CN101649298A - Bdellovibrio bacteriovorus bacterial strain eliminating aquatic product Gram-positive pathogenic bacterium and application thereof - Google Patents
Bdellovibrio bacteriovorus bacterial strain eliminating aquatic product Gram-positive pathogenic bacterium and application thereof Download PDFInfo
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Abstract
The invention discloses a bdellovibrio bacteriovorus bacterial strain eliminating aquatic product Gram-positive pathogenic bacterium and an application thereof. The invention separates to obtain bdellovibrio bacteriovorus BDS02 the length of flagellum of which is at least 1.8mu m. The bdellovibrio bacteriovorus with flagellum on the end is cambered single cell the size of which is 0.65*0.2 mu m when observed by an electron microscope. The two layer plating method is used for cultivating the bdellovibrio bacteriovorus bacterial strain at the temperature of 28 DEG C for four days to form transparent round negativecolony the diameter of which is 1-2mm. Before the microorganism preparation prepared by the bdellovibrio bacteriovorus BDS02 is eaten by aquatic products, common Gram-positive pathogenic bacteria comprising Mycobacterium tuberculosis, star-shaped nocardia asteroids, rhodococcus erythropolis, streptococcus faecalis, staphylococcus epidermidis, clostridium perfringens and corynebacterium glutamicum in the transportation process and cultivation process can be eliminated, which improves the safe factor of raw aquatic products for consumers, protects the health of the consumers and provides guarantee for greenly processing and producing the aquatic products.
Description
Technical field
The present invention relates to a kind of Bdellovibrio, particularly a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof of eliminating aquatic product Gram-positive pathogenic bacterium.
Background technology
In recent years, great food safety incident taking place in succession all over the world, not only bring enormous economic loss, still to the test of social stability and safety, makes food-safety problem become the problem of people's growing interest.To this, the relevant laws and regulations of numerous food safety have all been set with organizing in countries in the world, to reach the purpose of prevention and control food-safety problem.Microorganism is one of important factor of contaminated food products, because its individuality is small, widely distributed, viability is strong, breed characteristics such as rapid, makes how to avoid and reduce microbial contamination becoming the matter of utmost importance that links such as food-processing and circulation are considered.
Culture disease is one of bottleneck of restriction culture fishery sustainable and healthy development always, and only the aquatic products disease loss that monitors of China in 2004 just reaches 15,100,000,000 yuan.The abuse of microbiotic and chemical disinfection medicine makes culturing economic animal disease resistant ability obviously descend again, both has been unfavorable for the healthy growth of cultivated animals, does not also meet the needs of healthy aquaculture theory and Sustainable development.Along with to the raising of fishery products safety requirements with to the attention of Resistant strain harm, the bionomic control technology that meets environmental friendliness and Sustainable development then is one of most promising developing direction of aquiculture disease at present and in the future, and microorganism is then playing the part of most important role therein.But, still there are many defectives in some probioticses aspect the diseases prevention and treatment of reply hydrocoles, and bacteriophagic Bdellovibrio has the biological characteristics of unique cracking bacterium, for new approach has been opened up in the diseases prevention and treatment of hydrocoles, a kind of green, environmental protection, healthy natural " microbiotic " are come into the aquaculture industry just gradually.If can be during fishery products be being cultured, before transportation or edible, the processing; carry out the biology elimination work of pathogenic bacterium earlier; then can reduce or eliminate the probability of food poisoning on the one hand; reduce or eliminate the possibility of drug residue; the safety coefficient of improving the quality of products; protection human consumer's health, the processing that also can be fishery products on the other hand provide the facility in high-quality raw material and the processing, for foreign exchange earning provides strong guarantee.
The gram-positive microorganism that can cause the fishery products disease in the aquaculture mainly comprises the mycobacterium (Mycobacterium sp.) that can cause mycobacterial diseases, cause the Nocardia bacteria (Nocardia sp.) of nocardiasis, cause the suis (Streptococcus sp.) of streptococcicosis, cause the staphylococcus epidermidis (Staphylococcus epidermidis) of staphylococcosis, cause the clostridium (Clostridium sp.) of clostridium enteritis, cause the aquifer cultivation environment to change and influence the coryneform bacteria (Corynebacterium sp.) of the rhodococcus (Rhodococcus sp.) and the initiation corynebacteriosis of the healthy state of culturing object.
Summary of the invention
The shortcoming that primary and foremost purpose of the present invention is to overcome prior art provides a kind of Bdellovibrio bacteriovorus bacterial strain of eliminating aquatic product Gram-positive pathogenic bacterium with not enough.
Another object of the present invention is to provide the application of described Bdellovibrio bacteriovorus bacterial strain.
Purpose of the present invention is achieved through the following technical solutions: a kind of Bdellovibrio bacteriovorus bacterial strain of eliminating aquatic product Gram-positive pathogenic bacterium, name is called Bdellovibrio (Bdellovibrio sp.) BDS02, be preserved in Chinese typical culture collection center on August 7th, 2009, deposit number is CCTCC NO:M 209170;
Described Bdellovibrio BDS02 carried out after the negative staining morphologic observation (see figure 1): BDS02 is unicellular under electron microscope, arc, size is 0.65 * 0.2 μ m, end is given birth to flagellum, flagellum length at least 1.8 μ m; Described Bdellovibrio BDS02 cultivates the transparent circular plaque that can form diameter 1-2mm in four days with the double-layer plate method in 28 ℃;
BDS0216S-ITS rDNA ( SEQ ID NO.1 ) ：CAGGCCTAACACATGCAAGTCGAACGGGGTAGCAATACCTAGTGGCGCACGGGTGAGTAACGCGTGGATAATCTGCCTTGGAGTGGGGGATAACTAGTCGAAAGATTAGCTAATACCGCATAAGACCACAGGAGCTGCGGCTCTAGGGGTCAAAGGTTTTTCGTTCCAAGATGAGTCCGCGTAAGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCTTTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATATTGCACAATGGAGGAAACTCTGATGCAGCGACGCCGCGTGAGTGATGAAGGCCTTCGGGTCGTAAAGCTCTGTCGCAGGGGAATAACACAATGAATGTACCCTGTAAGAAAGGATCGGCTAACTTCGTGCCAGCAGCCGCGGTAAGACGAGGGATCCTAGCGTTGTTCGGAATTATTGGGCGTAAAGCGGATGTAGGTGGCTTTGTAAGTCAGATGTGAAAGCCCAGGGCTCAACCCTGGAAGTGCATTTGATACTGCGAAGCTTGAGTGTCGGAGAGGTTACTAGAATTGTTGGTGTAGTGGTGAAATACGTAGATATCAACAGGAATACCGGAGGCGAAGGCGGGTAACTGGCCGAACACTGACACTGAGATCCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGGATACTTGTTGTTAGAGGTATTGACCCCTTCAGTGACGAAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTAGGCTTGACATGTACTGGAAGATTGGCAGAAATGTCGTCGCCCGCAAGGGTCGGTACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGCATTTAGTTGCCAGCATTCAGTTCGGCACTCTAGATGGACTGCCGGTGTTAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGCCTAGGGCTACACACGTGCTACAATGGTAGTCACAGAGCGAAGCTAAGCCGCGAGGTAGAGCAAATCGCTTAAAAGCTATCTAAGTTCAGATTGATCTCTGCAACTCGAGATCATGAAGTTGGAATCGCTAGTAATCGCGGAACAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAAAGTCGGCTGTACCAGAAGTCGCTGCGCTAACCGTAAGGAGGCAGGCGCCCAAGGTATGGTCGATGATTGGGGTGAAGTCGTAACAAGGGAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTTTCTAAGGTTTATCCGGTCAATCTTCATCAAGACTTGTTCTTGATAAGTTAAAATGACCCAATCTTAGGTCAACTTACTCTTCCCGAGTAAGTGAGTCCCAAAAATCTATCTAGCTGTTTAGTTTTGAGAGAGTGAAGCCTAACGGGCCTGTAGCTCAGTTGGTTACAGCACACGCTTGATAAGCGTGGGGTCGGAAGTTCGAGTCTTCCCAGGCCCACCAAGTTCTACTGTACTGGAATGCGGTGTAAGTTAGAGTTTTGCTGAACGGTTTTCGTTCTTTGACATTTGAATAGATTGATTTAGTTGATTTTTAGCGAGGTTAGTTCCATTCTTTTAAGCTACAAAGGGCTTACGGTGGATGCCCTGGCAGTCA
A kind of microbial preparation of eliminating aquatic product Gram-positive pathogenic bacterium contains described Bdellovibrio BDS02;
Described microbial preparation also contains Bdellovibrio (Bdellovibrio sp.) BDS01;
Described Bdellovibrio BDS01 is preserved in Chinese typical culture collection center on August 7th, 2009, and deposit number is CCTCCNO:M209169; Morphologic observation under electron microscope: BDS01 was unicellular after described Bdellovibrio BDS01 carried out negative staining, arc, and size is 0.9 * 0.25 μ m, and end is given birth to flagellum, and flagellum length is 4 μ m; Described Bdellovibrio BDS01 cultivates the transparent circular plaque that can form diameter 1-2mm in four days with the double-layer plate method in 28 ℃;
Described Bdellovibrio BDS02 is preferably by application number " 200710031166.X ", and the fermentation process that name is called " the fermentation culture technology of high-density Bdellovibrio telotroch " disclosed high-density Bdellovibrio of national inventing patent application telotroch ferments;
Described Bdellovibrio BDS01 is preferably by application number " 200710031166.X ", and the fermentation process that name is called " the fermentation culture technology of high-density Bdellovibrio telotroch " disclosed high-density Bdellovibrio of national inventing patent application telotroch ferments;
Described Bdellovibrio BDS02 and Bdellovibrio BDS01 preferably press bacterial count and mix at 1: 1;
Described microbial preparation is applied to eliminate aquatic product Gram-positive pathogenic bacterium;
The preferred freshwater product of described fishery products;
Described aquatic product Gram-positive pathogenic bacterium comprises mycobacterium tuberculosis (Mycobacteri μ mtuberculosis), nocardia asteroide (Nocardia asteroides), Rhodococcus (Rhodococcuserythropolis), streptococcus faecium (Streptococcus faecalis), staphylococcus epidermidis (Staphylococcusepidermidis), clostridium perfringens (Clostridi μ m perfringens) and corynebacterium glutamicum (Corynebacteri μ m glutamic μ m);
Described microbial preparation is applied to eliminate Gram-positive pathogenic bacterium edible or that the preceding fishery products of processing carry, and the Bdellovibrio optimum concentration range is 10 during application
4～10
8Pfu/mL;
Described microbial preparation is applied to control the Gram-positive pathogenic bacterium in the fishery products transportation, and the Bdellovibrio optimum concentration range is 10 during application
3～10
6Pfu/mL;
Described microbial preparation is applied to control the Gram-positive pathogenic bacterium in the aquaculture water body, and the Bdellovibrio optimum concentration range then is 10 during application
2～10
5Pfu/mL;
The present invention has following advantage and effect with respect to prior art:
1, microbial preparation of the present invention is remarkable at the effect of eliminating the Gram-positive pathogenic bacterium that fishery products carry.
The effect of microbial preparation of the present invention Gram-positive pathogenic bacterium before eliminating the edible or processing of fishery products, in transportation and the aquaculture water thereof is remarkable, with tilapia, Penaeus vannamei, Corbicula fluminea, steamed crab example, shown in Fig. 2～13, Gram-positive pathogenic bacterium is all had higher elimination factor, and elimination factor all reaches more than 90%.
2, good with pathogenic gram-positive bacterial security in the Bdellovibrio elimination fishery products before edible
The Gram-positive pathogenic bacterium method is biological method in the Bdellovibrio elimination fishery products, Bdellovibrio can be infected, the characteristic of cracking host bacteria makes it to be suitable as the biopurification factor of restraining or removing pathogenic bacterium in organism and the environment thereof, and it is behind the intact host bacteria of cracking, can wither away automatically because of hungry, therefore humans and animals be had no side effect.Not only can improve the safety coefficient that the human consumer eats fishery products raw, protection human consumer's health also provides safeguard for the green processing production of fishery products.
3, apply the present invention in the fishery products transportation, convenient, safe and effective
Fishery products adopt Bdellovibrio to control Gram-positive pathogenic bacterium in transportation and can prevent water quality deterioration, improve the fishery products survival rate, avoid using microbiotic simultaneously, guarantee the high-quality of fishery products.
4, the present invention is fit to be applied to the breeding process of fishery products
During culturing, promptly carry out the elimination work of pathogenic bacterium, be particularly conducive to and reduce or eliminate chemicals such as antibiotic use and residual, guarantee the high-quality of fishery products, culturing with tilapia, Penaeus vannamei, Corbicula fluminea, steamed crab is example, the effect of when 2 strain Bdellovibrios mix the microbial preparation use that obtains the elimination of Gram-positive pathogenic bacterium in the fresh water fishes and shrimps shellfish crab aquaculture water being tested as shown in figure 13, the quantity of pathogenic bacterium can be controlled in the 100cfu/mL in the aquaculture water.
5, the present invention is for eliminating the entrained pathogenic bacterium of fishery products a kind of new method is provided and applicable in the whole process of aquaculture before edible, reduce or eliminate chemicals such as residues of antibiotics effectively, guarantee the high-quality of fishery products, fundamentally avoid the generation of fishery products food poisoning, for foreign exchange earning provides strong guarantee.
Description of drawings
Fig. 1 is the aspect graph that Bdellovibrio BDS02 observes under Electronic Speculum.
Fig. 2 is that the microbial preparation that only contains BDS02 is used to eliminate the design sketch that fishery products eat preceding Gram-positive pathogenic bacterium clostridium perfringens.
Fig. 3 is that the microbial preparation that only contains BDS02 is used to eliminate the design sketch that fishery products eat preceding Gram-positive pathogenic bacterium Rhodococcus.
Fig. 4 is that the microbial preparation that only contains BDS02 is used to eliminate the design sketch of the Gram-positive pathogenic bacterium corynebacterium glutamicum before fishery products eat.
The microbial preparation that Fig. 5 is made up of BDS01 and BDS02 is used to eliminate the design sketch of the Gram-positive pathogenic bacterium before fishery products eat.
Fig. 6 is the design sketch that the microbial preparation that only contains BDS02 is used for eliminating the Gram-positive pathogenic bacterium clostridium perfringens of fishery products transportation.
Fig. 7 is the design sketch that the microbial preparation that only contains BDS02 is used for eliminating the Gram-positive pathogenic bacterium Rhodococcus of fishery products transportation.
Fig. 8 is the design sketch that the microbial preparation that only contains BDS02 is used for eliminating the Gram-positive pathogenic bacterium corynebacterium glutamicum of fishery products transportation.
The microbial preparation that Fig. 9 is made up of BDS01 and BDS02 is used for eliminating the design sketch of the Gram-positive pathogenic bacterium of fishery products transportation.
Figure 10 only contains the design sketch that the BDS02 microbial preparation is used for eliminating the Gram-positive pathogenic bacterium clostridium perfringens of aquaculture process.
Figure 11 only contains the design sketch that the BDS02 microbial preparation is used for eliminating the Gram-positive pathogenic bacterium Rhodococcus of aquaculture process.
Figure 12 only contains the design sketch that the BDS02 microbial preparation is used for eliminating the Gram-positive pathogenic bacterium corynebacterium glutamicum of aquaculture process.
The microbial preparation that Figure 13 is made up of BDS01 and BDS02 is used for eliminating the design sketch of the Gram-positive pathogenic bacterium of aquaculture process.
The microbial preparation that Figure 14 is made up of BDS01 and BDS02 is used for eliminating the design sketch of the pathogenic bacterium of freshwater product breeding process.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
(1) separation and purification of Bdellovibrio BDS02
With host Aeromonas hydrophila (Aeromonas hydrophila, purchase in Guangdong Province DSMZ of Institute of Micro-biology, numbering GIM1.172) is inoculated in and contains 100mL sterilization LB (Luria-Bertani) liquid nutrient medium (peptone 10g, yeast extract paste 5g, distilled water 1000mL, pH 7.2) triangular flask in, placing rotating speed is 200rpm, temperature is to cultivate 16h in 28 ℃ the constant temperature shaking table, makes it be in logarithmic phase.Nutrient solution is under 4 ℃ of temperature condition, with the centrifugal 20min of 6000rpm speed, abandoning supernatant, the thalline that precipitates 1mL DNB (dilute nutrient broth) liquid nutrient medium (nutrient broth 0.8g, caseinic acid hydrolyzate 0.5g, yeast extract 0.1g is dissolved in the 1000mL distilled water, and the pH value is 7.2～7.6) suspend again that (concentration is about 3 * 10 in the back
10Cfu/mL) place 4 ℃ of refrigerators, stand-by.
The mud sample of gathering is carried out pre-treatment, getting the about 3g of mud sample joins in the triangular flask that contains 50mL sterilization DNB liquid nutrient medium, the Aeromonas hydrophila bacteria suspension 0.5mL that adds the propagation Bdellovibrio that has prepared simultaneously, it is 200rpm that triangular flask is placed rotating speed, temperature is to cultivate 36h in 28 ℃ the constant temperature shaking table, nutrient solution is earlier under 4 ℃ of conditions, the centrifugal body refuse that goes of the centrifugal 15min of 1500rpm, back 6000rpm is centrifugal, and 20min goes the host, supernatant liquor is again under 4 ℃ of temperature condition, with the centrifugal 20min of 16000rpm speed, abandoning supernatant, throw out suspends again with 1mL DNB liquid nutrient medium, becomes sample concentration liquid.
Adopt the double-layer plate method: get 0.5mL sample concentration liquid and 0.5mL Aeromonas hydrophila suspension and mix, this mixed solution again with DNB upper strata substratum (the distilled water 1000mL of 50 ℃ of 3～3.5mL, pH7.2, agar powder 7g) mixes, be poured into DNB lower floor flat board (nutrient broth 0.8g, caseinic acid hydrolyzate 0.5g, yeast extract 0.1g, distilled water 1000mL, pH7.2, agar powder 17g) in and pave.Place 28 ℃ of constant incubators to cultivate double-layer plate, every 12h observation experiment result.Wait to contain on the double-deck agar plate of host bacterium and plaque occurs, picking is single spot of expansion constantly, joins in the triangular flask that contains 50mL DNB liquid nutrient medium to suspend, and adds corresponding host bacterium concentrated solution simultaneously, placing rotating speed is 200rpm, and temperature is cultured continuously 30h in 28 ℃ the constant temperature shaking table.Then, nutrient solution is under 4 ℃ of temperature condition, with the centrifugal 20min of the speed of 6000rpm, supernatant liquor is under 4 ℃ of temperature condition, with the centrifugal 20min of the speed of 16000rpm, topple over double-deck agar plate check again, once more the continuous single spot that enlarges of picking, and repeated several times, be the pure bacterial strain of a strain Bdellovibrio to single plaque plaque that forms shape, size, transparent equal unanimity that goes down to posterity.Select regular shape, transparent and plaque that plaque is maximum, called after Bdellovibrio BDS02.It is carried out morphologic observation under electron microscope (Fig. 1) after the negative staining: Bdellovibrio is unicellular, arc, and size is 0.65 * 0.2 μ m, end is given birth to flagellum, flagellum length at least 1.8 μ m; Described Bdellovibrio (Bdellovibrio sp.) BDS02 cultivates the transparent circular plaque that can form diameter 1～2mm in four days with the double-layer plate method in 28 ℃.According to the description of uncle's Jie Shi handbook (Bergey ' s Manual) to Bdellovibrio: Bdellovibrio is the bacterium that parasitizes on other bacteriums, is the shaft-like of single, little, crooked, motion at parasitic stage, cell, and polar flagella moves, and flagellum has sheath.Can confirm that thus separating the Bdellovibrio BDS02 that obtains is Bdellovibrio sp..Use the genomic dna that the DNA purification kit extracts Bdellovibrio BDS02, its 16S rDNA increases.In GenBank, carry out the homology search by order-checking and splicing back (seeing sequence table SEQ ID NO.1), the 16S-ITS rDNA of comparison result shows Bdellovibrio BDS02 and other disclosed Bdellovibrio 16S rDNA sequence similarity degree height, but incomplete same, illustrate that Bdellovibrio BDS02 separates to obtain first.Described Bdellovibrio BDS02 is being positioned at the Chinese typical culture collection center preservation of Chinese Wuhan City Wuhan University on August 7th, 2009, and deposit number is CCTCCNO:M209170.
(2) preparation of the microbial preparation of elimination aquatic product Gram-positive pathogenic bacterium
Bdellovibrio bacterium liquid ferments by the fermentation process of the disclosed high-density Bdellovibrio of the national inventing patent application telotroch of application number " 200710031166.X ": at 2 100mL LB (peptone 10g is housed respectively, yeast extract paste 5g, distilled water 1000mL, pH 7.2) inoculate Aeromonas hydrophila (Aeromonas hydrophila in the Erlenmeyer flask of liquid nutrient medium, purchase in Guangdong Province DSMZ of Institute of Micro-biology, numbering GIM1.172), 250rpm, 30 ℃ of shaking tables were cultivated 24 hours, nutrient solution is abandoned supernatant respectively at 4 ℃, the centrifugal 15min of 6000rpm.Respectively precipitation is joined 2 100mL DNB liquid nutrient medium (nutrient broth 0.8g is housed, caseinic acid hydrolyzate 0.5g, yeast extract 0.1g, be dissolved in the 1000mL distilled water, the pH value is 7.2～7.6) Erlenmeyer flask in, simultaneously more respectively from the double-layer plate the plaque of picking Bdellovibrio BDS02 and Bdellovibrio BDS01 (be preserved in Chinese typical culture collection center in the Chinese Wuhan City Wuhan University on August 7th, 2009, deposit number is CCTCCNO:M209169) to adding in the DNB liquid nutrient medium of Aeromonas hydrophila.Constant temperature shaking table 200rpm, 30 ℃ of cultivation 24h.Nutrient solution is respectively at 4 ℃ of centrifugal 20min of 6000rpm, get supernatant liquor, again with supernatant liquor respectively at 4 ℃ of centrifugal 20min of 16000rpm, keep precipitation, add the DNB liquid nutrient medium Bdellovibrio throw out that suspends again, make the concentration of Bdellovibrio BDS02 bacterium liquid and Bdellovibrio BDS01 bacterium liquid be respectively 10
10Pfu/mL.
Microbial preparation is for being 10 by the Bdellovibrio number
10The Bdellovibrio BDS02 bacterium liquid of pfu/mL is formed.
(3) microbial preparation is used to eliminate the edible preceding Gram-positive pathogenic bacterium of fishery products
1., preparation aquatic product Gram-positive pathogenic bacterium liquid
Clostridium perfringens (Clostridi μ m perfringens ATCC13124 purchases in microbial strains preservation center, Guangdong Province) is with reinforced clostridial medium (yeast extract paste 3g, extractum carnis 10g, peptone 10g, Zulkovsky starch 1g, glucose 5g, cysteine hydrochloride 0.5g, NaCl 3g, NaAc 3g, distilled water 1000mL, pH8.5), anaerobic condition is cultivated 24h for following 28 ℃.Rhodococcus (Rhodococcuserythropolis ATCC4277 purchases in Chinese common micro-organisms DSMZ) is with ATYP substratum (KH
2PO
41g, CaCl
20.1g, NaHCO
33g, sodium acetate 1g, MgCl
20.5g, NH
4Cl 1g, NaCl 1g, trace element solution 1mL, vitamin solution 1mL, sodium succinate 1g, yeast extract 0.5g, peptone 0.5g, distilled water 1000mL; PH 6.8; Trace element formula is: FeCl
24H
2O 1.8g, CoCl
26H
2O 0.25g, NiCl
26H
2O 0.01g, CuCl
22H
2O 0.01g, MnCl
24H
2O 0.7g, ZnCl
20.1g, H
3BO
30.5g, NaMoO
42H
2O 0.03g, Na
2SeO
35H
2O 0.01g, distilled water 1000mL; The vitamin solution prescription is: vitamin H 0.1g, nicotinic acid 0.35g, vitamin 0.3g, para-amino benzoic acid 0.2g, Pyridoxamine hydrochloride 0.1g, calcium pantothenate 0.1g, vitamins B
120.05g, distilled water 1000mL) and the shaking table cultivation, 30 ℃ of 160rpm cultivate 48h.Corynebacterium glutamicum (Corynebacteri μ mglutamic μ m ATCC13032, purchase in microbial strains preservation center, Guangdong Province) with common nutrient broth medium (peptone 10g, extractum carnis 3g, sodium-chlor 5g, distilled water 1000mL, pH7.4) shaking table is cultivated, and 28 ℃ of 160rpm cultivate 24h.The nutrient solution of above-mentioned each bacterium through 6000rpm, 4 ℃ of centrifugal 20min, is abandoned supernatant liquor respectively, and the precipitation thalline suspends again with the DNB liquid nutrient medium, and adjusting separately respectively, concentration is 10
9Cfu/mL.Clostridium perfringens bacterium liquid, red string coccus bacterium liquid and corynebacterium glutamicum bacterium liquid obtain aquatic product Gram-positive pathogenic bacterium liquid with 1: 1: 1 mixed.
2. microbial preparation is used to eliminate the edible preceding Gram-positive pathogenic bacterium of fishery products
Test chamber is the aquarium of 25L, and (test group has 3 groups, and use therein Bdellovibrio final concentration is respectively 10 to be divided into test group
4Pfu/mL, 10
6Pfu/mL and 10
8Pfu/mL) and control group, every group establish 3 parallel.Test water is the breed fresh water of membrane filtration, and each aquarium injects the 16L filtered water.Through the sterilization of isocyanide dichloride uric acid sodium, water temperature is 26 ℃ to aquarium before experiment.Add step and 1. prepare aquatic product Gram-positive pathogenic bacterium liquid in each aquarium, final concentration is difference 1 * 10
4Cfu/mL.Each aquarium is put each 10 of tilapia, Penaeus vannamei, Corbicula fluminea and steamed crab then.Test used tilapia and nearly weigh 140～190g, tilapia, Penaeus vannamei body are about and are 15-20cm, and the Corbicula fluminea average body is long to be 40mm, and the steamed crab specification is 30～40g.Control group is for only adding step and 1. preparing aquatic product Gram-positive pathogenic bacterium liquid but do not add Bdellovibrio, and experimental group adds microbial preparation respectively, and the Bdellovibrio final concentration of use is respectively 10
4Pfu/mL, 10
6Pfu/mL and 10
8Pfu/mL.Cultivate after 72 hours, get the 1000ml water sample, with 0.22um filter membrane (Millipore) filtering and concentrating bacterium, then at the thalline that washes with the 10ml sterilized water on the filter membrane, by 10 times of dilution method dilutions, the water sample of drawing 0.2mL at last from each dilution gradient sample is coated with flat board and detects again.Making three parallel samples detects.
The detection of clostridium perfringens is adopted and is strengthened clostridium agar (add in the reinforced clostridial medium agar 15g get final product) selectivity and cultivate, and the improvement Raymond substratum (Na that adds Succinic Acid (1%W/V) is adopted in the detection of Rhodococcus
2CO
30.1g, CaCI
26H
2O 0.01g, MnCI
24H
2O0.007g, Na
2HPO
412H
2O 35.8g, KH
2PO
413.6g, MgCl
26H
2O 0.16g, NH
4Cl2.0g, NaCl 5.0g, agar 15g, distilled water 1000mL, Succinic Acid 1%W/V, pH 6.7) select to cultivate, CLED substratum (peptone 4g, beef powder 3.0g, Tryptones 4g are adopted in the detection of corynebacterium glutamicum, lactose 10g, halfcystine 0.128g, bromothymol blue 0.02g, distilled water 1000mL, agar 15g, pH 3.0) the selectivity cultivation.Calculate viable count.
From Fig. 2, among Fig. 3 and Fig. 4 as can be known, only add the microbial preparation 72h that makes by Bdellovibrio BDS02 after, each Gram-positive pathogenic bacterium clostridium perfringens, Rhodococcus and the corynebacterium glutamicum concentration of test group is all on a declining curve in the series of trials.Act on after 72 hours, Bdellovibrio concentration is 10
4During pfu/mL, the elimination factor of clostridium perfringens, Rhodococcus and corynebacterium glutamicum is respectively 98.42%, 97.48% and 98.00%.Experimental result shows that when the microbial preparation that application is only made by Bdellovibrio BDS02 was eliminated Gram-positive pathogenic bacterium, the concentration of Bdellovibrio was big more, and action time is long more, and it is just good more to eliminate effect.
(1) preparation of the microbial preparation of elimination aquatic product Gram-positive pathogenic bacterium
Preparation process is with embodiment 1 step (2), and difference only is to add the physiological saline Bdellovibrio throw out that suspends again, and the concentration of Bdellovibrio BDS01 bacterium liquid and Bdellovibrio BDS02 bacterium liquid is respectively 10
10Pfu/mL presses bacterial count mixing in 1: 1 with Bdellovibrio BDS01 bacterium liquid and Bdellovibrio BDS02 bacterium liquid, obtains microbial preparation.
(2) microbial preparation is used to eliminate the edible preceding Gram-positive pathogenic bacterium of fishery products
1. prepare the contained Gram-positive pathogenic bacterium liquid of fishery products
Mycobacterium tuberculosis (Mycobacteri μ m tuberculosis ATCC607, purchase in Chinese common micro-organisms DSMZ), with liquid nutrient medium (after getting tender coconut juice and filtering through pad of cotton gauze, again by No. 1 filter paper filtering of Whatmann, every 80mL tender coconut juice filtrate adds 20mL horse serum and 5mL glycerine, add benzene first penicillin again to mixed solution, make that benzene first penicillin final concn reaches 100IU/mL in the substratum, mixed solution is again through 0.22um cellulose acetate membrane filtration) the shaking table cultivation, 160rpm cultivates 7d for 37 ℃.Nocardia asteroide (Nocardia asteroids ATCC19247 purchases in microbial strains preservation center, Guangdong Province) is used ISP-2 substratum (yeast extract 4g, malt extract 10g, glucose 4g, distilled water 1000mL, pH7.3) shaking table is cultivated, and 160rpm cultivates 7d for 28 ℃.Streptococcus faecium (Streptococcus faecalisATCC29212, purchase in microbial strains preservation center, Guangdong Province) and staphylococcus epidermidis (Staphylococcus epidermidis ATCC12228, purchase in microbial strains preservation center, Guangdong Province) use common nutrient broth medium (peptone 10g respectively, extractum carnis 3g, sodium-chlor 5g, distilled water 1000mL, pH7.4) shaking table is cultivated, and 160rpm cultivates 36h for 28 ℃.The cultivation of clostridium perfringens (Clostridi μ mperfringens ATCC13124), Rhodococcus (Rhodococcus erythropolis ATCC4277) and corynebacterium glutamicum (Corynebacteri μ m glutamic μ m ATCC13032) with embodiment 1 step (3) 1..The nutrient solution of above-mentioned each bacterium through 6000rpm, 4 ℃ of centrifugal 20min, is abandoned supernatant liquor respectively, and the precipitation thalline suspends again with the DNB liquid nutrient medium, and adjusting separately respectively, concentration is 10
9Cfu/mL.Above-mentioned bacterium is mixed by same ratio, obtain aquatic product Gram-positive pathogenic bacterium liquid.
2. microbial preparation is used to eliminate the edible preceding Gram-positive pathogenic bacterium of fishery products
Experimental technique with embodiment 1 step (3) 2., in the use of microorganism reagent, the final concentration of Bdellovibrio is also 2. identical with embodiment 1 step (3).Cultivate after 72 hours, get the 1000ml water sample, with 0.22um filter membrane (Millipore) filtering and concentrating bacterium, then at the thalline that washes with the 10ml sterilized water on the filter membrane, by 10 times of dilution method dilutions, the water sample of drawing 0.2mL at last from each dilution gradient sample is coated with flat board and detects again.Making three parallel samples detects.
Middlebrook 7H10 (Difco is adopted in the detection of mycobacterium tuberculosis, DISEASES OFAQUATIC ORGANISMS, Vol.61:41-51,2004) compound nutrient agar select to cultivate, Sha Shi dextrose culture-medium (the peptone 10g that contains paraxin is adopted in the detection of nocardia asteroide, glucose 40g, agar powder 11g, paraxin 0.1g, distilled water 1000mL, pH 6.0 ± 0.2) to select to cultivate, KF suis agar (peptone 10g, yeast powder 10g are adopted in the detection of streptococcus faecium, NaCl 5g, Sodium Glycerophosphate 10g, maltose 20g, lactose 1.0g, sodiumazide 0.15g, purpurum bromocresolis 0.015g, agar 20g, pH 7.2) select to cultivate, manitol salt agar flat board (extracted beef powder 1g is adopted in the detection of staphylococcus epidermidis, multivalence peptone 10g, sodium-chlor 75g, N.F,USP MANNITOL 10g, phenol red 0.025g, agar 12g, distilled water 1000mL, PH 7.4 ± 0.2) select to cultivate.The detection of clostridium perfringens is adopted and is strengthened clostridium agar selection cultivation, and the detection of Rhodococcus adopts the improvement Raymond substratum that adds Succinic Acid (1%W/V) to select cultivation, and the detection employing CLED substratum of corynebacterium glutamicum is selected to cultivate.Calculate viable count.
As Fig. 5, when not adding Bdellovibrio, the gram-positive microorganism total concn has certain increase; After adding Bdellovibrio, the gram-positive microorganism total concn all reduces.Using final concentration concentration when Bdellovibrio is 10
4Pfu/mL, 10
6Pfu/mL, 10
8During pfu/mL, microbial preparation acts on edible tilapia, Penaeus vannamei, Corbicula fluminea and steamed crab, and the elimination factor of gram-positive microorganism total concn is respectively 99.90%, 99.95% and 99.98% after 72 hours.
The microbial preparation that only contains Bdellovibrio BDS02 is used for eliminating the Gram-positive pathogenic bacterium of fishery products transportation, and detailed process is as follows:
The used tilapia of test chamber, test water and test, Penaeus vannamei, Corbicula fluminea, steamed crab specification are all with embodiment 1.Put 4 intact grids in each aquarium, in each grid, put into tilapia, Penaeus vannamei, Corbicula fluminea and steamed crab respectively, immediately it is kept flat in the aquarium after sealing the grid lid, discharge unnecessary water in the case, make its water level just cover grid.Through the sterilization of isocyanide dichloride uric acid sodium, water temperature is 26 ℃ before experiment.The continuous charge of experimental session water.3 aquariums are organized in contrast, only add the aquatic product Gram-positive pathogenic bacterium liquid that embodiment 1 step (3) 1. prepares, and final concentration is 1 * 10
4Cfu/mL.In the test group, 3 aquariums are one group, totally four groups, except pathogenic bacterium add with control group is the same, also add the microbial preparation that embodiment 1 prepares respectively, and the Bdellovibrio final concentration of use reaches 10 respectively
3Pfu/mL, 10
4Pfu/mL, 10
5Pfu/mL, 10
6Pfu/mL.Cultivate after 72 hours, get the 1000ml water sample, with 0.22um filter membrane (Millipore) filtering and concentrating bacterium, then at the thalline that washes with the 10ml sterilized water on the filter membrane, by 10 times of dilution method dilutions, the water sample of drawing 0.2mL at last from each dilution gradient sample is coated with flat board and detects again.Making three parallel samples detects.
The detection method of clostridium perfringens (Clostridi μ m perfringens ATCC13124), Rhodococcus (Rhodococcus erythropolis ATCC4277) and corynebacterium glutamicum (Corynebacteri μ mglutamic μ m ATCC13032) is carried out live bacterial count with embodiment 1.
Eliminate test effect shown in Fig. 6～8.Fig. 6～8 are respectively 10 for Bdellovibrio final concentration in the series of trials
3Pfu/mL, 10
4Pfu/mL, 10
5Pfu/mL and 10
6Clostridium perfringens, Rhodococcus and the corynebacterium glutamicum logarithmic value of concentration separately during pfu/mL.All data are the mean value of three parallel samples (aquarium).When Bdellovibrio BDS02 concentration is 10
3During pfu/mL, the elimination factor of clostridium perfringens, Rhodococcus and corynebacterium glutamicum is respectively 96.84%, 96.02% and 94.99%.
Experimental result shows that Bdellovibrio BDS02 can effectively eliminate gram-positive microorganism.The microbial preparation that only contains Bdellovibrio BDS02 is used for eliminating the Gram-positive pathogenic bacterium of fishery products transportation, and it is 10 that Bdellovibrio is used final concentration
3～10
6During pfu/mL, the Gram-positive bacteria concentration all keeps downtrending.Along with the increase of Bdellovibrio BDS02 working concentration, the elimination factor of gram-positive microorganism is big more.
The microbial preparation that will contain Bdellovibrio BDS02 and BDS01 is used for eliminating the Gram-positive pathogenic bacterium of fishery products transportation, and detailed process is as follows:
Experimental technique is with embodiment 3, and difference only is that used microbial preparation and aquatic product Gram-positive pathogenic bacterium are respectively the microbial preparation and the aquatic product Gram-positive pathogenic bacterium of embodiment 2 preparations.
After 72 hours, adopt the method for embodiment 2 to detect, calculate the viable bacteria number.
As shown in Figure 9, compare with control series, the gram-positive microorganism total concn all keeps downtrending in the series of trials.Use final concentration concentration when Bdellovibrio and be respectively 10
3Pfu/mL, 10
4Pfu/mL, 10
5Pfu/mL, 10
6During pfu/mL, microbial preparation elimination factor to the gram-positive microorganism total concn in the transportation of tilapia, Penaeus vannamei, Corbicula fluminea and steamed crab is respectively 99.80%, 99.87%, 99.92% and 99.95%.
The microbial preparation that will contain Bdellovibrio BDS02 is used for eliminating the Gram-positive pathogenic bacterium of aquaculture process, and detailed process is as follows:
Before the test, build 12 temporarily and culture ponds (0.5m * 0.5m * 0.5m=125L).The pond be will culture and test group and control group will be divided into.Test water is not filtering breed fresh water, and 100L is injected in each pond.Culture the pond and sterilize through isocyanide dichloride uric acid sodium before experiment, the duration of test water temperature is 26 ℃, and each 20 of tilapia, Penaeus vannamei, Corbicula fluminea and steamed crab are put in each pond, and the every day of each bait throwing in sooner or later once.Test used tilapia, Penaeus vannamei, Corbicula fluminea, steamed crab specification all with embodiment 1.3 ponds are control group, only add the aquatic product Gram-positive pathogenic bacterium liquid that embodiment 1 step (3) 1. prepares, and final concentration is 1 * 10
4Cfu/mL.In the test group, 3 ponds are one group, except pathogenic bacterium add with control group is the same, also add the microbial preparation that embodiment 1 prepares respectively, and the Bdellovibrio final concentration of use reaches 10 respectively
2Pfu/mL, 10
3Pfu/mL, 10
4Pfu/mL and 10
5Pfu/mL.
After 72 hours, adopt the method for embodiment 1 to detect, calculate the viable bacteria number.
Eliminate test effect shown in Figure 10～12.Figure 10～12 are respectively 10 for Bdellovibrio final concentration in the series of trials
2Pfu/mL, 10
3Pfu/mL, 10
4Pfu/mL and 10
5Clostridium perfringens, Rhodococcus and the corynebacterium glutamicum logarithmic value of concentration separately during pfu/mL, all data are the mean value in three parallel samples (breed pond).When the concentration of Bdellovibrio BDS02 is 10
2During pfu/mL, the elimination factor of clostridium perfringens, Rhodococcus and corynebacterium glutamicum is respectively 94.99%, 92.05% and 90.00%.
Experimental result shows that Bdellovibrio BDS02 can effectively eliminate gram-positive microorganism.The microbial preparation that only contains Bdellovibrio BDS02 is used for eliminating the Gram-positive pathogenic bacterium of aquaculture process, and it is 10 that Bdellovibrio is used final concentration
2～10
5During pfu/mL, the Gram-positive bacteria concentration all keeps downtrending.Along with the increase of Bdellovibrio BDS02 working concentration, the elimination factor of gram-positive microorganism is big more.
Microbial preparation is used for eliminating the Gram-positive pathogenic bacterium of aquaculture process, and detailed process is as follows:
Experimental technique is with embodiment 5, and difference only is that used microbial preparation and aquatic product Gram-positive pathogenic bacterium are respectively the microbial preparation and the aquatic product Gram-positive pathogenic bacterium of embodiment 2 preparations.
After 72 hours, adopt the method for embodiment 2 to detect, calculate the viable bacteria number.
Eliminate test effect as shown in figure 13, all data are the mean value in three parallel samples (breed pond).
As shown in Figure 13, compare with control group, the gram-positive microorganism total concn all keeps downtrending in the series of trials.It is 10 that Bdellovibrio is used final concentration
2Pfu/mL, 10
3Pfu/mL, 10
4Pfu/mL and 10
5Pfu/mL, the elimination factor of gram-positive microorganism is respectively 99.00%, 99.60%, and 99.75% and 99.87%.The concentration of the gram-positive microorganism in the aquaculture water can be obviously eliminated in 2 strain Bdellovibrio couplings, has guaranteed the healthy growth of fishery products in the freshwater aquiculture process.
Embodiment 7
With the pathogenic bacterium that microbial preparation is used for eliminating the freshwater aquiculture water body, detailed process is as follows:
Aeromonas hydrophila (Aeromonas hydrophila, purchase in Guangdong Province DSMZ of Institute of Micro-biology, numbering GIM1.172), pseudomonas putida (Pseudomonas putida, purchase in Guangdong Province DSMZ of Institute of Micro-biology, the numbering GIM1.193) and intestinal bacteria (Escherichia coli purchases in Guangdong Province DSMZ of Institute of Micro-biology, numbering GIM1.42) respectively with cultivating with common nutrient broth medium shaking table, 160rpm cultivates 12h for 28 ℃.Nutrient solution is abandoned supernatant liquor respectively at 4 ℃ of centrifugal 20min of 6000rpm, and the precipitation thalline suspends again with sterile distilled water, these precipitation thalline is mixed again, and makes its final concentration reach 1 * 105cfu/mL respectively.RimLer-shotts selective medium (L-ornithine hcl 99 0.05%w/v is adopted in the detection of Aeromonas hydrophila, L-ornithine hcl 99 0.65%w/v, maltose 0.35%w/v, Sodium Thiosulfate 0.68%w/v, L-half deamination acid hydrochloride 0.03%w/v, dibromothymolsulfonphthalein 0.003%w/v, Ferric Ammonium Citrate 0.08%w/v, Sodium desoxycholate 0.1%w/v, Vulkamycin. PA-93 0.0005%w/v, yeast extract 0.3%w/v, sodium-chlor 0.5%w/v, agar 1.35%w/v, pH7.0), NMS selective medium (KH is adopted in the detection of pseudomonas putida
2PO
40.58g/l; Na
2HPO
412H
2O 2.17g/l; NaNO
20.85g/l; K
2SO
40.17g/l; MgSO
47H
2O 0.037g/l; FeSO
47H
2O 0.012g/l; Trace element solution 2mL, pH7.0), Wdwardsiella tarda (please provide bacterial strain and source, it is the bacterial strain that has more?) detection adopt HE selectivity nutrient agar (arteries and veins peptone 12g, extractum carnis 3g, lactose 12g, sucrose 12g, salicin 2g, cholate 20g sodium-chlor 5g is dissolved in 400mL distilled water, adds 20mL first liquid (Sulfothiorine 34g, ferric ammonium citrate 4g, distilled water 100mL), 20mL second liquid (sodium deoxycholate 10g, distilled water 100mL), 0.4% bromothymol blue solution 16mL, Andrade indicator 20mL (acid fuchsin 0.5g, 1mol/L sodium hydroxide solution 16mL, distillation 100mL, azaleine is dissolved in the distilled water, adds sodium hydroxide solution.It is incomplete to fade as azaleine after a few hours, repeated hydrogenation sodium hydroxide solution 1～2mL, pH7.5 mixes with agar again), TBX (a kind of color developing culture medium) selectivity nutrient agar is adopted in colibacillary detection.
The Gram-positive pathogenic bacterium of gram negative pathogenic bacteria and step (2) preparation was pressed 1: 1, obtained the pathogenic bacterium in the freshwater aquiculture water body.
The microbial preparation of embodiment 2 preparations is eliminated the experimental technique of the pathogenic bacterium experiment in the freshwater aquiculture water body with embodiment 6, and difference only is the pathogenic bacterium of used pathogenic bacterium for this examples preparation.
After 72 hours, carry out live bacterial count.
To the effect of the elimination of Gram-positive in the aquaculture water and gram negative pathogenic bacteria test as shown in figure 14.All data are the mean value in three parallel samples (breed pond).
As Figure 14, when not adding Bdellovibrio, the pathogenic bacterium concentration in the aquaculture water has certain increase; When adding concentration is 10
2During the pfu/mL Bdellovibrio, the concentration of pathogenic bacterium is initially 1 * 10
5Cfu/mL, during to 72h, the concentration of pathogenic bacterium is less than 100cfu/mL.As can be known this 2 strain Bdellovibrio share can better controlled the concentration of Gram-positive and gram negative pathogenic bacteria in the aquaculture water.It is 10 that Bdellovibrio sends out working concentration
2～10
5Pfu/mL, Bdellovibrio concentration is big more, and the effect of eliminating pathogenic bacterium is just good more.In the freshwater aquiculture water body, add described microbial preparation, can prevent various diseases comprehensively, guaranteed the high-quality of fishery products simultaneously again.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
SEQUENCE?LISTING
＜110〉South China Science ﹠ Engineering University
＜120〉a kind of Bdellovibrio bacteriovorus bacterial strain and application thereof of eliminating aquatic product Gram-positive pathogenic bacterium
<130>8
<160>1
<170>PatentIn?version?3.2
<210>1
<211>1835
<212>DNA
＜213〉Bdellovibrio (Bdellovibrio sp.)
<400>1
caggcctaac?acatgcaagt?cgaacggggt?agcaatacct?agtggcgcac?gggtgagtaa 60
cgcgtggata?atctgccttg?gagtggggga?taactagtcg?aaagattagc?taataccgca 120
taagaccaca?ggagctgcgg?ctctaggggt?caaaggtttt?tcgttccaag?atgagtccgc 180
gtaagattag?ctagttggtg?aggtaatggc?tcaccaaggc?gacgatcttt?aactggtctg 240
agaggatgat?cagtcacact?ggaactgaga?cacggtccag?actcctacgg?gaggcagcag 300
tagggaatat?tgcacaatgg?aggaaactct?gatgcagcga?cgccgcgtga?gtgatgaagg 360
ccttcgggtc?gtaaagctct?gtcgcagggg?aataacacaa?tgaatgtacc?ctgtaagaaa 420
ggatcggcta?acttcgtgcc?agcagccgcg?gtaagacgag?ggatcctagc?gttgttcgga 480
attattgggc?gtaaagcgga?tgtaggtggc?tttgtaagtc?agatgtgaaa?gcccagggct 540
caaccctgga?agtgcatttg?atactgcgaa?gcttgagtgt?cggagaggtt?actagaattg 600
ttggtgtagt?ggtgaaatac?gtagatatca?acaggaatac?cggaggcgaa?ggcgggtaac 660
tggccgaaca?ctgacactga?gatccgaaag?cgtggggatc?aaacaggatt?agataccctg 720
gtagtccacg?ccgtaaacga?tggatacttg?ttgttagagg?tattgacccc?ttcagtgacg 780
aagctaacgc?gttaagtatc?ccgcctgggg?agtacggtcg?caagattaaa?actcaaagaa 840
attgacgggg?gcccgcacaa?gcggtggagc?atgtggttta?attcgatgca?acgcgaagaa 900
ccttacctag?gcttgacatg?tactggaaga?ttggcagaaa?tgtcgtcgcc?cgcaagggtc 960
ggtacacagg?tgctgcatgg?ctgtcgtcag?ctcgtgtcgt?gagatgttgg?gttaagtccc 1020
gcaacgagcg?caacccctgc?atttagttgc?cagcattcag?ttcggcactc?tagatggact 1080
gccggtgtta?aaccggagga?aggtggggat?gacgtcaagt?cctcatggcc?cttatgccta 1140
gggctacaca?cgtgctacaa?tggtagtcac?agagcgaagc?taagccgcga?ggtagagcaa 1200
atcgcttaaa?agctatctaa?gttcagattg?atctctgcaa?ctcgagatca?tgaagttgga 1260
atcgctagta?atcgcggaac?agaatgccgc?ggtgaatacg?ttcccgggcc?ttgtacacac 1320
cgcccgtcac?accatgaaag?tcggctgtac?cagaagtcgc?tgcgctaacc?gtaaggaggc 1380
aggcgcccaa?ggtatggtcg?atgattgggg?tgaagtcgta?acaagggagc?cgtaggggaa 1440
cctgcggctg?gatcacctcc?tttctaaggt?ttatccggtc?aatcttcatc?aagacttgtt 1500
cttgataagt?taaaatgacc?caatcttagg?tcaacttact?cttcccgagt?aagtgagtcc 1560
caaaaatcta?tctagctgtt?tagttttgag?agagtgaagc?ctaacgggcc?tgtagctcag 1620
ttggttacag?cacacgcttg?ataagcgtgg?ggtcggaagt?tcgagtcttc?ccaggcccac 1680
caagttctac?tgtactggaa?tgcggtgtaa?gttagagttt?tgctgaacgg?ttttcgttct 1740
ttgacatttg?aatagattga?tttagttgat?ttttagcgag?gttagttcca?ttcttttaag 1800
ctacaaaggg?cttacggtgg?atgccctggc?agtca 1835
Claims (9)
1, a kind of Bdellovibrio bacteriovorus bacterial strain of eliminating aquatic product Gram-positive pathogenic bacterium, it is characterized in that, described Bdellovibrio bacteriovorus bacterial strain is Bdellovibrio (Bdellovibrio sp.) BDS02, is preserved in Chinese typical culture collection center on August 7th, 2009, and deposit number is CCTCCNO:M209170.
2, a kind of microbial preparation of eliminating aquatic product Gram-positive pathogenic bacterium is characterized in that, described microbial preparation contains the described Bdellovibrio BDS02 of claim 1.
3, microbial preparation according to claim 2 is characterized in that: described microbial preparation contains Bdellovibrio (Bdellovibrio sp.) BDS01.
4, microbial preparation according to claim 3 is characterized in that: Bdellovibrio BDS02 and Bdellovibrio BDS01 press bacterial count mixing in 1: 1 in the described microbial preparation.
5, the application of each described microbial preparation of claim 2～4 is characterized in that: described microbial preparation is used to eliminate aquatic product Gram-positive pathogenic bacterium.
6, according to the application of the described microbial preparation of claim 5, it is characterized in that: described fishery products are freshwater product.
7, application according to the described microbial preparation of claim 6 is characterized in that: described aquatic product Gram-positive pathogenic bacterium is mycobacterium tuberculosis (Mycobacteri μ m tuberculosis), nocardia asteroide (Nocardia asteroides), Rhodococcus (Rhodococcus erythropolis), streptococcus faecium (Streptococcus faecalis), staphylococcus epidermidis (Staphylococcusepidermidis), clostridium perfringens (Clostridi μ m perfringens) and corynebacterium glutamicum (Corynebacteri μ m glutamic μ m).
8, according to the application of the described microbial preparation of claim 6, it is characterized in that:
Described microbial preparation is applied to eliminate Gram-positive pathogenic bacterium edible or that the preceding fishery products of processing carry;
Described microbial preparation is applied to control the Gram-positive pathogenic bacterium in the fishery products transportation;
Described microbial preparation is applied to control the Gram-positive pathogenic bacterium in the aquaculture water body.
9, the application of described microbial preparation according to Claim 8 is characterized in that:
Described microbial preparation is applied to eliminate Gram-positive pathogenic bacterium edible or that the preceding fishery products of processing carry, and the Bdellovibrio final concentration of use is 10
4～10
8Pfu/mL;
Described microbial preparation is applied to control the Gram-positive pathogenic bacterium in the fishery products transportation, and the Bdellovibrio final concentration of use is 10
3～10
6Cfu/mL;
Described microbial preparation is applied to control the Gram-positive pathogenic bacterium in the aquaculture water body, and the Bdellovibrio final concentration of use is 10
2～10
5Pfu/mL.
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| CN106085913B (en) * | 2016-07-01 | 2019-02-22 | 马鞍山奥柯环保科技发展有限公司 | A kind of complex microorganism preparations and preparation method thereof for sewage treatment |
| CN115474565A (en) * | 2021-05-31 | 2022-12-16 | 华南理工大学 | Application of bdellovibrio bacteriovorus mutant strain in litopenaeus vannamei culture |
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