Improved TSA bouillon culture medium and application

Abstract

The invention relates to the technical field of microorganisms, and discloses an improved TSA broth culture medium and application, the improved TSA broth culture medium comprises tryptone, sodium chloride, soybean peptone, agar, Rpf recombinant protein and magnesium sulfate, and the mass concentration ratio of the tryptone to the sodium chloride to the soybean peptone to the agar to the Rpf recombinant protein to the magnesium sulfate is (10g / L-20g / L) to (4g / L-6g / L) to (3g / L-7g / L) to (13g / L-17g / L) to (10mu g / L-100mu g / L) to (0.5 g / L-0.7 g / L). The improved TSA bouillon culture medium obtained by the invention can be used for successfully culturing microorganisms in a VBNC state.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, in particular to an improved TSA broth culture medium and application thereof. Background Art

[0002] TSA broth medium, also known as tryptone soy agar medium, is a commonly used microbial culture medium, widely used in the culture and separation of microorganisms such as bacteria and fungi. Its main components include tryptone, soytone and agar. TSA broth medium is suitable for the cultivation of a variety of bacteria, fungi and yeasts. It does not require specific selection of cultured microorganisms. Therefore, it is often used for microbial inspection and analysis in drugs, chemicals, foods and other samples. However, the TSA broth medium in the prior art cannot cultivate VBNC state microorganisms present in the test environment, resulting in errors in detection. U.S. patent application US20200340040A1 discloses the use of PCR amplification technology to detect microorganisms in the VBNC state, but the technology is cumbersome and costly. Therefore, there is an urgent need for a TSA broth medium that can successfully detect microorganisms in the VBNC state. Summary of the invention

[0003] The invention provides an improved TSA broth culture medium and application thereof.

[0004] The technical solution adopted by the present invention is as follows.

[0005] A modified TSA broth culture medium comprises tryptone, sodium chloride, soytone, agar, Rpf recombinant protein and magnesium sulfate (MgSO4), wherein the mass concentration ratios of the tryptone, sodium chloride, soytone, agar, Rpf recombinant protein and magnesium sulfate are (10g / L-20g / L): (4g / L-6g / L): (3g / L-7g / L): (13g / L-17g / L): (10μg / L-100μg / L): (0.5g / L-0.7g / L).

[0006] Furthermore, the nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1.

[0007] Furthermore, the improved TSA broth culture medium also includes sodium pyruvate and vitamin B12, and the mass concentration ratios of the tryptone, sodium chloride, soytone, agar, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and vitamin B12 are (10g / L-20g / L): (4g / L-6g / L): (3g / L-7g / L): (13g / L-17g / L): (10μg / L-100μg / L): (0.5g / L-0.7g / L): (0.27g / L-4.4g / L): (0.25mg / L-2mg / L).

[0008] Furthermore, the mass concentration ratio of tryptone, sodium chloride, soytone, agar, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and vitamin B12 is 15g / L: 5g / L: 5g / L: 15g / L: 10μg / L: 0.6g / L: 1.1g / L: 0.5mg / L.

[0009] Furthermore, the pH value of the improved TSA broth medium is 7.2-7.6.

[0010] The present invention also provides application of the improved TSA broth culture medium in culturing VBNC state microorganisms.

[0011] The present invention also provides the use of the improved TSA broth culture medium in culturing VBNC Escherichia coli and / or VBNC Staphylococcus aureus.

[0012] Furthermore, the culture temperature is 36°C-38°C, and the culture time is 18h-24h.

[0013] The present invention has the following technical effects: Rpf recombinant protein has good peptidoglycan hydrolase activity, which can decompose bacterial peptidoglycan, making the cell wall loose. The degraded peptidoglycan can enter the cell through the loose cell wall, thereby awakening cells 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 cell state, but also act as an energy substance, playing an important role in the glycolysis process. After adding vitamin B12, the synergistic effect of Rpf recombinant protein and sodium pyruvate can be significantly enhanced, awakening cells in the VBNC state. DETAILED DESCRIPTION

[0014] 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 belong to the scope of protection of the present invention.

[0015] In the first aspect, some embodiments of the present invention provide a modified TSA broth culture medium, which includes tryptone, sodium chloride, soytone, agar, Rpf recombinant protein and magnesium sulfate, and the mass concentration ratio of tryptone, sodium chloride, soytone, agar, Rpf recombinant protein and magnesium sulfate is (10g / L-20g / L): (4g / L-6g / L): (3g / L-7g / L): (13g / L-17g / L): (10μg / L-100μg / L): (0.5g / L-0.7g / L).

[0016] In some embodiments, the nucleotide sequence of the Rpf recombinant protein is shown as SEQ ID NO.1.

[0017] In some embodiments, the improved TSA broth culture medium also includes sodium pyruvate and vitamin B12, and the mass concentration ratios of tryptone, sodium chloride, soytone, agar, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and vitamin B12 are (10g / L-20g / L): (4g / L-6g / L): (3g / L-7g / L): (13g / L-17g / L): (10μg / L-100μg / L): (0.5g / L-0.7g / L): (0.27g / L-4.4g / L): (0.25mg / L-2mg / L).

[0018] In some embodiments, the modified TSA broth medium further comprises water.

[0019] In some embodiments, the improved TSA broth medium includes peptone, sodium chloride, soytone, agar, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and vitamin B12, and the mass concentration ratio of peptone, sodium chloride, soytone, agar, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and vitamin B12 is 15g / L: 5g / L: 5g / L: 15g / L: 10μg / L: 0.6g / L: 1.1g / L: 0.5mg / L.

[0020] In some embodiments, the pH of the modified TSA broth medium is 7.2-7.6.

[0021] In a second aspect, some embodiments of the present invention also provide the use of improved TSA broth medium in culturing VBNC state microorganisms.

[0022] In a third aspect, some embodiments of the present invention further provide use of a modified TSA broth medium in culturing VBNC Escherichia coli and / or VBNC Staphylococcus aureus.

[0023] In some embodiments, the culture temperature is 36° C.-38° C., and the culture time is 18 h-24 h.

[0024] The following is described in detail with reference to some embodiments and comparative examples: 1. Preparation of VBNC Staphylococcus aureus: (1) Inoculate Staphylococcus aureus ATCC6538 into a tryptic soytone liquid medium (commercially available), incubate at 37°C for 14-16 hours, and store at 2°C-8°C. Inoculate into a tryptic soytone liquid medium at a volume ratio of 1:100 to expand the culture of Staphylococcus aureus ATCC6538.

[0025] (2) Counting of bacterial content: Take 1 mL of fresh culture medium and make 10-fold serial dilutions with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4). Take 100 μL of each dilution and apply it on tryptic soy agar medium, including 10 3 times, 10 4 times, 10 5 times, 10 6 Times, culture in a 37℃ biochemical incubator overnight, and observe and count the colonies the next day.

[0026] (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 64mg / L vancomycin and 150g / L NaCl. The pH value of the stress solution was adjusted to 2.8 with glacial acetic acid. After being evenly resuspended, it was placed in a refrigerator at 2℃~8℃ and incubated for 24 hours. Samples were taken to detect the number of culturable bacteria and the total number of viable bacteria.

[0027] (4) Determination of culturable bacterial count: 24 hours after stress induction, take 100 μL of bacterial solution and count the number of culturable bacteria according to the method in step (2) above to detect the number of culturable bacteria in the bacterial solution.

[0028] (5) Determination of total viable count: 24 hours after stress induction, take 1 mL of bacterial suspension. 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 the total viable count can be detected by flow cytometry. The detection result is 4.1±0.45×10 4cell / mL.

[0029] (6) Cultivable bacteria count: The test was performed using plate technology, and the result was 0 CFU / mL.

[0030] (7) The VBNC bacterial content was calculated according to the following formula: VBNC bacteria = total number of viable bacteria - number of culturable bacteria.

[0031] (8) Verification of VBNC-state Staphylococcus aureus: 1 mL of the prepared VBNC-state bacterial suspension was added to a tryptic soytone medium containing 10 mM sodium pyruvate and a tryptic soytone medium without sodium pyruvate, respectively, and cultured at 37°C for 24 hours. The results showed that the tryptic soytone medium containing sodium pyruvate was obviously turbid, while the tryptic soytone medium without sodium pyruvate was clear. It can be seen that VBNC-state Staphylococcus aureus was successfully obtained.

[0032] 2. Preparation of VBNC Escherichia coli: (1) Inoculate Escherichia coli ATCC25922 into LB medium (commercially available), incubate overnight (14 to 16 hours) at 37°C with shaking at 180 r / min, and store in a refrigerator at 2°C to 8°C. Inoculate into fresh LB medium at a volume ratio of 1:100 (V:V) of bacterial liquid to medium to expand the culture of Escherichia coli ATCC25922.

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

[0034] (3) Stress induction: Wash the bacteria twice with sterile PBS buffer solution (10 mmol, pH = 7.2-7.4). Dilute the bacteria to 1×10 7 CFU / mL, equal amount was packaged, 25mL / tube, centrifuged at 10000r / min for 10 minutes, the supernatant was removed, and the cells were resuspended with the stress solution equivalent to the original volume for induction treatment. The stress solution was a solution containing 6.4mg / L vancomycin and 150g / L NaCl. The pH value of the stress solution was adjusted to 2.8 with glacial acetic acid. After being evenly resuspended, it was placed in a refrigerator at 2℃~8℃ and incubated for 24 hours. Samples were taken to detect the number of culturable bacteria and the total number of viable bacteria.

[0035] (4) Determination of culturable bacterial count: 24 hours after stress induction, take 100 μL of bacterial solution and count the number of culturable bacteria according to the method in step (2) above to detect the number of culturable bacteria in the bacterial solution.

[0036] (5) Determination of total viable count: 24 hours after stress induction, take 1 mL of bacterial suspension. 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 the total viable count can be detected by flow cytometry. The detection result is 5.9±0.95×10 4 cell / mL.

[0037] (6) Cultivable bacteria count: The test was performed using plate technology, and the result was 0 CFU / mL.

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

[0040] Embodiment 1: Preparation of modified TSA broth medium: tryptone (15g / L), sodium chloride (5g / L), soytone (5g / L), agar (15g / L), Rpf recombinant protein (10μg / L), magnesium sulfate (0.6g / L), sodium pyruvate (1.1g / L) and vitamin B12 (0.5mg / L), and the balance is water. Magnesium sulfate, tryptone, sodium chloride, soytone, agar and water are mixed, the pH value of the medium is adjusted to 7.4, and autoclaved at 121℃ for 15 minutes; Rpf recombinant protein, sodium pyruvate and vitamin B12 are sterilized by filtration. When the culture medium after autoclaving at 121℃ is cooled to 45℃-50℃, the sterilized Rpf recombinant protein, sodium pyruvate and vitamin B12 are added.

[0041] Wherein, the nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1, and SEQ ID NO.1 is: .

[0042] 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.

[0043] The nucleotide sequence of the Rpf protein of Micrococcus luteus before mutation is shown in SEQ ID NO.2, which is: .

[0044] 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.

[0045] Embodiment 2: Preparation of modified TSA broth medium: tryptone (15g / L), sodium chloride (5g / L), soytone (5g / L), agar (15g / L), Rpf recombinant protein (10μg / L), magnesium sulfate (0.6g / L), sodium pyruvate (0.275g / L) and vitamin B12 (0.13mg / L), and the balance is water. Magnesium sulfate, tryptone, sodium chloride, soytone, agar and water are mixed, the pH value of the medium is adjusted to 7.4, and autoclaved at 121℃ for 15 minutes; Rpf recombinant protein, sodium pyruvate and vitamin B12 are sterilized by filtration. When the culture medium after autoclaving at 121℃ is cooled to 45℃-50℃, the sterilized Rpf recombinant protein, sodium pyruvate and vitamin B12 are added.

[0046] Embodiment 3: Preparation of modified TSA broth medium: tryptone (15g / L), sodium chloride (5g / L), soytone (5g / L), agar (15g / L), Rpf recombinant protein (100μg / L), magnesium sulfate (0.6g / L), sodium pyruvate (0.55g / L) and vitamin B12 (0.25mg / L), and the balance is water. Magnesium sulfate, tryptone, sodium chloride, soytone, agar and water are mixed, the pH value of the medium is adjusted to 7.4, and autoclaved at 121℃ for 15 minutes; Rpf recombinant protein, sodium pyruvate and vitamin B12 are sterilized by filtration. When the culture medium after autoclaving at 121℃ is cooled to 45℃-50℃, the sterilized Rpf recombinant protein, sodium pyruvate and vitamin B12 are added.

[0047] Embodiment 4: Preparation of modified TSA broth medium: tryptone (15g / L), sodium chloride (5g / L), soytone (5g / L), agar (15g / L), Rpf recombinant protein (100μg / L), magnesium sulfate (0.6g / L), sodium pyruvate (4.4g / L) and vitamin B12 (2mg / L), and the balance is water. Magnesium sulfate, tryptone, sodium chloride, soytone, agar and water are mixed, the pH value of the medium is adjusted to 7.4, and autoclaved at 121℃ for 15 minutes; Rpf recombinant protein, sodium pyruvate and vitamin B12 are sterilized by filtration. When the culture medium after autoclaving at 121℃ is cooled to 45℃-50℃, the sterilized Rpf recombinant protein, sodium pyruvate and vitamin B12 are added.

[0048] Embodiment 5: Preparation of modified TSA broth medium: tryptone (15g / L), sodium chloride (5g / L), soytone (5g / L), agar (15g / L), Rpf recombinant protein (10μg / L) and magnesium sulfate (0.6g / L), the balance is water. Magnesium sulfate, tryptone, sodium chloride, soytone, agar and water are mixed, the pH value of the medium is adjusted to 7.4, and autoclaved at 121℃ for 15 minutes; Rpf recombinant protein is sterilized by filtration. When the culture medium after autoclaving at 121℃ is cooled to 45℃-50℃, the sterilized Rpf recombinant protein is added.

[0049] Comparative Example 1: Preparation of modified TSA broth medium: tryptone (15g / L), sodium chloride (5g / L), soytone (5g / L), agar (15g / L) and sodium pyruvate (3.3g / L), the balance is water. Magnesium sulfate, tryptone, sodium chloride, soytone, agar and water are mixed, 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 culture medium after autoclaving at 121°C is cooled to 45°C-50°C, the sterilized sodium pyruvate is added.

[0050] Comparative Example 2: Preparation of modified TSA broth medium: tryptone (15g / L), sodium chloride (5g / L), soytone (5g / L), agar (15g / L), Rpf recombinant protein (10μg / L), magnesium sulfate (0.6g / L) and sodium pyruvate (3.3g / L), and the balance is water. Magnesium sulfate, tryptone, sodium chloride, soytone, agar and water are mixed, the pH value of the medium is adjusted to 7.4, and autoclaved at 121℃ for 15 minutes; Rpf recombinant protein and sodium pyruvate are sterilized by filtration. When the culture medium after autoclaving at 121℃ is cooled to 45℃-50℃, the sterilized Rpf recombinant protein and sodium pyruvate are added.

[0051] Comparative Example 3: Preparation of modified TSA broth medium: tryptone (15g / L), sodium chloride (5g / L), soytone (5g / L), agar (15g / L), Rpf recombinant protein (10μg / L), magnesium sulfate (0.6g / L) and vitamin B12 (1mg / L), the balance is water. Magnesium sulfate, tryptone, sodium chloride, soytone, agar and water are mixed, the pH value of the medium is adjusted to 7.4, and autoclaved at 121℃ for 15 minutes; Rpf recombinant protein and vitamin B12 are sterilized by filtration. When the culture medium after autoclaving at 121℃ is cooled to 45℃-50℃, the sterilized Rpf recombinant protein and vitamin B12 are added.

[0052] The improved TSA broth medium obtained in the examples and comparative examples was used to culture VBNC Escherichia coli and VBNC Staphylococcus aureus.

[0053] The improved TSA broth culture medium obtained in the examples and comparative examples was tested: (1) Determination of the composition of the improved TSA broth medium: Culture medium preparation: prepare the culture medium of the embodiment and comparative example respectively.

[0054] Bacterial solution dilution: Use sterile PBS buffer solution to adjust the total number of viable bacteria in the VBNC state of the VBNC state Escherichia coli working bacterial suspension to 10,000 cells / mL.

[0055] Inoculation and culture: 0.1 mL of VBNC Escherichia coli suspension was taken and inoculated into the culture medium plates of the embodiment and the comparative example respectively, and evenly spread. Three plates were inoculated for each treatment. The plates were incubated at 37°C for 24 hours, and the colonies were observed and counted the next day.

[0056] Counting: Count the number of colonies growing on the plate. The results are shown in Table 1.

[0057] (2) Growth rate quantitative test: Referring to GB4789.28-2024, the quantitative determination method for the growth rate of target bacteria in non-selective separation and counting solid culture medium was used to detect the growth rate of Escherichia coli and Staphylococcus aureus in the modified TSA broth medium, and the bacterial enrichment effect of Escherichia coli in the VBNC state and Staphylococcus aureus in the VBNC state was examined.

[0058] Culture medium preparation: unmodified TSA broth culture medium and modified TSA broth culture medium obtained in Example 1 were prepared.

[0059] Bacterial liquid dilution: Use sterile PBS buffer to adjust the total number of viable bacteria in the VBNC state of Escherichia coli working bacterial suspension to 1000 cells / mL. Use sterile PBS buffer to adjust the total number of viable bacteria in the VBNC state of Staphylococcus aureus working bacterial suspension to 1000 cells / mL.

[0060] Inoculation and culture: 0.1 mL of the bacterial suspension in the VBNC state was taken and inoculated into the national standard culture medium (i.e., unmodified TSA broth culture medium, the formula is 15 g / L tryptone, 5 g / L sodium chloride, 5 g / L soytone, 15 g / L agar, and the balance is water) and the modified TSA broth culture medium plate obtained in Example 1, and evenly spread. Two plates were inoculated for each treatment. Incubate at 37°C for 18 hours.

[0061] Counting: Count the number of colonies grown on the plate. Calculate the growth rate PR=Ns / No. Where, "PR" is the "growth rate", "Ns" is the "average number of colonies on the test medium plate", and "No" is the "average number of colonies on the reference medium plate". The growth rate of the culture medium for Escherichia coli or Staphylococcus aureus should not be less than 0.7. The appearance, size and morphology of the colonies grown on each plate should conform to the characteristics of Escherichia coli or Staphylococcus aureus. The results are shown in Table 2.

[0062] (3) Suitability test of modified TSA broth medium for microbial limit detection: In order to evaluate whether the modified TSA broth medium is suitable for microbial limit detection, this experiment was based on the General Rules 1108 of Part IV of the Chinese Pharmacopoeia (2020 Edition) and GB4789.28-2024 "Quality Requirements for Food Microbiology Test Culture Media and Reagents of National Food Safety Standards", and used Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis to evaluate the growth rate of aerobic bacteria in the modified TSA broth medium. Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis were prepared into 100 CFU / mL bacterial suspensions with PBS buffer solution. 1 mL of each bacterial suspension was inoculated in a sterile plate, and then 15 mL-20 mL of the national standard culture medium plate (TSA) or modified TSA broth medium melted at a temperature not exceeding 45 ° C was injected, mixed, solidified, and inverted for 3 days at 35 ° C. Two culture medium plates were prepared in parallel for each culture medium. The growth rate was calculated by calculating the ratio of the average number of colonies grown after each bacterial culture in the modified TSA broth medium and the national standard medium. The growth rate of each bacterium should be between 0.5 and 2.0, proving that the modified TSA broth medium can detect VBNC Escherichia coli and Staphylococcus aureus, and is also suitable for the detection of aerobic bacteria. The results are shown in Table 3.

[0063] (4) Perform microbial limit testing on the modified TSA broth medium: According to the "1108 Chinese Pharmacopoeia 2020 Edition Microbial Limit Test Method for Chinese Herbal Medicine Pieces", the total aerobic bacteria, Escherichia coli and Salmonella were tested for the extracted licorice slices. Parallel tests were performed using the modified TSA broth medium of Example 1 and the national standard medium. The results are shown in Table 4.

[0064] Pretreatment of test sample. Take licorice sample as test sample. Take 25g of test sample, chop it thoroughly, and add modified TSA broth medium without agar to a total volume of 250mL. Vortex and mix for 20 minutes to obtain the "1:10" test solution (i.e. 25g sample in 250mL modified TSA broth medium to obtain the test sample). At the same time, use TSB medium (trypticase soy broth) (commercially available) for parallel experiments.

[0065] Aerobic bacteria count test. Take 1mL of 1:10 test solution and place it in a sterile plate with a diameter of 90mm. Inject 15m-20mL of modified TSA broth medium melted at a temperature not exceeding 45℃, mix well, solidify, and invert and culture at 30℃-35℃ for 3-5 days. Observe the growth of colonies, count all colonies grown on the plate, count and report. At the same time, use TSA medium (commercially available) for parallel experiments.

[0066] Escherichia coli test: Take 10 mL of 1:10 test solution in 100 mL of the modified TSA broth medium of Example 1 of the present invention, mix well, and culture at 30°C-35°C for 18 hours-24 hours. At the same time, a parallel experiment was performed with TSA medium (commercially available). 1 mL of the culture of the above-mentioned modified TSA broth medium or TSA medium (commercially available) was inoculated into 100 mL of MacConkey liquid medium (commercially available) and cultured at 42°C-44°C for 24 hours-48 hours. Take the MacConkey liquid culture and inoculate it on the MacConkey agar medium plate, and culture it at 30°C-35°C for 18 hours-72 hours. Result judgment: If there is colony growth on the MacConkey agar medium plate, separation, purification and appropriate identification tests should be carried out to confirm whether it is Escherichia coli; if there is no colony growth on the MacConkey agar medium plate, or if there is colony growth but the identification result is negative, it is determined that the culture medium has not detected Escherichia coli.

[0067] Salmonella inspection: Take 10 mL of 1:10 test solution in 100 mL of the modified TSA broth medium of Example 1 of the present invention, mix well, and culture at 30°C-35°C for 18 hours-24 hours. At the same time, a parallel experiment was performed with TSA medium (commercially available). Take 0.1 mL of the culture of the above-mentioned modified TSA broth medium or TSA medium (commercially available) and inoculate it into 10 mL RV Salmonella enrichment liquid culture medium (commercially available) and culture it at 30°C-35°C for 18 hours-24 hours. Take a small amount of RV Salmonella enrichment liquid culture and streak it on a xylose lysine deoxycholate agar medium (commercially available) plate and culture it at 30°C-35°C for 18 hours-48 hours. Enteritidis Salmonella grows well on xylose lysine deoxycholate agar medium plates, and the colonies are light red or colorless, transparent or translucent, with or without black in the center. Determine whether Salmonella is detected in the culture medium.

[0068] (5) Application test of improved TSA broth medium: Applicability verification of the sedimentation bacteria test in clean rooms (areas): The sedimentation bacteria test was conducted in the seedling preparation room of the clean production area of ​​the inactivated vaccine GMP production line of a company in Yantai City in accordance with the "GB / T 16292 Test Method for Suspended Particles in Clean Rooms (Areas) in Pharmaceutical Industry". The cleanliness level of the seedling preparation room is 10,000, the indoor area is 15 square meters, and 2 detection sampling points are set. The sedimentation bacteria should be ≤3CFU / dish. Collect at the beginning of production, and the exposure time of the culture dish is 2 hours. After all sampling is completed, the culture dish is inverted in a constant temperature incubator at 30℃-35℃ and cultured for 48h. Two 90mm culture dishes are placed at the same time for negative control tests. At the same time, the national standard culture medium and the modified TSA broth culture medium plate of Example 1 are used for plate detection. The results are shown in Table 5.

[0069] (6) Culture medium repeatability test: Three boxes were randomly selected from three batches of products prepared in Example 1 to test the growth rate of Escherichia coli and Staphylococcus aureus using the modified TSA broth culture medium in Example 1, and to test the bacterial enrichment effect of VBNC Escherichia coli and VBNC Staphylococcus aureus. Each batch of test kits was tested three times in parallel.

[0070] Results and Analysis: Table 1 Composition of culture medium in Examples and Comparative Examples

[0071] Table 2 Growth rate quantitative test results

[0072] Table 3 Results of culture medium suitability test

[0073] Table 4 Test results of microbial limit test of Chinese herbal medicine pieces

[0074] Table 5 Test results of culture medium for sedimentation bacteria in clean rooms and clean areas of pharmaceutical industry

[0075] Table 6 Culture medium repeatability test results

[0076] By comparing the culture medium of Examples 1-5 with Comparative Examples 1-3, it is found that the improved TSA broth culture medium of the present invention can successfully activate VBNC state Escherichia coli, thereby detecting Escherichia coli. In the improved TSA broth culture medium of the present invention, the Rpf recombinant protein has good activity, can decompose the peptidoglycan of the cell wall of Escherichia coli, the cell wall becomes loose, and the degraded peptidoglycan can enter the cell through the loose cell wall, thereby resuscitating the VBNC state cells; on this basis, magnesium sulfate is added at the same time, and the magnesium ions provided by magnesium sulfate further activate the activity of the Rpf recombinant protein, which is beneficial to the effect of the Rpf recombinant protein on Escherichia coli; in Example 5, it can be found that the Rpf recombinant protein cooperates with magnesium sulfate to achieve the culturability of VBNC state Escherichia coli. In Comparative Example 1, sodium pyruvate is used alone. Sodium pyruvate is not only a scavenger of active oxygen free radicals, which removes active oxygen free radicals that damage DNA in cells and restores the cell state, but also can be used as an energy substance and play an important role in the glycolysis process.

[0077] In Comparative Example 1, the addition of sodium pyruvate can also achieve the culturability of VBNC state Escherichia coli. The study further found that when Rpf recombinant protein, magnesium sulfate and sodium pyruvate were used at the same time, although VBNC state Escherichia coli could be activated, the culturing effect was better than that of Comparative Example 1, but significantly lower than that of Example 5; when Rpf recombinant protein, magnesium sulfate and vitamin B12 were used, the effect of Comparative Example 3 was also significantly lower than that of Example 5. When Rpf recombinant protein, magnesium sulfate, sodium pyruvate and vitamin B12 were used at the same time, the culturing capacity of the culture medium was significantly improved, and the number of viable bacteria of VBNC state Escherichia coli obtained in Examples 1-4 was at least 9 times that of Comparative Examples 1-3.

[0078] It should be understood by those skilled in the art that the interaction and coordination of the culture medium components realize the activation of VBNC state Escherichia coli, thereby realizing the process of microbial culturing. Comparing Example 1 and Example 2, it is found that when the amount of sodium pyruvate and vitamin B12 added to the culture medium is increased, the number of culturable Escherichia coli in the VBNC state can be significantly increased; Comparing Example 2 and Example 3, it is found that although adding sodium pyruvate and vitamin B12 within a certain range is beneficial to increase the number of culturable VBNC state Escherichia coli, when an excessive amount of Rpf recombinant protein is added, the recovery and bacterial growth effect of VBNC state bacteria will be weakened. It can be seen that there is an interaction between Rpf recombinant protein, magnesium sulfate, sodium pyruvate and vitamin B12. Finally, comparing Example 3 and Example 4, it is found that when Rpf recombinant protein, sodium pyruvate and vitamin B12 are added in excess, the culturing capacity of the culture medium is significantly reduced. In summary, it is further preferred that the mass concentration ratio of trypsin, sodium chloride, soytone, agar, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and vitamin B12 is 15 g / L: 5 g / L: 5 g / L: 15 g / L: 10 μg / L: 0.6 g / L: 1.1 g / L: 0.5 mg / L.

[0079] The results in Table 2 show that the improved TSA broth medium of the present invention can be used to culture Escherichia coli, Staphylococcus aureus, VBNC Escherichia coli and VBNC Staphylococcus aureus; and the growth rates of the improved TSA broth medium of the present invention for Escherichia coli and Staphylococcus aureus are both greater than 0.7, which meets the growth rate requirements of the target bacteria in non-selective separation and counting solid culture medium. The colonies on each culture medium meet the characteristics of the respective strains.

[0080] The results in Table 3 show that the ratio of the average number of colonies on the modified TSA broth medium plate of the present invention to the average number of colonies on the national standard medium plate is between 0.5 and 2.0. Therefore, the modified TSA broth medium of the present invention is suitable for the cultivation and detection of Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis.

[0081] The results in Table 4 show that for the 10 samples of licorice slices, the number of positive Escherichia coli and Salmonella was 0 when the national standard culture medium was used for detection, but 2 samples were positive for Escherichia coli when the improved TSA broth culture medium of the present invention was used for detection.

[0082] The results in Table 5 show that the number of colonies reported by the national standard culture medium plate test in the clean room of the 10,000-level seedling room is 0.5 / plate; the result of the test using the improved TSA broth culture medium of the present invention is 5.5 CFU, which exceeds the national standard requirement (greater than 3 CFU / plate). It can be seen that the improved TSA broth culture medium of the present invention is suitable for the sedimentation bacteria test in the clean room (area).

[0083] The repeatability test results in Table 6 show that the growth rates of the three batches of modified TSA broth culture medium for Escherichia coli or Staphylococcus aureus are all greater than 0.7, indicating that the nutritional performance of the three batches of laboratory trial products is good. The three batches of modified TSA broth culture medium can achieve recovery culture for VBNC Escherichia coli and VBNC Staphylococcus aureus. The repeatability of the three batches of modified TSA broth culture medium is good.

Claims

1. A modified TSA broth medium, characterized in that: The improved TSA broth medium comprises tryptone, sodium chloride, soytone, agar, Rpf recombinant protein and magnesium sulfate, and the mass concentration ratio of the tryptone, sodium chloride, soytone, agar, Rpf recombinant protein and magnesium sulfate is (10g / L-20g / L): (4g / L-6g / L): (3g / L-7g / L): (13g / L-17g / L): (10μg / L-100μg / L): (0.5g / L-0.7g / L); The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.

1.

2. The improved TSA broth medium according to claim 1, characterized in that The improved TSA broth culture medium also includes sodium pyruvate and vitamin B12. The mass concentration ratios of the tryptone, sodium chloride, soytone, agar, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and vitamin B12 are (10g / L-20g / L): (4g / L-6g / L): (3g / L-7g / L): (13g / L-17g / L): (10μg / L-100μg / L): (0.5g / L-0.7g / L): (0.27g / L-4.4g / L): (0.25mg / L-2mg / L).

3. The improved TSA broth medium according to claim 2, characterized in that The mass concentration ratio of the tryptone, sodium chloride, soytone, agar, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and vitamin B12 is 15g / L: 5g / L: 5g / L: 15g / L: 10μg / L: 0.6g / L: 1.1g / L: 0.5mg / L.

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

6.

5. Use of the improved TSA broth medium according to any one of claims 1 to 4 in culturing VBNC state microorganisms.

6. Use of the improved TSA broth medium according to any one of claims 1 to 4 in culturing VBNC Escherichia coli and / or VBNC Staphylococcus aureus.

7. The use according to claim 6, characterized in that: The culture temperature is 36°C-38°C, and the culture time is 18h-24h.

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