Induction method of VBNC state shigella, resuscitation culture medium and application of VBNC state shigella

Abstract

The invention relates to the technical field of microorganisms, and discloses an induction method of VBNC state shigella, a resuscitation medium and application, and the resuscitation medium comprises Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, tryptone, glucose, dipotassium phosphate, monopotassium phosphate, sodium chloride and Tween 80. The resuscitation culture medium obtained by the invention can be used for successfully culturing Shigella in a VBNC state.

Description

Technical Field

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

[0002] Shigella Castellani is a type of Gram-negative short rod, commonly known as Shigella dysenteriae. It is cold-resistant and can grow on ordinary agar medium for 24 hours to form translucent smooth colonies with a diameter of 2 mm. It has no spores, no capsules, no flagella, and most of them have pili. It is an intestinal pathogen of humans and primates, causing bacillary dysentery.

[0003] Under existing technical conditions, Shigella 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 discloses the use of 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 contaminated with Shigella in a VBNC state, forming a blind spot in the field of Shigella detection in food and medicine. Therefore, there is an urgent need for a culture medium that can successfully detect Shigella in a VBNC state. Summary of the invention

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

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

[0006] The present invention provides a recovery medium, which comprises Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80, wherein the concentration ratios of the Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80 are (90ng / L-110ng / L): (0.5g / L-0.7g / L): (0.27g / L-2.2g / L): (0.13mg / L-1mg / L): (15g / L-25g / L): (0.5g / L-1.5g / L): (1.5g / L-2.5g / L): (1.5g / L-2.5g / L): (4g / L-6g / L): (1mL / L-2mL / L); The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1.

[0007] Furthermore, the recovery medium includes Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, trypsin, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80, and the concentration ratio of Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, trypsin, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80 is 100ng / L: 0.6g / L: 1.65g / L: 0.5mg / L: 20g / L: 1g / L: 2g / L: 2g / L: 5g / L: 1.5mL / L.

[0008] Furthermore, the pH value of the recovery medium is 7.2-7.6.

[0009] The present invention also provides application of the recovery culture medium in culturing Shigella in VBNC state.

[0010] Furthermore, the VBNC state Shigella is cultured in a recovery medium at a temperature of 36° C.-38° C. for 18 h-48 h.

[0011] The present invention also provides a method for inducing Shigella in the VBNC state, the induction method comprising: adjusting the pH value of a solution containing vancomycin and sodium chloride with glacial acetic acid, mixing the solution evenly, transferring Shigella that is not in the VBNC state to the evenly mixed solution, and incubating the mixture at a temperature of 2° C. to 8° C. for 35 h to 37 h; The mass concentration of vancomycin in the solution containing vancomycin and sodium chloride is 150 μg / mL-250 μg / mL.

[0012] Furthermore, before transferring the Shigella that is not in the VBNC state, the mixed solution is sterilized.

[0013] Furthermore, the pH value of the solution containing vancomycin and sodium chloride is adjusted to 2.6-3 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. The Rpf recombinant protein in the culture medium of the present invention has good peptidoglycan hydrolase activity, which can degrade the peptidoglycan of the Shigella cell wall, making the cell wall loose, and the degraded peptidoglycan can enter the cell through the loose cell wall, thereby awakening the cell, and the magnesium ions in the magnesium sulfate provide divalent metal ions for the activity of the Rpf recombinant protein. Sodium pyruvate can not only be used as an active oxygen free radical scavenger to remove the active oxygen free radicals that damage DNA in the cell and restore the cell to a state, but can also be used as an energy substance to play an important role in the glycolysis process. Vitamin B12 can promote the interaction between Rpf recombinant protein, magnesium sulfate and sodium pyruvate, and relieve the VBNC state of Shigella.

[0016] 2. The induction method of the present invention can prepare a Shigella suspension with a culturable number of 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 Shigella suspension can be used as a reference for evaluating the growth performance of the recovery culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the stress test results of the embodiments of the present invention and the comparative examples. 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 Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80, wherein the concentration ratio of Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80 is (9 0ng / L-110ng / L): (0.5g / L-0.7g / L): (0.27g / L-2.2g / L): (0.13mg / L-1mg / L): (15g / L-25g / L): (0.5g / L-1.5g / L): (1.5g / L-2.5g / L): (1.5g / L-2.5g / L): (4g / L-6g / L): (1mL / L-2mL / L).

[0020] In some embodiments, the nucleotide sequence of the Rpf recombinant protein is shown as SEQ ID NO.1.

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

[0022] In some embodiments, the recovery medium includes Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, trypsin, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80, and the concentration ratio of Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, trypsin, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80 is 100ng / L: 0.6g / L: 1.65g / L: 0.5mg / L: 20g / L: 1g / L: 2g / L: 2g / L: 5g / L: 1.5mL / 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 also provide the use of a recovery medium in culturing Shigella in a VBNC state.

[0025] In some embodiments, the VBNC state Shigella is cultured in a recovery medium at a temperature of 36° C.-38° C. for 18 h-48 h.

[0026] In a third aspect, some embodiments of the present invention also provide a method for inducing Shigella in a VBNC state, the induction method comprising: adjusting the pH value of a solution containing vancomycin and sodium chloride with glacial acetic acid, mixing evenly, transferring Shigella that is not in a VBNC state to the evenly mixed solution, and incubating at a temperature of 2°C-8°C for 35h-37h; The mass concentration of vancomycin in the solution containing vancomycin and sodium chloride is 150 μg / mL-250 μg / mL.

[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 the Shigella that is not in the 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 sodium chloride in the solution containing vancomycin and sodium chloride is 140 g / L-160 g / L.

[0031] The following is described in conjunction with the embodiments and comparative examples: Embodiment 1: 1. Preparation of VBNC Shigella: (1) Inoculate Shigella flexneri CMCCB51572 into nutrient broth medium (commercially available), culture overnight (18 to 24 hours) at 37°C, and store in a refrigerator at 2°C to 8°C. Inoculate into nutrient broth medium at a volume ratio of 1:100 (V:V) to expand the culture of Shigella flexneri CMCCB51572.

[0032] (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 each dilution for 10 3 times, 10 4 times, 10 5 times, 10 6 Spread the bacterial solution of 100 times on TSA plates, culture them in a 37℃ biochemical incubator overnight, and observe and count the colonies the next day.

[0033] (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 200μg / 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, the cells were placed in a refrigerator at 2℃~8℃, incubated, and samples were taken to detect the culturable count and total viable count.

[0034] (4) Determination of culturable bacterial counts: 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. The culturable bacterial count in the bacterial solution was detected every 12 hours until the culturable bacterial count was 0 CFU / mL.

[0035] (5) Cultivable bacterial count: The plate count method is used for testing. When the test result is 0 CFU / mL, the incubation time is 36 hours.

[0036] (6) Determination of total viable count: Take 1 mL of bacterial suspension 36 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.

[0037] (7) The VBNC bacterial content was calculated according to the following formula: VBNC bacteria = total number of viable bacteria - number of culturable bacteria.

[0038] 2. Cultivate Shigella in VBNC state: Prepare recovery medium: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (1.65 g / L), vitamin B12 (0.5 mg / L), tryptone (20 g / L), glucose (1 g / L), dipotassium hydrogen phosphate (2 g / L), potassium dihydrogen phosphate (2 g / L), sodium chloride (5 g / L) and Tween 80 (1.5 mL / L), and the balance is water. MgSO4, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, Tween 80 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 vitamin B12 are sterilized by filtration. When the autoclaved medium is cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate and vitamin B12 are added.

[0039] Among them, the nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1, and SEQ ID NO.1 is:.

[0040] 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.

[0041] The nucleotide sequence of the Micrococcus luteus Rpf protein before mutation is shown in SEQ ID NO.2, and SEQ ID NO.2 is:.

[0042] The enzyme activity of the Rpf recombinant protein of nucleotide sequence SEQ ID NO.1 and the Rpf protein of nucleotide sequence SEQ ID NO.2 was tested, and it was found that the Rpf recombinant protease activity of nucleotide sequence SEQ ID NO.1 was 317U / mL, while the Rpf protease activity of the original nucleotide sequence SEQ ID NO.2 without mutation was 83U / mL. It can be seen that the recombinant protease activity after mutation was increased by more than 2 times.

[0043] Embodiment 2: Recovery medium of Example 2: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (0.275 g / L), vitamin B12 (0.13 mg / L), tryptone (20 g / L), glucose (1 g / L), dipotassium hydrogen phosphate (2 g / L), potassium dihydrogen phosphate (2 g / L), sodium chloride (5 g / L) and Tween 80 (1.5 mL / L), the balance is water. MgSO4, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, Tween 80 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 vitamin B12 are sterilized by filtration. When the autoclaved medium is cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate and vitamin B12 are added.

[0044] Embodiment 3: Recovery medium of Example 3: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (1.1 g / L), vitamin B12 (0.25 mg / L), tryptone (20 g / L), glucose (1 g / L), dipotassium hydrogen phosphate (2 g / L), potassium dihydrogen phosphate (2 g / L), sodium chloride (5 g / L) and Tween 80 (1.5 mL / L), the balance is water. MgSO4, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, Tween 80 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 vitamin B12 are sterilized by filtration. When the autoclaved medium is cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate and vitamin B12 are added.

[0045] Embodiment 4: Recovery medium of Example 4: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (2.2 g / L), vitamin B12 (1 mg / L), tryptone (20 g / L), glucose (1 g / L), dipotassium hydrogen phosphate (2 g / L), potassium dihydrogen phosphate (2 g / L), sodium chloride (5 g / L) and Tween 80 (1.5 mL / L), the balance is water. MgSO4, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, Tween 80 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 vitamin B12 are sterilized by filtration. When the autoclaved medium is cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate and vitamin B12 are added.

[0046] Comparative Example 1: (1) Inoculate Shigella flexneri CMCCB51572 into nutrient broth medium (commercially available), culture overnight (18 to 24 hours) at 37°C, and store in a refrigerator at 2°C to 8°C. Inoculate into nutrient broth medium at a volume ratio of 1:100 (V:V) to expand the culture of Shigella flexneri CMCCB51572.

[0047] (2) Counting of bacterial content: Take 1 mL of fresh culture solution and make 10-fold serial dilutions with sterile PBS buffer (10 mmol, pH = 7.2-7.4). Take 100 μL of each dilution for 10 3 times, 10 4 times, 10 5 times, 10 6 Spread the bacterial solution of 100 times on TSA plates, culture them in a 37℃ biochemical incubator overnight, and observe and count the colonies the next day.

[0048] (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, and the pH value of the 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.

[0049] (4) Determination of culturable bacterial counts: 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. The culturable bacterial count in the bacterial solution was detected every 12 hours until the culturable bacterial count was 0 CFU / mL.

[0050] (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.

[0051] (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 1shown.

[0052] (7) The VBNC bacterial content was calculated according to the following formula: VBNC bacteria = total number of viable bacteria - number of culturable bacteria.

[0053] Comparative Example 2: (1) Inoculate Shigella flexneri CMCCB51572 into nutrient broth medium (commercially available), culture overnight (18 to 24 hours) at 37°C, and store in a refrigerator at 2°C to 8°C. Inoculate into nutrient broth medium at a volume ratio of 1:100 (V:V) to expand the culture of Shigella flexneri CMCCB51572.

[0054] (2) Counting of bacterial content: Take 1 mL of fresh culture solution and make 10-fold serial dilutions with sterile PBS buffer (10 mmol, pH = 7.2-7.4). Take 100 μL of each dilution for 10 3 times, 10 4 times, 10 5 times, 10 6 Spread the bacterial solution of 100 times on TSA plates, culture them in a 37℃ biochemical incubator overnight, and observe and count the colonies the next day.

[0055] (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 volume was divided into 25mL / tube, centrifuged at 10000r / min for 10 minutes, the supernatant was removed, and the cells were resuspended with a stress solution equivalent to the original volume for induction treatment. The stress solution was a 200μg / mL vancomycin aqueous solution, incubated at room temperature, and samples were taken to detect the culturable count and total viable count.

[0056] (4) Determination of culturable bacterial counts: 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. The culturable bacterial count in the bacterial solution was detected every 12 hours until the culturable bacterial count was 0 CFU / mL.

[0057] (5) Cultivable bacterial count: The plate count method is used for testing. When the test result is 0 CFU / mL, the incubation time is 21 days.

[0058] (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.

[0059] (7) The VBNC bacterial content was calculated according to the following formula: VBNC bacteria = total number of viable bacteria - number of culturable bacteria.

[0060] Comparative Example 3: The recovery medium of comparative example 3: Rpf recombinant protein (100ng / L), MgSO4 (0.6g / L), tryptone (20g / L), glucose (1g / L), dipotassium hydrogen phosphate (2g / L), potassium dihydrogen phosphate (2g / L), sodium chloride (5g / L) and Tween 80 (1.5mL / L), and the balance is water. MgSO4, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, Tween 80 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.

[0061] Comparative Example 4: The recovery medium of comparative example 4: sodium pyruvate (1.1 g / L), tryptone (20 g / L), glucose (1 g / L), dipotassium hydrogen phosphate (2 g / L), potassium dihydrogen phosphate (2 g / L), sodium chloride (5 g / L) and Tween 80 (1.5 mL / L), and the balance is water. Among them, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, Tween 80 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.

[0062] Comparative Example 5: The recovery medium of comparative example 5: vitamin B12 (2 mg / L), tryptone (20 g / L), glucose (1 g / L), dipotassium hydrogen phosphate (2 g / L), potassium dihydrogen phosphate (2 g / L), sodium chloride (5 g / L) and Tween 80 (1.5 mL / L), and the balance is water. Among them, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, Tween 80 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; vitamin B12 is sterilized by filtration. When the autoclaved medium is cooled to 45°C-50°C, the sterilized vitamin B12 is added.

[0063] Comparative Example 6: The recovery medium of comparative example 6: Rpf recombinant protein (100ng / L), MgSO4 (0.6g / L), sodium pyruvate (1.1g / L), tryptone (20g / L), glucose (1g / L), dipotassium hydrogen phosphate (2g / L), potassium dihydrogen phosphate (2g / L), sodium chloride (5g / L) and Tween 80 (1.5mL / L), and the balance is water. MgSO4, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, Tween 80 and water are mixed, and then the pH value of the culture 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 culture medium is cooled to 45°C-50°C, the sterilized Rpf recombinant protein and sodium pyruvate are added.

[0064] The recovery culture medium obtained in the example and the comparative example was used to culture the VBNC Shigella flexneri prepared in Example 1, and the culture temperature was 37° C. and the culture time was 18 h to 24 h.

[0065] The recovery culture medium obtained from the embodiment and the comparative example was tested: (1) Determination of the composition of the recovery medium: 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 VBNC viable bacteria in the VBNC Shigella flexneri working bacterial suspension to 10,000 cells / mL.

[0067] Inoculation culture: 0.1 mL of VBNC Shigella flexneri suspension was inoculated into the culture medium of the embodiment and the comparative example, and 3 replicates were set for each culture medium. Cultured at 37°C for 24 hours. Take 1 mL of the culture solution, make a 10-fold serial dilution 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] (2) Verification of VBNC state of Shigella flexneri: 1 mL of the VBNC bacterial suspension prepared in the Example and the Comparative Example was added to a nutrient broth medium containing 10 mM sodium pyruvate and a nutrient broth medium without sodium pyruvate, respectively, and cultured at 37°C for 24 hours. The results showed that the nutrient broth medium containing sodium pyruvate was obviously turbid, while the nutrient broth medium without sodium pyruvate was clear. It can be seen that the methods of the Example and the Comparative Example successfully obtained VBNC state of Shigella flexneri.

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

[0071] (2) Growth rate determination: The total number of viable bacteria in the VBNC Shigella flexneri suspension was adjusted to 1000 cells / mL with a sterile PBS buffer solution, and 0.1 mL of the VBNC bacterial suspension was inoculated into 9.9 mL of the national standard culture medium (Shigella enrichment broth-novobiocin culture medium) and the recovery culture medium of the present invention, respectively. Each culture medium or each component culture medium was inoculated into three parallel tubes in parallel, and cultured at 37° C. for 18 hours.

[0072] Use sterile PBS buffer to adjust the total number of live bacteria in the fresh bacterial suspension of Shigella flexneri overnight culture to less than 100 cells / mL, and take 0.1 mL of the bacterial suspension and inoculate it into 9.9 mL of national standard culture medium and recovery culture medium respectively. Inoculate three parallel tubes of each culture medium or each component culture medium in parallel and culture at 37°C for 18 hours.

[0073] After 18 hours of culture, 10 μL of the culture was taken from each culture medium and evenly spread on a TSA agar plate. If the Shigella flexneri colonies on the TSA plate were greater than 10 CFU, the growth rate of the liquid culture medium was judged to be good.

[0074] (3) Example 1 Medium Selectivity Test: 1×10 3 ~5×10 3CFU Staphylococcus aureus ATCC6538 was inoculated into 10mL of national standard culture medium and recovery culture medium, and three parallel tubes were inoculated in each parallel, and cultured at 37℃ for 18 hours. The culture medium should not be turbid. Take 10μL of the culture medium after 18 hours of culture and evenly spread it on TSA plates. The number of colonies on TSA was <100CFU, indicating that the liquid culture medium had good selectivity.

[0075] (4) Example 1 Culture medium repeatability test: A semi-quantitative test method was used to test 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 test the growth rate of Shigella flexneri and the number of VBNC Shigella flexneri resuscitations. Each batch of test kits was tested three times in parallel.

[0076] (5) Example 1 Culture medium stability test: The recovery culture medium was stored at 2°C to 8°C. Culture medium samples were taken after 3 months, 6 months, 9 months, 12 months and 15 months of storage, and the product was subjected to growth rate and selectivity tests according to the growth rate test method and selectivity test method.

[0077] Results and Analysis: Table 1 Test results of embodiments and comparative examples

[0078] Table 2 Growth rate 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 Shigella flexneri or Staphylococcus aureus is enriched, it is transferred to TSA plate for counting.

[0080] Table 3 Test results of culture medium repeatability

[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 Shigella flexneri or Staphylococcus aureus is enriched, it is transferred to TSA plate for counting.

[0082] Table 4 Culture medium stability test results

[0083] 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 Shigella flexneri or Staphylococcus aureus is enriched, it is transferred to TSA plate for counting.

[0084] Example 1, Comparative Example 1 and Comparative Example 2 were all tested using the same batch and the same concentration, so the total number of viable bacteria in Example 1, Comparative Example 1 and Comparative Example 2 before mutagenesis was the same. Figure 1 In the method of Example 1, on the basis of ensuring the survival rate of Shigella flexneri, more Shigella flexneri strains are prompted to enter the VBNC state in a short time, and the culturable number is detected to be 0 cell / mL. Compared with other induction conditions, the concentration of Shigella flexneri in the VBNC state is the best.

[0085] 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 Shigella cell wall, so that the cell wall becomes loose, and the degraded peptidoglycan can enter the cell through the loose cell wall to achieve cell recovery, and the magnesium ions in magnesium sulfate provide divalent metal ions for the activity of the Rpf recombinant protease. Sodium pyruvate is not only an active oxygen free radical scavenger, but also removes the active oxygen free radicals that damage DNA in the cell to restore the cell state, and can also be used as an energy substance to play an important role in the glycolysis process. It was found in Comparative Example 3 that adding Rpf recombinant protein and magnesium sulfate to the culture medium can relieve the VBNC state of Shigella. In Comparative Example 4, adding sodium pyruvate alone to the culture medium can also relieve the VBNC state of Shigella. In Comparative Example 6, Rpf recombinant protein, magnesium sulfate and sodium pyruvate are added to the culture medium, and its effect is the same as that of Comparative Example 1. Vitamin B12 alone cannot eliminate the VBNC state of Shigella in the culture medium. By comparing Examples 1 to 4 with Comparative Example 6, it is found that the addition of vitamin B12 can significantly eliminate the VBNC state of Shigella. The VBNC state Shigella cultured in Examples 1 to 4 is at least 9 times that of Comparative Example 3 to Comparative Example 6.

[0086] On this basis, the coordination between Rpf recombinant protein, magnesium sulfate, sodium pyruvate and glutamine was further studied. By comparing Examples 1 to 3, it was found that vitamin B12 increased with the increase in the amount of sodium pyruvate added, and the culture medium of the present invention allowed more VBNC state Shigella to be revived. Comparison between Example 1 and Example 4 showed that when the amount of sodium pyruvate added reached 2.2 g / L, even if the amount of vitamin B12 added was increased, the number of VBNC state Shigella that could be cultured in Example 4 was lower than that in Example 1. Therefore, the culture medium is further preferred: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (1.65 g / L), vitamin B12 (0.5 mg / L), tryptone (20 g / L), glucose (1 g / L), dipotassium hydrogen phosphate (2 g / L), potassium dihydrogen phosphate (2 g / L), sodium chloride (5 g / L) and Tween 80 (1.5 mL / L).

[0087] The growth rate and selectivity of the recovery medium of the present invention were tested, and the test results are shown in Table 2. Staphylococcus aureus, VBNC Shigella flexneri and Shigella flexneri were inoculated in the national standard medium and the recovery medium of the present invention, respectively, and the turbidity of the culture was observed after 18 hours of culture. The results in Table 2 show that Shigella flexneri showed severe turbidity in the national standard medium and in the recovery medium of the present invention; while VBNC Shigella flexneri and Staphylococcus aureus were not turbid in the national standard medium, indicating that VBNC Shigella flexneri and Staphylococcus aureus did not grow in the national standard medium, and also verified that the induction method of the present invention successfully obtained VBNC state Shigella flexneri; VBNC Shigella flexneri also showed severe turbidity in the recovery medium of the present invention. The culture solution after 18 hours of culture was inoculated on a TSA plate for counting, and the semi-quantitative detection method in GB4789.28-2024 was used. The method requires that after amplification from the liquid culture medium, 10 microliters of the culture solution is applied to the TSA plate for counting, and the number of colonies should be no less than 10 CFU, proving that the culture medium has a good growth rate. The results show that VBNC Shigella flexneri is freed from the VBNC state, and Shigella flexneri grows successfully. In summary, the recovery culture medium of the present invention not only has a good growth rate, but also has good selectivity.

[0088] In Table 3, 3 boxes of pilot products were randomly selected from each of the 3 batches of products for intra-batch and inter-batch repeatability testing. The results showed that after the recovery medium was used to enrich Shigella flexneri, the TSA count was transferred, and colonies >10 CFU appeared on the plate, indicating that the 3 batches of recovery medium had a good growth rate for Shigella flexneri. It can be seen that the recovery medium of the present invention has good repeatability.

[0089] In Table 4, three batches of products were stored according to the storage conditions. The growth rate and selectivity of the products were measured after 3 months, 6 months, 9 months, 12 months and 15 months of storage. The results showed that within 15 months of storage, the nutritional performance and selectivity of the recovery medium were good, meeting the requirements of GB4789.28-2024. It can be seen that the VBNC Shigella thiola culture medium of the present invention has good stability.

[0090] In summary, the sterility, nutritional capacity, selectivity and stability of the resuscitation medium of the present invention all meet the requirements of food microbiology testing.

Claims

1. A recovery medium, characterized in that The recovery medium comprises Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80, and the concentration ratios of the Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, tryptone, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80 are (90ng / L-110ng / L): (0.5g / L-0.7g / L): (0.27g / L-2.2g / L): (0.13mg / L-1mg / L): (15g / L-25g / L): (0.5g / L-1.5g / L): (1.5g / L-2.5g / L): (1.5g / L-2.5g / L): (4g / L-6g / L): (1mL / L-2mL / 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 culture medium includes Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, trypsin, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80, and the concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, vitamin B12, trypsin, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and Tween 80 is 100ng / L: 0.6g / L: 1.65g / L: 0.5mg / L: 20g / L: 1g / L: 2g / L: 2g / L: 5g / L: 1.5mL / L.

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

6.

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

5. The use according to claim 4, characterized in that: The VBNC state Shigella is cultured in a recovery medium at a temperature of 36°C-38°C for 18h-48h.

6. A method for inducing Shigella in VBNC state for use according to claim 5, characterized in that: The induction method comprises: adjusting the pH value of a solution containing vancomycin and sodium chloride with glacial acetic acid, mixing the mixture evenly, transferring Shigella that is not in the VBNC state into the evenly mixed solution, and incubating the mixture at a temperature of 2° C. to 8° C. for 35 h to 37 h; The mass concentration of vancomycin in the solution containing vancomycin and sodium chloride is 150 μg / mL-250 μg / mL.

7. The induction method according to claim 6, characterized in that: Before transferring Shigella that is 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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