A recovery culture medium for promoting live non-culturable state vibrio parahaemolyticus and a preparation method and application thereof

Abstract

The application belongs to the technical field of microorganisms, and discloses a recovery medium for promoting recovery of living non-culturable state Vibrio parahaemolyticus, a preparation method and application thereof. The recovery medium is constructed by optimizing the medium components, and can effectively activate the metabolic activity of VBNC Vibrio parahaemolyticus and promote the rapid recovery and proliferation of the VBNC Vibrio parahaemolyticus. A plurality of high-sarcosine lactone group quorum sensing signal molecules are added, including N-butyryl-L-sarcosine lactone, N-hexanoyl-L-sarcosine lactone, N-octanoyl-L-sarcosine lactone, N-3-oxohexanoyl sarcosine lactone and N-3-oxotetradecanoyl-L-sarcosine lactone. The medium significantly improves the recovery efficiency and growth rate of the VBNC cells, so that the VBNC cells are recovered from the dormant state to the cultivable active state in a short time. The detection efficiency and accuracy are greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, relates to a culture medium for promoting the resuscitation of live, non-culturable Vibrio parahaemolyticus, its preparation method, and its application. Background Technology

[0002] Due to its low cost and broad-spectrum antibacterial properties, NaClO is widely used as a disinfectant in water and on the surfaces of food processing equipment to eliminate pathogenic bacteria. As a food safety certified chemical disinfectant, it has been approved for use in the microbial control of refrigerated food substrates. In the processing of fresh aquatic products, soaking raw food materials in a 50-100 mg / L chlorine solution for 10-20 minutes can significantly reduce the initial total bacterial count and the abundance of specific spoilage bacteria, extending shelf life. However, during food processing, fluctuations in NaClO concentration and frequent use can lead to the development of microbial resistance. The reactive oxygen species generated by NaClO can induce microorganisms to enter a VBNC (Volatile Boreal State) state.

[0003] The viable but nonculturable (VBNC) state is a self-protective phenomenon exhibited by bacteria in response to external environmental stress. In this state, bacteria cannot grow on standard laboratory culture media but retain some characteristics of culturable cells, such as metabolic activity, drug resistance, and virulence. Furthermore, VBNC cells can recover under favorable conditions in food processing environments, meaning that foodborne pathogens in the VBNC state may continue to contaminate food.

[0004] Vibrio parahaemolyticus (Vp) is a Gram-negative, rod-shaped bacterium whose natural hosts are marine animals such as salmon, shrimp, and oysters. Ingesting raw or undercooked seafood contaminated with Vp can cause acute gastrointestinal symptoms such as abdominal pain and diarrhea. The World Health Organization has listed Vp, along with Vibrio cholerae, as a major cause of many foodborne illnesses in humans. During food processing, factors such as low temperatures, low salinity, and acid-base balance can force Vp into a VBNC (virtually volute-neutralized) state. Under appropriate conditions, its return to a normal state poses a risk to food safety, making it necessary to understand the induction conditions and resuscitation techniques related to VBNC cells. Summary of the Invention

[0005] In view of the above-mentioned defects in the existing technology, the present invention first provides a composition for promoting the resuscitation of live, unculturable Vibrio parahaemolyticus.

[0006] A second object of the present invention is to provide the use of the above composition in promoting the resuscitation of live, unculturable Vibrio parahaemolyticus.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A composition obtained by mixing the following substances in a molar ratio of 1:1:1:1:1:N-butyryl-L-homoserine lactone C4-HSL, N-hexanoyl-L-homoserine lactone C6-HSL, N-octanoyl-L-homoserine lactone C8-HSL, N-3-oxo-hexanoylhomoserine lactone OHHL, and N-(3-oxotetradecanoyl)-L-homoserine lactone 3-oxo-C14-HSL.

[0009] Our team found that NaClO-induced VBNC-Vp cells could significantly restore their culturability in 2 μM signaling molecule systems (C4-HSL, C6-HSL, C8-HSL, OHHL, and 3-oxo-C14-HSL). Therefore, it is necessary to understand VBNC-Vp cell resuscitation techniques.

[0010] The present invention also provides a culture medium containing the above composition, wherein the composition is used in the culture medium at an amount of 2 μM.

[0011] In one specific embodiment, the composition of the above-mentioned culture medium is as follows: per liter of culture medium, it contains 15 g of tryptone, 5 g of soybean peptone, 5 g of sodium chloride, 2.5 g of glucose, 0.342 g of N-butyryl-L-homoserine lactone C4-HSL, 0.398 g of N-hexanoyl-L-homoserine lactone C6-HSL, 0.454 g of N-octanoyl-L-homoserine lactone C8-HSL, 0.426 g of N-3-oxo-hexanoylhomoserine lactone OHHL, and 0.65 g of N-(3-oxotetradecanoyl)-L-homoserine lactone 3-oxo-C14-HSL.

[0012] This invention constructs a resuscitation culture system that effectively activates the metabolic activity of Vibrio parahaemolyticus (VBNC) and promotes its rapid recovery and proliferation by systematically optimizing the culture medium components. The culture medium uses tryptone, soybean peptone, sodium chloride, and glucose as basic nutrients, and adds various homoserine lactone quorum sensing signaling molecules, including: N-butyryl-L-homoserine lactone (C4-HSL); N-hexanoyl-L-homoserine lactone (C6-HSL); N-octanoyl-L-homoserine lactone (C8-HSL); N-3-oxohexanoylhomoserine lactone (3-oxo-C6-HSL); and N-(3-oxotetradecanoyl)-L-homoserine lactone (3-oxo-C14-HSL). The preparation method includes: weighing the above components according to the specified proportions, adding them to distilled water, and stirring thoroughly until completely dissolved to obtain a homogeneous resuscitation culture medium solution.

[0013] This culture medium, by mimicking the bacterial quorum sensing signal environment, synergistically activates the respiratory chain and energy metabolism pathways of Vibrio parahaemolyticus, thereby significantly improving the recovery efficiency and growth rate of VBNC cells, enabling them to recover from a dormant state to a culturable active state in a shorter time. Using the above composition and culture medium, VBNC-Vp can be significantly revived in a short period, greatly improving detection efficiency and accuracy.

[0014] Therefore, the present invention also provides the use of the composition or the culture medium in promoting the resuscitation of live, unculturable Vibrio parahaemolyticus.

[0015] Therefore, the present invention also provides the use of the composition or the culture medium in reducing the incidence of false negatives in Vibrio parahaemolyticus.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The VBNC-Vp resuscitation medium of the present invention can revive VBNC-Vp, improve the pathogen detection rate of food samples, and reduce the incidence of false negatives in Vp detection in food and environment disinfected by NaClO, thereby providing technical support for the precise control of VBNC-Vp resuscitation in aquatic food.

[0018] (2) The VBNC-Vp resuscitation medium of the present invention has optimized the nutritional composition of the medium and added a variety of N-acyl homoserine lactones. It can resuscitate VBNC-Vp through multiple synergistic mechanisms such as quorum sensing activation, metabolic restart, oxidative stress repair and biomembrane induction. In particular, OHHL is added, which forms a complex with Opar protein, upregulates the expression of ATP synthase, increases the intracellular ATP level, and thus promotes the activity of ATP-dependent pathways such as DNA replication and translation, and enhances the cell division ability. At the same time, OHHL may induce Vp to exit the dormant state by inhibiting the toxin-antitoxin system, thereby restoring its culturability.

[0019] (3) This invention adds tryptone, soy peptone, sodium chloride, glucose and other components. Among them, tryptone and soy peptone provide small molecule peptides and free amino acids, which can be directly absorbed by VBNC cells. Sodium chloride can regulate osmotic pressure and ion homeostasis, and glucose can restart the precise regulation of energy metabolism, thereby promoting the recovery of Vp from VBNC state to cultureable state.

[0020] (4) The culture medium provided by the present invention has the advantages of significant resuscitation effect, good stability and simple operation, and is suitable for rapid detection, risk assessment and related basic research and practical application of Vibrio parahaemolyticus. Attached Figure Description

[0021] Figure 1The effects of different resuscitation methods on the resuscitation growth curve of VBNC-Vp in the embodiments of this application are shown below: (A) TSB group; (B) heat treatment group; (C) TSB-sodium pyruvate group; (D) TSB-Tween 80 group; (E) TSB-Signe group.

[0022] Figure 2 The effects of different resuscitation methods on the colony morphology of VBNC-Vp in the embodiments of the present invention are shown below: (A) Normal group; (B) VBNC group; (C) TSB group; (D) Heat treatment group; (E) TSB-sodium pyruvate group; (F) TSB-Tween 80 group; (G) TSB-signaling group.

[0023] Figure 3 The effects of different resuscitation methods on the morphological characteristics of VBNC-Vp in the embodiments of the present invention are shown below: (A) Normal group; (B) VBNC group; (C) TSB group; (D) Heat treatment group; (E) TSB-sodium pyruvate group; (F) TSB-Tween 80 group; (G) TSB-Signe group.

[0024] Figure 4 The effects of different resuscitation methods on VBNC-Vp survival (or fluorescence staining) in the embodiments of the present invention are shown below: (A) Normal group; (B) VBNC group; (C) TSB group; (D) Heat treatment group; (E) TSB-sodium pyruvate group; (F) TSB-Tween 80 group; (G) TSB-signal group.

[0025] Figure 5 This invention illustrates the effect of different resuscitation methods on VBNC-Vp ATP concentration in various embodiments.

[0026] Figure 6 This invention illustrates the effect of different resuscitation methods on the activity of the VBNC-Vp respiratory chain in embodiments of the present invention. Detailed Implementation

[0027] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0028] In the following examples, the bacterial strain source is Vp ATCC17802 (tdh) - / trh + / tlh + For reagents or instruments that do not specify the manufacturer, they are all conventional products that can be purchased through legitimate channels.

[0029] A method for preparing VBNC-Vp resuscitation medium: 15 g of tryptone, 5 g of soybean peptone, 5 g of sodium chloride, 2.5 g of glucose, 0.342 g of N-butyryl-L-homoserine lactone (C4-HSL), 0.398 g of N-hexanoyl-L-homoserine lactone (C6-HSL), 0.454 g of N-octanoyl-L-homoserine lactone (C8-HSL), 0.426 g of N-3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL, OHHL), and 0.65 g of N-(3-oxotetradecanoyl)-L-homoserine lactone (3-oxo-C14-HSL) are added to a beaker containing 1 L of distilled water, stirred and dissolved to prepare a resuscitation solution.

[0030] Example 1

[0031] I. Subculture of Vibrio parahaemolyticus

[0032] The *Vp* ATCC17802 strain, frozen at -80 °C, was streaked onto tryptic soy agar (TSA) plates. The streaked plates were then inverted and incubated at 37 °C for 24 h. A single colony was picked and inoculated into 20 mL of sterile tryptic soy broth (TSB) medium and cultured in a shaking incubator at 37 °C and 160 r / min for 16 h. The culture was then removed from the shaking incubator and inoculated at 1% onto 10 mL of tryptic soy broth (TSB) liquid medium and cultured at 37 °C and 160 r / min for 4.5 h. At this point, *Vp* was in the logarithmic growth phase, with an initial concentration of approximately 2 × 10⁻⁶. 8 CFU / mL.

[0033] II. Induction of VBNC by sodium hypochlorite

[0034] Take the bacterial culture that has been cultured to the logarithmic growth phase on a shaker, divide the culture into 12 equal aliquots (15 mL each), centrifuge (8000 g, 20 min), wash and resuspend, and adjust the density to 10⁻⁶. 6 CFU / mL. Sodium hypochlorite (NaClO) stock solutions were diluted in distilled water to final concentrations of 10, 20, 30, 40, 50, 60, and 70 ppm, and detected using a total chlorine assay kit. Subsequently, 15 mL of bacterial culture was centrifuged, and the supernatant was removed. The bacterial precipitate was resuspended in equal volumes (15 mL) of NaClO solutions of different concentrations. The induction solution was allowed to react for 20 min, then immediately neutralized with 0.12% (v / v) sodium thiosulfate to terminate the reaction. A control group was prepared using the same procedure, but sterile phosphate-buffered saline (PBS) was used instead of NaClO to demonstrate the role of NaClO in the induction process.

[0035] III. Assay for the recovery status of Vibrio parahaemolyticus (VBNC)

[0036] After centrifugation, VBNC-Vp cells were washed with sterile PBS and then resuspended in five resuscitation media: TSB, heat-treated (heat shock at 72 °C for 10 s), TSB containing 2 mg / mL sodium pyruvate, TSB containing 5% Tween 80, and TSB containing 2 μM signaling molecule systems (C4-HSL, C6-HSL, C8-HSL, OHHL, 3-oxo-C14-HSL). Each experimental group underwent a tenfold serial dilution of the original solution and was cultured in a shaker at 37 °C for 48 h. Turbidity changes in the bacterial culture were monitored every 3 h using a spectrophotometer (λ=600 nm), and the resuscitation results were observed and recorded every 24 h.

[0037] IV. Detection of Vibrio parahaemolyticus (VBNC)

[0038] Colony analysis of culturable Vp in the induction and recovery systems was performed using thiosulfate citrate bile salt sucrose agar (TCBS).

[0039] V. Observation of the cell morphology of Vibrio parahaemolyticus (VBNC)

[0040] Vp bacterial suspensions in normal, VBNC, and resuscitation states were collected and centrifuged to collect the bacterial pellet. The bacterial cells were washed twice with PBS and fixed with 2.5% (v / v) glutaraldehyde at 4 °C for 12 h. After fixation, the samples were sequentially dehydrated using 30%, 50%, 70%, 80%, 90%, and 100% ethanol, each treatment lasting 10 min with shaking. The dehydrated samples were dried using the critical point carbon dioxide drying method. Finally, the samples were subjected to gold sputtering for 1 min and observed using a scanning electron microscope at an accelerating voltage of 20 kV.

[0041] VI. Determination of Vibrio parahaemolyticus (VBNC) cell viability

[0042] SYTO9 can penetrate the cell membrane and enter the cell, where it binds to DNA and emits green fluorescence; propidium iodide can only enter cells with damaged membrane structures, where it competitively binds to DNA and emits red fluorescence. A laser confocal scanning microscope was used to simultaneously acquire dual-channel fluorescence images to separately identify live cells (green fluorescence) and dead cells (red fluorescence).

[0043] VII. Determination of ATP Concentration in Vibrio parahaemolyticus (VBNC)

[0044] Intracellular ATP levels in bacteria were determined using an ATP assay kit. Vp bacterial suspensions in normal, VBNC, and resuscitation states were collected, centrifuged, and the supernatant was discarded. The bacterial pellet was washed with 0.85% NaCl solution and resuspended. The resuspended suspension was mixed with the reaction solution at a 1:1 (v / v) ratio, reacted at room temperature in the dark, and the chemiluminescence intensity was measured using a multi-mode microplate reader. Simultaneously, a standard curve was constructed using a series of ATP standards, and the absolute concentration of ATP in the samples was calculated accordingly.

[0045] VIII. Determination of respiratory chain activity of Vibrio parahaemolyticus (VBNC)

[0046] Respiratory chain activity was assessed in normal, VBNC, and resuscitated Vp cells using a rapid CTC staining kit. CTC formonitrile dye was added to each mL of bacterial suspension to a final concentration of 5 mmol / L, along with 1 μL of enhancer. After reduction via the respiratory chain, CTC formonitrile dye formed a fluorescent precipitate within the cells, with a maximum excitation wavelength of approximately 480 nm and a maximum emission wavelength of approximately 630 nm. Following staining, the fluorescence signal of each cell was detected using flow cytometry, and respiratory chain activity was quantitatively characterized by average fluorescence intensity. The final results are expressed as a percentage of respiratory chain activity relative to normal cells.

[0047] Results analysis:

[0048] 1. For example Figure 1 As shown, the TSB group had the lowest recovery rate, the longest lag period of 24 hours, and the lowest final biomass among all groups. Figure 1 A); Although the heat treatment group slightly shortened the recovery time, the improvement was limited. Figure 1 B). In contrast, the addition of sodium pyruvate can effectively eliminate oxidative stress, bringing the recovery time forward to 12 hours. Figure 1 C); The group supplemented with Tween 80 began to recover at 18 h and later showed a higher logarithmic growth rate (C). Figure 1 D). The TSB-signalin group showed the best recovery effect, with its VBNC cells entering the logarithmic growth phase within 12 hours, exhibiting the fastest recovery rate and ultimately reaching OD. 600nm The value was closest to the control group level and significantly better than other treatment groups. Figure 1 E). Experiments have shown that the 2μM serotonin system can effectively activate dormant cells, significantly shorten the recovery lag period, and increase the concentration of culturable cells, thus possessing practical application value.

[0049] 2. For example Figure 2 As shown, in the normal group, Vp colonies were abundant, morphologically intact, and typically smooth and blue-green. Figure 2 A); however, no colony growth was observed in the VBNC group ( Figure 2B) indicates that the bacteria have entered the VBNC state. After resuscitation using standard TSB medium ( Figure 2 C), only a small number of small colonies were observed, indicating limited recovery ability. After TSB heat treatment, the number of colonies increased, but the overall recovery level remained low. Figure 2 D). Adding sodium pyruvate or Tween 80 to TSB significantly increased the number of colonies, made their distribution more uniform, and resulted in larger colony morphologies. Figure 2 E and F indicate that these two additives have a good resuscitation promoting effect on bacteria in the VBNC state. Among them, the TSB-signalin group formed the most colonies, with the highest number, densest distribution, and stable morphology, close to the normal group, and its resuscitation effect was significantly better than the other groups. Figure 2 (G) indicates that the culture medium can effectively promote the recovery of culturability of VBNC-Vp.

[0050] 3. For example Figure 3 As shown, the normal group of Vp cells are typically rod-shaped, randomly distributed, with smooth cell surfaces and intact structures. Figure 3 A). The morphology of cells in the VBNC group changed significantly, with most cells transforming from rod-shaped to spherical. The cell surface showed obvious wrinkles and a rough structure, and some cells exhibited polymer attachment and aggregation. Figure 3 B) indicates that it has entered the VBNC state. After TSB resuscitation treatment, some cells recovered to a short rod shape, but surface irregularities still existed ( Figure 3 C); TSB heat treatment group showed further improvement in cell morphology, but the degree of recovery was limited ( Figure 3 D). After treatment with TSB-sodium pyruvate and TSB-Tween 80, the rod-like characteristics of cells were significantly enhanced, the surface smoothness was improved, and the aggregation phenomenon was reduced. Figure 3 E and F). Among them, the TSB-signalin group showed the most significant recovery in cell morphology, with most cells exhibiting regular rod-shaped patterns and smooth surfaces, highly similar to the normal group. Figure 3 (G) indicates that the resuscitation medium can effectively reverse morphological abnormalities in the VBNC state and significantly promote the recovery of cell structure and activity.

[0051] 4. For example Figure 4 As shown, the cells in the normal control group exhibit bright and uniformly distributed green rod-shaped fluorescence, and their morphology remains intact. Figure 4 A); The VBNC group cells were mostly spherical or irregular in shape, with significantly weakened green fluorescence, and some cells showed red fluorescence (A); Figure 4 B) indicates a decrease in cell viability. After routine TSB resuscitation treatment, the number of green fluorescent cells increased, and no red fluorescence was observed, but the resuscitation effect remained relatively limited. Figure 4 C); The green fluorescence intensity of the TSB heat-treated group was further enhanced compared to the TSB group ( Figure 4D). Compared with the above treatments, the number of green fluorescent cells in the TSB-sodium pyruvate group, TSB-Tween 80 group, and TSB-signalin group increased significantly, and the cell morphology gradually recovered to short rod-shaped. Figure 4 (E, F, and G). Among them, the TSB-signalin group showed the most significant recovery effect, indicating that this treatment can effectively promote the restoration of culturable activity of VBNC-state Vibrio parahaemolyticus.

[0052] 5. For example Figure 5 As shown, normal cells had the highest ATP concentration (5.82±0.12 nmol / L), while the ATP level in VBNC cells decreased significantly to 3.85±0.08 nmol / L, indicating extremely low metabolic activity and a VBNC state. After TSB resuscitation and TSB heat treatment, the ATP concentration only slightly increased, showing limited recovery. TSB medium supplemented with sodium pyruvate and Tween-80 could both increase ATP levels to some extent. After resuscitation using the TSB-signalin method, the ATP concentration showed a significant increase, approaching the level of normal cells, and the resuscitation effect was significantly better than other groups. This indicates that signalin can effectively activate the energy metabolism of VBNC cells and is a key factor in promoting their efficient resuscitation.

[0053] 6. For example Figure 6 As shown, the respiratory chain activity of VBNC cells was significantly reduced, only reaching (26.17±2.08)% of that of normal cells, indicating severe inhibition of respiratory chain activity. Among the various resuscitation treatment groups, the TSB group and TSB heat treatment group only induced limited metabolic activation; while the TSB-sodium pyruvate group and TSB-Tween 80 group showed some recovery of respiratory chain activity. The TSB-signalin group showed the most significant recovery of respiratory chain activity, significantly better than other resuscitation treatment methods, indicating that signalin can effectively promote the reactivation of respiratory chain-related enzyme systems. These results further confirm at the metabolic level that the culture medium constructed in this study and its preparation method can significantly promote the transition of VBNC cells from a dormant state to a metabolically active state, demonstrating good resuscitation potential.

Claims

1. A composition characterized in that, The composition is obtained by mixing the following substances in a molar ratio of 1:1:1:1:1:N-butyryl-L-homoserine lactone C4-HSL, N-hexanoyl-L-homoserine lactone C6-HSL, N-octanoyl-L-homoserine lactone C8-HSL, N-3-oxo-hexanoylhomoserine lactone OHHL, and N-(3-oxotetradecanoyl)-L-homoserine lactone 3-oxo-C14-HSL.

2. A culture medium comprising the composition of claim 1, characterized in that, The composition was used in the culture medium at a concentration of 2 μM.

3. The culture medium according to claim 2, characterized in that, The culture medium consists of the following components per liter: 15 g tryptone, 5 g soybean peptone, 5 g sodium chloride, 2.5 g glucose, 0.342 g N-butyryl-L-homoserine lactone C4-HSL, 0.398 g N-hexanoyl-L-homoserine lactone C6-HSL, 0.454 g N-octanoyl-L-homoserine lactone C8-HSL, 0.426 g N-3-oxo-hexanoylhomoserine lactone OHHL, and 0.65 g N-(3-oxotetradecanoyl)-L-homoserine lactone 3-oxo-C14-HSL.

4. The use of the composition of claim 1 or the culture medium of claim 2 in promoting the resuscitation of live, unculturable Vibrio parahaemolyticus.

5. The use of the composition of claim 1 or the culture medium of claim 2 in reducing the incidence of false negatives in Vibrio parahaemolyticus.

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