740624 B CN 103740624B
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- bacillus pumilus
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Abstract
The invention provides Bacillus pumilus which is a GY001 strain and has a collection number of CGMCC No.8639. The screened Bacillus pumilus is used for fermenting and producing exopolysaccharides. According to the screened Bacillus pumilus for exopolysaccharides, the acid and alkali resistance of the exopolysaccharides is high, and the unique activity structure has a unique effect of resisting prawn viruses and has an ecological effect of maintaining good prawn digestive tract balance. The nonspecific immunity level of prawn breeding can be obviously improved, the pathogen infection resistance of the bred prawns is greatly improved, particularly multiple biological functions, such as virus infection resistance, of the prawns can be obviously improved, and the Bacillus pumilus is low in cost and easy to transport and store.
Description
Technical field
The invention belongs to microbe to screen applied technical field, be specifically related to a kind of bacillus pumilus and preparing the application in exocellular polysaccharide, the exocellular polysaccharide that this bacillus pumilus produces is for the prevention of prawn WSSV and treatment.
Background technology
The development of prawn culturing big area is risen the 1970s and 1980s, and China coast is very large to northern prawn culturing output from south, serves very large promoter action to the development of coastal economy.At present, virus infection is the serious threat affecting prawn culturing, causes serious financial loss to prawn culturing family.Shrimp white spot syndrome virus (White Spot Syndrome Virus, WSSV) disease is one of current serious harm shrimp culture industry sound development virus disease.Prawn disease according to the World Bank is reported, within 1994, prawn virus disease causes the production loss of 74% to global shrimp culture industry, and its financial loss reaches more than 3,000,000,000 dollars.Nineteen ninety-five, this disease is classified as one of Aquatic animals virus epidemic disease needing report by International Office of Epizootics (OIE), Food and Argriculture OrganizationFAO (FAO) and Asian-Pacific area aquaculture development network center (NACA).
Shrimp white spot syndrome virus (WSSV) is sick, and in Late Cambrian in 1992 in Taiwan, 5 ~ August in 1993 breaks out in the coastal plant from south to north of China mainland, and the prawn culturing field of the Asian countries such as Japan has also broken out this disease in succession.At present, the popular countries and regions of this virus reported in Asia comprise coastal and Taiwan Province in Korea, Thailand, Korea S, Indonesia, Vietnam, Malaysia, India, Sri Lanka, Bangladesh, China's Mainland etc.Nineteen ninety-five, there is white spot syndrome virus in Texas, USA prawn culturing field, and other area is on the Western Hemisphere broken out in succession.So far, WSSV worldwide spreads, and causes the financial loss of hundreds of hundred million yuan of dollars every year to shrimp culture industry, becomes one of principal disease threatening shrimp culture industry.
Antibiotic discovery facilitates the huge advance of physianthropy, and microbiotic is also the animal farming industry especially antibacterial choice drug of curing the disease of culture fishery.But along with high-density, intensive, mass-producing, the developing rapidly of industrialized culture pattern, the problems such as disease is propagated in a large number, microbiotic is abused wantonly, breeding environment deterioration, the enhancing of cause of disease resistance, cultivated animals drug residue are also day by day by people are paid close attention to.Developing meets the active demand that environmentally friendly disease control product has become culture fishery.Probiotic bacterium is as antibiotic substitute, cultured product is had no side effect, noresidue and resistance, improve aquatic animal production performance and health level, ensure the Sustainable development of aquatic product quality and promotion culture fishery, good Social benefit and economic benefit can be obtained.
Summary of the invention
The object of this invention is to provide a kind of bacillus pumilus and preparing the application in exocellular polysaccharide, the antiviral exocellular polysaccharide product that this bacillus pumilus produces can improve the ability of prawn antagonism WSSV effect, reduces prawn mortality ratio.
The invention provides a kind of bacillus pumilus (Bacillus pumilus), be GY001 strain, preservation is numbered: CGMCC No.8639, preservation date on December 27th, 2013, and depositary institution is China General Microbiological culture presevation administrative center; Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
The bacillus pumilus that the present invention screens is for fermentative production exocellular polysaccharide;
Its suitableeest leavening temperature is 37 DEG C.
Bacillus pumilus of the present invention is also used as immunostimulant; May be used for the preparation of prawn culturing medicine, or for adding in prawn culturing feed as antiviral.
The opposing soda acid ability that the present invention filters out its exocellular polysaccharide of exocellular polysaccharide bacillus pumilus is strong, and the active structure of its uniqueness has the effect unique function of antagonism prawn ' s virus, has the Ecology Action safeguarding good prawn digestive system balance.Cultured prawn innate immune activity can be significantly improved, greatly strengthen cultured prawn anti-microbial pathogen infectivity, particularly can significantly improve prawn and resist the various biological functions such as virus infection ability, with low cost, be easy to transport, storage.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and experimental technique used in an embodiment if no special instructions, is ordinary method.The material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1: the screening of bacillus pumilus (Bacillus pumilus) GY001 bacterial strain
(1) separation and purification of bacterium:
Extract respectively from Jiangnan, Qingdao in February, 2013 from the disease-resistant Penaeus vannamei enteron aisle sample the pond of falling ill through WSSV, put into 100ml sterilized water, after vibration 20min, be bacterial suspension.Draw 1ml respectively, after diluting 1000 times, get 30 μ l and coat on LB flat board, after slightly dry, be inverted 30 DEG C of cultivations.After 48h, according to the feature of the colonial morphology on flat board, color, select representative colonies in the flat lining out purifying of LB, the bacterial isolates after purifying is inoculated in slant medium, and 4 DEG C of preservations are stand-by.Finally be divided into from gut of shrimp from obtaining 125 strain bacterial strains.
(2) antibacterial screening
Bacteriostatic experiment-double-layer plate, Odontothrips loti: every 5mL bactericidal nurishing nutrient agar (about 50 DEG C) adds 100 μ L pathogenic bacterium bacterium liquid (bacterium amounts 10
6the order of magnitude), mixing hypsokinesis makes double-layer plate down to nutrient agar plate, places Oxford cup, add 200 μ L cultured bacillus pumilus bacterium liquid in the cup of Oxford after solidifying on substratum, after the diffusion of bacterium liquid, put into 28 DEG C of incubators cultivate 20h, observe antibacterial circle diameter;
(3) mensuration of bacterium viscosity
The mensuration of viscosity: adopt rheometer (Bo Lifei company DV ?III+ type).Tested viscosity at 4 DEG C, rotor model is RV6, and measure three times under the speed of 10r/min respectively, once, each sample does two parallel samples to every 15s value, and result calculates arithmetical av.
Table 1: sieve strain characteristic evaluation result again
Select the obvious bacterial strain of inhibition zone as bacterial strain to be selected, carry out anti-biotic resistance screening.Through inhibition zone and antibiotic-screening, and viscosity assessment, final screening obtains 1 strain to intestinal bacteria (ATCC25922), Salmonellas (ATCC14028), the most obvious strain bacterial strain of streptococcus aureus (ATCC6538) growth-inhibiting effect.For GY001 strain, preservation is numbered: CGMCC No.8639, preservation date on December 27th, 2013, and depositary institution is China General Microbiological culture presevation administrative center; Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Embodiment 2: bacillus pumilus GY001 is external to antibiotic sensitivity experiments
1) microbiotic is selected: every large class microbiotic for animals choose conventional and be relatively easy to get 1 ?2 kinds.According to the conventional treatment consumption of various microbiotic in complete diet pellet, 2 times of proposed treatment consumption is severe consumption, and 1/2 for the treatment of consumption is prevention consumption.
2) bacterium solution preparation: bacillus pumilus GY001 is seeded to 28 DEG C of cultivation 48h in liquid 2216E substratum, is diluted to 10 with 2216E substratum
8the order of magnitude.
3) sensitivity experiments: get the GY001 bacterium liquid that 5mL is diluted, add 1 wherein) in selected microbiotic make its concentration reach severe consumption, treatment consumption and prevention consumption respectively, be placed in 37 DEG C cultivate 4h after, count with 2216E agar.Calculate the survival rate of bacillus pumilus GY001 under different antibiotic concentration.Result shows, except gentamicin, bacillus pumilus GY001 with the microbiotic effect 4h of other different concns after, its survival rate is all more than 80%, and majority is more than 60%.Can infer thus, bacillus pumilus GY001 can use with the most of microbiotic except gentamicin simultaneously and can not affect its effect within the scope of its safe medication.
2216E culture medium prescription is: peptone 10g, yeast extract 5g, NaCl10g, and agar 18g adds seawater to 1000ml, adjusts pH to 7.0,121 DEG C of sterilizing 20min.
GY001 bacterial strain of the present invention is through the microscopic examination of bacterium colony and form, staining reaction, the experiment of customary physiological biochemical characteristic, and 16S rDNA sequential analysis, and this identification of strains is a kind of bacillus pumilus, and its feature is as follows:
(A) morphological specificity: thalline is rod-short, Gram-positive, raw in gemma, atrichia;
(B) cultural characteristic: bacterium colony is rounded, light yellow, smooth surface, glossy;
(C) physiological and biochemical property: this bacterial strain as well supports bacterium, and growth temperature 14 ~ 60 DEG C, optimum growth temperature is 37 DEG C.The medium pH scope that can bear is 3 ~ 11, the most suitable growth pH is 8.0.
(D) 16S rDNA sequential analysis: result is carried out maximum homology and compared in Genbank, found that, this bacterial strain is the highest with the similarity of the 16S rDNA (accession number is EU447665.1) of the Bacillus pumilus reported, show that the sibship of this bacterial strain and bacillus pumilus is very near, therefore after qualification, substantially determine that this antagonistic strain belongs to Bacillus pumilus.
GY001 bacterial strain of the present invention proves himself do not have lethal effect to prawn, the also toxicological harmless effect of its exocellular polysaccharide through infection experiment.Compared with control group, the exocellular polysaccharide spice of antiviral bacillus pumilus fermented liquid significantly can reduce the mortality ratio that prawn infects WSSV, and immune protective rate reaches 40%, namely improves the Anti-infection to WSSV ability of prawn.
Embodiment 3, bacillus pumilus produce exocellular polysaccharide
(1) bacterial strain activation: get bacillus pumilus GY001 bacterial strain, bacterial strain on substratum 37 DEG C cultivate 2 days, 37 DEG C in the fermentation medium, 180rpm shake-flask culture 3 days obtains liquid spawn; Described substratum is: 1L distilled water, 10g glucose, 25g peptone, 5g sodium-chlor, 1.0g extractum carnis, 18g agar; Fermention medium is: Zulkovsky starch 10g, Semen Maydis powder 15g, lactose 10g, glucose 2.5g, yeast extract paste 0.5g, CaCl21g, MgSO47H2O1g, and deionized water is dissolved to 1000mL, pH7.6;
(2) bacterial strain spreads cultivation: be 10 by the liquid-spawn inoculation of step (1) in being equipped with inoculum size in fermention medium triangular flask
6the inoculum size of CFU/mL is inoculated in 100mL LB triangular flask, 37 DEG C 37 DEG C, 180rpm shake-flask culture 5 days, and 4 DEG C keep 16h.
2. the extraction of exocellular polysaccharide
(1) isoelectric point method is except casein: adjust sample liquid pH to 8.0 with saturated NaOH, 4000r/min, centrifugal 15min, and supernatant liquor HCL regulates the centrifugal 15min of pH to 4.6,3000 × g.
(2) enzymolysis: adjust pH to 7.5, add the trypsin solution (concentration is 3g/L, now with the current) of 1/10 volume, 40 DEG C of water-baths, after enzymolysis 2.5h, the centrifugal 15min of 2000 × g.
(3) Sevag deproteinated: the centrifugal 15min of Sevag liquid vibration 30min, 3000 × g adding 1/3 volume, removes precipitation, in triplicate.
(4) alcohol settling: add 3 ?5 times of volumes 95% ethanol, fully vibration (polysaccharide claims flocks to separate out), the centrifugal 15min of 3000 × g, collecting precipitation.
(5) washing is dry: by precipitation washing with acetone dehydration twice, vacuum-drying obtains Crude polysaccharides.
3. Crude polysaccharides purity testing
Adopt Ben Fen ?sulphate method measure EPS content, make typical curve with glucose.Accurate absorption Glucose standards solution (1mg/mL), 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12mL (is equivalent to glucose 0.0, 50, 60, 70, 80, 90, 100, 120 μ g), and each sample reserve liquid 0.1mL is placed in 10mL colorimetric cylinder, 1.0mL is supplied with distilled water, add 1.0mL5.0% phenol solution respectively, shake up rear static 2min, add rapidly the 5mL vitriol oil, static 10min is placed in 30 DEG C of water-baths and heats 15min, take out, put in cold water and cool, take blank tube as contrast, optical density(OD) is measured at wavelength 490nm place with ultraviolet spectrophotometer.Take glucose concn as X-coordinate, optical density value is ordinate zou drawing standard curve, calculates exocellular polysaccharide content.
Result of use:
Healthy Environment of Litopenaeus vannamei Low more than 500 tail, random packet is raised, and changes water every day 1 time, day quantity of exchanged water be 1/3; To throw something and feed every day feed 3 times, daily ration of feeding be about 1.5 ?2.0 grams/10 tails, suck residual feed after the 1h that throws something and feeds, and according to the number adjustment daily ration, feeding quantity of residual bait; Water temperature 22 ~ 25 DEG C, pH8.2 ± 0.2, and continuous charge, support 7d before experiment temporarily.
After supporting temporarily the phase, experiment prawn is divided into experimental group and control group 2 treatment group at random, each treatment group have 3 parallel, put prawn 90 tail in a suitable place to breed for each parallel group.Control group to be thrown something and fed basal feed in the whole experimental phase always, and throw something and feed immunization feedstuff (basal feed adds the exocellular polysaccharide of above-mentioned preparation), i.e. 1 day basal feed of throwing something and feeding in experimental group interval, 1 day immunization feedstuff of throwing something and feeding, until experiment terminates.21d after immunization feedstuff of throwing something and feeding, the direct oral infection prawn pathological material of disease of white spot syndrome virus, the prawn quantity of record virus infection stage death every day, and the cumulative mortality calculating that WSSV infects Environment of Litopenaeus vannamei Low in 14d.Result shows: compared with control group, and the exocellular polysaccharide spice of antiviral bacillus pumilus fermented liquid significantly can reduce the mortality ratio that prawn infects WSSV, and immune protective rate reaches 40%, namely improves the Anti-infection to WSSV ability of prawn.
The above results shows, the opposing soda acid ability of exocellular polysaccharide prepared by the bacillus pumilus that the present invention screens is strong, has the unique function that antagonism prawn ' s virus RNA turns over transcriptase interference, suppresses WSSV copying in prawn body.And its exocellular polysaccharide good water solubility, is dissolved in rapidly in aquaculture water, regularly splashes in water, can effectively improve prawn non-specific immunity, obviously reduce prawn sickness rate, improve prawn surviving rate.
Therefore, bacillus pumilus of the present invention can be used as excellent microbial forage additive for aquaculture field.
Claims (1)
1. an exocellular polysaccharide, is characterized in that, described exocellular polysaccharide is standby by bacillus pumilus fermentation; Described bacillus pumilus (Bacillus pumilus), be GY001 strain, preservation is numbered: CGMCC No.8639, preservation date on December 27th, 2013, and depositary institution is China General Microbiological culture presevation administrative center; Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
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| CN102326677A (en) * | 2011-10-21 | 2012-01-25 | 北京大北农科技集团股份有限公司 | Composite micro-ecological feed additive and preparation method and premix thereof |
| CN103409492A (en) * | 2013-08-23 | 2013-11-27 | 新疆阜丰生物科技有限公司 | Method for extracting transparent low-acyl gellan gum |
| CN103497906A (en) * | 2013-08-02 | 2014-01-08 | 国家***第三海洋研究所 | Microecological preparation, preparation method, and applications thereof |
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| CN102326677A (en) * | 2011-10-21 | 2012-01-25 | 北京大北农科技集团股份有限公司 | Composite micro-ecological feed additive and preparation method and premix thereof |
| CN103497906A (en) * | 2013-08-02 | 2014-01-08 | 国家***第三海洋研究所 | Microecological preparation, preparation method, and applications thereof |
| CN103409492A (en) * | 2013-08-23 | 2013-11-27 | 新疆阜丰生物科技有限公司 | Method for extracting transparent low-acyl gellan gum |
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