CN101845403A - Yeast strain and method for preparing phage bdellovibro preparation by using the same - Google Patents
Yeast strain and method for preparing phage bdellovibro preparation by using the same Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a novel strain XD03S (CCTCC NO: M 2010036) of yeast, which is capable of being the parasitichost of phage bdellovibro. The invention further discloses a method for preparing phage bdellovibro preparation by using the yeast strain, which comprises the following steps of: (1) phage bdellovibro purification: taking escherichia coli as target cell, adopting liquid cultivation and a double-layer tap water agar combination method to separate phage bdellovibro base strains from the nature, then adopting a single dental plague passage method to carry out liquid cultivation and the double-layer tap water agar combination method to carry out continuous single dental plague passage purification of at least 10 generations, and obtaining the phage bdellovibro inbred strains with strong phagocytosis capability; and (2) culturing the phage bdellovibro inbred strains obtained in the step (1) and the yeast strain XD03S (CCTCC NO: M 2010036) in a composite way, and obtaining the phage bdellovibro preparation. The preparation method simplifies the production technology of the phage bdellovibro, and reduces sewage discharge and energy consumption.
Description
Technical field
The present invention relates to a kind of yeast bacterial strain, the invention still further relates to the method for preparing phage bdellovibro preparation with this yeast bacterial strain simultaneously.
Background technology
Bacteriophagic Bdellovibrio is a kind of small-sized parasitics gram negative bacterium that U.S. scientist Stolp and Petzlod1962 find.It has unique life cycle, and the one, freely moving about the stage outside host cell, another is the growth and breeding stage in host cell.This peculiar mode of life of bacteriophagic Bdellovibrio has caused people's extensive interest.Be named as bacteriophagic Bdellovibrio in 1963.
Bacteriophagic Bdellovibrio is the parasitics bacterium of making a living with predator bacteria specially, and it is littler than common bacterium, and the effect of similar phage is arranged, but it is not a virus, and it has all morphological specificitys of bacterium.Bacteriophagic Bdellovibrio is one of natural self-cleaning factor, it is not easy to parasitize in the probiotics, but be easy to parasitize in the pathogenic bacterium, in aquaculture, the many Gram-negative bacterias in the water body all are the pathogeny bacterium of aquatic animal as Aeromonas hydrophila, vibrios, eel chytrid etc.
During the nearly last ten years aspect the application and development of bacteriophagic Bdellovibrio, domesticly many researchs have been carried out.Ministry of Agriculture's file in 1994 is listed bacteriophagic Bdellovibrio in the microorganism beneficial bacterium class of feed medicated premix permission use.Nineteen ninety-five, people such as Shanghai University's biological medicine center Shao's longan was the host bacterium with crucian hemorrhagic ascites disease pathogen bacterium point-like aerogenesis Zymomonas mobilis, and being separated to 6 strain bacteriophagic Bdellovibrios from nature all has bacteriolyze to this germ.And under experiment condition, aspect the self cleaning of pathogenic bacteria, study, for utilizing the generation of bacteriophagic Bdellovibrio control fish pond bacterial disease, carrying out the biological control new technology provides foundation and approach.Biology department of Xiamen University poplar in 1997 refined special wait the people in the Xiamen the coastal bacteriophagic Bdellovibrio that is separated to.The quantity of vibrios class becomes positive correlation in the quantity of bacteriophagic Bdellovibrio and the seawater.Only add bacteriophagic Bdellovibrio in the sterilization seawater, negative growth appears in its quantity, if add Vibrio anguillarum 8927 bacterial strains simultaneously, then bacteriophagic Bdellovibrio quantity increases and causes host bacterium number to reduce.They adopt double-deck agar plate method to carry out the phagocytosis test with bacteriophagic Bdellovibrio to 25 strain prawn pathogenic bacterias and intestinal bacteria, Bacillus subtilus, Pseudomonas aeruginosa, streptococcus aureus again later on, the result shows the parasitic scope difference of different bacteriophagic Bdellovibrio bacterial strains, but all can both be formed plaque clearly by some bacteriophagic Bdellovibrio parasitism for examination host bacterium.Their experiment provides evidence for utilizing bacteriophagic Bdellovibrio to carry out sea-food aquaculture organism control infectation of bacteria.Zhao Ming in 2002 is gloomy studies show that bacteriophagic Bdellovibrio strides to prevent or to reduce the generation of shrimp crab disease and spread in the disease control of shrimp crab, quite high to causing the morbific pathogenic bacteria clearance rate of shrimp crab, the Aeromonas hydrophila clearance rate is 97.2%, point-like Zymomonas mobilis clearance rate is 95.8%, the vibrios clearance rate is 93%, the intestinal bacteria clearance rate is 91.8%, the Salmonellas clearance rate is 97.3%, and bacteriophagic Bdellovibrio can improve the inside and outside environment of shrimp and crab body, promote shrimp crab healthy growth, strengthen the immunological competence of shrimp crab collective.Once just waited in 2004 and studies show that adding bacteriophagic Bdellovibrio in the water of fish pond can reduce total plate count in the pond water effectively.
As seen, along with domestic and international deepening continuously to the bacteriophagic Bdellovibrio research work, along with constantly applying of bacteriophagic Bdellovibrio product, will play increasing effect to antibiotic dependence, the aspect such as residual that reduces microbiotic, chemostefilant etc. changing people.
At present both at home and abroad the bacteriophagic Bdellovibrio product of the overwhelming majority all is that to adopt intestinal bacteria be the host bacterium, with after the intestinal bacteria deactivation as this production technique production of the host of bacteriophagic Bdellovibrio, there are several big problems in this technology:
1, most of intestinal bacteria are unwanted bacteria, will pollute because of carelessness, and risk is bigger, are the non-virulent bacterium though some engineering strains such as colibacillary C600 bacterial strain are also arranged, and also do not belong to probiotics, can not directly utilize as probiotic bacterium;
2, cultured intestinal bacteria need be carried out centrifugal concentrating, deactivation behind the taking-up thalline has produced a large amount of centrifuge waste waters like this, and inactivation technology needs very big energy consumption again;
3, the bacteriophagic Bdellovibrio product of terminal is single bacteriophagic Bdellovibrio product, and its function is very limited.
Summary of the invention
First purpose of the present invention has provided a kind of yeast bacterial strain, and this bacterial strain can be used as the host of bacteriophagic Bdellovibrio.
Second purpose of the present invention provides the above-mentioned yeast bacterial strain of a kind of usefulness to prepare the method for phage bdellovibro preparation.This preparation method has simplified the production technique of bacteriophagic Bdellovibrio, reduces sewage discharge and energy consumption.
The saccharomycetic new strain X D03S that first purpose of the present invention provides, it has the ability as the bacteriophagic Bdellovibrio parasitic host.This saccharomycetic new strain X D03S on January 29th, 2010 in China's typical culture collection center preservation, and receive preservation registration number CCTCC NO:M 2010036.
Second purpose of the present invention realizes by following technical measures: the above-mentioned yeast bacterial strain of a kind of usefulness prepares the method for phage bdellovibro preparation, and it may further comprise the steps:
(1) purifying of Bdellovibrio: with intestinal bacteria is target cell, adopt liquid culture to separate with the method that double-layer tap water agar combines and separate bacteriophagic Bdellovibrio basis bacterial strain from occurring in nature, adopt the single bacterial plaque method of going down to posterity to carry out method that liquid culture and double-layer tap water agar combine then and carry out the continuous single bacterial plaque at least 10 generations purifying that goes down to posterity, obtain the strong bacteriophagic Bdellovibrio pure lines bacterial strain of phagocytosis ability;
(2) the bacteriophagic Bdellovibrio pure lines bacterial strain of gained in the step (1) and yeast strain X D03S (CCTCC NO:M2010036) are carried out compound cultivation and obtain phage bdellovibro preparation.
The preferred substratum that uses adopts in the compound cultivation in the step of the present invention (2): 1000 milliliters of molasses 5～20 grams, egg-white powder 2～5 grams, water, the PH condition is 3.3-7.8.
23～36 ℃ of the temperature of the compound cultivation in the step of the present invention (2).
Preferred 1～5 day of the time that bacteriophagic Bdellovibrio pure lines bacterial strain and yeast strain X D03S cultivate in the step of the present invention (2).
It is comparatively single only to cultivate the function of the phage bdellovibro preparation that obtains with bacteriophagic Bdellovibrio pure lines bacterial strain and yeast strain X D03S, therefore the contriver filter out can with the symbiotic milk-acid bacteria of yeast strain X D03S, yeast and genus bacillus, set up the coordination cultural method for preparing phage bdellovibro preparation with above-mentioned yeast bacterial strain, be intended to obtain comparatively comprehensively phage bdellovibro preparation of function.Described method is specific as follows:
(1) purifying of Bdellovibrio: with intestinal bacteria is target cell, adopt liquid culture to separate with the method that double-layer tap water agar combines and separate bacteriophagic Bdellovibrio basis bacterial strain from occurring in nature, adopt the single bacterial plaque method of going down to posterity to carry out method that liquid culture and double-layer tap water agar combine then and carry out the continuous single bacterial plaque at least 10 generations purifying that goes down to posterity, obtain the strong bacteriophagic Bdellovibrio pure lines bacterial strain of phagocytosis ability;
(2) carry out compound cultivation with milk-acid bacteria, yeast and genus bacillus respectively with yeast strain X D03S (CCTCC NO:M 2010036), filter out can with the symbiotic milk-acid bacteria of yeast strain X D03S (CCTCC NO:M 2010036), yeast and genus bacillus;
(3) with gained in the step (2) can carry out compound cultivation earlier with the symbiotic milk-acid bacteria of yeast strain X D03S (CCTCC NO:M 2010036), yeast and genus bacillus and yeast strain X D03S (CCTCC NO:M 2010036), and then the bacteriophagic Bdellovibrio pure lines bacterial strain of gained is proceeded to cultivate and is obtained phage bdellovibro preparation in the inoculation step (1).
The preferred substratum that uses adopts in the compound cultivation in step of the present invention (2) and the step (3): 1000 milliliters of molasses 5～20 grams, egg-white powder 2～5 grams, water, the PH condition is 3.3-7.8.
23～36 ℃ of the temperature of the compound cultivation in step of the present invention (2) and the step (3).
Preferred 1～4 day of the time can carrying out compound cultivation with the symbiotic milk-acid bacteria of yeast XD03S (CCTCC NO:M 2010036), yeast and genus bacillus and yeast XD03S (CCTCC NO:M 2010036) in the step of the present invention (3).
Preferred 1～5 day of the time of proceeding to cultivate behind the bacteriophagic Bdellovibrio of the gained pure lines bacterial strain in the inoculation step (1) in the step of the present invention (3).
The present invention compared with prior art has the following advantages:
1, yeast strain X D03S provided by the invention (CCTCC NO:M 2010036) is a non-pathogenic bacteria, and is a kind of probiotic bacterium, but has the ability as the bacteriophagic Bdellovibrio parasitic host.Adopt this yeast bacterial strain to prepare phage bdellovibro preparation, can not need to carry out operations such as centrifugal, deactivation, simplified production process, and ensured production safety.
2, the present invention adopts to have as the yeast strain X D03S (CCTCCNO:M 2010036) of bacteriophagic Bdellovibrio parasitic host's ability and is prepared phage bdellovibro preparation as the host of bacteriophagic Bdellovibrio, need not to carry out production processes such as centrifugal, deactivation, simplified production process, improve production efficiency, and reduced sewage discharge and energy consumption.
3, the present invention passes through symbiosis culture, filter out can with the symbiotic milk-acid bacteria of yeast strain X D03S (CCTCC NO:M 2010036), yeast and genus bacillus, after yeast strain X D03S (CCTCC NO:M 2010036) cultivates for some time, adding bacteriophagic Bdellovibrio cultivates together, the compound phage bdellovibro preparation that finally obtains, not only has bacteriophagic Bdellovibrio in this product, go back profitable probliotics and its symbiosis simultaneously, can bring into play bacteriophagic Bdellovibrio simultaneously phagolysis of pathogenic bacterium and the regulating effect of probiotic bacterium.Increase and strengthen the function of the phage bdellovibro preparation of knowing clearly, will promote greatly and quicken it and apply.Can estimate, will be the main direction of foreign study and application applying to bacteriophagic Bdellovibrio aspect agricultural and the medical science as a kind of " microbiotic of living " from now on, so succeeding in developing of this product has boundless market outlook.
Compound phage bdellovibro preparation product of the present invention is in Guangdong Province, Hainan Province, ground such as Fujian Province have carried out large-area pilot scale to be promoted, company limited of Zhuhai City prawn study on the industrialization institute for example, the green treasured place bio tech ltd in Shenzhen, Gu Lao town Lv Yuan root plant, new pearl fishes and shrimps pharmacy, Zhongshan city's plate cottonrose hibiscus perseverance reaches fish pharmacy, also has the male fishing of Wenchang, Hainan Province Li Can pharmacy, Zhangzhou City, the Fujian long-living fishing of Lee pharmacies etc. prove through the pilot-scale experiment in 1 year, this product has improved fish, the surviving rate of aquatic products such as shrimp, be subjected to raiser's welcome deeply, the breed success ratio that improves them has been played critical effect.
Yeast strain X DS03 of the present invention on January 29th, 2010 in China's typical culture collection center preservation, and receive preservation registration number CCTCC NO:M 2010036.
Embodiment
Embodiment one:
(1) preparation of raw material:
1) collection of water sample
Collect and culture water sampling in the pool, bed mud is sealed the bacterium source of preparation as the separation test of bacteriophagic Bdellovibrio basis bacterial strain up for safekeeping.
2) preparation of host bacterium
Buy intestinal bacteria C600 as host bacterium in early stage from Guangdong Microbes Inst DSMZ, C600 is made the C600 bacteria suspension respectively, 3500r/min is centrifuged into bacterium mud, and bacterium mud made the 1010cfu/ml bacteria suspension, a part is unsterilised to be viable bacteria mud C600, part sterilization is inactivated bacteria mud C600, puts into 4 degree refrigerators after handling and preserves stand-by.
(2) separation of Bdellovibrio
Adopt liquid culture to separate with the method that double-layer tap water agar combines:
1) bacteria suspension, 300ml viable bacteria mud C600 and the 300ml inactivated bacteria mud C600 that gets 300ml C600 is contained in respectively in the different triangular flasks.The water sample part of gained at the centrifugal 15min of 3000r/min, get among bacteria suspension, viable bacteria mud C600 and the inactivated bacteria mud C600 that supernatant liquor joins the C600 in the triangular flask respectively and cultivate, every bottle adds the 20ml supernatant liquor, triplicate, under 28-32 ℃ temperature, leave standstill and cultivated 4-6 days, cultured bacterium liquid centrifugal 15min under 3000r/min, get supernatant liquor and do the bacterial plaque separating experiment again with the double-layer tap water agar method;
2) bacteria suspension, 300ml viable bacteria mud C600 and the 300ml inactivated bacteria mud C600 that gets 300ml C600 is contained in respectively in the different triangular flasks.The water sample of gained join respectively in the triangular flask bacteria suspension, viable bacteria mud C600 and the deactivation of C600 after C600 in cultivate, every bottle adds 20ml supernatant liquor, triplicate.Static cultivation is 4-6 days under 28-32 ℃ temperature.Cultured bacterium liquid centrifugal 15min under 3000r/min, get supernatant liquor and do the bacterial plaque separating experiment again with the double-layer tap water agar method;
3) double-layer tap water agar method: lower floor pours in the culture dish of diameter 18cm sterilization with 1.7% tap water agar of sterilization, the 50ml/ ware, put into after solidifying about the thermostat container insulation 3h about 45 degree, the upper strata installs to colorimetric cylinder to 0.8% tap water agar powder substratum branch of sterilization, the 7ml/ pipe, the bacterium mud 0.8ml that adds the C600 after the deactivation again, be placed in the water-bath about 45 degree and be incubated, to join at the water sample supernatant liquor 0.5ml of the centrifugal 15min of 3000r/min at last in the colorimetric cylinder about 40 degree and pour into rapidly on the ready bottom substratum after shaking up, make its uniform distribution in the above, the thermostat container of putting into after solidifying about 28 degree is inverted cultivation 4-6 days, observes continuously;
(3) purifying of Bdellovibrio
Utilize intestinal bacteria C600 to be the host, adopt the single bacterial plaque method of going down to posterity to carry out the method that liquid culture and double-layer tap water agar combine and carry out repeatedly the basic bacterial strain that purifying obtains bacteriophagic Bdellovibrio, because bacterial classification is impure, it is impure to cultivate the plaque that comes out, bacterial plaque is little, unintelligible, therefore need carry out the purifying seed selection to wild strain.Utilize the single bacterial plaque method of going down to posterity to carry out the method that liquid culture and double-layer tap water agar combine and carry out purifying, go down to posterity through the single bacterial plaque of 10 generation successive, select that to form plaque big, bacterial plaque is clear, the Bdellovibrio bacteriovorus bacterial strain XD03 that the bacterial plaque formation time is short.
(4) screening of non-escherichia coli host
Utilize XD03 respectively with 3 strains of lactic acid bacteria, 3 saccharomycetes, 8 bacillus are the host, utilize the single bacterial plaque method of going down to posterity to carry out the method that liquid culture and double-layer tap water agar combine and tame cultivation, filter out the host that can form bacterial plaque, to provoke plaque big at every turn, bacterial plaque is clear, form short bacterial plaque of bacterial plaque time and carry out screening experiment next time, go down to posterity, filter out a saccharomycete through the single bacterial plaque of 12 generation successive, it is big to reach the formation plaque, bacterial plaque is clear, the target that the bacterial plaque formation time is short, and we are with this saccharomycete called after XD03S (CCTCC NO:M 2010036).
(5) compound host's screening
Get 3 saccharomycete XD03S (CCTCC NO:M 2010036) respectively with 3 strains of lactic acid bacteria, 2 saccharomycetes, the compound culture experiment of 8 bacillus probiotic bacteriums, screening can with the symbiotic bacterial strain of XD03S, filter out at last can with symbiotic 1 strains of lactic acid bacteria of XD03S, 1 saccharomycete, 1 bacillus, this three strains bacterium is carried out compound cultivation obtains compound host bacterium, the culture temperature of above-mentioned compound cultivation is 23 ℃, the PH condition is 3.3, and substratum is: 1000 milliliters of molasses 5 grams, egg-white powder 2 grams, water.
(6) cultivation of phage bdellovibro preparation
This step adopts substratum to be: 1000 milliliters of molasses 5 grams, egg-white powder 2 grams, water, the PH condition is 3.3.To obtain and to be seeded in the substratum as compound host bacterium with symbiotic 1 strains of lactic acid bacteria of yeast XD03S (CCTCCNO:M 2010036), 1 saccharomycete and 1 bacillus in 1 saccharomycete XD03S (CCTCC NO:M 2010036) and the step (5), earlier under 23 ℃ of temperature, the PH condition is to cultivate 1 day under 3.3 the condition, and then the bacteriophagic Bdellovibrio XD03 bacterial classification that obtains in the inoculation step (3), then under 23 ℃ of temperature, the PH condition is to cultivate 1 day under 3.3 the condition, obtains phage bdellovibro preparation.
Embodiment two:
Different with embodiment one is:
(5) compound host's screening
Get saccharomycete XD03S CCTCC NO:M 2010036) and 3 strains of lactic acid bacteria, 2 saccharomycetes, the compound culture experiment of 8 bacillus probiotic bacteriums, screening can with the symbiotic bacterial strain of XD03S, filter out at last can with symbiotic 1 strains of lactic acid bacteria of XD03S, 1 saccharomycete, 1 bacillus, this three strains bacterium is carried out compound cultivation obtains compound host bacterium, the culture temperature of above-mentioned compound cultivation is 36 ℃, the PH condition is 7.8, and substratum is: 1000 milliliters of molasses 20 grams, egg-white powder 5 grams, water.
(6) cultivation of phage bdellovibro preparation
This step adopts substratum to be: 1000 milliliters of molasses 20 grams, egg-white powder 5 grams, water, the PH condition is 7.8.To obtain and to be seeded in the substratum as compound host bacterium with symbiotic 1 strains of lactic acid bacteria of yeast XD03S (CCTCCNO:M 2010036), 1 saccharomycete and 1 bacillus in 1 saccharomycete XD03S (CCTCC NO:M 2010036) and the step (5), earlier under 36 ℃ of temperature, the PH condition is to cultivate 4 days under 7.8 the condition, and then the bacteriophagic Bdellovibrio XD03 bacterial classification that obtains in the inoculation step (3), then under 36 ℃ of temperature, the PH condition is to cultivate 5 days under 7.8 the condition, obtains phage bdellovibro preparation.
Embodiment three:
Different with embodiment one is:
(5) compound host's screening
Get saccharomycete XD03S (CCTCC NO:M 2010036) and 3 strains of lactic acid bacteria, 2 saccharomycetes, the compound culture experiment of 8 bacillus probiotic bacteriums, screening can with the symbiotic bacterial strain of XD03S, filter out at last can with symbiotic 1 strains of lactic acid bacteria of XD03S, 1 saccharomycete, 1 bacillus, this three strains bacterium is carried out compound cultivation obtains compound host bacterium, the culture temperature of above-mentioned compound cultivation is 30 ℃, the PH condition is 5.5, and substratum is: 1000 milliliters of molasses 15 grams, egg-white powder 3.5 grams, water.
(6) cultivation of phage bdellovibro preparation
This step adopts substratum to be: 1000 milliliters of molasses 15 grams, egg-white powder 3.5 grams, water, the PH condition is 5.5.To obtain and to be seeded in the substratum as compound host bacterium with symbiotic 1 strains of lactic acid bacteria of yeast XD03S (CCTCC NO:M 2010036), 1 saccharomycete and 1 bacillus in 1 saccharomycete XD03S (CCTCC NO:M 2010036) and the step (5), earlier under 30 ℃ of temperature, the PH condition is to cultivate 3 days under 5.5 the condition, and then the bacteriophagic Bdellovibrio XD03 bacterial classification that obtains in the inoculation step (3), then under 30 ℃ of temperature, the PH condition is to cultivate 4 days under 5.5 the condition, obtains phage bdellovibro preparation.
Embodiment four:
Different with embodiment one is:
(5) compound host's screening
Get saccharomycete XD03S (CCTCC NO:M 2010036) and 3 strains of lactic acid bacteria, 2 saccharomycetes, the compound culture experiment of 8 bacillus probiotic bacteriums, screening can with the symbiotic bacterial strain of XD03S, filter out at last can with symbiotic 1 strains of lactic acid bacteria of XD03S, 1 saccharomycete, 1 bacillus, this three strains bacterium is carried out compound cultivation obtains compound host bacterium, the culture temperature of above-mentioned compound cultivation is 26 ℃, the PH condition is 4.0, and substratum is: 1000 milliliters of molasses 10 grams, egg-white powder 4 grams, water.
(6) cultivation of phage bdellovibro preparation
This step adopts substratum to be: 1000 milliliters of molasses 10 grams, egg-white powder 4 grams, water, the PH condition is 4.0.To obtain and to be seeded in the substratum as compound host bacterium with symbiotic 1 strains of lactic acid bacteria of yeast XD03S (CCTCCNO:M 2010036), 1 saccharomycete and 1 bacillus in 1 saccharomycete XD03S (CCTCC NO:M 2010036) and the step (5), earlier under 26 ℃ of temperature, the PH condition is to cultivate 2 days under 4.0 the condition, and then the bacteriophagic Bdellovibrio XD03 bacterial classification that obtains in the inoculation step (3), then under 26 ℃ of temperature, the PH condition is to cultivate 3 days under 4.0 the condition, obtains phage bdellovibro preparation.
Embodiment five:
Different with embodiment one～four is: present embodiment does not adopt compound host bacterium, only adopts yeast XD03S (CCTCC NO:M 2010036) as the host bacterium.
The cultivation of step (6) phage bdellovibro preparation
This step adopts substratum to be: 1000 milliliters of molasses 10 grams, egg-white powder 4 grams, water, the PH condition is 6.0.Be seeded in the substratum with 1 saccharomycete XD03S (CCTCC NO:M 2010036), earlier under 28 ℃ of temperature, the PH condition is to cultivate 3 days under 6.0 the condition, and then the bacteriophagic Bdellovibrio XD03 bacterial classification that obtains in the inoculation step (3), then under 28 ℃ of temperature, the PH condition is to cultivate 5 days under 6.0 the condition, obtains phage bdellovibro preparation.
Embodiment six: the application test of phage bdellovibro preparation provided by the invention in the Penaeus vannamei breeding process
Application test carries out in the Pearl River Delta, respectively at Jiangmen Da Ao, the Taishan, middle mountain plate cottonrose hibiscus, the winter canopy shrimp pool of the white any of several broadleaf plants of horizontal bar and Zhuhai experimentizes simultaneously, each area selects two cultural technique conditions relatively good, the raiser of the horizontal basically identical of breed tests, culture in the pool at 30 mouthfuls of Penaeus vannameis of 10 raisers in 5 zones altogether and test, it is the breed pool about 5 mu that each raiser selects 3 open area, phage bdellovibro preparation provided by the invention is adopted on the test pool, consumption is the per kilogram product with 1 mu of water body (calculating by one meter depth of water), usage is evenly to splash with this full pool, water dilution back, pool, uses once every 7 days.Contrast is cultured the pool one and is adopted common on the market bacteriophagic Bdellovibrio (the three phagocytosis kings that Nanjing Fu Run moral company produces), and usage and dosage is the same; Contrast is cultured the pool two and is adopted common biotechnological formulation (the EM bacterium that providence company in Jiangxi produces), and usage and dosage is the same.The pool is cultured in all tests and pool seedling is cultured in contrast, put the seedling time, the feed and other medicining condition basically identicals that use, the test-results on each test breed pool is very consistent after three months, 10 raisers' test is cultured pool surviving rate and is on average surpassed 95%, the surviving rate average 85% on the pool one is cultured in contrast, the surviving rate average out to 50% on the pool two is cultured in contrast, the sickness rate that whole breeding process is respectively tested the pool is very low, it is stable that cultivation water keeps always, illustrate that phage bdellovibro preparation of the present invention uses not only the control pathogenic bacteria is had better action in aquaculture water, to stablizing cultivation water, regulating the appearance micro-ecological environment also has good effect.The common bacteriophagic Bdellovibrio that is adopted on the pool one is cultured in contrast has better action to the control pathogenic bacteria, but owing to wherein there is not the effect of probiotics, the bacterial classification that does not have microecosystem regulation balance, stabilizing water quality, water quality is stable inadequately in the breeding process, and the phenomena of mortality that caused by water quality happen occasionally.The common EM bacterium that adopt on the contrast pool two is to there being the effect of stronger adjusting water quality, but powerless to deleterious pathogenic bacteria, therefore, bacteriosis and concurrent viral disease thereof happen occasionally in the breeding process, and the breed success ratio is totally lower.
The above embodiment of the present invention only is used for the present invention is set forth, yet protection scope of the present invention is not only to be confined to above embodiment.The person of an ordinary skill in the technical field only need get yeast, genus bacillus and milk-acid bacteria as raw material, according to content disclosed by the invention, all can realize purpose of the present invention.
Claims (10)
1. a saccharomycetic new strain X D03S (CCTCC NO:M 2010036), it has the ability as the bacteriophagic Bdellovibrio parasitic host.
2. one kind prepares the method for phage bdellovibro preparation with the described yeast bacterial strain of claim 1, it is characterized in that it may further comprise the steps:
(1) purifying of Bdellovibrio: with intestinal bacteria is target cell, adopt liquid culture to separate with the method that double-layer tap water agar combines and separate bacteriophagic Bdellovibrio basis bacterial strain from occurring in nature, adopt the single bacterial plaque method of going down to posterity to carry out method that liquid culture and double-layer tap water agar combine then and carry out the continuous single bacterial plaque at least 10 generations purifying that goes down to posterity, obtain the strong bacteriophagic Bdellovibrio pure lines bacterial strain of phagocytosis ability;
(2) the bacteriophagic Bdellovibrio pure lines bacterial strain of gained in the step (1) and yeast strain X D03S (CCTCC NO:M2010036) are carried out compound cultivation and obtain phage bdellovibro preparation.
3. the method for preparing phage bdellovibro preparation according to claim 2, it is characterized in that, the preferred substratum that uses adopts in the compound cultivation in the described step (2): 1000 milliliters of molasses 5～20 grams, egg-white powder 2～5 grams, water, the PH condition is 3.3-7.8.
4. the method for preparing phage bdellovibro preparation according to claim 2 is characterized in that, 23～36 ℃ of the temperature of the compound cultivation in the described step (2).
5. the method for preparing phage bdellovibro preparation according to claim 2 is characterized in that, the time that bacteriophagic Bdellovibrio pure lines bacterial strain and yeast strain X D03S cultivate in the described step (2) is 1～5 day.
6. one kind prepares the method for phage bdellovibro preparation with the described yeast bacterial strain of claim 1, it is characterized in that it may further comprise the steps:
(1) purifying of Bdellovibrio: with intestinal bacteria is target cell, adopt liquid culture to separate with the method that double-layer tap water agar combines and separate bacteriophagic Bdellovibrio basis bacterial strain from occurring in nature, adopt the single bacterial plaque method of going down to posterity to carry out method that liquid culture and double-layer tap water agar combine then and carry out the continuous single bacterial plaque at least 10 generations purifying that goes down to posterity, obtain the strong bacteriophagic Bdellovibrio pure lines bacterial strain of phagocytosis ability;
(2) carry out compound cultivation with milk-acid bacteria, yeast and genus bacillus respectively with yeast strain X D03S (CCTCC NO:M 2010036), filter out can with the symbiotic milk-acid bacteria of yeast strain X D03S (CCTCC NO:M 2010036), yeast and genus bacillus;
(3) with gained in the step (2) can carry out compound cultivation earlier with the symbiotic milk-acid bacteria of yeast strain X D03S (CCTCC NO:M 2010036), yeast and genus bacillus and yeast strain X D03S (CCTCC NO:M 2010036), and then the bacteriophagic Bdellovibrio pure lines bacterial strain of gained is proceeded to cultivate and is obtained phage bdellovibro preparation in the inoculation step (1).
7. the method for preparing phage bdellovibro preparation according to claim 6, it is characterized in that, the substratum that uses in the compound cultivation in described step (2) and the step (3) adopts: 1000 milliliters of molasses 5～20 grams, egg-white powder 2～5 grams, water, the PH condition is 3.3-7.8.
8. the method for preparing phage bdellovibro preparation according to claim 6 is characterized in that, 23～36 ℃ of the temperature of the compound cultivation in described step (2) and the step (3).
9. the method for preparing phage bdellovibro preparation according to claim 6, it is characterized in that the time that can carry out compound cultivation with the symbiotic milk-acid bacteria of yeast XD03S (CCTCC NO:M 2010036), yeast and genus bacillus and yeast XD03S (CCTCC NO:M 2010036) in the described step (3) is 1～4 day.
10. the method for preparing phage bdellovibro preparation according to claim 6 is characterized in that, the time of proceeding to cultivate behind the bacteriophagic Bdellovibrio of the gained pure lines bacterial strain in the inoculation step (1) in the described step (3) is 1～5 day.
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| CN103937673A (en) * | 2014-04-25 | 2014-07-23 | 浙江农林大学天目学院 | Soil microbial culture medium and preparation method thereof |
| CN111471622A (en) * | 2020-04-21 | 2020-07-31 | 上海海洋大学 | Fermentation production method of bdellovibrio bacteriovorus by using rhodobacter sphaeroides XR12 as host bacteria |
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| CN100394926C (en) * | 2006-04-06 | 2008-06-18 | 薛恒平 | Production process of bdellophage preparation |
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| CN1357618A (en) * | 2000-12-15 | 2002-07-10 | 徐钢 | Biological bacteriophagic Bdellovibrio prepn and its production process |
| CN100394926C (en) * | 2006-04-06 | 2008-06-18 | 薛恒平 | Production process of bdellophage preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103937673A (en) * | 2014-04-25 | 2014-07-23 | 浙江农林大学天目学院 | Soil microbial culture medium and preparation method thereof |
| CN103937673B (en) * | 2014-04-25 | 2016-06-08 | 浙江农林大学天目学院 | A kind of soil microorganism culture medium and preparation method thereof |
| CN111471622A (en) * | 2020-04-21 | 2020-07-31 | 上海海洋大学 | Fermentation production method of bdellovibrio bacteriovorus by using rhodobacter sphaeroides XR12 as host bacteria |
| CN111471622B (en) * | 2020-04-21 | 2023-03-31 | 上海海洋大学 | Fermentation production method of bdellovibrio bacteriovorus by using rhodobacter sphaeroides XR12 as host bacteria |
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