Induction method of VBNC state Listeria monocytogenes, resuscitation culture medium and application

Abstract

The invention relates to the technical field of microorganisms, and discloses a VBNC state Listeria monocytogenes induction method, a resuscitation medium and application, and the resuscitation medium comprises tryptone, multivalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, aesculin, nalidixic acid, acridine, Rpf recombinant protein and magnesium sulfate. The resuscitation culture medium obtained by the invention can be used for successfully culturing the Listeria in a VBNC state.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to an induction method and a recovery culture medium for Listeria in a VBNC state and applications thereof. Background Art

[0002] Listeria is a type of Gram-positive, short rod-shaped bacteria belonging to the family Listeriaceae. Listeria is commonly found in soil, water, plants and animal intestines. It is one of the important food-borne pathogens and can grow especially at refrigerated temperatures, posing a major threat to food safety.

[0003] Listeria enrichment broth (LB1), English name Listeria Enrichment Broth Base, is a commonly used enrichment culture medium for Listeria culture. The main components of Listeria enrichment broth culture medium include polyvalent peptone, disodium hydrogen phosphate, potassium dihydrogen phosphate, etc. There will be Listeria in VBNC state in the test environment, but the Listeria enrichment broth in the prior art cannot culture Listeria in VBNC state, resulting in errors in detection. U.S. patent application US20200340040A1 discloses the use of PCR amplification technology to detect microorganisms in VBNC state, but the technology is cumbersome and costly. Therefore, there is an urgent need for a culture medium that can successfully detect Listeria in VBNC state. Summary of the invention

[0004] The invention provides an induction method for Listeria in a VBNC state, a recovery culture medium and an application thereof.

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

[0006] A recovery medium, comprising tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein and magnesium sulfate (MgSO4), wherein the concentration ratio of the tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein and magnesium sulfate is (4g / L-6g / L): (4g / L -6g / L): (4g / L-6g / L): (11g / L-13g / L): (1.2g / L-1.6g / L): (19g / L-21g / L): (0.9g / L-1.1 g / L): (0.4mL / L-0.6mL / L): (0.2mL / L-0.4mL / L): (0.9μg / L-1.1μg / L): (0.5g / L-0.7g / L); The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1.

[0007] Furthermore, the recovery medium also includes sodium pyruvate and ferrous sulfate, and the concentration ratios of the tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and ferrous sulfate are (4g / L-6g / L): (4g / L-6g / L): (4g / L-6g / L): (11g / L-13g / L): (1. 2g / L-1.6g / L): (19g / L-21g / L): (0.9g / L-1.1g / L): (0.4mL / L-0.6mL / L): (0.2mL / L-0.4m L / L): (0.9μg / L-1.1μg / L): (0.5g / L-0.7g / L): (0.55g / L-2.2g / L): (0.76g / L-3.04g / L).

[0008] Furthermore, the concentration ratios of the tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and ferrous sulfate are 5g / L: 5g / L: 5g / L: 12g / L: 1.4g / L: 20g / L: 1g / L: 0.5mL / L: 0.3mL / L: 1μg / L: 0.6g / L: 1.1g / L: 1.52g / L.

[0009] Furthermore, the pH value of the recovery medium is 7.2-7.6.

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

[0011] Furthermore, the culture temperature is 36°C-38°C, and the culture time is 18h-36h.

[0012] The present invention also provides a method for inducing Listeria in a VBNC state, the induction method comprising: The pH value of the solution containing vancomycin and sodium chloride is adjusted with glacial acetic acid, mixed evenly, and Listeria that is not in the VBNC state is transferred to the mixed evenly solution, and incubated at a temperature of 2°C-8°C for 44h-52h; The mass concentration of the vancomycin in the solution containing vancomycin and sodium chloride is 45 μg / mL-55 μg / mL.

[0013] Furthermore, the pH value of the solution containing vancomycin and sodium chloride is adjusted to 2.6-3.0 using glacial acetic acid.

[0014] Furthermore, the mass concentration of sodium chloride in the solution containing vancomycin and sodium chloride is 140 g / L-160 g / L.

[0015] The present invention has the following technical effects: 1. Rpf recombinant protein has good activity and can decompose the peptidoglycan of Listeria, making the cell wall loose. The degraded peptidoglycan can enter the cell through the loose cell wall, thereby awakening Listeria in the VBNC state. The magnesium ions in magnesium sulfate provide divalent metal ions for the activity of Rpf recombinant protein, which is beneficial to the effect of Rpf recombinant protein on cells. Sodium pyruvate can not only act as a scavenger of reactive oxygen free radicals, remove reactive oxygen free radicals that damage DNA in cells, and restore the state of cells, but also act as an energy substance and play an important role in the glycolysis process. After adding ferrous sulfate, the synergistic effect of Rpf recombinant protein and sodium pyruvate can be significantly enhanced, awakening Listeria in the VBNC state.

[0016] 2. The induction method of the present invention successfully induces Listeria into the VBNC state on the basis of improving the survival rate of Listeria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a test result diagram of the embodiment of the present invention and the comparative example. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all 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.

[0019] In a first aspect, some embodiments of the present invention provide a recovery medium, which includes tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein and magnesium sulfate, and the concentration ratio of the tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein and magnesium sulfate is (4g / L-6g / L). L): (4g / L-6g / L): (4g / L-6g / L): (11g / L-13g / L): (1.2g / L-1.6g / L): (19g / L-21g / L): (0.9g / L -1.1g / L): (0.4mL / L-0.6mL / L): (0.2mL / L-0.4mL / L): (0.9μg / L-1.1μg / L): (0.5g / L-0.7g / L); The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1.

[0020] In some embodiments, the recovery medium further comprises sodium pyruvate and ferrous sulfate, and the concentration ratios of tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and ferrous sulfate are (4g / L-6g / L): (4g / L-6g / L): (4g / L-6g / L): (11g / L-13g / L): (1. 2g / L-1.6g / L): (19g / L-21g / L): (0.9g / L-1.1g / L): (0.4mL / L-0.6mL / L): (0.2mL / L-0.4m L / L): (0.9μg / L-1.1μg / L): (0.5g / L-0.7g / L): (0.55g / L-2.2g / L): (0.76g / L-3.04g / L).

[0021] In some embodiments, the recovery medium further comprises water.

[0022] In some embodiments, the recovery medium includes tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and ferrous sulfate, and the concentration ratio of the tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and ferrous sulfate is 5g / L: 5g / L: 5g / L: 12g / L: 1.4g / L: 20g / L: 1g / L: 0.5mL / L: 0.3mL / L: 1μg / L: 0.6g / L: 1.1g / L: 1.52g / L.

[0023] In some embodiments, the pH of the recovery medium is 7.2-7.6.

[0024] In a second aspect, some embodiments of the present invention further provide a use of a recovery medium in culturing Listeria in a VBNC state.

[0025] In some embodiments, the culture temperature is 36° C.-38° C., and the culture time is 18 h-36 h.

[0026] In a third aspect, some embodiments of the present invention further provide a method for inducing Listeria in a VBNC state, the induction method comprising: The pH value of the solution containing vancomycin and sodium chloride is adjusted with glacial acetic acid, mixed evenly, and Listeria that is not in the VBNC state is transferred to the mixed evenly solution, and incubated at a temperature of 2° C. to 8° C. for 44 h to 52 h.

[0027] In some embodiments, the solution containing vancomycin and sodium chloride is prepared by dispersing vancomycin and sodium chloride in water.

[0028] In some embodiments, the mixed solution is sterilized before transferring Listeria that are not in a VBNC state.

[0029] In some embodiments, the pH value of the solution containing vancomycin and sodium chloride is adjusted to 2.6-3 using glacial acetic acid.

[0030] In some embodiments, the mass concentration of vancomycin in the solution containing vancomycin and sodium chloride is 45 μg / mL-55 μg / mL.

[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 detail with reference to specific embodiments and comparative examples: Embodiment 1: 1. Preparation of VBNC Listeria: (1) Listeria monocytogenes ATCC19115 was inoculated into BHI liquid medium (commercially available), cultured at 37°C with shaking at 180 r / min overnight (14 to 16 hours), and stored in a refrigerator at 2 to 8°C. The bacterial liquid was inoculated into fresh BHI liquid medium at a volume ratio of 1:100 (V:V) for expansion culture.

[0033] (2) Counting bacterial content: Take 1 mL of the fresh culture solution from step (1) and make 10-fold serial dilutions with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4). Take 100 μL of the 10-fold dilution. 3 times, 10 4 times, 10 5 times, 10 6 The bacterial liquid was times the volume and spread on tryptic soy agar medium respectively, and cultured in a 37°C biochemical incubator overnight. 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 50μg / mL 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, it was placed in a refrigerator at 2℃~8℃, incubated statically, and samples were taken to detect the number of culturable bacteria and the total number of viable bacteria.

[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. 100 μL of the bacterial solution was taken and the bacterial count was performed according to the method in step (2) above to detect the number of culturable bacteria in the bacterial solution. The test was performed every 12 hours until the culturable bacterial count was 0 CFU / mL.

[0036] (5) Cultivable bacterial count: The plate count method is used for detection. When the test result is 0 CFU / mL, the incubation time is 48 h.

[0037] (6) Determination of total viable count: Take 1 mL of bacterial suspension 48 hours 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.

[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 Listeria in VBNC state: Prepare recovery medium: tryptone (5 g / L), polyvalent peptone (5 g / L), yeast extract (5 g / L), disodium hydrogen phosphate (12 g / L), potassium dihydrogen phosphate (1.4 g / L), sodium chloride (20 g / L), esculin (1 g / L), nalidixic acid (0.5 mL / L), acridine yellow (0.3 mL / L), Rpf recombinant protein (1 μg / L), magnesium sulfate (0.6 g / L), sodium pyruvate (1.1 g / L) and ferrous sulfate (1.52 g / L), the balance is water. Among them, magnesium sulfate, tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, aesculin and water are mixed, the pH value of the culture medium is adjusted to 7.4, and then it is sterilized at 121°C for 15 minutes; nalidixic acid, acridine yellow, Rpf recombinant protein, sodium pyruvate and ferrous sulfate are sterilized by filtration. When the culture medium sterilized at 121°C is cooled to 45°C-50°C, the sterilized nalidixic acid, acridine yellow, Rpf recombinant protein, sodium pyruvate and ferrous sulfate 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 Biotechnology (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 of sequence SEQ ID NO.1 and the Rpf protein of sequence SEQ ID NO.2 was tested, and it was found that the Rpf recombinant protein activity of sequence SEQ ID NO.1 of the present invention was 317U / mL, while the Rpf protein activity of the original sequence SEQ ID NO.2 without mutation was 83U / mL. It can be seen that the proteinase activity after mutation was increased by more than 2 times.

[0044] Embodiment 2: Prepare recovery medium: tryptone (5 g / L), polyvalent peptone (5 g / L), yeast extract (5 g / L), disodium hydrogen phosphate (12 g / L), potassium dihydrogen phosphate (1.4 g / L), sodium chloride (20 g / L), esculin (1 g / L), nalidixic acid (0.5 mL / L), acridine yellow (0.3 mL / L), Rpf recombinant protein (1 μg / L), magnesium sulfate (0.6 g / L), sodium pyruvate (0.55 g / L) and ferrous sulfate (3.04 g / L), the balance is water. Among them, magnesium sulfate, tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, aesculin and water are mixed, the pH value of the culture medium is adjusted to 7.4, and then it is sterilized at 121°C for 15 minutes; nalidixic acid, acridine yellow, Rpf recombinant protein, sodium pyruvate and ferrous sulfate are sterilized by filtration. When the culture medium sterilized at 121°C is cooled to 45°C-50°C, the sterilized nalidixic acid, acridine yellow, Rpf recombinant protein, sodium pyruvate and ferrous sulfate are added.

[0045] Embodiment 3: Prepare recovery medium: tryptone (5 g / L), polyvalent peptone (5 g / L), yeast extract (5 g / L), disodium hydrogen phosphate (12 g / L), potassium dihydrogen phosphate (1.4 g / L), sodium chloride (20 g / L), esculin (1 g / L), nalidixic acid (0.5 mL / L), acridine yellow (0.3 mL / L), Rpf recombinant protein (1 μg / L), magnesium sulfate (0.6 g / L), sodium pyruvate (2.2 g / L) and ferrous sulfate (0.76 g / L), the balance is water. Among them, magnesium sulfate, tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, aesculin and water are mixed, the pH value of the culture medium is adjusted to 7.4, and then it is sterilized at 121°C for 15 minutes; nalidixic acid, acridine yellow, Rpf recombinant protein, sodium pyruvate and ferrous sulfate are sterilized by filtration. When the culture medium sterilized at 121°C is cooled to 45°C-50°C, the sterilized nalidixic acid, acridine yellow, Rpf recombinant protein, sodium pyruvate and ferrous sulfate are added.

[0046] Embodiment 4: Prepare recovery medium: tryptone (5g / L), polyvalent peptone (5g / L), yeast extract (5g / L), disodium hydrogen phosphate (12g / L), potassium dihydrogen phosphate (1.4g / L), sodium chloride (20g / L), esculin (1g / L), nalidixic acid (0.5mL / L), acridine yellow (0.3mL / L), Rpf recombinant protein (1μg / L) and magnesium sulfate (0.6g / L), and the balance is water. Magnesium sulfate, tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin and water were mixed, the pH value of the medium was adjusted to 7.4, and then sterilized at 121℃ for 15 minutes; nalidixic acid, acridine yellow and Rpf recombinant protein were sterilized by filtration. When the culture medium sterilized at 121°C is cooled to 45°C-50°C, the sterilized nalidixic acid, acridine yellow and Rpf recombinant protein are added.

[0047] Comparative Example 1: Prepare recovery medium: tryptone (5g / L), polyvalent peptone (5g / L), yeast extract (5g / L), disodium hydrogen phosphate (12g / L), potassium dihydrogen phosphate (1.4g / L), sodium chloride (20g / L), esculin (1g / L), nalidixic acid (0.5mL / L), acridine yellow (0.3mL / L), and sodium pyruvate (1.1g / L), and the balance is water. Mix tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin and water, adjust the pH value of the medium to 7.4, and then sterilize at 121℃ for 15 minutes; nalidixic acid, acridine yellow, and sodium pyruvate are sterilized by filtration. When the culture medium sterilized at 121°C is cooled to 45°C-50°C, sterilized nalidixic acid, acridine yellow and sodium pyruvate are added.

[0048] Comparative Example 2: Prepare recovery medium: tryptone (5g / L), polyvalent peptone (5g / L), yeast extract (5g / L), disodium hydrogen phosphate (12g / L), potassium dihydrogen phosphate (1.4g / L), sodium chloride (20g / L), esculin (1g / L), nalidixic acid (0.5mL / L), acridine yellow (0.3mL / L), Rpf recombinant protein (1μg / L), magnesium sulfate (0.6g / L) and sodium pyruvate (1.1g / L), and the balance is water. Magnesium sulfate, tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin and water were mixed, the pH value of the medium was adjusted to 7.4, and then sterilized at 121℃ for 15 minutes; nalidixic acid, acridine yellow, Rpf recombinant protein and sodium pyruvate were sterilized by filtration. When the culture medium sterilized at 121° C. and autoclaved is cooled to 45° C.-50° C., sterilized nalidixic acid, acridine yellow, Rpf recombinant protein and sodium pyruvate are added.

[0049] Comparative Example 3: (1) Listeria monocytogenes ATCC19115 was inoculated into BHI liquid medium (commercially available), cultured overnight (14 to 16 hours) at 37°C with shaking at 180 r / min, and stored in a refrigerator at 2°C to 8°C. The bacterial liquid was inoculated into fresh BHI liquid medium at a volume ratio of 1:100 (V:V) for expansion culture.

[0050] (2) Counting bacterial content: Take 1 mL of the fresh culture solution from step (1) and make 10-fold serial dilutions with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4). Take 100 μL of the 10-fold dilution. 3 times, 10 4 times, 10 5 times, 10 6 The bacterial suspension was spread on tryptic soy agar medium, cultured in a 37°C biochemical incubator overnight, and the colonies were observed and counted the next day.

[0051] (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 150g / L NaCl (sodium chloride was dispersed in water), and the pH value of the stress solution was adjusted to 2.8 with glacial acetic acid. After being resuspended evenly, the cells were placed in a refrigerator at 2℃~8℃, incubated statically, and samples were taken to detect the number of culturable bacteria and the total number of viable bacteria.

[0052] (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. 100 μL of the bacterial solution was taken and the bacterial count was performed according to the method in step (2) above to detect the number of culturable bacteria in the bacterial solution. The test was performed every 12 hours until the culturable bacterial count was 0 CFU / mL.

[0053] (5) Cultivable bacterial count: The plate count method is used for testing. When the test result is 0 CFU / mL, the incubation time is 7 days.

[0054] (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.

[0055] (7) The VBNC bacterial content was calculated according to the following formula: VBNC bacteria = total number of viable bacteria - number of culturable bacteria.

[0056] Comparative Example 4: (1) Listeria monocytogenes ATCC19115 was inoculated into BHI liquid medium (commercially available), cultured at 37°C with shaking at 180 r / min overnight (14 to 16 hours), and stored in a refrigerator at 2 to 8°C. The bacterial liquid was inoculated into fresh BHI liquid medium at a volume ratio of 1:100 (V:V) for expansion culture.

[0057] (2) Counting bacterial content: Take 1 mL of the fresh culture solution from step (1) and make 10-fold serial dilutions with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4). Take 100 μL of the 10-fold dilution. 3 times, 10 4 times, 10 5 times, 10 6 The bacterial liquid was spread on tryptic soy agar medium and cultured in a 37°C biochemical incubator overnight. The colonies were observed and counted the next day.

[0058] (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 50μg / mL vancomycin (vancomycin was dispersed in water), and the pH value of the stress solution was adjusted to 2.8 with glacial acetic acid. After being evenly resuspended, it was placed in a refrigerator at 2-8℃, incubated statically, and samples were taken to detect the culturable count and total number of viable bacteria.

[0059] (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. 100 μL of the bacterial solution was taken and the bacterial count was performed according to the method in step (2) above to detect the number of culturable bacteria in the bacterial solution. The test was performed every 12 hours until the culturable bacterial count was 0 CFU / mL.

[0060] (5) Culturable bacterial count: The plate count method is used for testing. When the test result is 0 CFU / mL, the incubation time is 21 days.

[0061] (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 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.

[0062] (7) The VBNC bacterial content was calculated according to the following formula: VBNC bacteria = total number of viable bacteria - number of culturable bacteria.

[0063] Verification of VBNC state Listeria: 1 mL of the VBNC state bacterial suspension prepared in the examples and comparative examples was added to BHI medium containing 10 mM sodium pyruvate and BHI medium without sodium pyruvate, respectively, and cultured at 37°C for 24 hours. The results showed that the BHI medium containing sodium pyruvate was obviously turbid, while the BHI medium without sodium pyruvate was clear. It can be seen that the methods of the examples and comparative examples successfully obtained VBNC state Listeria.

[0064] The culture medium obtained in the example and the comparative example was used to culture the VBNC Listeria prepared in Example 1, the culture temperature was 37° C., and the culture time was 24 h: the recovery ability of the culture medium was further evaluated and the components of the recovery culture medium were determined.

[0065] Culture medium preparation: The culture medium of the embodiment and the comparative example were prepared respectively.

[0066] Bacterial solution dilution: Use sterile PBS buffer solution to adjust the total number of viable cells in the VBNC state of the Listeria monocytogenes working bacterial suspension to 10,000 cells / mL.

[0067] Inoculation culture: 0.1 mL of VBNC state Listeria monocytogenes working bacteria was inoculated into the culture medium of the embodiment and the comparative example respectively, and 3 replicates were set for each culture medium. Cultured at 37°C for 24 hours. Take 1 mL of the culture solution, make 10-fold serial dilutions with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4), take 100 μL of the appropriate dilution solution and apply it on the tryptic soy peptone agar medium, cultured in a 37°C 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] The culture medium obtained in Example 1 was further tested: (1) Nutritional capacity (semi-quantitative test of growth rate): refer to the semi-quantitative detection method of selective enrichment medium in GB4789.28-2024 to detect the enrichment effect of the resuscitation medium of the present invention on VBNC state Listeria monocytogenes and Listeria monocytogenes; Preparation and packaging of culture medium: Prepare national standard culture medium (unimproved recovery culture medium, formula: tryptone 5g / L, polyvalent peptone 5g / L, yeast extract 5g / L, disodium hydrogen phosphate 12g / L, potassium dihydrogen phosphate 1.4g / L, sodium chloride 20g / L, esculin 1g / L, nalidixic acid 0.5mL / L and acridine yellow 0.3mL / L), quantitatively package, 9mL / tube. Obtain the recovery culture medium prepared in Example 1, quantitatively package, 9mL / tube; Inoculation: Use sterile PBS buffer solution to adjust the VBNC monocytogenes ATCC19115 bacterial suspension to a total viable count of 1000 cells / mL, take 0.1 mL of the VBNC bacterial suspension and inoculate it into 9.9 mL of national standard culture medium and recovery culture medium, inoculate three parallel tubes of each culture medium or each component culture medium in parallel, and culture at 37°C for 18 hours; The total bacterial count of Listeria monocytogenes ATCC19115 suspension was adjusted to less than 100 CFU / mL with sterile PBS, and 0.1 mL of the bacterial suspension was taken and inoculated into 9.9 mL of national standard culture medium and Listeria enrichment broth recovery medium, respectively. Three parallel tubes were inoculated in each treatment group and cultured at 37°C for 18 hours; Plate count: Take 10 μL of each culture medium after 18 hours of culture and spread it evenly on TSA agar plates (commercially available). If the colonies on the TSA plate are greater than 10 CFU, the growth rate of the Listeria enrichment broth recovery medium is judged to be good.

[0070] (2) Selectivity: 1×10 3 ~5×10 3CFU Enterococcus faecalis strain CMCC (B) 32482 or Escherichia coli ATCC25922 were inoculated in 9.9 mL of national standard culture medium and recovery medium, respectively. Three parallel tubes (SaP001, SaP002, SaP003) were inoculated in each treatment group and cultured at 37°C for 18 hours. 10 μL of culture was taken from each culture medium after 18 hours of culture and evenly spread on TSA plates. The number of Enterococcus faecalis and / or Escherichia coli colonies on the TSA plates was <100 CFU, indicating that the recovery medium had good selectivity.

[0071] (3) Repeatability: A semi-quantitative test method was used to detect the intra-batch and inter-batch repeatability of the culture medium in accordance with GB4789.28-2024. Three boxes were randomly selected from each of the three batches of products to evaluate the nutritional performance of the culture medium for Listeria monocytogenes and VBNC Listeria monocytogenes, and to detect the selectivity for Enterococcus faecalis and Escherichia coli. Each batch of test kits was tested three times in parallel. The test method is as follows: Preparation and packaging of culture medium: Prepare national standard culture medium, quantitatively package, 9.9mL / tube. Take the recovery culture medium to be tested, quantitatively package, 9.9mL / tube.

[0072] Qualitative determination of growth rate; Selectivity assay.

[0073] (4) Stability test: Three batches of products were stored at 2-8°C. Culture medium samples were taken after 3 months, 6 months, 9 months, 12 months and 15 months of storage, and the nutritional capacity and selectivity of the products were tested.

[0074] (5) Clinical sample testing: Ten samples of milk from Shounong Dairy in Xingtai, Hebei (milk samples refer to milk samples collected from large refrigerated tanks used to store and cool freshly squeezed milk at dairy farms) were transported to the testing laboratory in cold storage within 2 hours after collection. According to the MPN counting method of GB4789.30 2016 National Food Safety Standard Food Microbiology Monocytogenes Listeria Test, Listeria was tested simultaneously with Listeria enrichment broth resuscitation medium and Listeria enrichment broth LB1 (commercially available national standard culture medium).

[0075] Results and analysis: Table 1 Culture medium of Examples and Comparative Examples

[0076] Table 2 Growth rate quantitative test results

[0077] Note: a: Turbidity calculation: 0 means no turbidity; 1 means very slight turbidity; 2 means severe turbidity, which is determined according to the evaluation standard of GB4789.28-2024; b: After enrichment of Listeria monocytogenes, Escherichia coli or Enterococcus faecalis, the cells were transferred to TSA plates for counting.

[0078] Table 3 Culture medium repeatability test results

[0079] Note: a: Turbidity calculation: 0 means no turbidity; 1 means very slight turbidity; 2 means severe turbidity, which is determined according to the evaluation standard of GB4789.28-2024; b: After enrichment of Listeria monocytogenes, Escherichia coli or Enterococcus faecalis, the cells were transferred to TSA plates for counting.

[0080] Table 4 Stability test results

[0081] Note: a: Turbidity calculation: 0 means no turbidity; 1 means very slight turbidity; 2 means severe turbidity, which is determined according to the evaluation standard of GB4789.28-2024; b: After enrichment of Listeria monocytogenes, Escherichia coli or Enterococcus faecalis, the cells were transferred to TSA plates for counting.

[0082] Table 5 Clinical trial results

[0083] The VBNC state Listeria was successfully obtained by the method of the present invention. Figure 1 The results show that compared with Comparative Examples 3 and 4, Example 1 not only improves the survival rate of Listeria, but also has more Listeria in the VBNC state in a shorter time. Compared with other induction conditions, the concentration of bacteria in the VBNC state is optimal.

[0084] In Table 1, the culture medium obtained in Examples 1 to 4 of the present invention can culture significantly more VBNC state Listeria than Comparative Examples 1 to 2. In the culture medium of Example 4, the Rpf recombinant protein and magnesium sulfate have a synergistic effect and can successfully awaken the VBNC state Listeria. Among them, the Rpf recombinant protein has good activity and can decompose the peptidoglycan of the cell wall of Listeria, and the cell wall becomes loose. The degraded peptidoglycan can enter the cell through the loose cell wall. The magnesium ions provided by magnesium sulfate are conducive to activating the activity of the Rpf recombinant protein and enhancing the effect of the Rpf recombinant protein on Listeria. The resurrection ability of the culture medium of Example 4 of the present invention is significantly better than that of Comparative Example 1 of the prior art.

[0085] By comparing Example 4 with Comparative Example 2, it was found that when sodium pyruvate was further added, the culture effect of the culture medium was significantly reduced, indicating that sodium pyruvate affected the activity of the Rpf recombinant protein. When Rpf recombinant protein, magnesium sulfate, sodium pyruvate and ferrous sulfate were used in the same culture medium (such as Examples 1-3), the culture effect of the culture medium on VBNC state Listeria was significantly improved, indicating that ferrous sulfate can relieve the inhibitory effect of sodium pyruvate on Rpf recombinant protein.

[0086] It should be understood by those skilled in the art that the interaction and coordination of the culture medium components activates Listeria in the VBNC state, thereby achieving a culturable process of the microorganism.

[0087] In the present invention, the effect of the culture medium is further enhanced by the mutual cooperation of sodium pyruvate and ferrous sulfate. For example, in Examples 1 to 3, when the molar concentration ratio of sodium pyruvate and ferrous sulfate is 1:1 (Example 1), the most VBNC state Listeria can be cultured. Therefore, it is further preferred that the concentration ratio of tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and ferrous sulfate is 5g / L: 5g / L: 5g / L: 12g / L: 1.4g / L: 20g / L: 1g / L: 0.5mL / L: 0.3mL / L: 1μg / L: 0.6g / L: 1.1g / L: 1.52g / L.

[0088] The results in Table 2 show that the bacterial content of the recovery culture medium enrichment liquid of the present invention is significantly higher than that of the national standard culture medium, so the recovery culture medium has a relatively high growth rate and good nutritional performance. And the recovery culture medium in the present invention can not only recover Listeria monocytogenes and Listeria monocytogenes in the VBNC state, but also has good selectivity. After Enterococcus faecalis and Escherichia coli were inoculated with the national standard culture medium and the recovery culture medium for enrichment, the enrichment liquid was not turbid. After plate transfer, the colony counts of Enterococcus faecalis and Escherichia coli in 10 μL of enrichment liquid were both 0, less than 100 CFU, indicating that the recovery culture medium has good selectivity.

[0089] The results in Table 3 show that after the three batches of recovery culture medium were used to enrich Listeria monocytogenes and its VBNC Listeria monocytogenes, 10 μL of the enrichment solution was transferred to MAC agar for counting, and colonies greater than 10 CFU appeared on the plate, indicating that the three batches of laboratory trial recovery culture medium had a good growth rate for Listeria monocytogenes. After the three batches of Listeria enrichment broth recovery culture medium were used to enrich Staphylococcus aureus, 10 μL of the enrichment solution was transferred to TSA plate for counting, and colonies less than 100 CFU appeared on the TSA plate, indicating that the three batches of laboratory trial recovery culture medium had good selectivity.

[0090] The results in Table 4 show that within 15 months of storage, the nutritional performance and selectivity of the Listeria enrichment broth resuscitation medium are good and meet the requirements of GB4789.28-2024.

[0091] The results in Table 5 show that Listeria monocytogenes was not detected in any of the 10 bulk milk samples when the Listeria enrichment broth LB1 (commercially available national standard culture medium) was used for fermentation testing, while Listeria monocytogenes was detected in 3 bulk milk samples when the Listeria enrichment broth resuscitation culture medium of the present invention was used for fermentation culture. This indicates that the Listeria enrichment broth resuscitation culture medium has a high sensitivity to Listeria monocytogenes in milk samples.

Claims

1. A recovery medium, characterized in that The recovery medium comprises tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein and magnesium sulfate, and the concentration ratio of the tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein and magnesium sulfate is (4g / L-6g / L): (4g / L-6g / L L): (4g / L-6g / L): (11g / L-13g / L): (1.2g / L-1.6g / L): (19g / L-21g / L): (0.9g / L-1.1g / L): (0.4mL / L-0.6mL / L): (0.2mL / L-0.4mL / L): (0.9μg / L-1.1μg / L): (0.5g / L-0.7g / L); The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.

1.

2. The recovery medium according to claim 1, characterized in that The recovery medium also includes sodium pyruvate and ferrous sulfate. The concentration ratios of the tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and ferrous sulfate are (4g / L-6g / L): (4g / L-6g / L): (4g / L-6g / L): (11g / L-13g / L): (1.2g / L-1.6g / L): (19g / L-21g / L): (0.9g / L-1.1g / L): (0.4mL / L-0.6mL / L): (0.2mL / L-0.4mL / L): (0.9μg / L-1.1μg / L): (0.5g / L-0.7g / L): (0.55g / L-2.2g / L): (0.76g / L-3.04g / L).

3. The recovery medium according to claim 2, characterized in that The concentration ratios of the tryptone, polyvalent peptone, yeast extract, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, esculin, nalidixic acid, acridine yellow, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and ferrous sulfate are 5g / L: 5g / L: 5g / L: 12g / L: 1.4g / L: 20g / L: 1g / L: 0.5mL / L: 0.3mL / L: 1μg / L: 0.6g / L: 1.1g / L: 1.52g / L.

4. The recovery medium according to claim 1, characterized in that The pH value of the recovery medium is 7.2-7.

6.

5. Use of the recovery medium according to any one of claims 1 to 4 in culturing Listeria in a VBNC state.

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

7. A method for inducing Listeria in VBNC state for use according to claim 6, characterized in that: The induction method comprises: The pH value of the solution containing vancomycin and sodium chloride is adjusted with glacial acetic acid, mixed evenly, and Listeria that is not in the VBNC state is transferred to the mixed evenly solution, and incubated at a temperature of 2°C-8°C for 44h-52h; The mass concentration of the vancomycin in the solution containing vancomycin and sodium chloride is 45 μg / mL-55 μg / mL.

8. The induction method according to claim 7, characterized in that: The pH of the solution containing vancomycin and sodium chloride was adjusted to 2.6-3.0 using glacial acetic acid.

9. The induction method according to claim 7, 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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