Improved LST broth media and uses

Abstract

The invention relates to the technical field of microorganisms, and discloses an improved LST broth culture medium and application, and the improved LST broth culture medium comprises tryptone, ammonium chloride, lactose, dipotassium phosphate, monopotassium phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and pyridoxal phosphate. The improved LST broth culture medium disclosed by the invention can be used for successfully enriching the VBNC-state escherichia coli, so that researchers can successfully detect the VBNC-state escherichia coli in a culture manner.

Description

Technical Field

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

[0002] LST broth medium is a commonly used culture medium, mainly used for microbial culture, especially in food microbial testing, for the growth and separation of bacteria. The main components of LST broth medium include tryptone, lactose, sodium chloride, sodium lauryl sulfate, etc.

[0003] LST broth medium is often used to detect Escherichia coli or other Gram-negative bacteria in food. Escherichia coli is cultured in LST broth medium to observe whether there is an acidic reaction or gas production, so as to determine whether there is an Escherichia coli group. LST broth medium is particularly suitable for the enrichment culture of Escherichia coli, which is conducive to the identification of Escherichia coli in food. However, conventional LST broth medium cannot enrich and culture Escherichia coli in the VBNC state, and Escherichia coli cannot be detected from food. U.S. patent application US20200340040A1 uses PCR amplification technology to detect microorganisms in the VBNC state, but the technology is cumbersome and costly. Therefore, there is an urgent need for an LST broth medium that can successfully detect Escherichia coli in the VBNC state. Summary of the invention

[0004] The invention provides an improved LST broth culture medium and application thereof.

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

[0006] A modified LST broth medium, comprising tryptone, ammonium chloride, lactose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate (MgSO4), sodium pyruvate and pyridoxal phosphate, wherein the mass concentration ratio of the tryptone, ammonium chloride, lactose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and pyridoxal phosphate is (15 g / L- 25g / L): (4g / L-6g / L): (3g / L-7g / L): (2.5g / L-3g / L): (2.5g / L-3g / L): (0.02g / L-0.04 g / L): (0.9μg / L-1.1μg / L): (0.5g / L-0.7g / L): (0.55g / L-3.3g / L): (0.25mg / L-2mg / L); The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.1.

[0007] Furthermore, the mass concentration ratio of trypsin, ammonium chloride, lactose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate, sodium pyruvate, and pyridoxal phosphate is 20g / L: 5g / L: 5g / L: 2.75g / L: 2.75g / L: 0.03g / L: 1μg / L: 0.6g / L: 2.2g / L: 1mg / L.

[0008] Furthermore, the pH value of the improved LST broth medium is 7.2-7.6.

[0009] The present invention also provides application of the improved LST broth culture medium in culturing Gram-negative bacteria in the VBNC state.

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

[0011] Furthermore, the culture temperature is 36°C-38°C, and the culture time is 18h-24h.

[0012] The present invention has the following technical effects: Rpf recombinant protein has good peptidoglycan hydrolase activity, which can decompose the peptidoglycan of the Escherichia coli cell wall, making the cell wall loose. The degraded peptidoglycan can enter the cell through the loose cell wall, thereby awakening the 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 to remove reactive oxygen free radicals that damage DNA in cells, but also as an energy substance, playing an important role in the glycolysis process. After adding pyridoxal phosphate, the synergistic effect of Rpf recombinant protein and sodium pyruvate can be significantly enhanced, awakening cells in the VBNC state. DETAILED DESCRIPTION

[0013] 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.

[0014] In a first aspect, some embodiments of the present invention provide a modified LST broth medium, wherein the modified LST broth medium comprises tryptone, ammonium chloride, lactose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and pyridoxal phosphate, and the mass concentration of tryptone, ammonium chloride, lactose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and pyridoxal phosphate is 2.347 kcal / cm3. The concentration ratio is (15g / L-25g / L): (4g / L-6g / L): (3g / L-7g / L): (2.5g / L-3g / L): (2.5g / L-3g / L): (0.02g / L-0.04g / L): (0.9μg / L-1.1μg / L): (0.5g / L-0.7g / L): (0.55g / L-3.3g / L): (0.25mg / L-2mg / L).

[0015] In some embodiments, the nucleotide sequence of the Rpf recombinant protein is shown as SEQ ID NO.1.

[0016] In some embodiments, the modified LST broth medium further comprises water.

[0017] In some embodiments, the improved LST broth medium includes tryptone, ammonium chloride, lactose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate, sodium pyruvate, and pyridoxal phosphate. The mass concentration ratio of tryptone, ammonium chloride, lactose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate, sodium pyruvate, and pyridoxal phosphate is 20 g / L: 5 g / L: 5 g / L: 2.75 g / L: 2.75 g / L: 0.03 g / L: 1 μg / L: 0.6 g / L: 2.2 g / L: 1 mg / L.

[0018] In some embodiments, the pH of the modified LST broth medium is 7.2-7.6.

[0019] In a second aspect, some embodiments of the present invention also provide the use of a modified LST broth medium in culturing Gram-negative bacteria in a VBNC state.

[0020] In a third aspect, in some embodiments of the present invention, the present invention also provides the use of a modified LST broth medium in culturing Escherichia coli in a VBNC state.

[0021] In some embodiments, the culture temperature is 36° C.-38° C., and the culture time is 18 h-24 h.

[0022] The following is described in conjunction with the embodiments and comparative examples: Preparation of VBNC Escherichia coli: (1) Inoculate Escherichia coli ATCC25922 into LB medium (commercially available), incubate at 37°C with shaking at 180 rpm overnight (14 to 16 hours), and store at 2 to 8°C. Inoculate into fresh LB medium at a volume ratio of 1:100 (V:V) to expand the culture of Escherichia coli ATCC25922.

[0023] (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.

[0024] (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.

[0025] (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.

[0026] (5) Determination of total viable count: 1 mL of bacterial solution was taken 24 h after stress induction. A large number of strains died during the induction process. Therefore, after staining with the CTC Bacterial Viability Detection Kit (Tongren Chemical Research Institute), the total number of remaining viable bacteria was detected by flow cytometry. The result was 5.9±0.95×10 4 cell / mL.

[0027] (6) Number of culturable bacteria: The plate count method was used for testing, and the test result was 0 CFU / mL.

[0028] (7) The VBNC bacterial content was calculated according to the following formula: VBNC bacteria = total number of viable bacteria - number of culturable bacteria.

[0029] (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.

[0030] Example 1: Cultivating Escherichia coli in VBNC state: Improved LST broth medium was prepared: Rpf recombinant protein (1 μg / L), magnesium sulfate (0.6 g / L), pyridoxal phosphate (1 mg / L), sodium pyruvate (2.2 g / L), tryptone (20 g / L), lactose (5 g / L), sodium chloride (5 g / L), dipotassium hydrogen phosphate (2.75 g / L), potassium dihydrogen phosphate (2.75 g / L), sodium lauryl sulfate (0.03 g / L), and the balance was water. Magnesium sulfate, tryptone, lactose, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate and water were mixed, and then the pH value of the medium was adjusted to 7.4 and sterilized at 121°C for 15 minutes; Rpf recombinant protein, sodium pyruvate and pyridoxal phosphate were sterilized by filtration. When the culture medium sterilized at 121° C. is cooled to 45° C.-50° C., the sterilized Rpf recombinant protein, sodium pyruvate and pyridoxal phosphate are added.

[0031] Among them, the nucleotide sequence of the Rpf recombinant protein (SEQ ID NO.1) is: .

[0032] 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.

[0033] The nucleotide sequence of the Rpf protein of Micrococcus luteus before mutation (SEQ ID NO.2) is: .

[0034] 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 protease activity of the Rpf recombinant protein of sequence SEQ ID NO.1 in the present invention was 317U / mL, while the protease activity of the original sequence SEQ ID NO.2 without mutation was 83U / mL. It can be seen that the protease activity after mutation is increased by more than 2 times.

[0035] Embodiment 2: Improved LST broth medium of Example 2: Rpf recombinant protein (1 μg / L), magnesium sulfate (0.6 g / L), pyridoxal phosphate (0.25 mg / L), sodium pyruvate (0.55 g / L), tryptone (20 g / L), lactose (5 g / L), sodium chloride (5 g / L), dipotassium hydrogen phosphate (2.75 g / L), potassium dihydrogen phosphate (2.75 g / L), sodium lauryl sulfate (0.03 g / L), and the balance is water. Magnesium sulfate, tryptone, lactose, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate and water are mixed, and then the pH value of the medium is adjusted to 7.4, and sterilized at 121°C for 15 minutes; Rpf recombinant protein, sodium pyruvate and pyridoxal phosphate are sterilized by filtration. When the culture medium sterilized at 121° C. is cooled to 45° C.-50° C., the sterilized Rpf recombinant protein, sodium pyruvate and pyridoxal phosphate are added.

[0036] Embodiment 3: Improved LST broth medium of Example 3: Rpf recombinant protein (1 μg / L), magnesium sulfate (0.6 g / L), pyridoxal phosphate (0.5 mg / L), sodium pyruvate (1.1 g / L), tryptone (20 g / L), lactose (5 g / L), sodium chloride (5 g / L), dipotassium hydrogen phosphate (2.75 g / L), potassium dihydrogen phosphate (2.75 g / L), sodium lauryl sulfate (0.03 g / L), and the balance is water. Magnesium sulfate, tryptone, lactose, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate and water are mixed, and then the pH value of the medium is adjusted to 7.4, and sterilized at 121°C for 15 minutes; Rpf recombinant protein, sodium pyruvate and pyridoxal phosphate are sterilized by filtration. When the culture medium sterilized at 121° C. is cooled to 45° C.-50° C., the sterilized Rpf recombinant protein, sodium pyruvate and pyridoxal phosphate are added.

[0037] Embodiment 4: The improved LST broth medium of Example 4: Rpf recombinant protein (1 μg / L), magnesium sulfate (0.6 g / L), pyridoxal phosphate (2 mg / L), sodium pyruvate (3.3 g / L), tryptone (20 g / L), lactose (5 g / L), sodium chloride (5 g / L), dipotassium hydrogen phosphate (2.75 g / L), potassium dihydrogen phosphate (2.75 g / L), sodium lauryl sulfate (0.03 g / L), and the balance is water. Magnesium sulfate, tryptone, lactose, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate and water are mixed, and then the pH value of the medium is adjusted to 7.4, and sterilized at 121°C for 15 minutes; Rpf recombinant protein, sodium pyruvate and pyridoxal phosphate are sterilized by filtration. When the culture medium sterilized at 121° C. is cooled to 45° C.-50° C., the sterilized Rpf recombinant protein, sodium pyruvate and pyridoxal phosphate are added.

[0038] Comparative Example 1: Improved LST broth medium of comparative example 1: Rpf recombinant protein (1 μg / L), magnesium sulfate (0.6 g / L), tryptone (20 g / L), lactose (5 g / L), sodium chloride (5 g / L), dipotassium hydrogen phosphate (2.75 g / L), potassium dihydrogen phosphate (2.75 g / L), sodium lauryl sulfate (0.03 g / L), and the balance is water. Magnesium sulfate, tryptone, lactose, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate and water are mixed, and then the pH value of the culture medium is adjusted to 7.4, and sterilized at 121°C for 15 minutes; Rpf recombinant protein is sterilized by filtration. When the culture medium after high-pressure sterilization at 121°C is cooled to 45°C-50°C, the sterilized Rpf recombinant protein is added.

[0039] Comparative Example 2: The improved LST broth medium of comparative example 2: sodium pyruvate (1.1 g / L), tryptone (20 g / L), lactose (5 g / L), sodium chloride (5 g / L), dipotassium hydrogen phosphate (2.75 g / L), potassium dihydrogen phosphate (2.75 g / L), sodium lauryl sulfate (0.03 g / L), and the balance is water. Among them, tryptone, lactose, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate 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; 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.

[0040] Comparative Example 3: Improved LST broth medium of comparative example 3: pyridoxal phosphate (1 mg / L), tryptone (20 g / L), lactose (5 g / L), sodium chloride (5 g / L), dipotassium hydrogen phosphate (2.75 g / L), potassium dihydrogen phosphate (2.75 g / L), sodium lauryl sulfate (0.03 g / L), and the balance is water. Among them, tryptone, lactose, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate 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; pyridoxal phosphate is sterilized by filtration. When the culture medium after autoclaving at 121°C is cooled to 45°C-50°C, the sterilized pyridoxal phosphate is added.

[0041] Comparative Example 4: Improved LST broth medium of comparative example 4: Rpf recombinant protein (1 μg / L), magnesium sulfate (0.6 g / L), sodium pyruvate (1.1 g / L), tryptone (20 g / L), lactose (5 g / L), sodium chloride (5 g / L), dipotassium hydrogen phosphate (2.75 g / L), potassium dihydrogen phosphate (2.75 g / L), sodium lauryl sulfate (0.03 g / L), and the balance is water. Magnesium sulfate, tryptone, lactose, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate and water are mixed, and then the pH value of the culture medium is adjusted to 7.4, and sterilized at 121°C for 15 minutes; Rpf recombinant protein and sodium pyruvate are sterilized by filtration. When the culture medium after high-pressure sterilization at 121°C is cooled to 45°C-50°C, the sterilized Rpf recombinant protein and sodium pyruvate are added.

[0042] Comparative Example 5: Improved LST broth medium of comparative example 5: Rpf recombinant protein (1 μg / L), magnesium sulfate (0.6 g / L), pyridoxal phosphate (1 mg / L), tryptone (20 g / L), lactose (5 g / L), sodium chloride (5 g / L), dipotassium hydrogen phosphate (2.75 g / L), potassium dihydrogen phosphate (2.75 g / L), sodium lauryl sulfate (0.03 g / L), and the balance is water. Magnesium sulfate, tryptone, lactose, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate and water are mixed, and then the pH value of the culture medium is adjusted to 7.4, and sterilized at 121°C for 15 minutes; Rpf recombinant protein and pyridoxal phosphate are sterilized by filtration. When the culture medium after high-pressure sterilization at 121°C is cooled to 45°C-50°C, the sterilized Rpf recombinant protein and pyridoxal phosphate are added.

[0043] Comparative Example 6: Improved LST broth medium of comparative example 6: pyridoxal phosphate (1 mg / L), sodium pyruvate (1.1 g / L), tryptone (20 g / L), lactose (5 g / L), sodium chloride (5 g / L), dipotassium hydrogen phosphate (2.75 g / L), potassium dihydrogen phosphate (2.75 g / L), sodium lauryl sulfate (0.03 g / L), and the balance is water. Among them, tryptone, lactose, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate and water are mixed, and then the pH value of the culture medium is adjusted to 7.4, and sterilized at 121°C for 15 minutes; sodium pyruvate and pyridoxal phosphate are sterilized by filtration. When the culture medium after high-pressure sterilization at 121°C is cooled to 45°C-50°C, the sterilized sodium pyruvate and pyridoxal phosphate are added.

[0044] The improved LST broth medium obtained in the examples and comparative examples was used to culture VBNC Escherichia coli. The culture conditions for VBNC Escherichia coli were as follows: the culture temperature was 36° C.-38° C., and the culture time was 24 hours.

[0045] The improved LST broth culture medium obtained from the examples and comparative examples was tested: (1) Determination of the composition of the improved LST broth medium: Culture medium preparation: The modified LST broth culture medium of the embodiment and the comparative example was prepared respectively.

[0046] Bacterial solution dilution: Use sterile PBS buffer solution to adjust the working bacterial suspension of Escherichia coli in the VBNC state, and adjust the total number of viable bacteria in the VBNC state to 10,000 cells / mL.

[0047] Inoculation culture: 0.1 mL of the working bacterial suspension of VBNC Escherichia coli was taken and 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 of the bacterial solution and apply it on the tryptic soy peptone agar medium, culture it in a 37°C biochemical incubator overnight, and observe and count the colonies the next day.

[0048] Counting: Count the number of colonies growing on the plate. The results are shown in Table 1.

[0049] Taking the improved LST broth culture medium of Example 1 as an example, further research was conducted.

[0050] (2) Nutritional capacity of modified LST broth culture medium (semi-quantitative test of growth rate): Refer to the semi-quantitative detection method of GB4789.28-2024 to detect the bacterial growth effect of modified LST broth culture medium on VBNC Escherichia coli.

[0051] The prepared VBNC state Escherichia coli ATCC25922 bacterial suspension was adjusted with a sterile PBS buffer solution to adjust the total number of viable bacteria to 1000 cells / mL, and 0.1 mL of the VBNC state Escherichia coli bacterial suspension was inoculated into 9.9 mL of the national standard culture medium (i.e., LST broth culture medium, with a formula of 20 g / L tryptone, 5 g / L lactose, 5 g / L sodium chloride, 2.75 g / L dipotassium hydrogen phosphate, 2.75 g / L potassium dihydrogen phosphate, and 0.03 g / L sodium lauryl sulfate) and the improved LST broth culture medium obtained in the embodiment of the present invention. Three parallel tubes were inoculated in parallel for each culture medium or each component culture medium, and cultured at 37° C. for 18 hours.

[0052] Plate count: Get 10 μL of the culture after enrichment and spread it evenly on the TSA agar plate. If the colony on the TSA agar plate is greater than 10 CFU, the growth rate of the culture medium is judged to be good.

[0053] (3) Improved LST broth medium selectivity test: 1×10 3 ~5×10 3 CFU Enterococcus faecalis CMCC (B) 32482 strain or Staphylococcus aureus ATCC 6538 strain were inoculated into 10mL national standard culture medium and modified LST broth culture medium, respectively, and three parallel tubes were inoculated in parallel. They were cultured at 37℃ for 18 hours, and the culture medium should be free of turbidity. Take 10μL of the culture after enrichment and spread it evenly on the TSA plate. The colony count of Enterococcus faecalis and / or Staphylococcus aureus on the TSA plate should be less than 100CFU, indicating that the culture medium has good selectivity.

[0054] (4) Repeatability test of modified LST broth medium: A semi-quantitative test method was used to detect the intra-batch and inter-batch repeatability of the culture medium in accordance with GB4789.28-2024. Three boxes were randomly selected from each of the three batches of modified LST broth medium to evaluate the nutritional performance of the culture medium for Escherichia coli and VBNC state Escherichia coli and the selectivity for Staphylococcus aureus and Enterococcus faecalis. Each batch of test kits was tested in parallel three times. The test method is as follows: Preparation and packaging of culture medium: Prepare national standard culture medium, quantitatively package, 9.9mL / tube. Take the modified LST broth culture medium to be tested, quantitatively package, 9.9mL / tube; Qualitative determination of growth rate: refer to the semi-quantitative detection method of GB4789.28-2024; Medium selectivity assay.

[0055] (5) Culture medium stability experiment: Three batches of modified LST broth culture medium products were stored at 2°C-8°C. Culture medium samples were taken after 3 months, 6 months, 9 months, 12 months and 15 months of storage, and the culture medium was tested for nutritional performance and selectivity.

[0056] (6) Clinical sample testing: 10 water samples were collected from different sampling points of tap water in a canteen in Yantai City and different sampling points of water reservoirs (10 water samples). The samples were tested according to the MPN counting method of GB4789.3-2016 National Food Safety Standard for Food Microbiology Examination of Coliform Bacteria. According to the National Standard for Drinking Water Hygiene (GB / T5749-2022), total coliform bacteria should not be detected.

[0057] Sample dilution and inoculation fermentation: dilute the water sample 10 times in series, select 3 suitable sample homogenates with continuous dilutions, take 1 mL of each dilution and inoculate into 3 9 mL national standard culture media or modified LST broth culture media. At the same time, take 1 mL of sterile saline as a blank control. Incubate at 37°C for 24 hours. Observe whether bubbles are generated in the catheter. If gas is produced, perform a multiple fermentation test (confirmation test); if no gas is produced, continue to culture for 48 hours. The tube that produces gas is the coliform-positive tube, and the tube that does not produce gas is the coliform-negative tube.

[0058] Repeated fermentation test (confirmation test): Use an inoculation loop to take one loop of culture from the gas-producing modified LST broth medium, transfer it to a brilliant green lactose bile salt broth (BGLB) tube, culture it at 37℃ for 48 hours, and observe the gas production. The gas-producing tube is the coliform-positive tube.

[0059] Reporting of Most Probable Coliform Number (MPN): Retrieve the MPN table and report the MPN value of coliform bacteria per mL of sample.

[0060] Results and Analysis: Table 1 Test results of culture medium of examples and comparative examples

[0061] Table 2 Quantitative test results of growth rate in culture medium of Example 1

[0062] 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 GB4789.28-2013; b: After bacterial enrichment, transfer to TSA plates for counting.

[0063] Table 3 Results of culture medium repeatability test in Example 1

[0064] 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 GB4789.28-2013; b: After bacterial enrichment, transfer to TSA plates for counting.

[0065] Table 4 Results of culture medium stability test in Example 1

[0066] 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 GB4789.28-2013; b: After bacterial enrichment, transfer to TSA plates for counting.

[0067] Table 5 Results of clinical water sample testing using culture medium in Example 1

[0068] Among them, “+” represents that the microorganism was detected.

[0069] The improved LST broth medium of the present invention is added with Rpf recombinant protein, which has good activity and can decompose the peptidoglycan of the cell wall of Escherichia coli in the VBNC state, so that the cell wall becomes loose, and the degraded peptidoglycan can enter the Escherichia coli cell through the loose cell wall, thereby awakening the Escherichia coli. In the culture medium, magnesium ions of magnesium sulfate provide divalent metal ions for the activity of Rpf recombinant protease, thereby facilitating the action of Rpf recombinant protein on Escherichia coli in the VNBC state.

[0070] As can be seen from Table 1, due to the addition of Rpf recombinant protein and magnesium sulfate in the culture medium of Comparative Example 1, VBNC state Escherichia coli was successfully cultured. Sodium pyruvate was added to the culture medium of Comparative Example 2. The sodium pyruvate in the culture medium can not only be used 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 be used as an energy substance, playing an important role in the glycolysis process. Therefore, in Comparative Example 2, the addition of sodium pyruvate alone can also promote Escherichia coli to release the VBNC state. Rpf recombinant protein, magnesium sulfate and sodium pyruvate were added to the culture medium of Comparative Example 4 at the same time, and its effect was not much different from that of Comparative Example 2. On this basis, the addition of pyridoxal phosphate significantly promoted the release of Escherichia coli from the VBNC state. Compared with Comparative Example 4, the Escherichia coli cultured in Examples 1-4 was at least 9 times that of Comparative Example 4.

[0071] Further study of Example 1-Example 3 found that pyridoxal phosphate increased with the increase of sodium pyruvate, and the number of Escherichia coli cells in the VBNC state of culture increased. However, with reference to the comparative verification of Example 3 and Example 4, when the addition amount of pyridoxal phosphate was further increased, the number of Escherichia coli cells in the VBNC state of culture began to decline, which shows that pyridoxal phosphate can regulate the expression of cell transcription factors and genes related to recovery by regulating the Rpos gene. This mechanism has a synergistic effect on sodium pyruvate regulation of intracellular redox reactions, and by regulating intracellular ROS levels and antiperoxidase systems, it synergistically regulates the formation and recovery of the VBNC state of cells. When sodium pyruvate is added in excess, it will cause an imbalance in the reaction, thereby affecting the recovery effect. Therefore, the mass concentration ratio of trypsin, ammonium chloride, lactose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate, sodium pyruvate, and pyridoxal phosphate is further preferably 20 g / L: 5 g / L: 5 g / L: 2.75 g / L: 2.75 g / L: 0.03 g / L: 1 μg / L: 0.6 g / L: 2.2 g / L: 1 mg / L.

[0072] The growth rate test results in Table 2 show that the improved LST broth medium of the present invention can successfully culture Escherichia coli and VBNC state Escherichia coli.

[0073] Enterococcus faecalis and Staphylococcus aureus were inoculated into the improved LST broth medium of the present invention respectively, spread evenly, and cultured the plate at 37°C for 18 to 24 hours. As a result, it was found that Enterococcus faecalis and Staphylococcus aureus were completely inhibited and no colony grew. It can be seen that the improved LST broth medium of the present invention has good selectivity.

[0074] The results in Table 3 show that the improved LST broth medium of the present invention has good repeatability, can successfully culture Escherichia coli and VBNC state Escherichia coli, and has stable selectivity, and has no culture effect on Enterococcus faecalis and Staphylococcus aureus.

[0075] The results in Table 4 show that the improved LST broth medium of the present invention still has good nutritional performance and selectivity after being stored for 12 months.

[0076] The results in Table 5 show that the LST broth culture medium obtained in Example 1 of the present invention can be successfully used to detect VBNC Escherichia coli in water samples.

Claims

1. A modified LST broth medium, characterized in that: The improved LST broth medium comprises tryptone, ammonium chloride, lactose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and pyridoxal phosphate, and the mass concentration ratios of the tryptone, ammonium chloride, lactose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate, sodium pyruvate and pyridoxal phosphate are (15g / L-25g / L): (4g / L-6g / L): (3g / L-7g / L): (2.5g / L-3g / L): (2.5g / L-3g / L): (0.02g / L-0.04g / L): (0.9μg / L-1.1μg / L): (0.5g / L-0.7g / L): (0.55g / L-3.3g / L): (0.25mg / L-2mg / L); The nucleotide sequence of the Rpf recombinant protein is shown in SEQ ID NO.

1.

2. The improved LST broth medium according to claim 1, characterized in that The mass concentration ratio of the trypsin, ammonium chloride, lactose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium lauryl sulfate, Rpf recombinant protein, magnesium sulfate, sodium pyruvate, and pyridoxal phosphate is 20 g / L: 5 g / L: 5 g / L: 2.75 g / L: 2.75 g / L: 0.03 g / L: 1 μg / L: 0.6 g / L: 2.2 g / L: 1 mg / L.

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

6.

4. Use of the improved LST broth medium according to any one of claims 1 to 3 in culturing Gram-negative bacteria in a VBNC state.

5. Use of the improved LST broth medium according to any one of claims 1 to 3 in culturing Escherichia coli in a VBNC state.

6. The use according to claim 5, characterized in that: The culture temperature is 36°C-38°C, and the culture time is 18h-24h.

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