CN105200034B - A kind of induced synthesis method of VBNC states salmonella - Google Patents
A kind of induced synthesis method of VBNC states salmonella Download PDFInfo
- Publication number
- CN105200034B CN105200034B CN201510724454.8A CN201510724454A CN105200034B CN 105200034 B CN105200034 B CN 105200034B CN 201510724454 A CN201510724454 A CN 201510724454A CN 105200034 B CN105200034 B CN 105200034B
- Authority
- CN
- China
- Prior art keywords
- salmonella
- vbnc
- states
- synthesis method
- vbnc states
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention discloses a kind of induced synthesis method of VBNC states salmonella, including salmonella is suspended in liquid, is uniformly mixed, it is 10 to make its bacterial concentration7~108CFU/mL, by bacterium solution, 190~570W of ultrasonic wave handles 5~25min under 42 DEG C~55 DEG C constant temperatures, detects whether that induction salmonella enters VBNC states.The present invention has expanded the understanding to VBNC state salmonellas, for ultrasonic heat technology is preferably applied to food processing, control of microorganisms and food security guarantee in terms of provide fundamental basis and technical support.
Description
Technical field
The invention belongs to food microorganisms culture and detection field, more specifically, being related to a kind of VBNC states Salmonella
The induced synthesis method of bacterium.
Background technology
(Viable but nonculturable, VBNC) state of cultivating that microorganism lives but non-refers to that it loses
The ability of growth and breeding in conventional medium, but can recover again under suitable conditions and recover a kind of special dormancy of Culturability
State.Pardon to wait from nineteen eighty-two Xu Huai and find existence but not educable comma bacillus in river mouth and marine environment for the first time
(Vibrio cholerae) and Escherichia coli (Escherichia coli), until Colwell in 1985 formally proposes that VBNC is general
It reads, finds VBNC states in 90 various bacterias of 20 kinds so far.In food processing and storage, mainly there is object
Reason Stress Factors and two major class Induction of bacterial of chemical induction factor enter VBNC states.Physical coercion factor is mainly included in food
The technologies such as low temperature, freezing, drying, irradiation and the high pressure that processing and storage process is used.Current study show that low temperature can induce
Nearly all bacterium enters VBNC states.Chemical induction factor is mainly caused due to improper using disinfectant and preservative, is wrapped
Include chlorine-containing disinfectant, sulfur dioxide and some acid preservatives.Food-borne pathogens and spoilage organisms, will once into VBNC states
Food-safe and publilc health safety is very unfavorable.It is mainly reflected in incubation period long, poor controllability and potential pathogenic.
Salmonella typhi is a kind of enteric bacilli, and salmonella disease caused by infection is that have on public hygienics
One of zoonosis of significance easily causes human gi-tract inflammation.From the point of view of food security angle, salmonella is drawn
Fac vegetable juice, fresh-cut fruit and vegetable and egg milk product poisoning form the threat to human health in world wide and safety.
The content of the invention
The purpose of this part is to summarize some aspects of the embodiment of the present invention and briefly introduce some preferably to implement
Example.It may do in this section and the description of the application and the title of the invention a little simplified or omit to make our department
Point, the purpose of abstract of description and denomination of invention obscure, and this simplification or omit and cannot be used for limiting the scope of the invention.
In view of problem present in the induced synthesis method of above-mentioned and/or existing VBNC state salmonellas, it is proposed that this
Invention.
Therefore, it is an object of the present invention to provide a kind of method of salmonella VBNC state induced synthesis, especially
A kind of method that salmonella typhimurium is induced to enter VBNC states.
In order to solve the above technical problems, the present invention provides following technical solution：A kind of induction of VBNC states salmonella
Forming method, including salmonella is suspended in liquid, is uniformly mixed, it is 10 to make its bacterial concentration7~108CFU/mL,
By bacterium solution, 190~570W of ultrasonic wave handles 5~25min under 42 DEG C~55 DEG C constant temperatures, detects whether induction salmonella
Into VBNC states.
A kind of preferred embodiment of induced synthesis method as VBNC states salmonella of the present invention, wherein：Institute
It states and detects whether that induction salmonella enters VBNC states, method is：It will treated salmonella suspension centrifugal treating
Obtain bacterial sediment, rinsed with sterile distilled water, retain bacterial sediment, be then resuspended with sterile water, add in dyed blended liquid into
Row dyeing, observes on laser confocal microscope by laser excitation, if with the presence of the cell of fluoresced green, illustrates
It is induced to enter VBNC states.
A kind of preferred embodiment of induced synthesis method as VBNC states salmonella of the present invention, wherein：Institute
Salmonella is stated as salmonella typhimurium.
A kind of preferred embodiment of induced synthesis method as VBNC states salmonella of the present invention, wherein：Also
Including the method that salmonella is made to recover, the method that salmonella is made to recover is by the salmonella in VBNC states
Bacterium solution centrifugation extraction bacterial sediment object, is rinsed with sterile saline, is resuspended with culture medium, concentration after culture can be recovered
For 107~108The bacterium solution of CFU/mL.
A kind of preferred embodiment of induced synthesis method as VBNC states salmonella of the present invention, wherein：Institute
Centrifugation extraction bacterial sediment object is stated, centrifugal condition is 3500 × g, centrifuges 10min.
A kind of preferred embodiment of induced synthesis method as VBNC states salmonella of the present invention, wherein：Institute
It states and is resuspended with culture medium, culture medium includes：Beef extract 5.0g L-1, peptone 10.0gL-1, yeast extract 5.0gL-1, Portugal
Grape sugar 5.0gL-1, sodium chloride 5.0gL-1, pH 7.0.
A kind of preferred embodiment of induced synthesis method as VBNC states salmonella of the present invention, wherein：Institute
State culture can be recovered after concentration be 107~108In the bacterium solution of CFU/mL, the condition of culture is 20~37 DEG C of temperature, is trained
Support 6~72h.
A kind of preferred embodiment of induced synthesis method as VBNC states salmonella of the present invention, wherein：Also
Including, the method whether recovered of detection salmonella, it by concentration after the recovery is 10 to be7~108The bacterium solution of CFU/mL is mixed
It closes uniformly, is sampled using steriling test mode, and be uniformly coated on SS agar plates, be upside down in biochemical cultivation case, in 37
DEG C culture for 24 hours, if grow dark brown have metallic luster circle bacterium colony, show successful resuscitation.
It is a further object to provide a kind of induced synthesis method of VBNC states salmonella in the micro- life of food
The application in analyte detection field.
Beneficial effects of the present invention：Salmonella induction can be entered by VBNC states in 30min by the present invention, and
It can the more rapidly recovery under the conditions of suitable recovery.The understanding to VBNC state salmonellas has been expanded, it is micro- to make up tradition
The defects of biological plate count method cannot detect VBNC states bacterium and cause " missing inspection " has evaded the risk of pollution, to incite somebody to action
TS technologies are provided fundamental basis and technology branch in terms of being preferably applied to food processing, control of microorganisms and food security guarantee
It holds.
Description of the drawings
Fig. 1 is VBNC salmonella typhimurium recovery schematic diagrames in the embodiment of the present invention 3；
Fig. 2 is that ultrasonic heat processing induction salmonella typhimurium enters VBNC states and quick in the embodiment of the present invention 4
Recovery schematic diagram；
Fig. 3 is VBNC salmonella typhimurium recovery schematic diagrames in the embodiment of the present invention 4.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to Figure of description pair
The specific embodiment of the present invention is described in detail.
Many details are elaborated in the following description to facilitate a thorough understanding of the present invention, still the present invention can be with
Implemented using other different from other manner described here, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is from the limitation of following public specific embodiment.
Secondly, " one embodiment " or " embodiment " referred to herein refers to may be included at least one realization side of the present invention
A particular feature, structure, or characteristic in formula." in one embodiment " that different places occur in the present specification not refers both to
Same embodiment, nor the individual or selective embodiment mutually exclusive with other embodiment.
Embodiment 1：Salmonella typhimurium in induction BPY culture mediums is handled using ultrasonic heat and enters VBNC states
Take can breeding bacteria be 107~108Salmonella typhimurium (bacterium numbering CMCC50115) BPY cultures of CFU/mL
Base suspension bacteria liquid 60mL sterile workings are transferred in chuck beaker.Circulator bath device is connected, design temperature is 52 DEG C.At balance
5min is managed, it is 52 DEG C to make chuck beaker center BPY bacterium solutions temperature.Ultrasonic unit is opened immediately, using 190W power.Ultrasonic wave
It is heat-treated 20min.Sample is taken out, is cooled to 4 DEG C.
By salmonella suspension before and after the processing, taken using steriling test mode with vortex mixed instrument after mixing
Sample, and be uniformly coated on SS agar plates, it is upside down in biochemical cultivation case, with 37 DEG C of cultures for 24 hours, if without any salmonella
Colony growth, then it was initially believed that successfully being induced into VBNC states.And then salmonella suspension 1mL after TS is handled
Centrifugal treating obtains bacterial sediment, and is rinsed more than twice with sterile distilled water, retains bacterial sediment, finally with sterile water weight
It is outstanding.Add in the LIVE/DEAD@BacLight of 30 μ LTMBacterial Viability kits (Molecular Probes)
The middle dyed blended liquid of SYTO9/PI is dyed, and is observed by 488nm laser excitations on laser confocal microscope, if there is hair
The cell of green fluorescence is in the presence of (SYTO9 makes all Bacterial stains that green be presented, and PI is only capable of the killed bacterial for making cell membrane breakage
Dye and finally go out SYTO9 green fluorescences and that killed bacterial is presented is red), illustrate to have induced it into VBNC states.Treating can
Breeding bacteria is prescribed a time limit less than 1CFU/mL detections and viable count is prescribed a time limit higher than detection, illustrates that salmonella typhimurium enters VBNC shapes
State.And reverse transcription quantitative PCR (RT-qPCR) can be carried out using salmonella characterizing gene as primer to quantify detection VBNC shapes
State salmonella.
Embodiment 2：Salmonella typhimurium in induction red bayberry juice is handled using ultrasonic heat and enters VBNC states
Take can breeding bacteria be 107~108Salmonella typhimurium (bacterium numbering CMCC50115) BPY cultures of CFU/mL
Base suspension bacteria liquid 60mL, using 6000rpm centrifuge 5min, collect bacterial sediment sterile working be transferred to have been loaded with sterilizing after
In the chuck beaker of 60mL red bayberry juices.Circulator bath device is connected, design temperature is 45 DEG C.Balance Treatment 2min, makes chuck
Beaker center cranberry juice bacterium solution temperature is 45 DEG C.Ultrasonic unit is opened immediately, using 190W power.Ultrasonic heat processing
28min.Sample is taken out, is cooled to 4 DEG C.
By salmonella suspension before and after the processing, taken using steriling test mode with vortex mixed instrument after mixing
Sample, and be uniformly coated on SS agar plates, it is upside down in biochemical cultivation case, with 37 DEG C of cultures for 24 hours, if without any salmonella
Colony growth, then it was initially believed that successfully being induced into VBNC states.And then salmonella suspension 1mL after TS is handled
Centrifugal treating obtains bacterial sediment, and is rinsed more than twice with sterile distilled water, retains bacterial sediment, finally with sterile water weight
It is outstanding.Add in the LIVE/DEAD@BacLight of 30 μ LTMBacterial Viability kits (Molecular Probes)
The middle dyed blended liquid of SYTO9/PI is dyed, and is observed by 488nm laser excitations on laser confocal microscope, if there is hair
The cell of green fluorescence is in the presence of (SYTO9 makes all Bacterial stains that green be presented, and PI is only capable of the killed bacterial for making cell membrane breakage
Dye and finally go out SYTO9 green fluorescences and that killed bacterial is presented is red), illustrate to have induced it into VBNC states.Treating can
Breeding bacteria is prescribed a time limit less than 1CFU/mL detections and viable count is prescribed a time limit higher than detection, illustrates that salmonella typhimurium enters VBNC shapes
State.And reverse transcription quantitative PCR (RT-qPCR) can be carried out using salmonella characterizing gene as primer to quantify detection VBNC shapes
State salmonella.
Embodiment 3：VBNC salmonella typhimuriums recover
Into BPY bacterium solutions 10mL, the 8000rpm centrifugation 5min of VBNC states in Example 1.It is rushed with sterile saline
It washes thalline twice, is finally suspended in bacterial sediment in the BPY culture mediums that 10mL has sterilized, cultivated in 37 DEG C of constant temperature, 200rpm
36h.100 μ L of bacterium solution are spread evenly across on SS agar plates after taking recovery, are inverted in incubator, and 37 DEG C are cultivated for 24 hours, by bacterium colony
Salmonella number of viable after counting method record recovery, the result is shown in Figure 1.
Embodiment 4：Ultrasonic heat processing induction salmonella typhimurium enters VBNC states and rapid fluid resuscitation
60mL is contained 107~108The salmonella typhimurium BPY culture medium suspension bacteria liquids of CFU/mL are placed in chuck beaker
In, using ultrasonic heat processing system in 380W, 50 DEG C of processing 25min.Sample is taken out, is cooled to 4 DEG C.SS agar is used immediately
Medium culture can cultivate salmonella typhimurium quantity, be determined as less than 1CFU/mL detection limits.And it is contaminated with SYTO9 and PI is double
Salmonella after processing, is observed using laser co-focusing, obtains still having part salmonella typhimurium in sample in green
Fluorescence.By treated, bacterium solution is rinsed twice by centrifugation and with physiological saline.Bacterial sediment is resuspended with BPY culture mediums,
Hatching recovery culture is carried out respectively at 25 DEG C and 37 DEG C, salmonella typhimurium can be cultivated by being detected respectively at 48h and 12h, knot
Fruit sees Fig. 2 and Fig. 3.
It can be seen that it is induced the present invention provides a kind of salmonella VBNC easy to operate, at low cost, that recovery is fast, is multiple
Soviet Union and detection method.By suitable for ultrasonic heat processing salmonella being made to be induced in 30min into VBNC states, and pass through one
The more rapidly recovery under the conditions of suitable recovery of the special culture medium of kind.By the invention it is possible to it expands husky to VBNC states
The understanding of door Salmonella；For ultrasonic heat is applied in food processing process, more accurate pathogenic bacteria, especially sramana are provided
The testing result of Salmonella is able to detect that traditional microorganism plate count method can't detect still active or even pathogenic
The salmonella of property, has evaded pathogenic bacteria risk hidden danger, for ultrasonic heat technology is preferably applied to food processing, microorganism
It controls and food security guarantee aspect is provided fundamental basis and technical support.
It should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to preferable
The present invention is described in detail in embodiment, it will be understood by those of ordinary skill in the art that, it can be to the technology of the present invention
Scheme is modified or replaced equivalently, and without departing from the spirit and scope of technical solution of the present invention, should all be covered in this hair
Among bright right.
Claims (4)
- A kind of 1. induced synthesis method of VBNC states salmonella, it is characterised in that：Including,Salmonella is suspended in liquid, is uniformly mixed, it is 10 to make its bacterial concentration7~108CFU/mL, by bacterium solution at 52 DEG C Ultrasonic wave 190W handles 20min under constant temperature, detects whether that induction salmonella enters VBNC states, the salmonella is Salmonella typhimurium.
- 2. the induced synthesis method of VBNC states salmonella as described in claim 1, it is characterised in that：It is described to detect whether Induction salmonella enters VBNC states, and method is：By treated, salmonella suspension centrifugal treating obtains bacterial sediment, is rinsed with sterile distilled water, retains thalline and sinks It forms sediment, is then resuspended with sterile water, adds in dyed blended liquid and dyed, seen by laser excitation on laser confocal microscope It examines, if with the presence of the cell of fluoresced green, illustrates it has been induced to enter VBNC states.
- 3. the induced synthesis method of VBNC states salmonella as claimed in claim 2, it is characterised in that：It further includes, makes sand The method of door Salmonella recovery,The method that salmonella is made to recover is that the salmonella bacterium solution centrifugation in VBNC states is extracted bacterial sediment object, It is rinsed with sterile saline, is resuspended with culture medium, concentration is 10 after culture can be recovered7~108The bacterium solution of CFU/mL.
- 4. a kind of induced synthesis method of VBNC state salmonellas as described in claims 1 to 3 is any is in food microorganisms The application of detection field.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510724454.8A CN105200034B (en) | 2015-10-28 | 2015-10-28 | A kind of induced synthesis method of VBNC states salmonella |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510724454.8A CN105200034B (en) | 2015-10-28 | 2015-10-28 | A kind of induced synthesis method of VBNC states salmonella |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105200034A CN105200034A (en) | 2015-12-30 |
| CN105200034B true CN105200034B (en) | 2018-05-25 |
Family
ID=54947996
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510724454.8A Active CN105200034B (en) | 2015-10-28 | 2015-10-28 | A kind of induced synthesis method of VBNC states salmonella |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105200034B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838705B (en) * | 2016-05-27 | 2019-07-23 | 浙江大学 | A kind of rapid induction staphylococcus aureus enter it is living can not cultivation conditions method |
| CN113528401B (en) * | 2021-08-20 | 2023-08-22 | 山东大学 | Method for accurately obtaining VBNC-state bacteria |
| CN115094017B (en) * | 2022-06-30 | 2023-07-25 | 江南大学 | Method for inducing yeast to enter VBNC state |
| CN115232779A (en) * | 2022-07-18 | 2022-10-25 | 浙江省农业科学院 | Method for inducing salmonella enteritidis to enter VBNC state by utilizing sodium hypochlorite |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423808A (en) * | 2008-12-09 | 2009-05-06 | 吉林农业大学 | Resuscitation fluids of VBNC salmonella and preparation method thereof and resuscitation method |
| CN103031360A (en) * | 2012-10-22 | 2013-04-10 | 吉林农业大学 | VBNC (viable but noncultivable)-like cultivable bacteria and preparation method thereof |
-
2015
- 2015-10-28 CN CN201510724454.8A patent/CN105200034B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423808A (en) * | 2008-12-09 | 2009-05-06 | 吉林农业大学 | Resuscitation fluids of VBNC salmonella and preparation method thereof and resuscitation method |
| CN103031360A (en) * | 2012-10-22 | 2013-04-10 | 吉林农业大学 | VBNC (viable but noncultivable)-like cultivable bacteria and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Quantification of viable but nonculturable Salmonella spp.and Shigella spp. during sludge anaerobic digestion and their reactivation during cake storage;B. Fu 等;《Journal of Applied Microbiology》;20150907;第119卷(第4期);1138-1147 * |
| Resuscitation of Salmonella enterica Serovar Typhimurium and Enterohemorrhagic Escherichia coli from the Viable but Nonculturable State by Heat-Stable Enterobacterial Autoinducer;R. Reissbrodt 等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20021231;第68卷;4788-4794 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105200034A (en) | 2015-12-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105200034B (en) | A kind of induced synthesis method of VBNC states salmonella | |
| Ananthanarayan | Ananthanarayan and Paniker's textbook of microbiology | |
| Parveen et al. | Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products | |
| Gabriel et al. | Inactivation of Salmonella, E. coli and Listeria monocytogenes in phosphate-buffered saline and apple juice by ultraviolet and heat treatments | |
| CN101287843B (en) | Method and kit for detection of microorganism | |
| Mahony et al. | Stable L-forms of Clostridium perfringens and their growth on glass surfaces | |
| CN105838705B (en) | A kind of rapid induction staphylococcus aureus enter it is living can not cultivation conditions method | |
| CN110184222A (en) | A kind of bacteriophagic Bdellovibrio freeze-dried powder preparation and its application | |
| CN106498087A (en) | The dry powdered LAMP quick detection kits of C.perfringens and its using method | |
| CN102643919B (en) | Detection kit and detection method for viable bacteria in vibrio parahaemolyticus in food | |
| AU2015249274A1 (en) | Microbiological growth media and methods of using the same | |
| Gaurav et al. | Isolation and identification of Shigella spp. from human fecal samples collected from Pantnagar, India | |
| Zhang et al. | Putative inactivation mechanism and germicidal efficacy of induced electric field against Staphylococcus aureus | |
| Wang et al. | Effects of pulsed electric fields on the survival behaviour of Saccharomyces cerevisiae suspended in single solutions of low concentration | |
| CN111808787B (en) | Method for improving germination rate of clostridium sporogenes | |
| Palilu et al. | Isolation and identification of Staphylococcus sp. in powdered infant milk | |
| CN113005054A (en) | Bacillus amyloliquefaciens SS-ZC-26 and preparation method and application thereof | |
| CN103436623A (en) | Rapid detection kit for viable salmonella in food and use method thereof | |
| CN103981270A (en) | Photobacterium damsela rapid detection primer, kit and application | |
| CN107677817A (en) | A kind of salmonella typhimurium quick determination method based on immune magnetic Nano material photo-thermal effect | |
| CN106520755A (en) | Inducing method of staphylococcus aureus in viable but nonculturable state | |
| CN106511990A (en) | A concentration process and preparing process for a freeze-dried brucellosis live vaccine for veterinary use | |
| CN101705298B (en) | Quick bacterium examination kit and detection method thereof | |
| CN111718976A (en) | Method for detecting thermophilic bacteria in CIP cleaning residual water | |
| Wang et al. | Recovery of Salmonella from internally and externally contaminated whole tomatoes using several different sample preparation procedures |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| DD01 | Delivery of document by public notice |
Addressee: Zhang Luyi Document name: Notification of Passing Examination on Formalities Addressee: Wang Xiaodong Document name: Notification of Passing Examination on Formalities |
|
| DD01 | Delivery of document by public notice |
Addressee: Zhang Luyi Document name: Notice of conformity |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |