VBNC Escherichia coli induction method, VBNC Escherichia coli recovery culture medium and application

Abstract

The invention relates to the technical field of microorganisms, and discloses a VBNC Escherichia coli induction method, a VBNC Escherichia coli resuscitation medium and application, and the VBNC Escherichia coli resuscitation medium comprises Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, cholate, neutral red, crystal violet and agar. The VBNC escherichia coli resuscitation culture medium provided by the invention can be used for successfully culturing escherichia coli in a VBNC state.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to an induction method of VBNC Escherichia coli and a VBNC Escherichia coli recovery culture medium and application thereof. Background Art

[0002] Escherichia coli (scientific name: Escherichia coli), also known as Escherichia coli, is a common prokaryotic organism that can be divided into six categories based on its biological characteristics: enteropathogenic, enterotoxigenic, enteroinvasive, enterohemorrhagic, enteroadhesive, and diffusely adherent Escherichia coli. Escherichia coli is classified as a Gram-negative bacterium that mainly parasitizes in the large intestine of humans and animals, accounting for about 1% of intestinal bacteria, and does not cause disease under normal circumstances.

[0003] Under existing technical conditions, Escherichia coli may enter a non-culturable (VBNC) state. This characteristic poses a challenge to detection methods based on conventional culture media and can easily lead to false negative results. U.S. patent application US20200340040A1 uses PCR amplification technology to detect microorganisms in a VBNC state, but the method is cumbersome and costly. Existing culture detection technology cannot effectively detect samples in a VBNC Escherichia coli contaminated state, forming a blind spot in the field of Escherichia coli detection in food and medicine, posing a serious threat to public health. Therefore, there is an urgent need for a culture medium that can successfully detect VBNC Escherichia coli. Summary of the invention

[0004] The invention provides a VBNC Escherichia coli induction method, a VBNC Escherichia coli recovery culture medium and an application thereof.

[0005] The technical solution adopted by the present invention is as follows.

[0006] The invention provides a VBNC Escherichia coli recovery medium, wherein the VBNC Escherichia coli recovery medium comprises Rpf recombinant protein, MgSO4, sodium pyruvate, NAD (nicotinamide adenine dinucleotide), peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet and agar, wherein the mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet and agar is (90 ng / L-11 0ng / L): (0.5g / L-0.7g / L): (0.27g / L-2.2g / L): (0.5g / L-3g / L): (5g / L-20g / L): (2g / L-4g / L): (8g / L-1 2g / L): (4g / L-6g / L): (1.2g / L-1.7g / L): (0.02g / L-0.04g / L): (0.001g / L-0.003g / L): (14g / L-16g / L).

[0007] Furthermore, the nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1.

[0008] Furthermore, the VBNC Escherichia coli recovery medium includes Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, bile salts, neutral red, crystal violet and agar, and the mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, bile salts, neutral red, crystal violet and agar is 100ng / L: 0.6g / L: 1.1g / L: 2g / L: 10g / L: 3g / L: 10g / L: 5g / L: 1.5g / L: 0.03g / L: 0.002g / L: 15g / L.

[0009] Furthermore, the pH value of the VBNC Escherichia coli recovery culture medium is 7-7.5.

[0010] The present invention also provides application of the VBNC Escherichia coli recovery culture medium in culturing VBNC state Escherichia coli.

[0011] Further, the VBNC Escherichia coli is cultured in a VBNC Escherichia coli recovery medium at a temperature of 36° C.-38° C. for 18 h-38 h.

[0012] The present invention also provides an induction method for the VBNC Escherichia coli, the induction method comprising: The pH value of the solution containing vancomycin and sodium chloride is adjusted with glacial acetic acid, and the mixture is mixed evenly. The Escherichia coli that is not in the VBNC state is transferred into the mixed solution, and incubated at a temperature of 2°C to 8°C for 22h to 24h. The mass concentration of vancomycin in the solution containing vancomycin and sodium chloride is 6 mg / L-7 mg / L.

[0013] Furthermore, before transferring the E. coli that is not in the VBNC state, the mixed solution is sterilized.

[0014] Furthermore, the pH value of the solution containing vancomycin and sodium chloride is adjusted to 2.6-3 using glacial acetic acid.

[0015] Furthermore, the mass concentration of sodium chloride in the solution containing vancomycin and sodium chloride is 140 g / L-160 g / L.

[0016] The present invention has the following technical effects: 1. The Rpf recombinant protein proposed in the present invention has good peptidoglycan hydrolase activity, which can degrade the peptidoglycan of the Escherichia coli cell wall, thereby making the cell wall loose, and the degraded peptidoglycan can enter the cell through the loose cell wall, thereby resuscitating the cell, and the magnesium ions in the magnesium sulfate provide divalent metal ions for the activity of the Rpf recombinant protein. Sodium pyruvate is not only a scavenger of active oxygen free radicals, but also removes the active oxygen free radicals that damage DNA in the cell, which is beneficial to the cell recovery state, and can also be used as an energy substance, playing an important role in the glycolysis process. The addition of NAD can promote the joint action of Rpf recombinant protein, magnesium sulfate and sodium pyruvate, which can significantly promote Escherichia coli to release the VBNC state.

[0017] 2. The induction method of the present invention can prepare a culturable Escherichia coli suspension with a 0 CFU / mL in a relatively short period of time, while maximizing the total number of viable bacteria in the bacterial suspension, ensuring that the prepared VBNC state Escherichia coli suspension can be used as a reference for evaluating the growth performance of the recovery culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the induced data results provided by the embodiments and comparative examples of the present invention. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] In the first aspect, some embodiments of the present invention provide a VBNC Escherichia coli recovery medium, wherein the VBNC Escherichia coli recovery medium comprises Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, bile salts, neutral red, crystal violet and agar, wherein the mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, bile salts, neutral red, crystal violet and agar is (90 ng / L-11 0ng / L): (0.5g / L-0.7g / L): (0.27g / L-2.2g / L): (0.5g / L-3g / L): (5g / L-20g / L): (2g / L-4g / L): (8g / L-1 2g / L): (4g / L-6g / L): (1.2g / L-1.7g / L): (0.02g / L-0.04g / L): (0.001g / L-0.003g / L): (14g / L-16g / L).

[0021] In some embodiments, the nucleotide sequence of the Rpf recombinant protein is shown as SEQ ID NO.1.

[0022] In some embodiments, the VBNC Escherichia coli recovery medium further comprises water.

[0023] In some embodiments, the VBNC Escherichia coli recovery medium includes Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, bile salts, neutral red, crystal violet and agar, and the mass concentration ratio of Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, bile salts, neutral red, crystal violet and agar is 100ng / L: 0.6g / L: 1.1g / L: 2g / L: 10g / L: 3g / L: 10g / L: 5g / L: 1.5g / L: 0.03g / L: 0.002g / L: 15g / L.

[0024] In some embodiments, the pH value of the VBNC Escherichia coli recovery medium is 7-7.5.

[0025] In a second aspect, some embodiments of the present invention also provide use of the VBNC Escherichia coli recovery medium in culturing VBNC Escherichia coli.

[0026] In some embodiments, the VBNC Escherichia coli is cultured in a VBNC Escherichia coli recovery medium at a temperature of 36° C.-38° C. for 18 h-38 h.

[0027] In a third aspect, the present invention further provides an induction method for the VBNC Escherichia coli, the induction method comprising: The pH value of the solution containing vancomycin and sodium chloride is adjusted with glacial acetic acid, and the mixture is mixed evenly. The Escherichia coli that is not in the VBNC state is transferred into the mixed solution, and incubated at a temperature of 2°C to 8°C for 22h to 24h. The mass concentration of vancomycin in the solution containing vancomycin and sodium chloride is 6 mg / L-7 mg / L.

[0028] In some embodiments, the solution containing vancomycin and sodium chloride is prepared by dispersing vancomycin and sodium chloride in water.

[0029] In some embodiments, the mixed solution is sterilized before transferring the E. coli that is not in the VBNC state.

[0030] In some embodiments, the pH value of the solution containing vancomycin and sodium chloride is adjusted to 2.6-3.0 using glacial acetic acid.

[0031] In some embodiments, the mass concentration of sodium chloride in the solution containing vancomycin and sodium chloride is 140 g / L-160 g / L.

[0032] The following is described in conjunction with the embodiments and comparative examples: Embodiment 1: 1. Preparation of VBNC Escherichia coli: (1) Inoculate E. coli ATCC25922 into LB medium (commercially available), culture overnight (14-16 hours) at 37°C and 180 rpm with shaking, and store at 2-8°C. Inoculate into fresh LB medium at a volume ratio of 1:100 (V:V) of bacterial liquid to medium to expand the culture of E. coli ATCC25922.

[0033] (2) Counting of bacterial content: Take 1 mL of fresh culture solution and make 10-fold serial dilutions with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4). Take 100 μL of the diluted 10 3 times, 10 4 times, 10 5 times, 10 6 The bacterial liquid was spread on LB agar medium, cultured in a 37℃ biochemical incubator overnight, and the colonies were observed and counted the next day.

[0034] (3) Stress induction: Wash the bacteria twice with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4). Dilute the bacteria to 1×10 7 CFU / mL, equal amount was packaged, 25mL / tube, centrifuged at 10000r / min for 10 minutes, the supernatant was removed, and the cells were resuspended with the stress solution equivalent to the original volume for induction treatment. The stress solution was a solution containing 6.4mg / L vancomycin and 150g / L NaCl. The pH value of the stress solution was adjusted to 2.8 with glacial acetic acid. After being evenly resuspended, the cells were placed in a refrigerator at 2-8℃, incubated, and samples were taken to detect the culturable count and total viable count.

[0035] (4) Determination of culturable bacterial count: After stress induction, the culturable microbial colonies in the bacterial solution were detected. The incubation time was determined based on the time when the culturable bacterial count was 0 CFU / mL. The detection method was to take 100 μL of the bacterial solution and count the bacteria according to the method in step (2) above. The culturable bacterial count in the bacterial solution was detected every 12 hours until the culturable bacterial count was 0 CFU / mL.

[0036] (5) Cultivable bacterial count: The plate technique is used for detection. When the test result is 0 CFU / mL, the incubation time is 24 h.

[0037] (6) Determination of total viable count: Take 1 mL of bacterial suspension 24 hours after stress induction. A large number of strains will die during the induction process. After induction, the bacterial suspension contains three states of bacteria: dead, culturable, and VBNC. The CTC Bacterial Viability Detection Kit (Tongren Chemical Research Institute) can be used to stain cells with respiratory activity, and then the total viable count can be detected by flow cytometry. The results are as follows: Figure 1 shown.

[0038] (7) The VBNC bacterial content was calculated according to the following formula: VBNC bacteria = total number of viable bacteria - number of culturable bacteria.

[0039] 2. Cultivate E. coli in VBNC state: Preparation of VBNC Escherichia coli recovery medium: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (1.1 g / L), NAD (2 g / L), peptone (10 g / L), yeast extract (3 g / L), lactose (10 g / L), sodium chloride (5 g / L), bile salt (1.5 g / L), neutral red (0.03 g / L), crystal violet (0.002 g / L) and agar (15 g / L), the balance is water. MgSO4, peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet, agar and water were mixed, and then the pH value of the medium was adjusted to 7.4 and sterilized at 121 ° C for 15 minutes; Rpf recombinant protein, sodium pyruvate and NAD were sterilized by filtration. When the culture medium after high-pressure sterilization is cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate and NAD are added.

[0040] Among them, the nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1, and SEQ ID NO.1 is:.

[0041] Source of Rpf recombinant protein: The Rpf recombinant protein of the present invention is obtained by mutating the amino acid sequence of the Rpf protein of Micrococcus luteus, and sending the mutated nucleotide sequence to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0042] The nucleotide sequence of the Micrococcus luteus Rpf protein before mutation is shown in SEQ ID NO.2, and SEQ ID NO.2 is:.

[0043] The enzyme activity of the Rpf recombinant protein with the nucleotide sequence of SEQ ID NO.1 and the Rpf protein with the nucleotide sequence of SEQ ID NO.2 was tested, and it was found that the enzyme activity of the Rpf recombinant protein with the nucleotide sequence of SEQ ID NO.1 was 317U / mL, while the enzyme activity of the protein with the original nucleotide sequence of SEQ ID NO.2 was 83U / mL. The protease activity after mutation was increased by more than 2 times.

[0044] Embodiment 2: VBNC Escherichia coli recovery medium of Example 2: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (0.55 g / L), NAD (1 g / L), peptone (10 g / L), yeast extract (3 g / L), lactose (10 g / L), sodium chloride (5 g / L), bile salt (1.5 g / L), neutral red (0.03 g / L), crystal violet (0.002 g / L) and agar (15 g / L), and the balance is water. MgSO4, peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet, agar and water are mixed, and then the pH value of the medium is adjusted to 7.4, and autoclaved at 121°C for 15 minutes; Rpf recombinant protein, sodium pyruvate and NAD are sterilized by filtration. When the culture medium after high-pressure sterilization is cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate and NAD are added.

[0045] Embodiment 3: The VBNC Escherichia coli recovery medium of Example 3: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (0.275 g / L), NAD (0.5 g / L), peptone (10 g / L), yeast extract (3 g / L), lactose (10 g / L), sodium chloride (5 g / L), bile salt (1.5 g / L), neutral red (0.03 g / L), crystal violet (0.002 g / L) and agar (15 g / L), and the balance is water. MgSO4, peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet, agar and water are mixed, and then the pH value of the medium is adjusted to 7.4, and sterilized at 121°C for 15 minutes; Rpf recombinant protein, sodium pyruvate and NAD are sterilized by filtration. When the culture medium after high-pressure sterilization is cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate and NAD are added.

[0046] Embodiment 4: VBNC Escherichia coli recovery medium of the embodiment: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (2.2 g / L), NAD (3 g / L), peptone (10 g / L), yeast extract (3 g / L), lactose (10 g / L), sodium chloride (5 g / L), bile salt (1.5 g / L), neutral red (0.03 g / L), crystal violet (0.002 g / L) and agar (15 g / L), and the balance is water. MgSO4, peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet, agar and water are mixed, and then the pH value of the medium is adjusted to 7.4, and autoclaved at 121°C for 15 minutes; Rpf recombinant protein, sodium pyruvate and NAD are sterilized by filtration. When the culture medium after high-pressure sterilization is cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate and NAD are added.

[0047] Comparative Example 1: (1) Inoculate E. coli ATCC25922 into LB medium (commercially available), culture overnight (14-16 hours) at 37°C and 180 rpm with shaking, and store in a refrigerator at 2-8°C. Inoculate fresh LB medium at a volume ratio of 1:100 (V:V) to expand the culture of E. coli ATCC25922.

[0048] (2) Counting of bacterial content: Take 1 mL of fresh culture solution and make 10-fold serial dilutions with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4). Take 100 μL of the diluted 10 3 times, 10 4 times, 10 5 times, 10 6 The bacterial liquid was spread on LB agar medium, cultured in a 37℃ biochemical incubator overnight, and the colonies were observed and counted the next day.

[0049] (3) Stress induction: Wash the bacteria twice with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4). Dilute the bacteria to 1×10 7 CFU / mL, equal amount was packaged, 25mL / tube, centrifuged at 10000r / min for 10 minutes, the supernatant was removed, and the cells were resuspended with the stress solution equivalent to the original volume for induction treatment. The stress solution was an aqueous solution containing 150g / L NaCl (sodium chloride was dispersed in the aqueous solution), and the pH value of the stress solution was adjusted to 2.8±0.2 with glacial acetic acid. The cells were placed in a refrigerator at 2-8℃, incubated, and samples were taken to detect the culturable count and total viable count.

[0050] (4) Determination of culturable bacterial count: After stress induction, the culturable microbial colonies in the bacterial solution were detected. The incubation time was determined based on the time when the culturable bacterial count was 0 CFU / mL. The detection method was to take 100 μL of the bacterial solution and count the bacteria according to the method in step (2) above. The culturable bacterial count in the bacterial solution was detected every 12 hours until the culturable bacterial count was 0 CFU / mL.

[0051] (5) Cultivable bacterial count: The plate technique is used for detection. When the test result is 0 CFU / mL, the incubation time is 7 days.

[0052] (6) Determination of total viable count: Take 1 mL of bacterial suspension 7 days after stress induction. A large number of strains will die during the induction process. After induction, the bacterial suspension contains three states of dead, culturable and VBNC bacteria. The CTC Bacterial Viability Detection Kit (Tongren Chemical Research Institute) can be used to stain cells with respiratory activity, and then the total viable count can be detected by flow cytometry. The results are as follows: Figure 1 shown.

[0053] (7) The VBNC bacterial content was calculated according to the following formula: VBNC bacteria = total number of viable bacteria - number of culturable bacteria.

[0054] Comparative Example 2: (1) Inoculate E. coli ATCC25922 into LB medium (commercially available), culture overnight (14-16 hours) at 37°C and 180 rpm with shaking, and store at 2-8°C. Inoculate into fresh LB medium at a volume ratio of 1:100 (V:V) to expand the culture of E. coli ATCC25922.

[0055] (2) Counting of bacterial content: Take 1 mL of fresh culture solution and make 10-fold serial dilutions with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4). Take 100 μL of the diluted 10 3 times, 10 4 times, 10 5 times, 10 6 The bacterial liquid was spread on LB agar medium, cultured in a 37℃ biochemical incubator overnight, and the colonies were observed and counted the next day.

[0056] (3) Stress induction: Wash the bacteria twice with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4). Dilute the bacteria to 1×10 7CFU / mL, equal volume was divided into 25mL / tube, centrifuged at 10000r / min for 10 minutes, the supernatant was removed, and the cells were resuspended with the stress solution equivalent to the original volume for induction treatment. The stress solution was 6.4mg / L vancomycin aqueous solution (vancomycin dissolved and dispersed in water), incubated at room temperature, and samples were taken to detect the culturable count and total viable count.

[0057] (4) Determination of culturable bacterial count: After stress induction, the culturable microbial colonies in the bacterial solution were detected. The incubation time was determined based on the time when the culturable bacterial count was 0 CFU / mL. The detection method was to take 100 μL of the bacterial solution and count the bacteria according to the method in step (2) above. The culturable bacterial count in the bacterial solution was detected every 12 hours until the culturable bacterial count was 0 CFU / mL.

[0058] (5) Cultivable bacterial count: The plate technique is used for detection. When the test result is 0 CFU / mL, the incubation time is 21 days.

[0059] (6) Determination of total viable count: Take 1 mL of bacterial suspension 21 days after stress induction. A large number of strains will die during the induction process. After induction, the bacterial suspension contains three states of dead, culturable and VBNC bacteria. The CTC Bacterial Viability Detection Kit (Tongren Chemical Research Institute) can be used to stain cells with respiratory activity, and then the total viable count can be detected by flow cytometry. The results are as follows: Figure 1 shown.

[0060] (7) The VBNC bacterial content was calculated according to the following formula: VBNC bacteria = total number of viable bacteria - number of culturable bacteria.

[0061] Comparative Example 3: VBNC Escherichia coli recovery medium in Comparative Example 3: Rpf recombinant protein (100ng / L), MgSO4 (0.6g / L), peptone (10g / L), yeast extract (3g / L), lactose (10g / L), sodium chloride (5g / L), bile salt (1.5g / L), neutral red (0.03g / L), crystal violet (0.002g / L) and agar (15g / L), the balance is water. MgSO4, peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet, agar and water are mixed, and then the pH value of the medium is adjusted to 7.4, and autoclaved at 121°C for 15 minutes; Rpf recombinant protein is sterilized by filtration. When the autoclaved medium is cooled to 45°C-50°C, the sterilized Rpf recombinant protein is added.

[0062] Comparative Example 4: Comparative Example 4 VBNC Escherichia coli recovery medium: sodium pyruvate (0.275g / L), peptone (10g / L), yeast extract (3g / L), lactose (10g / L), sodium chloride (5g / L), bile salt (1.5g / L), neutral red (0.03g / L), crystal violet (0.002g / L) and agar (15g / L), the balance is water. Peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet, agar and water are mixed, and then the pH value of the medium is adjusted to 7.4, and autoclaved at 121°C for 15 minutes; sodium pyruvate is sterilized by filtration. When the autoclaved medium is cooled to 45°C-50°C, the sterilized sodium pyruvate is added.

[0063] Comparative Example 5: Comparative Example 5 VBNC Escherichia coli recovery medium: Rpf recombinant protein (100ng / L), MgSO4 (0.6g / L), sodium pyruvate (0.275g / L), peptone (10g / L), yeast extract (3g / L), lactose (10g / L), sodium chloride (5g / L), bile salt (1.5g / L), neutral red (0.03g / L), crystal violet (0.002g / L) and agar (15g / L), the balance is water. MgSO4, peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet, agar and water are mixed, and then the pH value of the medium is adjusted to 7.4, and autoclaved at 121°C for 15 minutes; Rpf recombinant protein and sodium pyruvate are sterilized by filtration. When the autoclaved medium is cooled to 45°C-50°C, the sterilized Rpf recombinant protein and sodium pyruvate are added.

[0064] The VBNC Escherichia coli recovery culture medium obtained in the examples and comparative examples was used to culture the prepared VBNC Escherichia coli, and the culture temperature was 37° C. and the culture time was 24 h.

[0065] The VBNC E. coli recovery culture medium obtained in the examples and comparative examples was tested: (1) Determination of the composition of VBNC E. coli recovery medium: Medium preparation: VBNC Escherichia coli recovery medium of the embodiment and comparative example were prepared respectively.

[0066] Bacterial liquid dilution: The total number of VBNC viable bacteria in the VBNC Escherichia coli working bacterial suspension obtained in Example 1 was adjusted to 10,000 cells / mL using a sterile PBS buffer solution.

[0067] Inoculation culture: Take 0.1mL of VBNC Escherichia coli suspension and inoculate it into the culture medium of the embodiment and the comparative example respectively, and set 3 replicates for each culture medium. Incubate at 37℃ for 24 hours. Take 1mL of culture solution, make 10-fold serial dilutions with sterile PBS buffer solution (10mmol, pH=7.2~7.4), take 100μL of the appropriate dilution solution and apply it on the tryptic soy peptone agar medium, incubate it in a 37℃ biochemical incubator overnight, and observe and count the colonies the next day.

[0068] Counting: Count the number of colonies growing on the plate. The results are shown in Table 1.

[0069] (2) Verification of VBNC Escherichia coli: 1 mL of the VBNC bacterial suspension prepared in the Example and the Comparative Example was added to LB medium containing 10 mM sodium pyruvate and LB medium without sodium pyruvate, respectively, and cultured at 37°C for 24 hours. The results showed that the LB medium containing sodium pyruvate was obviously turbid, while the LB medium without sodium pyruvate was clear. It can be seen that the methods of the Example and the Comparative Example successfully obtained VBNC Escherichia coli.

[0070] Taking the VBNC Escherichia coli recovery culture medium of Example 1 as an example, further research was conducted.

[0071] (3) Qualitative determination of growth rate: According to "SN / T 1538.2-2016 Guide to Culture Medium Preparation Part 2: Practical Guide to Culture Medium Performance Test", the growth rate test is used to detect the nutritional capacity of the culture medium for Escherichia coli and VBNC Escherichia coli. Inoculate no more than 100 cells of VBNC Escherichia coli liquid or Escherichia coli liquid to the reference plate (prepared according to the national standard method, the difference between the reference plate and the culture medium of the embodiment of the present invention is that the reference plate does not contain Rpf recombinant protein, sodium pyruvate, magnesium sulfate and NAD) and the VBNC Escherichia coli recovery medium of the present invention, wherein the VBNC Escherichia coli recovery medium obtained in Example 1 is prepared into a product, and three batches of products (P001, P002, P001) are extracted, and 3 samples (sample 1, sample 2, sample 3) are randomly extracted from each batch; and cultured at 37°C for 18 hours. Under ultraviolet light, observe and count the number of bacteria growing on the plate, and the growth rate PR=Ns / No. Wherein, PR represents the growth rate, Ns represents the average value of the total number of colonies on the test medium plate, and No represents the average value of the total number of colonies on the reference medium plate. The growth rate of the culture medium of the present invention for Escherichia coli should be not less than 0.7. The appearance, size and morphology of the colonies grown on each plate should all meet the characteristics of Escherichia coli, and the results are shown in Table 2.

[0072] (4) Example 1 Culture medium selectivity determination: 10 4CFU Enterococcus faecalis is inoculated on the VBNC Escherichia coli recovery culture medium of the present invention, evenly spread, and the plate is placed at 37° C. for 18 to 24 hours.

[0073] (5) Repeatability test of culture medium in Example 1: The VBNC E. coli recovery culture medium obtained in Example 1 was prepared into products, and three batches of products (P001, P002, P001) were selected. Three samples (sample 1, sample 2, sample 3) were randomly selected from each batch to detect the growth rate of E. coli and the number of VBNC E. coli recovery. Each product was tested three times in parallel. The differences between different batches of products and the same batch of products (coefficient of variation CV% = mean / standard deviation) are shown in Tables 3 and 4.

[0074] (6) Culture medium stability test of Example 1: The VBNC Escherichia coli recovery culture medium of the present invention was stored at 2°C-8°C. Culture medium samples were taken after 1 month, 2 months, 3 months, 6 months, 9 months, 12 months and 15 months of storage, and the product was tested for growth rate. The results are shown in Table 5.

[0075] (7) The VBNC Escherichia coli recovery culture medium obtained in Example 1 of the present invention was applied to the detection of tap water in a canteen in Yantai City. 10 water samples from different sampling points of tap water in a canteen in Yantai City and 10 water samples from different sampling points of a reservoir were collected. The test was carried out according to the plate counting method of "GB 4789.38-2012 National Food Safety Standard Food Microbiology Inspection Escherichia coli Count". The water sample was diluted 10 times in series, and 2 to 3 suitable sample homogenates of continuous dilution were selected. Each dilution was inoculated into 2 sterile plates, with 1 mL in each plate. At the same time, 1 mL of sterile physiological saline was added to the sterile plates as a blank control. At the same time, the VBNC Escherichia coli recovery culture medium of the present invention was prepared, and 10 to 15 mL of the VBNC Escherichia coli recovery culture medium was cooled to 45°C to 50°C, and then the cooled culture medium was poured into each plate. The plate was carefully rotated to fully mix the culture medium and the sample homogenate. After the agar solidifies, add 3-4 mL VRBA-MUG (crystal violet neutral red bile salt MUG agar medium) to cover the surface of the plate. After solidification, turn the plate over and incubate at 37°C for 18-24 hours. Select a plate with a colony count between 10 CFU and 100 CFU, and observe and count the number of bacteria growing on the plate under ultraviolet light. Set crystal violet neutral red bile salt agar medium as the national standard medium control.

[0076] Results and Analysis: Table 1 Test results of embodiments and comparative examples

[0077] Table 2 Growth rate test results

[0078] Table 3 Repeatability test results

[0079] Table 4 Repeatability test results of E. coli recovery medium

[0080] Table 5 Product stability test results

[0081] Among them, “+” indicates the detection of VBNC Escherichia coli.

[0082] Table 6 VBNC E. coli recovery medium test water sample results

[0083] First, the VBNC Escherichia coli was obtained by the induction method of the present invention, and other methods were compared. The results are as follows Figure 1 In the present invention, Example 1, Comparative Example 1 and Comparative Example 2 all use the same batch of Escherichia coli with the same concentration for testing, so the total number of viable bacteria in Example 1, Comparative Example 1 and Comparative Example 2 before induction is the same. Figure 1 It can be seen that the method of Example 1 can promote more E. coli ATCC25922 strains to enter the VBNC state in a shorter time while ensuring the survival rate of E. coli, and the culturable number is detected to be 0 cell / mL. Compared with other induction conditions, its VBNC bacteria concentration is the best.

[0084] As shown in Table 1, the Rpf recombinant protein in the culture medium of the present invention has good enzyme activity, can degrade the peptidoglycan of the Escherichia coli cell wall, so that the cell wall becomes loose, and the degraded peptidoglycan can enter the cell through the loose cell wall, thereby awakening the cell recovery, and the magnesium ions in magnesium sulfate provide divalent metal ions for the activity of the Rpf recombinant protein. It can be found in Comparative Example 3 that the addition of Rpf recombinant protein and magnesium sulfate to the culture medium can relieve the VBNC state of Escherichia coli. In Comparative Example 4, sodium pyruvate is not only a scavenger of active oxygen free radicals, but also removes the active oxygen free radicals that damage DNA in the cell, and the cell recovers the state, and can also be used as an energy substance, playing an important role in the glycolysis process, and the addition of sodium pyruvate alone can also prompt Escherichia coli to relieve the VBNC state. Compared with Comparative Example 4, the Rpf recombinant protein obtained by the present invention has good activity and can increase the number of VBNC Escherichia coli that can be cultivated. In Comparative Example 5, it was found that when Rpf recombinant protein, magnesium sulfate and sodium pyruvate were used together to culture VBNC Escherichia coli, the improvement effect was not obvious compared with Comparative Example 3. When NAD was added to the culture medium, the VBNC state of Escherichia coli could be significantly relieved. Compared with Comparative Example 5, the Escherichia coli cultured in Examples 1 to 4 were at least twice that of Comparative Example 5.

[0085] On this basis, the composition formula of the VBNC Escherichia coli recovery medium was further studied. It was found through Examples 1 to 3 that NAD increased with the increase of sodium pyruvate, which was beneficial to increase the number of VBNC Escherichia coli that could be cultivated in the medium; and it was found through Examples 1 and 4 that when sodium pyruvate was further increased, even if the amount of NAD added was increased, the increase in the number of VBNC Escherichia coli that could be cultivated could not be achieved. Therefore, it is further preferred that the mass concentration ratio of Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet and agar is 100ng / L: 0.6g / L: 1.1g / L: 2g / L: 10g / L: 3g / L: 10g / L: 5g / L: 1.5g / L: 0.03g / L: 0.002g / L: 15g / L.

[0086] The growth rate test results in Table 2 show that the VBNC E. coli recovery medium obtained in the present invention can successfully culture E. coli and VBNC E. coli, and the appearance, size and morphology of the colonies grown on each plate are consistent with the characteristics of E. coli, and are purple-red colonies with precipitation rings.

[0087] Enterococcus faecalis was inoculated on the VBNC Escherichia coli recovery medium of the present invention, evenly coated, and the plate was cultured at 37°C for 18 to 24 hours. The results showed that the Enterococcus faecalis was completely inhibited and no colony grew. It can be seen that the VBNC Escherichia coli recovery medium of the present invention has good selectivity.

[0088] The results in Table 3 show that the coefficient of variation of the growth rate within and between batches of the VBNC E. coli resuscitation medium of the present invention is not higher than 15%. The results in Table 4 show that the VBNC E. coli resuscitation medium of the present invention is used to detect the VBNC E. coli suspension, and the number of colonies grown is counted, and the coefficient of variation of the colonies within and between batches is calculated, and both are not higher than 15%. It can be seen that the VBNC E. coli resuscitation medium of the present invention has good repeatability.

[0089] The results in Table 5 show that within 15 months of storage, the growth rate of E. coli in the VBNC E. coli resuscitation medium was greater than 0.7, and VBNC E. coli could be detected, which met the requirements.

[0090] The results in Table 6 show that, when tested with VBNC E. coli resuscitation medium, E. coli was detected in 2 of 10 tap water samples and in 4 of 10 reservoir water samples. Therefore, the VBNC E. coli resuscitation medium prepared by the present invention has a higher detection rate for E. coli than the national standard medium and can be applied to actual inspections.

Claims

1. A VBNC Escherichia coli recovery medium, characterized in that: The VBNC Escherichia coli recovery medium comprises Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet and agar, and the mass concentration ratio of Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet and agar is (90ng / L-110ng / L): (0.5g / L-0 .7g / L): (0.27g / L-2.2g / L): (0.5g / L-3g / L): (5g / L-20g / L): (2g / L-4g / L): (8g / L-12g / L): (4 g / L-6g / L): (1.2g / L-1.7g / L): (0.02g / L-0.04g / L): (0.001g / L-0.003g / L): (14g / L-16g / L); The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.

1.

2. The VBNC Escherichia coli recovery medium according to claim 1, characterized in that The mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, NAD, peptone, yeast extract, lactose, sodium chloride, bile salt, neutral red, crystal violet and agar is 100 ng / L: 0.6 g / L: 1.1 g / L: 2 g / L: 10 g / L: 3 g / L: 10 g / L: 5 g / L: 1.5 g / L: 0.03 g / L: 0.002 g / L: 15 g / L.

3. The VBNC Escherichia coli recovery medium according to claim 1, characterized in that The pH value of the VBNC Escherichia coli recovery culture medium is 7-7.

5.

4. Use of the VBNC Escherichia coli recovery medium according to any one of claims 1 to 3 in culturing VBNC Escherichia coli.

5. The use according to claim 4, characterized in that: The VBNC Escherichia coli is cultured in a VBNC Escherichia coli recovery medium at a temperature of 36° C.-38° C. for 18 h-38 h.

6. A method for inducing VBNC Escherichia coli for use according to claim 5, characterized in that: The induction method comprises: The pH value of the solution containing vancomycin and sodium chloride is adjusted with glacial acetic acid, and the mixture is mixed evenly. The Escherichia coli that is not in the VBNC state is transferred to the mixed solution and incubated at 2°C-8°C for 22h-24h. The mass concentration of vancomycin in the solution containing vancomycin and sodium chloride is 6 mg / L-7 mg / L.

7. The induction method according to claim 6, characterized in that: Before transferring E. coli that are not in the VBNC state, the mixed solution is sterilized.

8. The induction method according to claim 6, characterized in that: The pH of the solution containing vancomycin and sodium chloride was adjusted to 2.6-3 using glacial acetic acid.

9. The induction method according to claim 6, characterized in that: The mass concentration of the sodium chloride in the solution containing vancomycin and sodium chloride is 140 g / L-160 g / L.

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