CN114395517B - Method for improving proportion of bacteria entering living non-culturable state - Google Patents

Method for improving proportion of bacteria entering living non-culturable state Download PDF

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CN114395517B
CN114395517B CN202210292267.7A CN202210292267A CN114395517B CN 114395517 B CN114395517 B CN 114395517B CN 202210292267 A CN202210292267 A CN 202210292267A CN 114395517 B CN114395517 B CN 114395517B
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CN114395517A (en
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饶雷
杨东
廖小军
王永涛
赵靓
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China Agricultural University
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Abstract

The invention provides a method for increasing the proportion of bacteria that enter a viable but uncultureable state, the method comprising: pretreating target bacteria to obtain pretreated target bacteria; subjecting the pretreated target bacteria to high-pressure carbon dioxide treatment so as to bring the pretreated target bacteria into a viable non-culturable state; wherein the pretreatment comprises an acid treatment and/or a heat treatment. The method can be used for quickly obtaining a large amount of bacteria in a living non-culturable state, is beneficial to the research on the bacteria in the living non-culturable state, and has good application prospect.

Description

Method for improving proportion of bacteria entering living non-culturable state
Technical Field
The invention relates to the field of food biology. In particular, the invention relates to a method of increasing the proportion of bacteria that enter a viable, non-culturable state.
Background
It is found that the vital signs of the bacteria are changed under the stimulation of the external environment, and the bacteria enter a special self-protection survival State, namely a living non-culturable State (VBNC). Under the state, the volume of the bacteria is continuously reduced, the ratio of the surface area to the volume is enlarged, the affinity to nutrient substances is improved, the starved nutrient environment can be endured, and the protective capability to other environmental stress factors such as preservatives, temperature shock, oxidation environment, osmotic pressure change and the like is enhanced. The bacteria in the VBNC state are characterized in that the growth and reproduction capacity on the plate is lost, the vital signs are alive, and the virulence and pathogenicity of the original bacteria are retained. Therefore, bacteria in the VBNC state can recover growth and reproduction under the suitable growth condition, and quickly reach the pathogenic dose, thereby becoming a 'recessive infectious source' for escaping detection and constituting a potential threat to safety. The development of studies on the characteristics and formation mechanism of live bacteria in a non-culturable state is important for effectively killing inactivated bacteria in a non-culturable state. However, the studies for obtaining viable non-culturable bacteria have been limited due to the low content and purity of the non-culturable bacteria obtained.
Therefore, at present, methods for increasing the ratio of bacteria into viable non-culturable state to obtain a large amount of viable non-culturable state bacteria have yet to be studied.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides a method capable of improving the proportion of bacteria entering the living non-culturable state, and by utilizing the method, a large number of bacteria in the living non-culturable state can be quickly obtained, so that the study on the bacteria in the living non-culturable state is facilitated, and the application prospect is good.
The present invention provides a method for increasing the proportion of bacteria that enter a viable, non-culturable state. According to an embodiment of the invention, the method comprises: pretreating the target bacteria to obtain pretreated target bacteria; subjecting the pretreated target bacteria to high-pressure carbon dioxide treatment so as to bring the pretreated target bacteria into a viable non-culturable state; wherein the pretreatment comprises an acid treatment and/or a heat treatment.
Earlier studies found that bacteria can be induced to enter a viable but non-culturable state by high pressure carbon dioxide treatment, but the proportion of bacteria in the viable but non-culturable state obtained is low and does not meet the purity requirements for viable but non-culturable bacteria well. Furthermore, the inventor finds that the bacteria are subjected to acid treatment and/or heat treatment in advance before the high-pressure carbon dioxide treatment, so that the bacteria can be effectively induced to enter a living non-culturable state, the ratio of the number of the living non-culturable state bacteria in the total bacteria obtained after the final treatment to the number of the total bacteria is increased, the yield of the living non-culturable bacteria is increased, the study on the living non-culturable state bacteria is facilitated, and the application prospect is good.
According to an embodiment of the invention, the method for increasing the proportion of bacteria that enter a viable, non-culturable state may also have the following additional technical features:
according to the embodiment of the invention, the acid treatment is carried out for 2-6 hours under the condition that the pH value is 3.5-5.5. The inventors have conducted a large number of experiments to obtain the above-mentioned preferable treatment conditions, whereby it is possible to efficiently induce bacteria to enter a viable but non-culturable state, and also to prevent bacteria from dying due to excessively low activity during the acid treatment process or from failing to return to a viable but non-culturable state. Specifically, the acid treatment is carried out at normal temperature and pressure.
According to an embodiment of the present invention, the acid treatment is performed by placing the target bacteria in an acid solution, wherein the pH value of the physiological saline solution is adjusted to 3.5-5.5 by hydrochloric acid. The acid liquor can provide acidic conditions and prevent bacteria from dying due to too low activity in the acid treatment process.
According to the embodiment of the invention, the heat treatment is carried out at 42-60 ℃ for 2-6 hours. The inventors have conducted a large number of experiments to obtain the above-mentioned preferable treatment conditions, whereby it is possible to efficiently induce bacteria to enter a viable but non-culturable state, and also to prevent the bacteria from dying due to excessively low activity during the heat treatment or from failing to return to a viable but non-culturable state after entering the viable but non-culturable state.
According to the embodiment of the invention, the high-pressure carbon dioxide treatment is carried out at the temperature of 25-37 ℃ and under the pressure of 5-7 MPa for 5-60 min. The inventor obtains the better treatment conditions through a large number of experiments, so that the bacteria can be efficiently induced to enter a living non-culturable state, and the death of the bacteria caused by too low activity in the high-pressure carbon dioxide treatment process can be avoided.
According to an embodiment of the present invention, the pretreated target bacteria are washed or suspended to obtain a cell suspension before the high pressure carbon dioxide treatment. Thereby facilitating removal of the culture/treatment solution of bacteria, especially the acid solution used for the acid treatment.
According to an embodiment of the present invention, the washing and the suspending are performed using physiological saline. Thereby, the subsequent high pressure carbon dioxide treatment is not disturbed.
According to an embodiment of the invention, the bacteria of interest are in log phase. Thus, the bacteria can be better induced to enter a viable but non-culturable state, and can also be prevented from dying due to low activity during the treatment process.
According to an embodiment of the invention, the bacterium of interest is Escherichia coli. The treatment conditions can better induce the escherichia coli to enter a living non-culturable state, and can also avoid the escherichia coli from dying due to low activity in the treatment process or being incapable of recovering to a culturable state after entering the living non-culturable state.
According to an embodiment of the invention, the bacteria of interest areEscherichia coliO157: H7. better induction can be achieved by adopting the treatment conditionsEscherichia coliO157: h7 can be in a living non-culturable state, so that it can be prevented from dying due to low activity in the treatment process or being incapable of being restored to a culturable state after being in the living non-culturable state.
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.
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The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows the ratio of high pressure carbon dioxide induction of E.coli and S.aureus into viable non-culturable bacteria after 4 hours of pretreatment;
FIG. 2 shows the ratio of the induction of E.coli into viable non-culturable bacteria with high pressure carbon dioxide after 24 hours of pretreatment.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
In this example, E.coli was induced in the following mannerEscherichia coliO157: h7 or Staphylococcus aureusS.aureusInto living beingNon-culturable state:
the log phase bacteria were centrifuged at 25 ℃ and 6000 g for 10 minutes. Washing twice with 5 mL of 0.85% NaCl, pretreating bacteria with acid solution (hydrochloric acid-normal saline) with pH of 5.5 for 4 hr and 24 hr, washing with normal saline, suspending thallus, and treating with high pressure carbon dioxide at 25 deg.C and 5 MPa for 25 min to make it enter into viable but non-culturable state.
Example 2
In this example, E.coli was induced in the following mannerEscherichia coliO157: h7 or Staphylococcus aureusS.aureusEntering a viable, non-culturable state:
the log phase bacteria were centrifuged at 25 ℃ and 6000 g for 10 minutes. Washing twice with 5 mL of 0.85% NaCl, pretreating bacteria at 45 deg.C for 4 hr and 24 hr, washing with physiological saline, suspending, and treating with high pressure carbon dioxide at 25 deg.C and 5 MPa for 25 min to make it in a living non-culturable state.
Comparative example 1
In this comparative example, E.coli was induced in the following mannerEscherichia coliO157: h7 or Staphylococcus aureusS.aureusEntering a viable, non-culturable state:
the log phase bacteria were centrifuged at 25 ℃ and 6000 g for 10 minutes. The cells were washed twice with 5 mL of 0.85% NaCl, left at room temperature (25 ℃) for 4 hours and 24 hours, respectively, washed with physiological saline, suspended, and then treated with high-pressure carbon dioxide at 25 ℃ and 5 MPa for 25 minutes to bring the cells into a viable but non-culturable state.
Comparative example 2
In this comparative example, E.coli was induced in the following mannerEscherichia coliO157: h7 or Staphylococcus aureusS.aureusEntering a viable, non-culturable state:
the log phase bacteria were centrifuged at 25 ℃ and 6000 g for 10 minutes. Washing twice with 5 mL of 0.85% NaCl, pretreating bacteria at 4 deg.C for 4 hr and 24 hr, washing with physiological saline, suspending the bacteria, and treating with high pressure carbon dioxide at 25 deg.C and 5 MPa for 25 min to make them in a living non-culturable state.
Comparative example 3
In this comparative example, E.coli was induced in the following mannerEscherichia coliO157: h7 or Staphylococcus aureusS.aureusEntering a viable, non-culturable state:
the log phase bacteria were centrifuged at 25 ℃ and 6000 g for 10 minutes. Washed twice with 5 mL of 0.85% NaCl and the bacteria were plated at 100. mu. M H2O2After pretreatment for 4 hours and 24 hours, respectively, the thalli are washed and suspended by normal saline, and then are treated by high-pressure carbon dioxide at 25 ℃ and 5 MPa for 25 min to be in a living non-culturable state.
Comparative example 4
In this comparative example, E.coli was induced in the following mannerEscherichia coliO157: h7 or Staphylococcus aureusS.aureusEntering a viable, non-culturable state:
stationary phase bacteria were centrifuged at 25 ℃ and 6000 g for 10 minutes. Washing twice with 5 mL of 0.85% NaCl, pretreating bacteria in 0.3M NaCl solution for 4 hr and 24 hr, washing with normal saline, suspending the bacteria, and treating with high pressure carbon dioxide at 25 deg.C and 5 MPa for 25 min to make them in a living non-culturable state.
Example 3
The bacterial liquids obtained in the embodiment 1, the embodiment 2 and the comparative examples 1 to 4 are respectively analyzed, and the specific experimental steps are as follows:
respectively counting the bacterial liquid obtained by the high-pressure carbon dioxide treatment by using a flow cytometer and a plate, and calculating the VBNC ratio A of the non-culturable bacteria according to the following formula:
A=（C-B）/C×100%，
wherein B is the number of bacteria counted by the plate and C is the number of bacteria counted by the flow cytometer.
As shown in FIG. 1, the VBNC ratio after pretreatment for Escherichia coli for 4 hours and room temperature pretreatment as no pretreatment was changed significantly compared with the control group, and the VBNC ratios after cold pretreatment (comparative example 2), oxidative stress (comparative example 3) and high-osmotic pretreatment (comparative example 4) were all changed significantly as compared with the control group, indicating that the VBNC ratios could not be increased significantly by the three treatment methods. The ratio of VBNC after acid pretreatment (example 1) was 79% and the ratio of VBNC after heat pretreatment (example 2) was 77%, and these two treatment methods were able to significantly increase the ratio of viable non-culturable bacteria of escherichia coli compared to the control group.
Compared with a control group, cold pretreatment, oxidation stress, high-permeability pretreatment, acid pretreatment and heat pretreatment have no significant change after the staphylococcus aureus is treated for 4 hours. Thus, it was shown that neither staphylococcus aureus could significantly increase the proportion of viable non-culturable bacteria after the above pretreatment.
As shown in fig. 2, after pretreatment for 24 hours, no significant change was observed in cold pretreatment, oxidative stress, high-osmotic pretreatment, acid pretreatment, and heat pretreatment, as compared with the control group. Thus, it was shown that the pretreatment time was too long and the proportion of viable non-culturable bacteria could not be increased.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean 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 invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer 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, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (7)

1. A method of increasing the proportion of bacteria that enter a viable, non-culturable state, comprising:
pretreating target bacteria to obtain pretreated target bacteria;
subjecting the pretreated target bacteria to high-pressure carbon dioxide treatment so as to bring the pretreated target bacteria into a viable non-culturable state;
wherein the pre-treatment comprises an acid treatment and/or a heat treatment;
the acid treatment is carried out for 2-6 hours under the condition that the pH value is 3.5-5.5;
the heat treatment is carried out for 2-6 hours at 42-60 ℃;
the target bacterium is Escherichia coli.
2. The method according to claim 1, wherein the acid treatment is carried out by placing the target bacteria in an acid solution obtained by adjusting a physiological saline solution to a pH of 3.5 to 5.5 with hydrochloric acid.
3. The method according to claim 1, wherein the high pressure carbon dioxide treatment is carried out at a temperature of 25 to 37 ℃ and a pressure of 5 to 7MPa for a period of 5 to 60 min.
4. The method according to claim 1, wherein the pretreated target bacteria are washed or suspended to obtain a cell suspension before the high pressure carbon dioxide treatment.
5. The method according to claim 4, wherein the washing and suspending are both performed with physiological saline.
6. The method of claim 1, wherein the bacteria of interest are in log phase.
7. The method of claim 1, wherein the method is performed in a batch processThe target bacterium isEscherichia coli O157：H7。
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