CN101356927A - Use of Bdellovibrio in eliminating pathogenicity vibrio in marine products and breeding water body thereof - Google Patents
Use of Bdellovibrio in eliminating pathogenicity vibrio in marine products and breeding water body thereof Download PDFInfo
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Abstract
The invention discloses an application of bdellovibrios in removing pathogenic vibrio in marine food products and the culture water. The bdellovibrio concentrated solution is added into the marine food products and/or the culture water so as to lead the concentration of the bdellovibrio to reach at least 10<2>pfu /ml. When the invention is applied before eating the marine food products, in the transportation process and in the culture water, the concentration range of bdellovibrio is respectively 10<4>-10<12> pfu /ml, 10<3>-10<11> pfu /ml, and 10<2>-10<6 > pfu /ml. More than 90% pathogenic vibrio carried by marine food products before eating and/or in the transportation process is cleared by adopting a biological method, and the pathogenic vibrio in the culture water can also be controlled within10cfu/ml. The invention is suitable for the pretreatment process before being eaten or processed, the transportation process and the culture process of marine food products, especially facilitating the reduction or elimination of chemical medicine residues such as antibiotics, thus fundamentally avoiding the occurrence of food poisoning of the marine food products.
Description
Technical field
The invention belongs to biological technical field, relate to bacterium and application thereof, specifically be meant the application of Bdellovibrio kinds of pathogenic vibrio in eliminating marine product and breeding water body thereof, be specially adapted to eliminate the edible or processing of marine product before, the common food poisoning vibrios in transportation and the breeding water body thereof.
Background technology
Kinds of pathogenic vibrio is to cause the human marine product common pathogenic bacteria the most of poisoning by food, in often being present in the body surface of marine product shellfish, fish and the body, in food poisoning, occupy sizable ratio, mainly cause acute gastroenteritis, particularly vibrio parahaemolytious (Vibrio parahaemolyticus) etc., it is pathogenic strong, morbidity is anxious, propagate soon, the source of infection is many, popular longer duration in the crowd.
There are some researches show have 9 kinds of vibrio frequenses can cause human foods approximately and poison.Vibrio parahaemolytious (V.parahaemolyticus), Vibrio flurialis (V.fluvialis) etc. can cause gastroenteritis, and Vibrio vulnificus (V.vulnificus), vibrio alginolyticus (V.alginolyticus), vibrio mimicus (V.mimicus), Vibrio cincinnatiensis (V.cincinnatiensis) etc. cause that wound infection and septicemia enteron aisle infect outward.In recent years, be obvious ascendant trend by the caused intestines problem of these kinds of pathogenic vibrio, infectious disease and food poisoning generation scale and crowd's exposure scale, all above salmonella food poisoning, leap to the first place of food posioning incident, become the key factor that influences food security, cause widely and pay close attention to.For this reason, all there is quite strict regulation countries in the world to the import and export of marine product and the vibrios content in the food hygiene quarantine.How accurate detection goes out vibrios in the food, how to remove vibrios and then prevention are become Recent study by its food source epidemic disease that causes focus.Nowadays the ripe vibrios detection method of an existing cover, how this eliminates it but detection finds that vibrios is arranged, then still there is not the effective removing method of a cover, only scholar of the U.S. once studied application Bdellovibrio pathogeny bacterium in eliminating food, illustrated the using value of Bdellovibrio aspect food sanitation safe.Along with development of biology, adopt the suitable biological cleaning factor to remove food-borne pathogenic vibrios in the marine product, will be that following effectively prevention is by the trend of intestines problem, infectious disease and food poisoning that it caused.
And marine product to be its food source sexuality dye of paramount importance communication media, the edible polluted bacteria of people Duo Yin and cause food poisoning without the marine product of well processed.The kinds of pathogenic vibrio that marine product is entrained except causing the human foods poisoning, also can cause this disease problem during culturing of marine product.In order to control fish vibrios disease, culture producer and have to use antibiotic, so cause the residual of fishing medicine again.Exceeding standard of the residual and vibrios of medicine brought obstacle for the outlet of China's aquatic products.
If can marine product be eaten or process preceding in addition culturing during carry out the elimination work of kinds of pathogenic vibrio earlier; then can reduce or eliminate the probability of food poisoning on the one hand; reduce or eliminate the possibility of medicament residue; the safety coefficient of improving the quality of products; protection consumer's health; the processing that also can be marine product on the other hand provides the facility in high-quality raw material and the processing, for foreign exchange earning provides strong guarantee.So can avoid the annual numerous marine product food poisoning that is taken place, can be international trade smoothly again and escort.
Handle the work of marine product, at home, mainly concentrate in the research and development of detection method and technology at present, abroad,, also just begun to use various physics or chemical means to reduce or eliminate the entrained germ of marine product except detection method and Study on Technology.
Removing for kinds of pathogenic vibrio, all still adopt the method for physics or chemicals at present both at home and abroad, but the method for physics or chemicals all exists than significant disadvantages, but also can only be applied to the process of food, be difficult to expand to the breeding process of marine product, and the biological method of research is removed kinds of pathogenic vibrio in edible marine product and the breeding water body thereof, just can provide a kind of safe and effective new technique for the overall process of " from culturing dining table ".
Summary of the invention
The objective of the invention is to overcome the deficiency that above-mentioned prior art exists, be suitable for the biological technology for eliminating of marine product kinds of pathogenic vibrio, the application of a kind of Bdellovibrio kinds of pathogenic vibrio in eliminating marine product and breeding water body thereof is provided by exploitation.
In order to achieve the above object, main technical schemes of the present invention is as follows:
The application of Bdellovibrio kinds of pathogenic vibrio in eliminating marine product and breeding water body thereof, application method comprises following concrete steps and condition thereof:
Step 1: the separation and purification of Bdellovibrio
Seawater or the bed mud of gathering carried out preliminary treatment, the vibrio parahaemolytious of inoculation propagation Bdellovibrio, fall double-layer plate, plaque appears on the double-layer plate that contains the host bacterium, single spot that picking constantly enlarges, again by micro-biological process liquid enrichment culture, the double-layer plate check, single spot of constantly enlarging of picking once more, and repeated several times, to single plaque formation shape that goes down to posterity, size, the plaque that transparency is all consistent is the pure bacterial strain of a strain Bdellovibrio, by the big subtotal of single spot in the same time, separablely go out the pure bacterial strain of 2 strain Bdellovibrios, described Bdellovibrio is being positioned at the Chinese typical culture collection center preservation of Chinese Wuhan City Wuhan University on January 13rd, 2008, and Bdellovibrio sp.BDJ01 deposit number is that CCTCC NO:M 208011 and Bdellovibrio sp.BDJ02 deposit number are CCTCC NO:M 208012.
Step 2: the preparation of Bdellovibrio concentrate
2 strain Bdellovibrios are carried out fermented and cultured respectively, and making concentration respectively is 10
11～10
13The Bdellovibrio concentrate of pfu/ml, stand-by;
Step 3: the application of Bdellovibrio kinds of pathogenic vibrio before the elimination marine product is edible and in the breeding water body
In the edible marine product that has polluted kinds of pathogenic vibrio and/or its breeding water body, add the Bdellovibrio concentrate of step 2 preparation, make the concentration of Bdellovibrio reach 10 at least
2Pfu/ml.
Described application method adopts 2 strain Bdellovibrios to mix and uses or independent respectively the use, and it is better that 2 strain Bdellovibrios mix result of use.
Described application method is specially adapted to eliminate kinds of pathogenic vibrio edible or that the preceding marine product of processing carries, and the Bdellovibrio optimum concentration range is 10 during application
4～10
12Pfu/ml.
Described application method also is applicable to the kinds of pathogenic vibrio of eliminating in the marine product transportation, and the Bdellovibrio optimum concentration range is 10 during application
3～10
11Pfu/ml.
Described application method also is applicable to the kinds of pathogenic vibrio of eliminating in the marine product breeding water body, and the Bdellovibrio optimum concentration range is 10 during application
2-10
6Pfu/ml.
Described kinds of pathogenic vibrio is meant vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios, Vibrio metchnikovii, Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus and Vibrio flurialis.
The present invention compared with prior art has following advantage:
1, Bdellovibrio of the present invention is remarkable at the effect of eliminating the kinds of pathogenic vibrio that marine product carries
The effect of Bdellovibrio of the present invention kinds of pathogenic vibrio before eliminating the edible or processing of marine product, in transportation and the breeding water body thereof is remarkable, is example with oyster, Penaeus Vannmei and perch, and extremely shown in Figure 9 as Fig. 1, kinds of pathogenic vibrio all has higher elimination factor.Particularly 2 strain Bdellovibrios mix when using, and elimination factor all reaches more than 99%.
2, good with kinds of pathogenic vibrio safety in the Bdellovibrio elimination marine product before edible
The method that Bdellovibrio is eliminated kinds of pathogenic vibrio in the marine product is biological method, Bdellovibrio can be infected, the characteristic of cracking host bacteria makes it to be suitable as the biological cleaning factor of restraining or removing pathogenic bacteria 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 consumer eats marine product raw, protection consumer's health also provides safeguard for the green processing production of marine product.
3, apply the present invention in the marine product transportation, convenient, safe and effective
Marine product adopts Bdellovibrio to eliminate pathogenic bacteria in transportation and can prevent water quality deterioration, improve survival rate, is particularly conducive to and reduces or eliminates chemicals such as residues of antibiotics, guarantees the high-quality of marine product.
4, be fit to be applied to the breeding process of marine product
During culturing, promptly carry out the elimination work of kinds of pathogenic vibrio, be particularly conducive to and reduce or eliminate chemicals such as residues of antibiotics, guarantee the high-quality of marine product, fundamentally avoid the generation of marine product food poisoning, for foreign exchange earning provides strong guarantee.Bdellovibrio is to effect such as Fig. 8, shown in Figure 9 of the elimination test of kinds of pathogenic vibrio in the marine product breeding water body, and kinds of pathogenic vibrio also can be controlled in the 10cfu/ml in the breeding water body.
5, the present invention provides a kind of new method for eliminating the entrained food-borne pathogenic vibrios of marine product, and be fit to be applied to that marine product is edible, preliminary treatment, transportation and breed overall process thereof before the processing, be particularly conducive to and reduce or eliminate chemicals such as residues of antibiotics, guarantee the high-quality of marine product, fundamentally avoid the generation of marine product food poisoning, for foreign exchange earning provides strong guarantee.
Description of drawings
When Fig. 1 handles edible preceding oyster, Penaeus Vannmei and perch for using 1 strain Bdellovibrio (BDJ01), vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios, Vibrio metchnikovii concentration logarithm value-time diagram;
Fig. 2 is that 2 strain Bdellovibrios are share when handling edible preceding oyster, Penaeus Vannmei and perch vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios, Vibrio metchnikovii concentration logarithm value-time diagram;
Fig. 3 is that 2 strain Bdellovibrios are share when handling edible preceding oyster, Penaeus Vannmei and perch Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis concentration logarithm value-time diagram;
When Fig. 4 handles oyster in the transportation, Penaeus Vannmei and perch for using 1 strain Bdellovibrio (BDJ02), Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis concentration logarithm value-time diagram;
Fig. 5 is that 2 strain Bdellovibrios are share when handling oyster in the transportation, Penaeus Vannmei and perch vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios, Vibrio metchnikovii concentration logarithm value-time diagram;
Fig. 6 is that 2 strain Bdellovibrios are share when handling oyster in the transportation, Penaeus Vannmei and perch Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis concentration logarithm value-time diagram;
Fig. 7 is 1 a strain Bdellovibrio (BDJ01) when being used for oyster, Penaeus Vannmei and perch breeding water body, vibrio alginolyticus, vibrio parahaemolytious, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis, vibrio mimicus concentration logarithm value-time diagram;
Fig. 8 is that 2 strain Bdellovibrios are share when oyster, Penaeus Vannmei and perch breeding water body vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios, Vibrio metchnikovii concentration logarithm value-time diagram;
Fig. 9 is that 2 strain Bdellovibrios are share when oyster, Penaeus Vannmei and perch breeding water body Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis concentration logarithm value-time diagram.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiment is not limited in this, kinds of pathogenic vibrio such as described vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios, Vibrio metchnikovii, Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus and Vibrio flurialis occur all effective simultaneously, but be that the expression of experimental result and chart data is clear, nine kinds of common kinds of pathogenic vibrio adopt the expression of any several combinations.
Embodiment 1
The application of the Bdellovibrio kinds of pathogenic vibrio that marine product carries before elimination is edible, marine product is an example with oyster, Penaeus Vannmei and perch, application method comprises following concrete steps and condition thereof:
Step 1: the separation and purification of Bdellovibrio
Seawater or the bed mud of gathering carried out preliminary treatment, the vibrio parahaemolytious of inoculation propagation Bdellovibrio, fall double-layer plate, plaque appears on the double-layer plate that contains the host bacterium, single spot that picking constantly enlarges, again by micro-biological process liquid enrichment culture, double-layer plate check, single spot of constantly enlarging of picking once more, and repeated several times, going down to posterity to single plaque, to form all consistent plaque of shape, size, transparency be the pure bacterial strain of a strain Bdellovibrio, by the big subtotal of single spot in the same time, isolate the pure bacterial strain of Bdellovibrio.
Separablely go out the pure bacterial strain of 2 strain Bdellovibrios, and to name it be BDJ01 bacterial strain and BDJ02 bacterial strain; Carry out the pcr amplification reaction and the gene sequencing of specificity 16SrRNA gene, obtain its partial sequence and be respectively 811bp and 774bp, its GenBank number of landing (accession number) is respectively EF011103 and EF094471:
Bacterial strain BDJ01
LOCUS?BDHSH0616S?811bp?GenBank?accession?number：EF011103ORIGIN?5’
1 CAAGTCGAAC?CGAGAAANGC?CNTTNCGGGG?TGGAGTACAGTGGCGCACGG?GTGAATAACG
61 CGTAGGTGAC?GTGCCTTTTA?GTGGGGGACA?ACATCGGGAAACCGGTGCTA?ATACCGCATA
121?AGTTAAGCGA?CATTGAAAAA?GCTTAAGAAA?GTGGGCTTCGGCTCACGCTG?AAAGATCGGC
181?CTGCGTATCA?TTAGCTTGTT?GGTGGGGTAA?CGGCCTACCAAGGCTACGAT?GATTAACTGG
241?TCTGAGAGGA?TGATCAGTCA?CACTGGAACT?GAGACACGGTCCAGACTCCT?ACGGGAGGCA
301?GCAGTAGGGA?ATATTGCGCA?ATGGGGGAAA?CCCTGACGCAGCAATGCCAC?GTGAGTGAGG
361?AAGGCCCTTG?GGTTGTAAAG?CTCTGTCCTA?TGGGAAGAACTGCATTACGG?TTAATACCCG
421?TAGTGTTTGA?CGGTACCATA?GAAGAAAGCA?CCGGCTAACTCCGTGCCAGC?AGCCGCGGTA
481?ATACGGAGGG?TGCAAGCGTT?GTTCGGATTT?ACTGGGCGTAAAGCGCGCGC?AGGCGGATTG
541?GCAAGTCAGA?TGTGAAATCT?CGGGGCTCAA?CCCCGAAACTGCGTCTGAAA?CTATCAGTCT
601?AGAGTCTCAT?AGGGGGCAGG?GGAATTTCAC?GTGTAGGGGTAAAATCCGTA?GAGATGTGAA
661?GGAACACCCG?TGGCGAAGGC?GCCTGCCTGG?ATGAGCACTGACGCTGAGGC?GCGAAAGCGT
721?GGGGAGCAAA?CAGGATTAGA?TACCCTGGTA?GTCCACGCCGTAAACGATGA?GTACTAGCCC
781?TTGGAGTATG?CCCCCNCCCC?CNGGNACGAA?A
Bacterial strain BDJ02
LOCUS?BDH1216S?774bp?GenBank?accession?number：EF094471ORIGIN?5’
1 gtggcgcacg?nntnantaac?gcgtaggtga?cgtgcctttt?agtgggggac?aacatcggga
61 aaccggtgct?aataccgcat?aagttaagcg?acattgaaaa?agcttaagaa?agtgggcttc
121?ggctcacgct?gaaagatcgg?cctgcgtatc?attagcttgt?tggtggggta?acggcctacc
181?aaggctacga?tgattaactg?gtctgagagg?atgatcagtc?acactggaac?tgagacacgg
241?tccagactcc?tacgggaggc?agcagtaggg?aatattgcgc?aatgggggaa?accctgacgc
301?agcaatgcca?cgtgagtgag?gaaggccctt?gggttgtaaa?gctctgtcct?atgggaagaa
361?ctgcattacg?gttaataccc?gtagtgtttg?acggtaccat?agaagaaagc?accggctaac
421?tccgtgccag?cagccgcggt?aatacggagg?gtgcaagcgt?tgttcggatt?tactgggcgt
481?aaagcgcgcg?caggcggatt?ggcaagtcag?atgtgaaatc?tcggggctca?accccgaaac
541?tgcgtctgaa?actatcagtc?tagagtctca?tagggggcag?gggaatttca?cgtgtagggg
601?taaaatccgt?agagatgtga?aggaacaccc?gtggcgaagg?cgcctgcctg?gatgagcact
661?gacgctgagg?cgcgaaagcg?tggggagcaa?acaggattag?ataccctggt?agtccacgcc
721?gtaaacgatg?agtactagcc?cttggaggta?ttgccccccn?tccagtgacc?gaaa
Described Bdellovibrio is being positioned at the Chinese typical culture collection center preservation of Chinese Wuhan City Wuhan University on January 13rd, 2008, and Bdellovibrio sp.BDJ01 deposit number is that CCTCC NO:M 208011 and Bdellovibriosp.BDJ02 deposit number are CCTCC NO:M 208012.
Step 2: the preparation of Bdellovibrio concentrate
Picking list spot Bdellovibrio from the double-layer plate and joins in the erlenmeyer flask that contains the DNB medium after the vibrio parahaemolytious concentrate mixes, and constant temperature shaking table 250rpm cultivates 24h for 28 ℃.Culture is got supernatant through 5000rpm, 4 ℃ of centrifugal 20min, and centrifugal 20min under 16000rpm, the 4 ℃ of conditions abandons supernatant again, adds an amount of DNB liquid nutrient medium Bdellovibrio sediment that suspends again, and making concentration respectively is 10
11～10
13The Bdellovibrio concentrate of pfu/ml.
Step 3: Bdellovibrio is tested the elimination of the kinds of pathogenic vibrio that edible marine product carries
(1) vibrio parahaemolytious (Vibrio parahaemolyticus), vibrio alginolyticus (Vibrio alginolyticus), vibrio mimicus (Vibrio mimicus), Fu Nisishi vibrios (Vibrio furnissii), Vibrio metchnikovii (Vibriometschnikovii), Vibrio cincinnatiensis (Vibrio cincinnatiensis), Kazakhstan Vickers (Vibrio harveyi), the preparation of Vibrio vulnificus (Vibrio vulnificus) and Vibrio flurialis (Vibrio fluvialis)
Cultivate with basic peptone water medium shaking table, 160rpm, 28 ℃ cultivate 12～16h and breed above-mentioned vibrios respectively, and culture fluid is abandoned supernatant through 5000rpm, 4 ℃ of centrifugal 20min, and precipitation thalline salinity is that 15 ‰ aseptic seawater suspends again, and making its concentration is 10
9Cfu/ml.
(2) the elimination test of Bdellovibrio kinds of pathogenic vibrio that marine product before edible is carried
Test chamber is the aquarium of 25L, is divided into test and contrasts two big series.Test water is a seawater, and each aquarium injects the 16L seawater.Testing used oyster average body length is 3.9 * 1.9cm, heavy 10.21g; The Penaeus Vannmei average body is long to be 12.1cm, heavy 12.5g; The perch average body is long to be 24cm, heavy 400g.Each aquarium is put each 10 of oyster, Penaeus Vannmei and perches.The duration of test water temperature is 27 ℃.3 aquariums are serial in contrast, only add kinds of pathogenic vibrio, do not add Bdellovibrio.In the experimental series, 3 aquariums are one group, add a certain amount of Bdellovibrio concentrate, make it reach a predetermined Bdellovibrio final concentration.So have the Bdellovibrio final concentration of 5 different gradients, that is: 10
4Pfu/ml, 10
6Pfu/ml, 10
8Pfu/ml, 10
10Pfu/ml and 10
12Pfu/ml.
During test, in corresponding aquarium, add vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios, the Vibrio metchnikovii that above-mentioned steps three (1) prepares, make its concentration reach 1.0 * 10 respectively
4Cfu/ml, then oyster, Penaeus Vannmei and perch are put into wherein adapt to after, in the experimental series aquarium, add the Bdellovibrio concentrates of different amounts again, make them reach different Bdellovibrio final concentrations.The specific detection of vibrios all adopts real-time fluorescence quantitative PCR to detect.Use 1 strain Bdellovibrio (BDJ01) as shown in Figure 1 to the elimination test effect of vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios, Vibrio metchnikovii in the edible marine product.Among Fig. 1-and ■-be Fu Nisishi vibrios concentration logarithm value curve in the experimental series,-◆-be the concentration logarithm value curve of Vibrio metchnikovii in the experimental series,-▲-is the concentration logarithm value curve of vibrio mimicus in the experimental series,-●-be vibrio parahaemolytious concentration logarithm value curve in the experimental series ,-*-be the concentration logarithm value curve of vibrio alginolyticus in the experimental series.Be minimum Bdellovibrio final concentration group (10 in the experimental series among Fig. 1
4Pfu/ml) the vibrios concentration logarithm value curve in.Because the elimination better effects if of other high concentration Bdellovibrio group is for for simplicity, so expression no longer in the drawings.All data are the mean value of three parallel samples (aquarium).
As can be known from Fig. 1, the initial concentration of kinds of pathogenic vibrio is 1.0 * 10
4Cfu/ml.After beginning to add Bdellovibrio, minimum Bdellovibrio final concentration group (10 in the experimental series
4Pfu/ml) each vibrios concentration is all on a declining curve.Experimental result shows, use 1 strain Bdellovibrio (BDJ01) when above vibrios is eliminated, the elimination factor of vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios, Vibrio metchnikovii is respectively 98.00%, 99.21%, 96.84%, 92.06%, 94.99%.Single as seen from the figure can the elimination to a certain extent with 1 strain Bdellovibrio (BDJ01) eats the kinds of pathogenic vibrio that marine product carries.
2 strain Bdellovibrios are share and eliminate vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios and the Vibrio metchnikovii that edible preceding oyster, Penaeus Vannmei and perch are carried, and other steps are with embodiment 1.Among Fig. 2-and ■-be Fu Nisishi vibrios concentration logarithm value curve in the experimental series,-◆-be the concentration logarithm value curve of Vibrio metchnikovii in the experimental series,-▲-is the concentration logarithm value curve of vibrio mimicus in the experimental series,-●-be vibrio parahaemolytious concentration logarithm value curve in the experimental series ,-*-be the concentration logarithm value curve of vibrio alginolyticus in the experimental series.Be minimum Bdellovibrio final concentration group (10 in the experimental series among Fig. 2
4Pfu/ml) the vibrios concentration logarithm value curve in.Because the elimination better effects if of other high concentration Bdellovibrio group is for for simplicity, so expression no longer in the drawings.All data are the mean value of three parallel samples (aquarium).
As can be known from Fig. 2, the initial concentration of kinds of pathogenic vibrio is 1.0 * 10
4Cfu/ml.After beginning to add Bdellovibrio, each vibrios concentration is all on a declining curve in the experimental series.Experimental result shows, use 2 strain Bdellovibrios above kinds of pathogenic vibrio is eliminated, the elimination factor of vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios, Vibrio metchnikovii is respectively 99.98%, 99.99%, 99.96%, 99.89%, 99.94%.Present embodiment and embodiment 1 more as can be known, the elimination effect that 2 strain Bdellovibrios are used kinds of pathogenic vibrio with obviously is better than single with 1 strain Bdellovibrio (BDJ01).
Embodiment 3
2 strain Bdellovibrios are share and eliminate Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, the Vibrio flurialis that edible preceding oyster, Penaeus Vannmei and perch are carried, and other steps are with embodiment 1.Among Fig. 3-and ■-be Vibrio harveyi concentration logarithm value curve in the experimental series,-◆-be the concentration logarithm value curve of Vibrio flurialis in the experimental series,-▲-is the concentration logarithm value curve of Vibrio cincinnatiensis in the experimental series ,-●-be Vibrio vulnificus concentration logarithm value curve in the experimental series.Be minimum Bdellovibrio final concentration group (10 in the experimental series among Fig. 3
4Pfu/ml) the vibrios concentration logarithm value curve in.Because the elimination better effects if of other high concentration Bdellovibrio group is for for simplicity, so expression no longer in the drawings.All data are the mean value of three parallel samples (aquarium).
As can be known from Fig. 3, the initial concentration of kinds of pathogenic vibrio is 1.0 * 10
4Cfu/ml.After beginning to add Bdellovibrio, each vibrios concentration is all on a declining curve in the experimental series.Experimental result shows, uses 2 strain Bdellovibrios above vibrios is eliminated, and the elimination factor of Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis is respectively 99.98%, 99.94%, 99.99%, 99.96%.
Adopt the method identical, use Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis in 1 strain Bdellovibrio (BDJ02) elimination marine product oyster, Penaeus Vannmei and the perch transportation with embodiment 1.In the experimental series, 3 aquariums are one group, add a certain amount of Bdellovibrio concentrate, make it reach a predetermined Bdellovibrio final concentration, so have the Bdellovibrio final concentration of 5 different gradients, that is: 10
3Pfu/ml, 10
6Pfu/ml, 10
8Pfu/ml, 10
10Pfu/ml and 10
11Pfu/ml.Eliminate test effect as shown in Figure 4, among Fig. 4-and ■-be Vibrio harveyi concentration logarithm value curve in the experimental series,-◆-be the concentration logarithm value curve of Vibrio flurialis in the experimental series,-▲-is the concentration logarithm value curve of Vibrio cincinnatiensis in the experimental series ,-●-be Vibrio vulnificus concentration logarithm value curve in the experimental series.Be minimum Bdellovibrio final concentration group (10 in the experimental series among Fig. 4
3Pfu/ml) the vibrios concentration logarithm value curve in.Because the elimination better effects if of other high concentration Bdellovibrio group is for for simplicity, so expression no longer in the drawings.All data are the mean value of three parallel samples (aquarium).
Experimental result shows that the initial concentration of vibrios is 1 * 10
4Cfu/ml.Vibrios concentration all keeps downward trend in the experimental series.The elimination factor of Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis is respectively 96.02%, 87.41%, 97.49%, 94.99%.It is effective to the kinds of pathogenic vibrio of eliminating in the marine product transportation to use 1 strain Bdellovibrio (BDJ02) as shown in Figure 4, but elimination factor is lower slightly.
Embodiment 5
Adopt the method identical with embodiment 1, use 2 strain Bdellovibrios and eliminate vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios and Vibrio metchnikovii in marine product oyster, Penaeus Vannmei and the perch transportation, other implementation condition is identical with embodiment 4, add the Bdellovibrio concentrate of variable concentrations, make it reach a predetermined Bdellovibrio final concentration.Eliminate test effect as shown in Figure 5, among Fig. 5-and ■-be Fu Nisishi vibrios concentration logarithm value curve in the experimental series,-◆-be the concentration logarithm value curve of Vibrio metchnikovii in the experimental series,-▲-is the concentration logarithm value curve of vibrio mimicus in the experimental series,-●-be vibrio parahaemolytious concentration logarithm value curve in the experimental series ,-*-be the concentration logarithm value curve of vibrio alginolyticus in the experimental series.Be minimum Bdellovibrio final concentration group (10 in the experimental series among Fig. 5
3Pfu/ml) the vibrios concentration logarithm value curve in.Because the elimination better effects if of other high concentration Bdellovibrio group is for for simplicity, so expression no longer in the drawings.All data are the mean value of three parallel samples (aquarium).
Experimental result shows that the initial concentration of kinds of pathogenic vibrio is 1 * 10
4Cfu/ml.Vibrios concentration is consistent in the experimental series keeps downward trend.Relatively initial concentration of each vibrios and the final concentration of 7h all have higher elimination factor as can be known, and the elimination factor of vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios and Vibrio metchnikovii is respectively 99.97%, 99.99%, 99.94%, 99.80%, 99.90%.Use 2 strain Bdellovibrios as shown in Figure 5 and can effectively remove kinds of pathogenic vibrio in the marine product transportation basically.
Adopt the method identical, use Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis in 2 strain Bdellovibrios elimination marine product oyster, Penaeus Vannmei and the perch transportation with embodiment 1.Other implementation condition adds the Bdellovibrio concentrate of variable concentrations with embodiment 4, makes it reach a predetermined Bdellovibrio final concentration.Eliminate test effect as shown in Figure 6, among Fig. 6-and ■-be Vibrio harveyi concentration logarithm value curve in the experimental series,-◆-be the concentration logarithm value curve of Vibrio flurialis in the experimental series,-▲-is the concentration logarithm value curve of Vibrio cincinnatiensis in the experimental series ,-●-be Vibrio vulnificus concentration logarithm value curve in the experimental series.Be minimum Bdellovibrio final concentration group (10 in the experimental series among Fig. 6
3Pfu/ml) the vibrios concentration logarithm value curve in.Because the elimination better effects if of other high concentration Bdellovibrio group is for for simplicity, so expression no longer in the drawings.All data are the mean value of three parallel samples (aquarium).
Experimental result shows that the initial concentration of vibrios is 1 * 10
4Cfu/ml.Vibrios concentration all keeps downward trend in the experimental series.The elimination factor of Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis is respectively 99.98%, 99.92%, 99.99%, 99.95%.Shown in Figure 6ly use 2 strain Bdellovibrios and can effectively remove kinds of pathogenic vibrio in the marine product transportation basically.The experimental result of comparing embodiment 4 is used 2 strain Bdellovibrios as can be known and the removing effect of the morbid vibrio in the marine product transportation obviously is better than single with 1 strain Bdellovibrio (BDJ02).
Embodiment 7
The application of Bdellovibrio kinds of pathogenic vibrio in eliminating the marine product breeding water body, marine product is an example with oyster, Penaeus Vannmei and perch.
To culture pond (125L) is divided into test and contrasts two big series.Test water is a seawater, and 100L is injected in each pond.Other implementation condition is with embodiment 1.In the experimental series, 3 ponds are one group, add a certain amount of Bdellovibrio concentrate, make it reach a predetermined Bdellovibrio final concentration, so have the Bdellovibrio final concentration of 3 different gradients, that is: 10
2Pfu/ml, 10
4Pfu/ml and 10
6Pfu/ml.
Use 1 strain Bdellovibrio (BDJ01) and eliminate vibrio alginolyticus, vibrio parahaemolytious, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis, vibrio mimicus in oyster, Penaeus Vannmei and the perch breeding water body, adopt the method identical with embodiment 1.Eliminate test effect as shown in Figure 7, among Fig. 7-and ■-be Vibrio harveyi concentration logarithm value curve in the experimental series,-◆-be the concentration logarithm value curve of vibrio mimicus in the experimental series,-▲-is the concentration logarithm value curve of Vibrio flurialis in the experimental series,-●-be vibrio parahaemolytious concentration logarithm value curve in the experimental series,-*-is the concentration logarithm value curve of Vibrio vulnificus in the experimental series ,-zero-be the concentration logarithm value curve of vibrio alginolyticus in the experimental series.Be minimum Bdellovibrio final concentration group (10 in the experimental series among Fig. 7
2Pfu/ml) the vibrios concentration logarithm value curve in.Because the elimination better effects if of other high concentration Bdellovibrio group is for for simplicity, so expression no longer in the drawings.All data are the mean value of three parallel samples.
As can be known from Fig. 7, the initial concentration of vibrios is 10
2The cfu/ml level.Each vibrios concentration also presents downward trend in the experimental series, use 1 strain Bdellovibrio (BDJ01) when above vibrios is eliminated, the elimination factor of vibrio alginolyticus, vibrio parahaemolytious, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis, vibrio mimicus is followed successively by 90.00%, 80.05%, 60.19%, 84.15%, 74.88%, 68.38%.
Adopt the method identical, adopt 2 strain Bdellovibrios to be applied to the marine product breed, promptly handle vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios and Vibrio metchnikovii in oyster, Penaeus Vannmei and the perch breeding water body with embodiment 1.Adopt method and the implementation condition identical with embodiment 7, the Bdellovibrio concentrate of adding variable concentrations makes it reach predetermined Bdellovibrio final concentration.Eliminate test effect as shown in Figure 8.Among Fig. 8-and ■-be Fu Nisishi vibrios concentration logarithm value curve in the experimental series,-◆-be the concentration logarithm value curve of Vibrio metchnikovii in the experimental series,-▲-is the concentration logarithm value curve of vibrio mimicus in the experimental series,-●-be vibrio parahaemolytious concentration logarithm value curve in the experimental series ,-*-be the concentration logarithm value curve of vibrio alginolyticus in the experimental series.Be minimum Bdellovibrio final concentration group (10 in the experimental series among Fig. 8
2Pfu/ml) the vibrios concentration logarithm value curve in.Because the elimination better effects if of other high concentration Bdellovibrio group is for for simplicity, so expression no longer in the drawings.All data are the mean value of three parallel samples.
As can be known from Fig. 8, the initial concentration of vibrios is 10
2The cfu/ml level.Each vibrios concentration also presents downward trend in the experimental series, uses 2 strain Bdellovibrios when above vibrios is eliminated, the concentration of vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios and Vibrio metchnikovii at 7h all less than 10cfu/ml.
Embodiment 9
Adopt the method identical, adopt 2 strain Bdellovibrios to be applied to the marine product breed, promptly handle Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis in oyster, Penaeus Vannmei and the perch breeding water body with embodiment 1.Adopt method and the implementation condition identical with embodiment 7, the Bdellovibrio concentrate of adding variable concentrations makes it reach predetermined Bdellovibrio final concentration.Eliminate test effect as shown in Figure 9.Among Fig. 9-and ■-be Vibrio harveyi concentration logarithm value curve in the experimental series,-◆-be the concentration logarithm value curve of Vibrio flurialis in the experimental series,-▲-is the concentration logarithm value curve of Vibrio cincinnatiensis in the experimental series ,-●-be Vibrio vulnificus concentration logarithm value curve in the experimental series.Be minimum Bdellovibrio final concentration group (10 in the experimental series among Fig. 9
2Pfu/ml) the vibrios concentration logarithm value curve in.Because the elimination better effects if of other high concentration Bdellovibrio group is for for simplicity, so expression no longer in the drawings.All data are the mean value of three parallel samples.
As can be known from Fig. 9, the initial concentration of vibrios is 10
2The cfu/ml level.Each vibrios concentration also presents downward trend in the experimental series, uses 2 strain Bdellovibrios when above vibrios is eliminated, the concentration of Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus, Vibrio flurialis at 7h all less than 10cfu/ml.
Sequence table is as follows:
＜110〉South China Science ﹠ Engineering University
＜120〉application of Bdellovibrio kinds of pathogenic vibrio in eliminating marine product and breeding water body thereof
<140>200810027132.8
<141>2008-03-31
<160>2
<210>1
<211>811
<212>DNA
＜213〉Bdellovibrio kind (Bdellovibrio sp.)
<220>
<221>16S?rDNA
<222>(1)…(811)
＜223〉n=a or g or c or t
<400>1
caagtcgaac?cgagaaangc?cnttncgggg?tggagtacag?tggcgcacgg?gtgaataacg?60
cgtaggtgac?gtgcctttta?gtgggggaca?acatcgggaa?accggtgcta?ataccgcata?120
agttaagcga?cattgaaaaa?gcttaagaaa?gtgggcttcg?gctcacgctg?aaagatcggc?180
ctgcgtatca?ttagcttgtt?ggtggggtaa?cggcctacca?aggctacgat?gattaactgg?240
tctgagagga?tgatcagtca?cactggaact?gagacacggt?ccagactcct?acgggaggca?300
gcagtaggga?atattgcgca?atgggggaaa?ccctgacgca?gcaatgccac?gtgagtgagg?360
aaggcccttg?ggttgtaaag?ctctgtccta?tgggaagaac?tgcattacgg?ttaatacccg?420
tagtgtttga?cggtaccata?gaagaaagca?ccggctaact?ccgtgccagc?agccgcggta?480
atacggaggg?tgcaagcgtt?gttcggattt?actgggcgta?aagcgcgcgc?aggcggattg?540
gcaagtcaga?tgtgaaatct?cggggctcaa?ccccgaaact?gcgtctgaaa?ctatcagtct?600
agagtctcat?agggggcagg?ggaatttcac?gtgtaggggt?aaaatccgta?gagatgtgaa?660
ggaacacccg?tggcgaaggc?gcctgcctgg?atgagcactg?acgctgaggc?gcgaaagcgt?720
ggggagcaaa?caggattaga?taccctggta?gtccacgccg?taaacgatga?gtactagccc?780
ttggagtatg?cccccncccc?cnggnacgaa?a?811
<210>2
<211>774
<212>DNA
＜213〉Bdellovibrio kind (Bdellovibrio sp.)
<220>
<221>16S?rDNA
<222>(1)…(774)
＜223〉n=a or g or c or t
<400>2
gtggcgcacg?nntnantaac?gcgtaggtga?cgtgcctttt?agtgggggac?aacatcggga?60
aaccggtgct?aataccgcat?aagttaagcg?acattgaaaa?agcttaagaa?agtgggcttc?120
ggctcacgct?gaaagatcgg?cctgcgtatc?attagcttgt?tggtggggta?acggcctacc?180
aaggctacga?tgattaactg?gtctgagagg?atgatcagtc?acactggaac?tgagacacgg?240
tccagactcc?tacgggaggc?agcagtaggg?aatattgcgc?aatgggggaa?accctgacgc?300
agcaatgcca?cgtgagtgag?gaaggccctt?gggttgtaaa?gctctgtcct?atgggaagaa?360
ctgcattacg?gttaataccc?gtagtgtttg?acggtaccat?agaagaaagc?accggctaac?420
tccgtgccag?cagccgcggt?aatacggagg?gtgcaagcgt?tgttcggatt?tactgggcgt?480
aaagcgcgcg?caggcggatt?ggcaagtcag?atgtgaaatc?tcggggctca?accccgaaac?540
tgcgtctgaa?actatcagtc?tagagtctca?tagggggcag?gggaatttca?cgtgtagggg?600
taaaatccgt?agagatgtga?aggaacaccc?gtggcgaagg?cgcctgcctg?gatgagcact?660
gacgctgagg?cgcgaaagcg?tggggagcaa?acaggattag?ataccctggt?agtccacgcc?720
gtaaacgatg?agtactagcc?cttggaggta?ttgccccccn?tccagtgacc?gaaa?774
Claims (6)
1, the application of Bdellovibrio kinds of pathogenic vibrio in eliminating marine product and breeding water body thereof, application method comprises following concrete steps and condition thereof:
Step 1: the separation and purification of Bdellovibrio
Seawater or the bed mud of gathering carried out preliminary treatment, the vibrio parahaemolytious of inoculation propagation Bdellovibrio, fall double-layer plate, plaque appears on the double-layer plate that contains the host bacterium, single spot that picking constantly enlarges, again by micro-biological process liquid enrichment culture, double-layer plate check, single spot of constantly enlarging of picking once more, and repeated several times, going down to posterity to single plaque, to form all consistent plaque of shape, size, transparency be the pure bacterial strain of a strain Bdellovibrio, by the big subtotal of single spot in the same time, separablely go out the pure bacterial strain of 2 strain Bdellovibrios;
Step 2: the preparation of Bdellovibrio concentrate
2 strain Bdellovibrios are carried out fermented and cultured respectively, and making concentration respectively is 10
11～10
13The Bdellovibrio concentrate of pfu/ml, stand-by;
Step 3: the application of Bdellovibrio kinds of pathogenic vibrio before the elimination marine product is edible and in the breeding water body
In the edible marine product that has polluted kinds of pathogenic vibrio and/or its breeding water body, add the Bdellovibrio concentrate of step 2 preparation, make the concentration of Bdellovibrio reach 10 at least
2Pfu/ml.
2, the application of Bdellovibrio according to claim 1 kinds of pathogenic vibrio in eliminating marine product and breeding water body thereof is characterized in that: described application method adopts 2 strain Bdellovibrios to mix and uses or independent respectively the use.
3, the application of Bdellovibrio according to claim 1 and 2 kinds of pathogenic vibrio in eliminating marine product and breeding water body thereof, it is characterized in that: described application method is used to eliminate kinds of pathogenic vibrio edible or that the preceding marine product of processing carries, and the Bdellovibrio concentration range is 10 during application
4～10
12Pfu/ml.
4, the application of Bdellovibrio according to claim 1 and 2 kinds of pathogenic vibrio in eliminating marine product and breeding water body thereof, it is characterized in that: described application method also is used for eliminating the kinds of pathogenic vibrio of marine product transportation, and the Bdellovibrio concentration range is 10 during application
3～10
11Pfu/ml.
5, the application of Bdellovibrio according to claim 1 and 2 kinds of pathogenic vibrio in eliminating marine product and breeding water body thereof, it is characterized in that: described application method also is used for eliminating the kinds of pathogenic vibrio of marine product breeding water body, and the Bdellovibrio concentration range is 10 during application
2～10
6Pfu/ml.
6, the application of Bdellovibrio according to claim 1 kinds of pathogenic vibrio in eliminating marine product and breeding water body thereof, it is characterized in that: described kinds of pathogenic vibrio is meant vibrio parahaemolytious, vibrio alginolyticus, vibrio mimicus, Fu Nisishi vibrios, Vibrio metchnikovii, Vibrio cincinnatiensis, Vibrio harveyi, Vibrio vulnificus and Vibrio flurialis.
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