Induction method of staphylococcus aureus in VBNC state, resuscitation culture medium and application

Abstract

The invention relates to the technical field of microorganisms, and discloses a VBNC state staphylococcus aureus induction method, a resuscitation medium and application, the resuscitation medium comprises Rpf recombinant protein, MgSO4, peptone, beef extract and sodium chloride, and the mass concentration ratio of the Rpf recombinant protein to the MgSO4 to the peptone to the beef extract to the sodium chloride is (90 ng / L to 110 ng / L): (0.5 g / L to 0.7 g / L): (9 g / L to 11 g / L): (4 g / L to 6 g / L): (70 g / L to 80 g / L); the nucleotide sequence of the Rpf recombinant protein is as shown in SEQ ID NO. 1. The resuscitation medium provided by the invention can successfully resuscitate staphylococcus aureus in a VBNC state.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for inducing Staphylococcus aureus in the VBNC state, a resuscitation medium, and its application. Background Technology

[0002] Staphylococcus aureus (S. aureus), also known as "Staphylococcus aureus," belongs to the genus Staphylococcus and is a representative Gram-positive bacterium, a common foodborne pathogen. Its optimal growth temperature is 37°C, and its optimal pH is 7.4. It is tolerant of high salt concentrations, growing in environments with salt concentrations approaching 10%. Staphylococcus aureus commonly resides on the skin, nasal cavity, pharynx, gastrointestinal tract, boils, and abscesses of humans and animals, and is also present in air and sewage. When testing samples of food, pharmaceuticals, and process water, the low bacterial contamination, low bacterial count, and the presence of difficult-to-culture VBNC bacteria make the detection sensitivity insufficient to meet the requirements for pathogen control, creating a detection blind spot and seriously threatening the microbial control of food and pharmaceuticals.

[0003] In existing technologies, the detection of Staphylococcus aureus in food and pharmaceutical samples mainly relies on culture methods. However, because Staphylococcus aureus can exist in a VBNC (virtual bacterial count), conventional microbial culture methods cannot detect it. US patent application US20200340040A1 uses PCR amplification technology to detect microorganisms in the VBNC state. However, PCR detection is costly, and its sensitivity is insufficient for most detection requirements; the sensitivity of PCR methods generally requires a viable count of 10⁻⁶ in the sample. 4 VBNC contamination in food and pharmaceutical samples is typically above 0–10 cells / mL, while the level of VBNC contamination is usually between 0 and 10. 4 Therefore, there is an urgent need for a culture medium that can successfully detect Staphylococcus aureus in the VBNC state (cells / mL). Summary of the Invention

[0004] This invention provides a method for inducing Staphylococcus aureus in the VBNC state, a resuscitation culture medium, and its application.

[0005] The technical solution adopted in this invention is as follows.

[0006] This invention provides a resuscitation medium comprising Rpf recombinant protein, MgSO4, peptone, beef extract, and sodium chloride, wherein the mass concentration ratio of Rpf recombinant protein, MgSO4, peptone, beef extract, and sodium chloride is (90 ng / L-110 ng / L): (0.5 g / L-0.7 g / L): (9 g / L-11 g / L): (4 g / L-6 g / L): (70 g / L-80 g / L). The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1.

[0007] Furthermore, the resuscitation medium also includes sodium pyruvate and glutamine, and the mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract and sodium chloride is (90 ng / L-110 ng / L): (0.5 g / L-0.7 g / L): (0.27 g / L-2.2 g / L): (1 g / L-3 g / L): (9 g / L-11 g / L): (4 g / L-6 g / L): (70 g / L-80 g / L).

[0008] Furthermore, the mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract, and sodium chloride is 100 ng / L: 0.6 g / L: 1.1 g / L: 2 g / L: 10 g / L: 5 g / L: 75 g / L.

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

[0010] The present invention also provides the application of the resuscitation medium in culturing Staphylococcus aureus in VBNC state: Staphylococcus aureus in VBNC state was cultured in resuscitation medium at a temperature of 36℃-38℃ for 18h-48h.

[0011] The present invention also provides a method for inducing Staphylococcus aureus 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 them evenly, transferring Staphylococcus aureus not in the VBNC state into the evenly mixed solution, and incubating it at a temperature of 2℃-8℃ for 22h-24h.

[0012] Furthermore, the pH of the solution containing vancomycin and sodium chloride was adjusted to 2.6-3 using glacial acetic acid.

[0013] Furthermore, the vancomycin concentration in the solution containing vancomycin and sodium chloride is 60 mg / L-68 mg / L.

[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 resuscitation medium of this invention has good peptidoglycan hydrolase activity, which can degrade the peptidoglycan in the cell wall of Staphylococcus aureus, making the cell wall loose. The degraded peptidoglycan can enter the cell through the loose cell wall, thereby reviving the cell. Magnesium ions in magnesium sulfate provide divalent metal ions for the activity of Rpf recombinant protein. Sodium pyruvate can not only act as a scavenger of reactive oxygen free radicals, which can remove reactive oxygen free radicals that damage DNA in the cell and put the cell in a recoverable state, but also act as an energy substance, playing an important role in glycolysis. When Rpf recombinant protein and sodium pyruvate are present in the culture medium, glutamine can be added to enhance the synergistic effect of Rpf recombinant protein and sodium pyruvate, which can relieve the VBNC state of Staphylococcus aureus. In addition, glutamine can also provide sufficient nutrition and energy for the resuscitation process, promoting the transformation of bacteria from the VBNC state to the culturable state.

[0016] 2. This invention also successfully provides a method for inducing Staphylococcus aureus to be in a VBNC state, which can successfully prepare bacteria with a total viable count of not less than 10. 4 Staphylococcus aureus suspension in VBNC state with cell / mL and culturable number of 0 cells / mL. Attached Figure Description

[0017] Figure 1 These are the stress experiment results of the embodiments and comparative examples of the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] In a first aspect, some embodiments of the present invention provide a resuscitation culture medium comprising Rpf recombinant protein, MgSO4, peptone, beef extract, and sodium chloride, wherein the mass concentration ratio of the Rpf recombinant protein, MgSO4, peptone, beef extract, and sodium chloride is (90 ng / L-110 ng / L): (0.5 g / L-0.7 g / L): (9 g / L-11 g / L): (4 g / L-6 g / L): (70 g / L-80 g / L). The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1.

[0020] In some embodiments, the resuscitation medium further includes sodium pyruvate and glutamine, and the mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract and sodium chloride is (90 ng / L-110 ng / L): (0.5 g / L-0.7 g / L): (0.27 g / L-2.2 g / L): (1 g / L-3 g / L): (9 g / L-11 g / L): (4 g / L-6 g / L): (70 g / L-80 g / L).

[0021] In some embodiments, the resuscitation medium also includes water.

[0022] In some embodiments, the resuscitation medium comprises recombinant Rpf protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract, and sodium chloride, wherein the mass concentration ratio of recombinant Rpf protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract, and sodium chloride is 100 ng / L: 0.6 g / L: 1.1 g / L: 2 g / L: 10 g / L: 5 g / L: 75 g / L.

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

[0024] Secondly, some embodiments of the present invention also provide the application of a resuscitation medium in culturing Staphylococcus aureus in the VBNC state.

[0025] In some embodiments, VBNC-state Staphylococcus aureus is cultured in resuscitation medium at a temperature of 36-38°C for 18-24 hours.

[0026] Thirdly, some embodiments of the present invention also provide a method for inducing Staphylococcus aureus in the VBNC state, the induction method comprising: The pH of the solution containing vancomycin and sodium chloride was adjusted with glacial acetic acid, mixed thoroughly, and Staphylococcus aureus not in the VBNC state was transferred into the well-mixed solution and incubated at 2℃-8℃ for 22h-24h.

[0027] In some embodiments, the well-mixed solution is sterilized before transferring Staphylococcus aureus that is not in the VBNC state.

[0028] In some embodiments, the method for preparing the solution containing vancomycin and sodium chloride is to disperse vancomycin and sodium chloride in water.

[0029] In some embodiments, glacial acetic acid is used to adjust the pH of the solution containing vancomycin and sodium chloride to 2.6-3.0.

[0030] In some embodiments, the vancomycin concentration in a solution containing vancomycin and sodium chloride is 60 mg / L to 68 mg / L.

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

[0032] The following detailed explanation is provided with reference to specific embodiments and comparative examples: Example 1: 1. Preparation of Staphylococcus aureus in VBNC state: (1) Staphylococcus aureus ATCC6538 was inoculated into tryptic soy liquid medium (commercially available) and cultured at 37°C for 14-16 hours, then stored at 2°C-8°C. Staphylococcus aureus ATCC6538 was then inoculated into tryptic soy liquid medium at a volume ratio of 1:100 to expand the culture.

[0033] (2) Bacterial cell count: Take 1 mL of fresh culture and make a 10-fold serial dilution with sterile PBS buffer (10 mmol, pH=7.2~7.4). Take 100 μL of each dilution and spread it onto tryptic soy agar medium, including 10... 3 times, 10 4 times, 10 5 times, 10 6 Double the volume and incubate overnight in a 37°C biochemical incubator. Observe and count the colonies the next day.

[0034] (3) Stress induction: The bacteria were washed twice with sterile PBS buffer (10 mmol, pH=7.2-7.4). The bacteria were then diluted to 1×10⁻⁶ with sterile PBS buffer (10 mmol, pH=7.2-7.4). 7 CFU / mL, aliquoted into 25mL tubes, centrifuged at 10000 rpm for 10 minutes, and the supernatant was removed. The tubes were then resuspended in an equivalent volume of stress solution for induction treatment. The stress solution contained 64 mg / L vancomycin and 150 g / L NaCl. The pH of the stress solution was adjusted to 2.8 with glacial acetic acid. After resuspending thoroughly, the tubes were incubated at 2℃–8℃. Samples were taken to determine 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 are detected. The incubation time is determined based on the culturable bacterial count being 0 CFU / mL. Take 100 μL of bacterial solution and count the bacteria according to the method in step (2) above. Detect the culturable bacterial count in the bacterial solution. Detect once every 12 hours until the detected culturable bacterial count is 0 CFU / mL.

[0036] (5) Number of culturable bacteria: The plate count method was used for detection. When the detection result was 0 CFU / mL, the incubation time was 24h.

[0037] (6) Total viable count determination: After 24 hours of stress induction, 1 mL of bacterial suspension was taken. During the induction process, a large number of bacterial strains died. After induction, the bacterial suspension contained bacteria in three states: dead, culturable, and VBNC. The total viable count could be determined by flow cytometry after staining the respiratory cells with the CTC bacterial viability assay kit (Tongren Chemical Research Institute). The results are as follows: Figure 1 As shown.

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

[0039] 2. Culture Staphylococcus aureus in the VBNC state: Preparation of resuscitation medium: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (1.1 g / L), glutamine (2 g / L), peptone (10 g / L), beef extract (5 g / L), sodium chloride (75 g / L), with the remainder being water. MgSO4, peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. Rpf recombinant protein, sodium pyruvate, and glutamine were sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate, and glutamine were added.

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

[0041] The Rpf recombinant protein was obtained by mutating the amino acid sequence of Micrococcus luteus Rpf protein. The mutated nucleotide sequence was then sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

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

[0043] Enzyme activity assays were performed on the recombinant Rpf protein with SEQ ID NO.1 and the Rpf protein with SEQ ID NO.2. The results showed that the Rpf protein with SEQ ID NO.1 had a protease activity of 317 U / mL, while the unmutated Rpf protein with SEQ ID NO.2 had a protease activity of 83 U / mL. This indicates that the mutated protease activity was increased by more than twofold.

[0044] Example 2: The resuscitation medium for Example 2 consisted of: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (0.55 g / L), glutamine (1 g / L), peptone (10 g / L), beef extract (5 g / L), sodium chloride (75 g / L), with the remainder being water. MgSO4, peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. Rpf recombinant protein, sodium pyruvate, and glutamine were sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate, and glutamine were added.

[0045] Example 3: The resuscitation medium for Example 3 consisted of: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (0.275 g / L), glutamine (2 g / L), peptone (10 g / L), beef extract (5 g / L), sodium chloride (75 g / L), with the remainder being water. MgSO4, peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. Rpf recombinant protein, sodium pyruvate, and glutamine were sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate, and glutamine were added.

[0046] Example 4: The resuscitation medium for Example 4 consisted of: Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (2.2 g / L), glutamine (3 g / L), peptone (10 g / L), beef extract (5 g / L), sodium chloride (75 g / L), with the remainder being water. MgSO4, peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. Rpf recombinant protein, sodium pyruvate, and glutamine were sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized Rpf recombinant protein, sodium pyruvate, and glutamine were added.

[0047] Example 5: The resuscitation medium for Example 5 consisted of Rpf recombinant protein (100 ng / L), MgSO4 (0.6 g / L), peptone (10 g / L), beef extract (5 g / L), and sodium chloride (75 g / L), with the remainder being water. The MgSO4, peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. The Rpf recombinant protein was sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized Rpf recombinant protein was added.

[0048] Comparative Example 1: (1) Staphylococcus aureus ATCC6538 was inoculated into tryptic soy liquid medium (commercially available) and cultured at 37°C for 14-16 hours, then stored at 2°C-8°C. Staphylococcus aureus ATCC6538 was then inoculated into tryptic soy liquid medium at a volume ratio of 1:100 to expand the culture.

[0049] (2) Counting of bacterial cells: Take 1 mL of fresh cultured bacterial solution and make a 10-fold serial dilution with sterile PBS buffer solution (10 mmol, pH=7.2~7.4). Take 100 μL of the appropriately diluted bacterial solution and spread it on tryptic soy agar medium. Incubate overnight in a biochemical incubator at 37℃. Observe and count the colonies the next day.

[0050] (3) Stress induction: The bacteria were washed twice with sterile PBS buffer (10 mmol, pH=7.2-7.4). The bacteria were then diluted to 1×10⁻⁶ with sterile PBS buffer (10 mmol, pH=7.2-7.4). 7 CFU / mL, aliquoted into 25mL tubes, centrifuged at 10000 rpm for 10 minutes, and then resuspended in an equivalent volume of stress solution for induction treatment. The stress solution was an aqueous solution containing 150 g / L NaCl, with the pH adjusted to 2.8 ± 0.2 using glacial acetic acid. The solution was incubated at 2℃–8℃, and samples were taken to determine the culturable count and total viable count.

[0051] (4) Determination of culturable bacterial count: After stress induction, the culturable microbial colonies in the bacterial solution are detected. The incubation time is determined based on the culturable bacterial count being 0 CFU / mL. Take 100 μL of bacterial solution and count the bacteria according to the method in step (2) above. Detect the culturable bacterial count in the bacterial solution. Detect once every 12 hours until the detected culturable bacterial count is 0 CFU / mL.

[0052] (5) Number of culturable bacteria: The plate count method was used for detection. When the detection result was 0 CFU / mL, the incubation time was 7 days.

[0053] (6) Total viable count determination: Seven days after stress induction, 1 mL of bacterial culture was taken. Since a large number of bacterial strains die during the induction process, the total viable count was determined by flow cytometry after staining with a CTC bacterial viability assay kit (Tongren Chemical Research Institute). The results are as follows: Figure 1 As shown.

[0054] (7) The content of VBNC bacteria is calculated according to the following formula: VBNC status bacteria = total number of viable bacteria - number of culturable bacteria.

[0055] Comparative Example 2: (1) Staphylococcus aureus ATCC6538 was inoculated into tryptic soy liquid medium (commercially available) and cultured at 37°C for 14-16 hours, then stored at 2°C-8°C. Staphylococcus aureus ATCC6538 was then inoculated into tryptic soy liquid medium at a volume ratio of 1:100 to expand the culture.

[0056] (2) Counting of bacterial cells: Take 1 mL of fresh cultured bacterial solution and make a 10-fold serial dilution with sterile PBS buffer solution (10 mmol, pH=7.2~7.4). Take 100 μL of the appropriately diluted bacterial solution and spread it on tryptic soy agar medium. Incubate overnight in a biochemical incubator at 37℃. Observe and count the colonies the next day.

[0057] (3) Stress induction: The bacteria were washed twice with sterile PBS buffer (10 mmol, pH=7.2-7.4). The bacteria were then diluted to 1×10⁻⁶ with sterile PBS buffer (10 mmol, pH=7.2-7.4). 7 CFU / mL, aliquoted into 25mL tubes, centrifuged at 10000 r / min for 10 minutes, and then resuspended in an equivalent volume of stress solution for induction treatment. The stress solution was 64 mg / L vancomycin aqueous solution. The tubes were incubated at room temperature, and samples were taken to determine the culturable count and total viable count.

[0058] (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 culturable bacterial count being 0 CFU / mL. 100 μL of bacterial solution was taken and the bacterial count was performed according to the method described in (2) above. The culturable bacterial count in the bacterial solution was detected once every 12 hours until the detected culturable bacterial count was 0 CFU / mL.

[0059] (5) Number of culturable bacteria: The plate technique was used for detection. When the detection result was 0 CFU / mL, the incubation time was 21 days.

[0060] (6) Total viable count determination: 21 days after stress induction, 1 mL of bacterial suspension was taken. During the induction process, a large number of bacterial strains died. After induction, the bacterial suspension contained bacteria in three states: dead, culturable, and VBNC. The CTC bacterial viability assay kit (Tongren Chemical Research Institute) was used to stain cells with respiratory activity, and the total viable count was detected by flow cytometry. The results are as follows: Figure 1 As shown.

[0061] (7) The content of VBNC bacteria is calculated according to the following formula: VBNC status bacteria = total number of viable bacteria - number of culturable bacteria.

[0062] Comparative Example 3: The resuscitation medium for Comparative Example 3 consisted of sodium pyruvate (1.1 g / L), peptone (10 g / L), beef extract (5 g / L), and sodium chloride (75 g / L), with the remainder being water. The peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. Sodium pyruvate was sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized sodium pyruvate was added.

[0063] Comparative Example 4: The resuscitation medium for Comparative Example 4 consisted of glutamine (2 g / L), peptone (10 g / L), beef extract (5 g / L), and sodium chloride (75 g / L), with the remainder being water. The peptone, beef extract, sodium chloride, and water were mixed, and the pH was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. Glutamine was sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized glutamine was added.

[0064] Comparative Example 5: The resuscitation medium for Comparative Example 5 consisted of recombinant Rpf protein (100 ng / L), MgSO4 (0.6 g / L), sodium pyruvate (1.1 g / L), peptone (10 g / L), beef extract (5 g / L), and sodium chloride (75 g / L), with the remainder being water. MgSO4, peptone, beef extract, sodium chloride, and water were mixed, and the pH of the medium was adjusted to 7.4 ± 0.2. The medium was then autoclaved at 121°C for 15 minutes. The recombinant Rpf protein and sodium pyruvate were sterilized by filtration. After the autoclaved medium cooled to 45°C-50°C, the sterilized recombinant Rpf protein and sodium pyruvate were added.

[0065] The resuscitation medium obtained in the examples and comparative examples was used to culture Staphylococcus aureus in VBNC state prepared in Example 1. The culture temperature was 37°C and the culture time was 24 h.

[0066] The resuscitation media obtained in the examples and comparative examples were tested: (1) Determination of the composition of the resuscitation medium: Culture medium preparation: Prepare culture media for the examples and comparative examples respectively.

[0067] Bacterial suspension dilution: The total number of viable VBNC-state Staphylococcus aureus working bacterial suspension was adjusted to 10,000 cells / mL using sterile PBS buffer solution.

[0068] Inoculation and Culture: 0.1 mL of VBNC-state Staphylococcus aureus suspension was inoculated into the culture media of the examples and comparative examples, and spread evenly. Three plates were inoculated for each treatment. The plates were incubated at 37°C for 18–24 hours. 1 mL of the culture was serially diluted 10-fold with sterile PBS buffer (10 mmol, pH 7.2–7.4). 100 μL of the appropriately diluted culture was spread onto tryptic soy agar and incubated overnight at 37°C. Colonies were observed and counted the following day.

[0069] Counting: The number of colonies grown on the plates was counted, and the results are shown in Table 2.

[0070] (2) Verification of VBNC-state Staphylococcus aureus: 1 mL of the VBNC bacterial suspension prepared in the examples and comparative examples was added to tryptic soy medium containing 10 mM sodium pyruvate and tryptic soy medium without sodium pyruvate, respectively, and incubated at 37°C for 24 hours. The results showed that the tryptic soy medium containing sodium pyruvate was significantly turbid, while the tryptic soy medium without sodium pyruvate was clear. It can be seen that the methods of the examples and comparative examples successfully obtained VBNC-state Staphylococcus aureus.

[0071] Further research was conducted using the resuscitation culture medium from Example 1 as an example.

[0072] (3) Qualitative determination of growth rate of culture medium in Example 1: Adjust the total viable count of VBNC Staphylococcus aureus suspension to 1000 cells / mL using sterile PBS buffer, and adjust the total viable count of Staphylococcus aureus suspension to below 100 cells / mL. Inoculate 1 mL of VBNC Staphylococcus aureus suspension and 1 mL of Staphylococcus aureus suspension into 9 mL of 7.5% sodium chloride broth (commercially available) and the resuscitation medium of this invention, respectively. Inoculate each medium or each component of the medium into three parallel tubes (SaP001, SaP002, SaP003), and incubate at 37°C for 18 hours. Counting: After 18 hours of incubation, take 10 μL of culture from each medium and spread it evenly on a TSA (tryptophan-soybean agar) plate. Count the microbial colonies. If the number of Staphylococcus aureus colonies on the plate is greater than 10 CFU, the resuscitation medium of this invention is considered to have good growth rate.

[0073] (4) Selectivity determination of culture medium in Example 1: 0.1 mL of culture medium containing 1×10 3 ~5×10 3 CFU of Escherichia coli ATCC25922 was inoculated into 9.9 mL of 7.5% sodium chloride broth and the resuscitation medium of this invention, respectively, in triplicate (SaP001, SaP002, SaP003) and incubated at 37°C for 18 hours. After 18 hours of incubation, 10 μL of culture from each medium was evenly spread onto a TSA plate, and the number of microbial colonies was counted. GB4789.28-2024 states that the optimization of the culture medium should not affect the selectivity of the original national standard culture medium. Therefore, the resuscitation medium of this invention has good selectivity only when the number of Escherichia coli colonies on the TSA plate is <100 CFU.

[0074] The resuscitation medium obtained in Example 1 was prepared into a product, as shown in Table 1.

[0075] Table 1. Components of the finished product: resuscitation culture medium (homogenized bag)

[0076] (4) Example 1 Culture medium stability test: Three batches of homogenized bags were stored according to the storage conditions in Table 1. Culture medium samples were taken after 3 months, 6 months, 9 months, 12 months and 15 months of storage, and the growth rate and selectivity of the products were determined.

[0077] Results and Analysis: Table 2 Test results of the examples and comparative examples

[0078] Table 3. Test results of growth rate in Example 1

[0079] Note: a: Turbidity calculation: 0 indicates no turbidity; 1 indicates very slight turbidity; 2 indicates severe turbidity, judged according to the GB4789.28-2024 evaluation standard; b: After enrichment with Staphylococcus aureus ATCC6538 or Escherichia coli ATCC25922, transfer to TSA plates for counting.

[0080] Table 4. Repeatability test results of culture media prepared from three batches of reagent kits.

[0081] Note: a: Turbidity calculation: 0 indicates no turbidity; 1 indicates very slight turbidity; 2 indicates severe turbidity, judged according to the GB4789.28—2024 evaluation standard; b: After enrichment of Staphylococcus aureus ATCC6538 or Escherichia coli ATCC25922, transfer to TSA for counting.

[0082] Table 5. Stability test results of the examples

[0083] Note: a: Turbidity calculation: 0 indicates no turbidity; 1 indicates very slight turbidity; 2 indicates severe turbidity, judged according to the GB4789.28—2024 evaluation standard; b: After enrichment with Staphylococcus aureus ATCC6538 or Escherichia coli ATCC25922, transfer to TSA and count.

[0084] First, comparing Example 1 with Comparative Examples 1 and 2 revealed that, after induction with different solution stresses, Example 1, Comparative Examples 1 and 2 all achieved a culturable Staphylococcus aureus count of 0 CFU / mL, but the number of Staphylococcus aureus deaths caused by different methods varied. Figure 1 It was found that Example 1 not only achieved a culturable Staphylococcus aureus count of 0 CFU / mL, but also increased the number of viable bacteria in the VBNC state. Figure 1 Example 1 showed the highest number of viable bacteria in the VBNC state. Therefore, the method of the present invention, while ensuring the survival rate of Staphylococcus aureus, induces more Staphylococcus aureus ATCC6538 to enter the VBNC state in a shorter time, with a culturable count of 0 cells / mL. Compared with other induction conditions, its VBNC state bacterial concentration is optimal.

[0085] As shown in Table 1, the Rpf recombinant protein in the culture medium of this invention has good enzymatic activity, capable of degrading peptidoglycan in the cell wall of Staphylococcus aureus, making the cell wall loose. The degraded peptidoglycan can then enter the cell through the loose cell wall, thereby achieving cell recovery. The magnesium ions in magnesium sulfate provide divalent metal ions for the activity of the Rpf recombinant protein. In Example 5, the addition of Rpf recombinant protein and magnesium sulfate to the culture medium can relieve the VBNC state of Staphylococcus aureus. Sodium pyruvate is not only a scavenger of reactive oxygen free radicals, clearing reactive oxygen free radicals that damage DNA in cells and enabling cell recovery, but it can also serve as an energy source, playing an important role in glycolysis. In Comparative Example 3, the addition of sodium pyruvate alone can also promote the relieving of the VBNC state of Staphylococcus aureus. However, using the Rpf recombinant protein and magnesium sulfate of this invention, the number of Staphylococcus aureus in the VBNC state that can be cultured is significantly higher than that of sodium pyruvate in the prior art. In Comparative Example 5, the use of recombinant Rpf protein, magnesium sulfate, and sodium pyruvate can also promote the recovery of Staphylococcus aureus, but its effect is significantly lower than that of using recombinant Rpf protein and magnesium sulfate alone (Example 5). Adding glutamine to the culture medium can significantly relieve the VBNC state of Staphylococcus aureus, which can be verified by comparing Examples 1-4 with Comparative Examples 3-5.

[0086] Based on this, further research was conducted on the synergistic effects between Rpf recombinant protein, magnesium sulfate, sodium pyruvate, and glutamine. It was found that glutamine levels increased with increasing sodium pyruvate content, further enhancing the number of culturable Staphylococcus aureus. Comparison of Examples 1-3 confirmed that Example 1 showed a more significant effect. However, when the amount of sodium pyruvate was further increased, even with further increases in glutamine, the number of culturable Staphylococcus aureus in the culture medium decreased, as verified by comparisons in Examples 1 and 4. Therefore, the optimal mass concentration ratio of Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract, and sodium chloride was 100 ng / L: 0.6 g / L: 1.1 g / L: 2 g / L: 10 g / L: 5 g / L: 75 g / L.

[0087] The growth rate and selectivity of the resuscitation medium of the present invention were tested, and the results are shown in Table 3. Staphylococcus aureus, VBNC Staphylococcus aureus, and Escherichia coli were inoculated into 7.5% sodium chloride broth and the resuscitation medium of the present invention, respectively. After 18 hours of incubation, the turbidity was observed. Table 3 shows that Staphylococcus aureus showed slight turbidity in the 7.5% sodium chloride broth and severe turbidity in the resuscitation medium of the present invention; while VBNC Staphylococcus aureus and Escherichia coli did not show turbidity in the 7.5% sodium chloride broth, indicating that VBNC Staphylococcus aureus and Escherichia coli did not grow in the 7.5% sodium chloride broth, further verifying that the induction method of the present invention successfully prepared VBNC-state Staphylococcus aureus; VBNC Staphylococcus aureus also showed severe turbidity in the resuscitation medium of the present invention. Referring to the semi-quantitative detection method in GB4789.28-2024, the cultured bacterial suspension after 18 hours of incubation was inoculated onto TSA plates for counting. This method requires that after amplification from the liquid medium, 10 μL of the culture suspension be spread onto a TSA plate for counting; the colony count should be no less than 10 CFU, demonstrating that the medium has a good growth rate. The results show that VBNC Staphylococcus aureus was freed from the VBNC state, and Staphylococcus aureus successfully grew. In summary, the resuscitation medium of this invention not only has a good growth rate but also good selectivity.

[0088] Table 4 shows that three boxes of pilot-scale product were randomly selected from each of the three batches for intra-batch and inter-batch repeatability testing. The results showed that after enrichment of Staphylococcus aureus on the resuscitation medium, TSA counting was performed, and colonies with >10 CFU appeared on all plates, indicating good growth rate of Staphylococcus aureus on the three batches of resuscitation medium. Therefore, the resuscitation medium of this invention has good repeatability.

[0089] Table 5 shows that three batches of resuscitation medium were stored according to the storage conditions in Table 1. Growth rate and selectivity were tested after 3, 6, 9, 12, and 15 months of storage. The results showed that the nutritional performance and selectivity of the resuscitation medium were good within 15 months of storage, meeting the requirements of GB4789.28-2024. Therefore, the resuscitation medium of this invention has good stability.

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

Claims

1. A recovery medium, characterized in that The recovery medium comprises Rpf recombinant protein, MgSO4, peptone, beef extract and sodium chloride, and the mass concentration ratio of the Rpf recombinant protein, MgSO4, peptone, beef extract and sodium chloride is (90ng / L-110ng / L): (0.5g / L-0.7g / L): (9g / L-11g / L): (4g / L-6g / L): (70g / L-80g / 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 glutamine. The mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract and sodium chloride is (90ng / L-110ng / L): (0.5g / L-0.7g / L): (0.27g / L-2.2g / L): (1g / L-3g / L): (9g / L-11g / L): (4g / L-6g / L): (70g / L-80g / L).

3. The recovery medium according to claim 2, characterized in that The mass concentration ratio of the Rpf recombinant protein, MgSO4, sodium pyruvate, glutamine, peptone, beef extract and sodium chloride is 100 ng / L: 0.6 g / L: 1.1 g / L: 2 g / L: 10 g / L: 5 g / L: 75 g / 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 Staphylococcus aureus in a VBNC state, characterized in that: The VBNC Staphylococcus aureus was cultured in a recovery medium at a temperature of 36°C-38°C for 18h-48h.

6. A method for inducing Staphylococcus aureus in VBNC state 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. Staphylococcus aureus 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 22 h to 24 h.

7. 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.

8. The induction method according to claim 6, characterized in that: The mass concentration of vancomycin in the solution containing vancomycin and sodium chloride is 60 mg / L-68 mg / L.

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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