CN102038121A - Application of bdellovibrio mixture bacterial liquid in removing pathogenic bacteria in vegetables - Google Patents
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Abstract
The invention discloses application of bdellovibrio mixture bacterial liquid in removing pathogenic bacteria in vegetables. The application method comprises that: the vegetables are soaked in the bdellovibrio mixture bacterial liquid, or cleaned, drained and sprayed with the bdellovibrio mixture bacterial liquid; the bdellovibrio mixture bacterial liquid is sprayed in the vegetable processing environment, and the spray amount is 10-100mL/m<2>; the concentration of a mixture in the bdellovibrio mixture bacterial liquid is at least 10<1>pfu/mL, preferably 10<1>-10<6>pfu/mL; and in the application, the removal rate for the pathogenic bacteria is over 99 percent. The bdellovibrio mixture bacterial liquid is suitable to be applied to all vegetables and fruits, can improve the safety factor of instant vegetables and fruits for consumers to protect the health of the consumers, and provides guarantee for green processing and production of vegetables.
Description
Technical field
The invention belongs to biological technical field, relate to the application of bacterium, particularly Bdellovibrio leech plastid and the telotroch mixed bacteria liquid new application in eliminating the vegetables pathogenic bacteria.
Background technology
In recent years, global food security malignant event takes place frequently, and food origin disease has become the major issue that influences publilc health, causes the main cause of people's unusual death often.Major part all is that pathogenic microorganisms causes in thousands of the food poisonings that the Ministry of Public Health reports every year in China.Vegetables often pollute EHEC, salmonella, Listeria monocytogenes, staphylococcus aureus etc., and China's food contaminant monitoring net data show, salmonella, EHEC, 3 kinds of common food-borne pathogens of monocyte hyperplasia listeria spp detect positive rate and rise year by year in the vegetables, are three kinds of major control pathogenic bacteria.
Vegetables are rich in various vitamins and dietary fiber, are the requisite food of people's daily life.In recent years, occur a kind of instant vegetable on the market, this victual has not only satisfied modern's fast pace work requirements, and has more catered to citizen and be particular about the psychology of health care and convenient leisure.This instant vegetable raw material can adopt the various vegetables except that leaf vegetables, comprises winter bamboo shoot, lotus root, potato, Sheep's-parsley, carrot or the like.Its processing flow process mainly is: fresh vegetables-cleaning-processing-vacuum packaging.
Existing cleaning degerming method is mainly by acid solution, alkali lye, heating degerming, promptly chemical degerming and heat kill bacterium in the production process.The chemistry degerming very easily causes chemical residual and human body is produced greatly harm, and the heat kill bacterium has not only been destroyed the nutriments such as vitamin in the vegetables, and the strong hear resistance of some pathogenic bacteria gemma also easily causes sterilization not thorough, finally causes the generation of poisoning by food.And some eat raw vegetables as romaine lettuce, green onion, tomato, cucumber etc. because people are just directly edible through the simple back of cleaning, so cause food posioning equally easily.In addition, because the use of some chemical bactericides as chlorine dioxide, hypochlorous acid, bleaching powder, peroxide etc., contains a large amount of chlorine in the waste water of feasible discharging, the waste residue, environment is caused very big harm.
Bacteriophagic Bdellovibrio is the parasitics bacterium that a class is made a living with predator bacteria specially.Its thalline size is also littler than common bacterium, is 0.2～0.6 * 0.8～1.2 microns, and the effect of similar bacteriophage is arranged, but is not virus, but real bacterium promptly has all various characteristicses of general bacterium.Parasitic and cracking bacterium is the characteristic of Bdellovibrio uniqueness.Also be that it keeps the most outstanding function of own vital movement.Our current research shows that the Bdellovibrio bacteriovorus bacterial strain that has can cracking comprise some common food-borne pathogens such as Escherichia coli, salmonella, staphylococcus aureus.
Bdellovibrio is divided two stages history of life: free living, can move telotroch stage of not breeding and the leech plastid stage of in the periplasmic space of specific host bacterium, carrying out growth and breeding.Bdellovibrio is in the telotroch stage, and identification also with the host irreversible the combination is taken place, and invades the host then.After Bdellovibrio enters host's bacterial cell periplasmic space, the host bacteria effect that in 30 minutes, promptly ceases breathing, death; Bdellovibrio then secretes plurality of enzymes, and the big molecule by the degraded host cell becomes monomer, takes in then to be used for the synthetic of its large biological molecule.Along with cell proliferation and hydrolase produce, make host cell wall film destroyed, thereby cracking discharge Bdellovibrio.And that the mixed bacteria liquid of telotroch and leech plastid can have telotroch concurrently is rapid-action, energetic and leech plastid preparation, preserves easily the more of a specified duration and stronger advantage of environment tolerance of bactericidal action time.
No matter existing studies show that as a kind of effective microorganism preparation, is in food industry or in fields such as (ocean) aquacultures, the application of Bdellovibrio all is safe.For example: abroad, Lenz and Hespell (1978) discovers, Bdellovibrio and animal and people's cell do not had an infectivity [Lenz R.W., Hespell R.B.Attempts to growbedellovibrios micurgically-injected into animal cells.Archives ofMicrobiology, 1978,119 (3): 245-248].At home, Lin Mao etc. (2006) have studied the effect of Bdellovibrio to fish cell, find that it does not have dissemination [Lin Mao, Yang Xianle, Xue Hui, Cao Haipeng, Qiu Junqiang to the fish bacterium.Bdellovibrio BDH2102 is to the effect of fish cell and pathogen.The microbiology circular, 2006,33 (1): 7-11].
Therefore, utilization Bdellovibrio mixed bacteria liquid is used to eliminate this biological method that vegetables carry total plate count and pathogenic bacteria and just seems safe and convenient as cleaning bacteria remover.Bdellovibrio has the special bacteriums of cracking pathogenic bacteria as a class, can use not only that instant vegetable is edible, the sterilization processing before the processing, has avoided again simultaneously in the instant vegetable gastronomical process because the harm that radix is excessive, pasteurization does not thoroughly cause of bacterium amount.And can not cause bacterium resistance to occur; there is not the too single-minded shortcoming of host in the phagotherapy yet, will be dead after killing pathogenic bacteria because of hunger, will not be residual; when guaranteeing food security, also effectively reduce power consumption, play the effect of protection environment.Therefore this green degerming method of Bdellovibrio has sizable advantage.
Summary of the invention
The objective of the invention is to overcome the drawback of prior art, the new application of Bdellovibrio leech plastid and telotroch mixed bacteria liquid pathogenic bacteria in eliminating vegetables cleaning process, process equipment and processing environment is provided, its feature is according to green non-pollution vegetables microbial standard, reduce equipment investment cost, improve the preceding security of instant vegetable processing, reduce the incidence of food-borne pathogens food poisoning.
Purpose of the present invention realizes by following proposal:
The application of Bdellovibrio mixture bacterium liquid in eliminating the vegetables pathogenic bacteria.
Described Bdellovibrio mixture bacterium liquid is that Bdellovibrio leech plastid bacterium liquid and telotroch bacterium liquid mix.
Described Bdellovibrio was Bdellovibrio (Bdellovibrio sp.) BDFM05, was preserved in Chinese typical culture collection center on August 7th, 2009, and the preservation address is Chinese Wuhan Wuhan University, and deposit number is CCTCC NO:M 209172.
Described Bdellovibrio mixture bacterium liquid is that leech plastid bacterium liquid mixes by bacterial population with telotroch bacterium liquid at 1: 1.
The application process of described Bdellovibrio mixture bacterium liquid in eliminating the vegetables pathogenic bacteria is: vegetables are soaked in Bdellovibrio mixture bacterium liquid sprayed Bdellovibrio mixture bacterium liquid in 15-45min or the processing environment vegetables, the sprinkling consumption is 10～100mL/m
2
The mixture bacterial concentration is at least 10 in the described Bdellovibrio mixture bacterium liquid
1Pfu/mL, preferred 10
1～10
6Pfu/mL.
The preparation method of described Bdellovibrio mixture bacterium liquid is as follows:
Bdellovibrio leech plastid bacterium liquid prepares in accordance with the following methods: the host is in the nutrient broth fluid nutrient medium in inoculation, at 35 ℃ and 200rpm shaking table cultivation 12h, nutrient solution is through 4 ℃, behind the centrifugal 10～20min of 5000～8000rpm, precipitation is added in the DNB fluid nutrient medium, insert Bdellovibrio BDFM05, under 25～35 ℃ of temperature, cultivate 36～48h with 150～300rpm shaking table, obtain containing the nutrient solution of Bdellovibrio leech plastid and Bdellovibrio telotroch, nutrient solution behind centrifugal 15～20min, is removed the supernatant that contains the Bdellovibrio telotroch with 6000～8000rpm under 4 ℃ of temperature, the precipitation that obtains is Bdellovibrio leech plastid; Is that 7.2～7.6 phosphate buffer suspends with Bdellovibrio leech plastid with DNB fluid nutrient medium, water, physiological saline or 0.2mol/L pH value, and obtaining concentration is 10
6～10
9The Bdellovibrio leech plastid bacterium liquid of pfu/mL;
Bdellovibrio telotroch bacterium liquid prepares in accordance with the following methods: with the supernatant that contains the Bdellovibrio telotroch among the Bdellovibrio leech plastid preparation method behind the centrifugal 15～20min of 16000～18000rpm, precipitation is the suspension of 7.2～7.6 phosphate buffer with DNB fluid nutrient medium, water, physiological saline or 0.2mol/L pH value, and obtaining concentration is 10
6～10
9The Bdellovibrio telotroch bacterium liquid of pfu/mL.
With Bdellovibrio leech plastid bacterium liquid and telotroch bacterium liquid mix Bdellovibrio mixture bacterium liquid.
Bdellovibrio BDFM05 is obtained through ultraviolet mutagenesis by wild type Bdellovibrio BDS02, Bdellovibrio BDFM05 carries out carrying out morphologic observation: BDFM05 after the negative staining under electron microscope and is unicellular, ellipse, and size is 1.43 * 0.53 μ m, end is given birth to flagellum, flagellum length at least 2 μ m; Described Bdellovibrio BDFM05 cultivates the transparent circular plaque that can form diameter 2～3mm in three days with the double-layer plate method in 28 ℃.
The present invention has following advantage and effect with respect to prior art:
1, of the present inventionly clean fresh vegetables with the Bdellovibrio mixed concentrated liquid, remarkable to its elimination effect of carrying total plate count and pathogenic bacteria.
As Figure 1-3, the Bdellovibrio mixed bacteria liquid can reach more than 99% the elimination factor of pathogenic bacteria.
2, security is good in the application of Bdellovibrio mixed bacteria liquid of the present invention total plate count and pathogenic bacteria in eliminating fresh vegetables.
The method that Bdellovibrio mixed bacteria liquid elimination fresh vegetables carries pathogenic bacteria is biological method, Bdellovibrio have can infect, the characteristic of cracking host bacteria, make it to be suitable as the biological cleaning factor of restraining or removing the vegetable surface pathogenic bacteria, 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.It is residual to reduce or eliminate the pathogenic bacteria that chemical residual that chemical degerming method causes and pasteurization not exclusively cause effectively, guarantees the high-quality of vegetables, fundamentally avoids the generation of greengrocery product food poisoning.Improve the safety coefficient of consumer's edible vegetable, protected consumer's health.
3, Bdellovibrio mixed bacteria liquid of the present invention can farthest be protected the nutritive value of vegetables to the degerming of fresh vegetables.
Use the Bdellovibrio mixed bacteria liquid and eliminate the common pathogen that fresh vegetables carries, avoided some vegetables must high temperature sterilization and to the destruction of thermal sensitivity vitamins and other nutritious components in the vegetables, to the nutrition value preserving of instant vegetable, tool has great advantage.
4, Bdellovibrio mixture bacterium liquid of the present invention can energy savings to the degerming method of fresh vegetables, simplifies the instant vegetable procedure, increases economic efficiency the protection environment.
5, also extensible other victual and the fruit of being applied to of the present invention, be particularly conducive to the use that reduces or eliminates chemicals such as agricultural chemicals and residual, guarantee the high-quality of vegetables and fruit,, ensure that human health has crucial meaning improving edible safety; Escort for the export trade simultaneously.
Advantages such as vegetables were carried the new method of pathogenic bacteria before 6, the mixed bacteria liquid of used Bdellovibrio leech plastid and telotroch ate, processes as elimination, and it is effective to possess elimination simultaneously, acts on lastingly, and tolerance is strong.
Description of drawings
Elimination factor-Bdellovibrio concentration map when Fig. 1 eliminates total plate count for Bdellovibrio mixes body fluid in vegetables are cleaned.
Elimination factor-time diagram when Fig. 2 eliminates it and carries pathogenic bacteria for Bdellovibrio mixture bacterium liquid in vegetables are cleaned.
Total plate count residual rate-sprinkling spirogram when Fig. 3 is process equipment and environment sprinkling Bdellovibrio mixture bacterium liquid.
Logarithm value-the time diagram of each pathogenic bacteria concentration mean value when Fig. 4 eliminates it and carries pathogenic bacteria for each conventional sterilization agent in vegetables are cleaned.
Pathogenic bacteria concentration logarithm value-time diagram when Fig. 5 eliminates it and carries Escherichia coli (Escherichia coli), salmonella (Salmonella), Listeria monocytogenes (Listeriamonocytohenes), staphylococcus aureus (Staphylococcus aureus) for Bdellovibrio mixture bacterium liquid in vegetables are cleaned.
The specific embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiment is not limited in this.
Bdellovibrio leech plastid and telotroch mixture bacterium liquid carry the elimination of total plate count to it when tomato is cleaned.
Application process comprises following concrete steps and condition thereof:
Step 1: the preparation of Bdellovibrio mixture bacterium liquid
The preparation of Bdellovibrio leech plastid: 100mL NB (nutrient broth) fluid nutrient medium (peptone 10g is being housed, yeast extract 5g, distilled water 1000mL, pH 7.2) conical flask in inoculate Aeromonas hydrophila (Aeromonas hydrophila, purchase in Guangdong Province DSMZ of Institute of Micro-biology numbering GIM1.172), 200rpm, 28 ℃ of shaking tables were cultivated 18 hours, nutrient solution is abandoned supernatant respectively at 4 ℃, the centrifugal 15min of 5000rpm.Respectively precipitation is joined 100mL DNB fluid nutrient medium (nutrient broth 0.8g is housed, caseinic acid hydrolysate 0.5g, yeast extract 0.1g, be dissolved in the 1000mL distilled water, the pH value is 7.4) conical flask in, add the plaque of Bdellovibrio (Bdellovibrio sp.) BDFM05 of picking from the double-layer plate again.Constant temperature shaking table 250rpm, 28 ℃ of cultivation 36h.Nutrient solution is removed the supernatant that contains the Bdellovibrio telotroch respectively at 4 ℃ of centrifugal 20min of 6000rpm, keeps precipitation (being Bdellovibrio leech plastid), adds physiological saline suspension Bdellovibrio leech plastid.Making concentration is 10
12The Bdellovibrio leech plastid concentrate of pfu/mL, stand-by;
The preparation of Bdellovibrio telotroch: with the supernatant that contains the Bdellovibrio telotroch among the Bdellovibrio leech plastid preparation method behind the centrifugal 20min of 16000rpm, the precipitation that obtains promptly is the Bdellovibrio telotroch, add DNB fluid nutrient medium (nutrient broth 0.8g, caseinic acid hydrolysate 0.5g, yeast extract 0.1g, be dissolved in the 1000mL distilled water, the pH value is 7.4) suspension Bdellovibrio telotroch.Making concentration is 10
12The Bdellovibrio telotroch concentrate of pfu/mL, stand-by;
Bdellovibrio telotroch bacterium liquid and Bdellovibrio leech plastid bacterium liquid are pressed bacterial population and are mixed at 1: 1.
Step 2: it carries the application experiment of total plate count to Bdellovibrio mixture bacterium liquid when eliminating vegetables processing cleaning
(1) preparation of pathogenic bacteria
Cultivate with common nutrient broth medium shaking table, 160rpm cultivates 12-16h propagation Escherichia coli (Escherichia coli for 28 ℃, purchase in the Guangdong Microbes Inst, numbering GIM1.137), salmonella typhimurium (Salmonella typhimurium, purchase in the Guangdong Microbes Inst, numbering GIM1.237), Listeria monocytogenes (Listeriamonocytohenes, purchase in the Guangdong Microbes Inst, numbering GIM1.228), staphylococcus aureus (Staphylococcus aureus, purchase in the Guangdong Microbes Inst, numbering GIM1.142), nutrient solution is through 6000rpm, 4 ℃ of centrifugal 20min, abandon supernatant, the precipitation thalline suspends with the PBS (PBS) of aseptic pH7.2, and obtaining concentration is 10
9The stoste of cfu/ml prepares the mixed liquor of above-mentioned four kinds of bacterium at last, and the final concentration of every kind of bacterium is 10
5Cfu/ml.
(2) elimination of pathogenic bacteria
Get fresh tomato, control group and test group are established in test.Three tomatoes of control group are three parallel samples, and remaining tomato is a test group, and three of every groups are three parallel samples.The tomato of all control groups and test group is earlier through 75% alcohol sterilization, after be dipped into 1min in the 500mL mixture bacterium liquid for preparing in (1), take out drain after, with the tomato direct impregnation of test group in 10
1Pfu/mL, 10
3Pfu/mL, 10
5Pfu/mL, 10
6In the 500mL Bdellovibrio mixture bacterium liquid of pfu/mL, every concentration all has three parallel samples, and the tomato of control group is soaked with clear water (conventional method), and soak time is 30min.Concrete detection method is: take out tomato and put into the sterile distilled water that aseptic pulper adds 50mL simultaneously and blend from every group of sample, get after the concussion evenly after stoste carries out 10 times of volume dilution with sterile distilled water successively, get the 0.1mL coating and detect.Detect with nutrient agar according to the GB4789.2-2010 total plate count.
The result as shown in Figure 1, simple cleaning can't degerming, can only rely on the effect that adds bacteria remover.And various concentration Bdellovibrio mixture bacterium liquid are respectively 94%, 99.99%, 99.99%, 99.99% to the elimination factor of total plate count behind the 30min.Therefore, Bdellovibrio concentration is high more, and it is good more to eliminate effect.
Bdellovibrio mixture bacterium liquid is applied to the disinfection of celery process application apparatus, processing environment.
Test comprises following concrete steps and condition thereof:
Step 1: with the step 1 of embodiment 1
Step 2: the application experiment of pathogenic bacteria mixed concentrated liquid sterilization.Is what vegetables (this example? process what vegetables?)
To celery job shop ground and body of wall, the conveyer belt of transportation celery and the surface of celery cleaning equipment, evenly spray concentration be Bdellovibrio telotroch and the leech plastid of 10pfu/mL by the mixed liquor that mixes at 1: 1, the sprinkling use amount then is respectively 10mL/m by the difference of test group
2, 30mL/m
2, 50mL/m
2, 70mL/m
2, or 100mL/m
2, spray once every day, sprayed continuously two days, and clear water is sprayed in contrast.Concrete detection method is with the obliterating sampling on process equipment surface or ground etc. of wet aseptic cotton carrier, again with the dull and stereotyped counting of cultivating.Detection method is with example 1.
The result as shown in Figure 2, in subsequently environment germ detected, the control group that sprays clear water had various pathogenic bacteria to detect, the residual rate of total plate count still reaches 78%.And in the test group of spraying Bdellovibrio mixture bacterium liquid, be 10mL/m in the sprinkling amount only
2Detect the salmonella of 2～3cfu/mL in the group, the test group of other sprinkling amounts does not detect common pathogen.The result of this experimental study shows that Bdellovibrio mixture bacterium liquid effect aspect the pathogenic bacteria in eliminating the celery process is remarkable, can guarantee celery product quality and safety.This shows that Bdellovibrio can be applied in the cleaning process of celery widely as bactericide.
The application of Bdellovibrio mixture bacterium liquid in Sheep's-parsley is cleaned.
Bdellovibrio mixture bacterium liquid and preparation method thereof is with example 1.
The preparation of pathogenic bacteria mixture bacterium liquid is with example 1, and concentration is 10
5Cfu/mL.
Get fresh celery, experiment divides control group and experimental group two big series, and three every group parallel, each parallel celery.The bright lotus root of all control groups and test group is earlier through 75% alcohol sterilization, after be dipped into 1min in the 500mL mixed bacteria liquid for preparing, after taking-up drains, is experimental group sprayed 20mL10pfu/mL Bdellovibrio liquid (mixture bacterium liquid?), control group spray equivalent clear water, room temperature is placed 40min, detect once every the 10min sampling, concrete grammar is to learn from else's experience the moistening sterilization swab (swab) of SPSS after smearing 4 times uniformly on the Sheep's-parsley surface, put vibration, mixing in the aseptic centrifuge tube that contains SPSS, adopt dull and stereotyped coating process to detect then.Specifically: detect, detect, detect, detect with the Baird-Parker agar plate with the listeria spp chromogenic culture medium with BS (bismuth sulfite agar) culture medium with VRB-MUG (dimethyl diaminophenazine chloride-bile salt culture-medium) culture medium according to GB 4789.10-2010 staphylococcus aureus according to GB 4789.30-2010 Listeria monocytogenes according to GB 4789.4-2010 salmonella according to GB 4789.38-2008 Escherichia coli.Control group bacterium number is each pathogenic bacteria sum mean value.
Testing result is sprayed the control group of clear water as shown in Figure 3, and various pathogenic bacteria bases are originally constant, and the pathogenic bacteria number average is on a declining curve in the experimental group of sprinkling BD, and the elimination factor of various pathogenic bacteria all reaches 99.99%.
Embodiment 4
Bdellovibrio mixture bacterium liquid and conventional disinfectant for vegetables bactericidal effect are relatively
It is that bactericide (clorox, bleaching powder, bleaching powder etc.), electrolytic acid ionized water, chlorine dioxide, ozone, peroxide mix with oxychloride that conventional disinfectant for vegetables has chlorine.
This example is an example with fresh pakchoi, Bdellovibrio (Bdellovibrio sp.) is adopted BDFM05, vegetables with 75% alcohol sterilization after, earlier fresh vegetables is dipped into 1min in the 500mL pathogenic bacteria mixed liquor for preparing, taking-up drains, fresh vegetables is dipped in various bactericide and the Bdellovibrio mixture bacterium liquid 40min.Initially detect once, behind the 10min, detect once every 10min every 1min.Concrete detection method is with example 3, and Bdellovibrio mixture bacterium liquid prepares with example 1, and concentration is 10pfu/mL.Pathogenic bacteria mixed liquor preparation method is with example 1.Concentration is 10
5Cfu/mL.Pathogenic bacteria are got each pathogenic bacteria and count mean value among the conventional sterilization agent sterilization figure.
The result is shown in Fig. 4-5.As can be seen from Figure 4 the conventional sterilization agent is rapid-action, can kill pathogen basically in the general 2min.As can be seen from Figure 5 can kill 99% pathogenic bacteria in the individual plant Bdellovibrio mixture bacterium liquid 30min, during 40min pathogenic bacteria detected less than.Bdellovibrio mixture bacterium liquid is eliminated pathogenic bacteria by the bacterium cracking needs a process, so onset is slow, but can kill big 70% pathogen in the 20min.Bdellovibrio is eliminated pathogenic bacteria by cracking and belongs to biological control, safe in utilizationly can not pollute environment, also can not impact quality of vegetable.Compare common bactericide and be more suitable for vegetables sterilization use.
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 spiritual essence 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.
Claims (8)
1. the application of Bdellovibrio mixture bacterium liquid in eliminating the vegetables pathogenic bacteria is characterized in that: described Bdellovibrio mixture bacterium liquid is that Bdellovibrio leech plastid bacterium liquid and telotroch bacterium liquid mix.
2. according to the application of the described Bdellovibrio mixture of claim 1 bacterium liquid in eliminating the vegetables pathogenic bacteria, it is characterized in that: described Bdellovibrio is Bdellovibrio (Bdellovibrio sp.) BDFM05, be preserved in Chinese typical culture collection center on August 7th, 2009, deposit number is CCTCC NO:M 209172.
3. according to the application of the described Bdellovibrio mixture of claim 1 bacterium liquid in eliminating the vegetables pathogenic bacteria, it is characterized in that: described Bdellovibrio mixture bacterium liquid is that leech plastid bacterium liquid mixes by bacterial population with telotroch bacterium liquid at 1: 1.
4. according to the application of the described Bdellovibrio mixture of claim 1 bacterium liquid in eliminating the vegetables pathogenic bacteria, it is characterized in that: the application of described Bdellovibrio mixture bacterium liquid in eliminating the vegetables pathogenic bacteria is that vegetables are soaked 15-45min in Bdellovibrio mixture bacterium liquid.
5. according to the application of the described Bdellovibrio mixture of claim 1 bacterium liquid in eliminating the vegetables pathogenic bacteria, it is characterized in that: the application of described Bdellovibrio mixture bacterium liquid in eliminating the vegetables pathogenic bacteria is to spray Bdellovibrio mixture bacterium liquid in the processing environment vegetables, and the sprinkling consumption is 10～100mL/m
2
6. the application in eliminating the vegetables pathogenic bacteria according to claim 4 or 5 described Bdellovibrio mixture bacterium liquid, it is characterized in that: bacterial concentration is at least 10 in the described Bdellovibrio mixture bacterium liquid
1Pfu/mL.
7. according to the application of the described Bdellovibrio mixture of claim 6 bacterium liquid in eliminating the vegetables pathogenic bacteria, it is characterized in that: described Bdellovibrio bacterial concentration is 10
1～10
6Pfu/mL.
8. according to the application of the described Bdellovibrio mixture of claim 1 bacterium liquid in eliminating the vegetables pathogenic bacteria, it is characterized in that: the preparation method of described Bdellovibrio mixture bacterium liquid is as follows:
Described Bdellovibrio leech plastid bacterium liquid prepares in accordance with the following methods: the host is in the nutrient broth fluid nutrient medium in inoculation, at 35 ℃ and 200rpm shaking table cultivation 12h, nutrient solution is through 4 ℃, behind the centrifugal 10～20min of 5000～8000rpm, precipitation is added in the DNB fluid nutrient medium, insert Bdellovibrio BDFM05, under 25～35 ℃ of temperature, cultivate 36～48h with 150～300rpm shaking table, obtain containing the nutrient solution of Bdellovibrio leech plastid and Bdellovibrio telotroch, nutrient solution behind centrifugal 15～20min, is removed the supernatant that contains the Bdellovibrio telotroch with 6000～8000rpm under 4 ℃ of temperature, the precipitation that obtains is Bdellovibrio leech plastid; Is that 7.2～7.6 phosphate buffer suspends with Bdellovibrio leech plastid with DNB fluid nutrient medium, water, physiological saline or 0.2mol/L pH value, and obtaining concentration is 10
6～10
9The Bdellovibrio leech plastid bacterium liquid of pfu/mL;
Described Bdellovibrio telotroch bacterium liquid prepares in accordance with the following methods: with the supernatant that contains the Bdellovibrio telotroch among the Bdellovibrio leech plastid preparation method behind the centrifugal 15～20min of 16000～18000rpm, precipitation is the suspension of 7.2～7.6 phosphate buffer with DNB fluid nutrient medium, water, physiological saline or 0.2mol/L pH value, and obtaining concentration is 10
6～10
9The Bdellovibrio telotroch bacterium liquid of pfu/mL.
With Bdellovibrio leech plastid bacterium liquid and telotroch bacterium liquid mix Bdellovibrio mixture bacterium liquid.
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| CN103898230A (en) * | 2014-04-18 | 2014-07-02 | 北京农学院 | Method for assessing food-borne pathogenic microbe infected leafy vegetables |
| CN103898230B (en) * | 2014-04-18 | 2016-02-17 | 北京农学院 | A kind of food-borne pathogenic microorganism infects the appraisal procedure of leafy vegetable |
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