CN116970541A - Method for preparing living non-culturable bacteria by using ultrahigh pressure - Google Patents
Method for preparing living non-culturable bacteria by using ultrahigh pressure Download PDFInfo
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- CN116970541A CN116970541A CN202311219832.8A CN202311219832A CN116970541A CN 116970541 A CN116970541 A CN 116970541A CN 202311219832 A CN202311219832 A CN 202311219832A CN 116970541 A CN116970541 A CN 116970541A
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- 241000894006 Bacteria Species 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims abstract description 32
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 15
- 241000191967 Staphylococcus aureus Species 0.000 claims description 15
- 241000588724 Escherichia coli Species 0.000 claims description 14
- 230000005526 G1 to G0 transition Effects 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 5
- 230000001580 bacterial effect Effects 0.000 description 15
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 229910002092 carbon dioxide Inorganic materials 0.000 description 5
- 239000001569 carbon dioxide Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000002411 adverse Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000002095 exotoxin Substances 0.000 description 1
- 231100000776 exotoxin Toxicity 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 235000006286 nutrient intake Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses a method for preparing living non-culturable bacteria. The method comprises the following steps: subjecting the bacteria of interest to ultra-high pressure in order to obtain said bacteria in a viable, non-culturable state; wherein the ultrahigh pressure treatment is carried out under the condition of 200-600MPa, the temperature of the ultrahigh pressure treatment is 4-37 ℃, and the time of the ultrahigh pressure treatment is 1-40min. The method provided by the invention can be used for treating bacteria, so that the bacteria can quickly enter a living non-culturable state within 5min, the preparation time of the living non-culturable state bacteria is shortened, and the preparation efficiency of the related living non-culturable state bacteria is improved.
Description
Technical Field
The invention relates to the technical field of foods and biological medicines, in particular to a method for preparing bacteria in a living non-culturable state.
Background
The viable, non-culturable (viable but nonculturable, VBNC) state, also known as the viable, non-culturable state, is a special form of bacteria in an adverse environment, meaning a special physiological state in which the bacteria, when in an adverse environment, shrink into spheres, are cultured by conventional methods and cannot grow and reproduce, but still recover under appropriate conditions and possess biological activity, toxicity or pathogenicity. Although the living non-culturable bacteria still have metabolic activity, they cannot grow and form colonies on the non-selective medium, so that the conventional bacteria detection method cannot detect the existence of the living non-culturable bacteria, and thus underestimating the number of bacteria in the detection sample makes the bacteria become a "invisible infectious source" for evading detection, and brings potential safety hazards to people. Therefore, research on characteristics, formation mechanisms, and the like of living non-culturable bacteria is important for effectively killing and inactivating the non-culturable bacteria. However, it takes a long time to obtain live bacteria in a non-culturable state, thereby limiting the progress of the study thereof.
Thus, there is a need to develop a method for rapidly preparing living bacteria in a non-culturable state.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides a method for preparing the living non-culturable bacteria, which can rapidly prepare the living non-culturable bacteria by adopting ultrahigh pressure and provides a basis for the subsequent study of the living non-culturable bacteria.
The present invention has been completed based on the following findings by the inventors:
the ultrahigh pressure mainly acts to destroy bacterial cell membranes, and the formation condition of the living non-culturable bacteria (for short, the living non-culturable bacteria) is to have complete cell membranes, so that it has great difficulty to achieve the formation of the living non-culturable bacteria induced by the ultrahigh pressure.
However, the inventors of the present invention have unexpectedly found that under milder ultra-high pressure treatment conditions, induction of bacteria in a normal state into viable, non-culturable bacteria can be achieved. Therefore, the method can prepare the living bacteria in the non-culturable state, has the advantages of short preparation time and the like, and can provide a basis for the subsequent research on the living bacteria in the non-culturable state.
In a first aspect of the invention, the invention provides a method of preparing bacteria in a viable, non-culturable state. According to an embodiment of the invention, the method comprises: subjecting the bacteria of interest to an ultra-high pressure treatment so as to obtain said bacteria in a viable, non-culturable state. The method can prepare the living bacteria in the non-culturable state, has the advantages of short preparation time and the like, and can provide a basis for the subsequent research of the living bacteria in the non-culturable state.
According to an embodiment of the present invention, the ultra-high pressure treatment is performed under a condition of 200-600 MPa.
According to the embodiment of the invention, the temperature of the ultrahigh pressure treatment is 4-37 ℃, and the time of the ultrahigh pressure treatment is 1-40min.
According to an embodiment of the present invention, the ultra-high pressure treatment is performed under 200-500 MPa.
According to the embodiment of the invention, the ultrahigh pressure treatment is carried out for 5-20 min under the conditions of 200-400MPa and 20-30 ℃.
According to the embodiment of the invention, the target bacteria are subjected to pre-culture treatment before the ultra-high pressure treatment to obtain bacteria in a logarithmic phase or a stationary phase, and the pre-culture treatment product is subjected to centrifugal treatment before the ultra-high pressure treatment and after the pre-culture treatment, wherein the centrifugal treatment is performed under the condition of 5000 Xg to 8000 Xg.
According to the embodiment of the invention, before the ultrahigh pressure treatment and after the centrifugation treatment, the centrifugation treatment precipitate is subjected to a cleaning treatment, wherein the cleaning treatment is performed by using physiological saline or PBS solution, and the mass percentage of NaCl in the physiological saline is 0.85 percent.
According to an embodiment of the invention, the bacteria of interest are bacteria in the log phase or stationary phase.
According to an embodiment of the invention, the bacteria of interest are selected from at least one of E.coli, staphylococcus aureus and Bacillus amyloliquefaciens.
In a second aspect of the invention, the invention provides a bacterium in a viable, non-culturable state. According to an embodiment of the invention, the bacteria in a viable, non-culturable state are prepared according to the method of the first aspect of the invention. The living non-culturable state bacteria of the present invention may provide a basis for subsequent studies on living non-culturable state bacteria.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is the time required for the ultra-high pressure and high pressure carbon dioxide treatment to induce bacteria to enter a viable, non-culturable state in example 1 of the present invention.
FIG. 2 shows the number of viable, non-culturable bacteria of E.coli, staphylococcus aureus, and Bacillus amyloliquefaciens treated at different pressures for 5 minutes in example 1 of the present invention.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The terms "comprising," "including," or "comprising" are used herein in an open-ended fashion, i.e., to include what is indicated by the present invention, and not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
The present invention proposes a method for producing a bacterium in a viable but non-culturable state and a bacterium in a viable but non-culturable state, which will be described in detail below, respectively.
Method for producing bacteria in viable, non-culturable state
In a first aspect of the invention, the invention provides a method of preparing bacteria in a viable, non-culturable state, comprising: the bacteria of interest are subjected to ultra-high pressure in order to obtain said bacteria in a viable, non-culturable state. The method can rapidly prepare the living non-culturable bacteria by adopting ultrahigh pressure, and provides a basis for the subsequent study of the living non-culturable bacteria.
In some alternative embodiments of the invention, the ultra-high pressure treatment is performed under conditions of 200-600MPa, such as 200MPa, 300MPa, 400MPa, 500MPa, 600MPa, and values ranging between any two of these values, such as 200MPa-500MPa, 300MPa-400MPa, 400MPa-500MPa.
In some alternative embodiments of the invention, the ultra-high pressure treatment is at a temperature of 4 ℃ to 37 ℃, such as 4 ℃, 5 ℃,6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 11 ℃, 12 ℃, 13 ℃, 14 ℃, 15 ℃, 16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃ and ranges between any two of the values thereof, such as 5 ℃ to 37 ℃, 8 ℃ to 34 ℃, 11 ℃ to 37 ℃, 11 ℃ to 34 ℃, 11 ℃ to 31 ℃, 14 ℃ to 14 ℃, 28 ℃, 17 ℃ to 25 ℃, 20 ℃ to 25 ℃, the time of the ultrahigh pressure treatment is 1-40min, such as 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, and a range between any two of these points, such as 4min-40min, 4min-30min, 4min-20min, and 4min-10min.
In some alternative embodiments of the invention, the ultrahigh pressure treatment is performed at 200-400MPa and 20-30 ℃ for 5-20 min.
In some alternative embodiments of the present invention, the bacteria of interest are pre-cultured to obtain bacteria in log phase or stationary phase prior to the ultra-high pressure treatment, and the pre-cultured product is centrifuged at 5000×g to 8000×g, for example 5000g, 6000g, 7000g, 8000g, and any range between any two of these values, such as 6000g to 8000g, 6000g to 70000 g, 5000g to 5000g, 5000g to 6000g, and 5000g to 6000g.
As used herein, the term "log phase" refers to the period of time in which the growth curve of the bacteria exhibits a linear rise in the number of viable bacteria. The log phase bacteria grow very rapidly in a stable geometric progression, which can last from several hours to several days (depending on the culture conditions and bacterial generation). Moreover, the bacterial morphology, staining, and bioactivity of the log phase are all typical and sensitive to the effects of external environmental factors.
In this context, the term "stationary phase" refers to a period in which the total number of growing colonies of bacteria is in a flat phase, but the bacterial population viability of the stationary phase is greatly changed. Due to nutrient consumption in the culture medium, toxic products (organic acids, H) 2 O 2 Etc.) the accumulated pH decreases, the bacterial growth rate gradually decreases, and the relative bacterial death rate begins to gradually increase, during which time the bacterial growth rate and death rate gradually equilibrate. The bacterial morphology, staining, biological activity of the stationary phase may be altered and corresponding metabolites such as exotoxins, endotoxins, antibiotics, spores, etc. may be produced.
In some alternative embodiments of the invention, the centrifuged sediment is subjected to a washing treatment prior to the ultra-high pressure treatment and after the centrifugation.
In some alternative embodiments of the invention, the washing treatment is performed with physiological saline or PBS solution.
In some alternative embodiments of the invention, the NaCl in the physiological saline is 0.85% by mass of the physiological saline.
In some alternative embodiments of the invention, the bacteria of interest are bacteria in the log phase or stationary phase.
In some alternative embodiments of the invention, the bacteria of interest are selected from at least one of E.coli, staphylococcus aureus and Bacillus amyloliquefaciens.
Bacteria in viable, non-culturable state
In a second aspect of the invention, the invention provides a bacterium in a viable, non-culturable state. According to an embodiment of the invention, the bacteria in a viable, non-culturable state are prepared according to the method of the first aspect of the invention. The living bacteria in the non-culturable state can provide a basis for subsequent researches on the living bacteria in the non-culturable state.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: coli, staphylococcus aureus and bacillus amyloliquefaciens into a viable, non-culturable state
In this example, a method for bringing E.coli, staphylococcus aureus and Bacillus amyloliquefaciens into a viable, non-culturable state is provided, comprising the steps of:
1. ultrahigh pressure treatment of different bacteria
Preparation of suspension of Escherichia coli, staphylococcus aureus and Bacillus amyloliquefaciens in logarithmic phase were cultured to stationary phase, centrifuged at 6000 Xg and washed twice with 0.85% NaCl aqueous solution or PBS, and then washed with 0.85% NaCl aqueous solutionThe cells were resuspended in NaCl aqueous solution or PBS to a cell concentration of about 10 8 cfu/ml。
Experimental group: ultrahigh pressure treatment
Placing the bacterial liquid of escherichia coli, staphylococcus aureus and bacillus amyloliquefaciens in a stable period after resuspension in a plastic bag, placing the plastic bag in a fast-in-origin middle-day CQC-600 ultrahigh pressure treatment bin, and treating for 5min at the temperature of 25 ℃ and the pressure of 0, 200, 300, 400 and 500MPa, and immediately releasing pressure after the parameters are reached to obtain the bacterial liquid of escherichia coli, staphylococcus aureus and bacillus amyloliquefaciens after ultrahigh pressure treatment.
Control group: high pressure carbon dioxide treatment
And (3) carrying out high-pressure carbon dioxide treatment on bacterial liquids of escherichia coli, staphylococcus aureus and bacillus amyloliquefaciens at a stable period after the resuspension in the plastic bag, wherein the high-pressure carbon dioxide treatment pressure is 5MPa, and the treatment temperature is 25 ℃.
The results in FIG. 1 show that ultra-high pressure for 5min induces bacteria into a viable, non-culturable state, whereas high pressure carbon dioxide takes 40min to induce into a viable, non-culturable state.
2. Detection of
The total cell count and the viable cell count of viable, non-culturable cells of the bacterial fluids of E.coli, staphylococcus aureus, bacillus amyloliquefaciens after the ultra-high pressure treatment in step one of this example were first measured with the Live/Dead BacLight bacterial viability kit (Molecular Probes Inc., eugene, OR, USA). 50. mu.L of bacteria were stained with 0.15. Mu.L of dye (SYTO 9:PI=1:1) for 15min in the dark. The final concentrations of SYTO 9 and PI were 10 and 60. Mu.M, respectively. The samples were then diluted 20-fold with phosphate buffer and analyzed by Flow Cytometry (FCM). The flow cytometer measurements were performed on a BD Accuri ™ C6 Plus FCM, with the threshold set to Side Scatter (SSC) >10000 and the flow rate set to low speed. At least 50000 cells were measured for all samples to ensure that the statistical Coefficient of Variation (CV) was less than 1%. The optical filter was set to measure the green fluorescence of SYTO 9 at 520 nm (FL 1) and the red fluorescence of PI at 630 nm (FL 3).
The plate counting method is adopted: referring to GB 4789.2-2010 method, the bacterial solutions of the escherichia coli, staphylococcus aureus and bacillus amyloliquefaciens treated by ultra-high pressure in the first step of the embodiment are subjected to 10-time stepwise gradient dilution by using NaCl aqueous solution with the mass percent of 0.85%, 1ml of 3 dilution samples with continuous dilution degree are sucked into a sterilization dish, about 20ml of TSA culture medium is poured into the sterilization dish to shake evenly, after the culture medium is solidified, the culture medium is inverted and is cultured in a culture box at 37 ℃ for 24 hours, and then the number of the culturable bacterial colonies is recorded. The number of viable, non-culturable cells is the difference between the number of viable cells detected by flow cytometry and the number of culturable cells detected by plate counting.
The number of viable bacteria and the number of culturable bacteria in the experimental group and the control group were detected by the above-described method, respectively, to thereby calculate the number of viable, non-culturable state bacteria=the number of viable bacteria-culturable bacteria. According to the method, whether the escherichia coli, staphylococcus aureus and bacillus amyloliquefaciens enter a living non-culturable state is detected, and when the number of culturable bacteria is zero and the number of living bacteria is not zero, the escherichia coli, staphylococcus aureus and bacillus amyloliquefaciens enter the living non-culturable state.
3. Results
The results of detecting the number of bacteria in different kinds of living non-culturable states are shown in FIG. 2. The result shows that when the pressure of the ultrahigh pressure treatment is 200-500MPa, the number of the live escherichia coli, staphylococcus aureus and bacillus amyloliquefaciens in the non-culturable state is 10 8 cfu/ml or so.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. A method of preparing bacteria in a viable, non-culturable state, comprising:
subjecting the bacteria of interest to an ultra-high pressure treatment so as to obtain said bacteria in a viable, non-culturable state.
2. The method according to claim 1, wherein the ultra-high pressure treatment is performed under conditions of 200-600 MPa.
3. The method according to claim 1, wherein the temperature of the ultra-high pressure treatment is 4-37 ℃ and the time of the ultra-high pressure treatment is 1-40min.
4. The method according to claim 1, wherein the ultra-high pressure treatment is performed under conditions of 200-500 MPa.
5. The method according to claim 1, wherein the ultra-high pressure treatment is performed at 200-400MPa and 20-30 ℃ for 5-20 min.
6. The method according to any one of claims 1 to 5, wherein the bacteria of interest are subjected to a pre-culture treatment prior to the ultra-high pressure treatment, resulting in bacteria in log phase or stationary phase;
centrifuging the pre-culture treatment product before the ultrahigh pressure treatment and after the pre-culture treatment;
the centrifugal treatment is carried out under the condition of 5000 Xg-8000 Xg.
7. The method according to claim 6, wherein the centrifugally treated precipitate is subjected to a washing treatment before the ultra-high pressure treatment and after the centrifugal treatment;
the cleaning treatment is carried out by using normal saline or PBS solution;
the mass percentage of NaCl in the physiological saline is 0.85 percent.
8. The method of any one of claims 1-5, wherein the bacteria of interest are bacteria in a log phase or stationary phase.
9. The method of any one of claims 1-5, wherein the bacteria of interest are selected from at least one of escherichia coli, staphylococcus aureus, and bacillus amyloliquefaciens.
10. A living non-culturable bacterium, characterized in that it is produced according to the method of any one of claims 1-9.
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2023
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