Preparation method of freshwater vibrio high-efficiency culture compound culture medium containing nucleotide complex

Abstract

The present application relates to the technical field of aquatic pathogenic bacteria culture, and particularly relates to a preparation method of a freshwater vibrio high-efficiency culture composite medium containing a nucleotide compound.A freshwater vibrio high-efficiency culture composite medium containing a nucleotide compound, in terms of weight parts, includes the following functional components: 2000-5000 parts of a carbon source, 1500-3000 parts of a nitrogen source, 225-405 parts of a precursor substance, 65-125 parts of an amino acid, 55-110 parts of an energy system, 650-1300 parts of an ion regulator, 35-85 parts of a membrane stabilizer, and 10-20 parts of an antioxidant.The present application solves the technical problem that the prior art lacks a culture method that can effectively promote the growth of freshwater vibrio and ensure the motility activity of the freshwater vibrio.Through reasonable formulation and process design, the present application breaks through the bottleneck of the existing freshwater vibrio culture technology, provides support for the research, targeted prevention and control, and efficient detection method establishment of aquatic pathogenic bacteria, and has a significant application prospect.

Description

Technical Field

[0001] This invention relates to the field of aquatic pathogenic bacteria culture technology, specifically to a method for preparing a high-efficiency culture medium for freshwater Vibrio containing nucleotide complexes. Background Technology

[0002] In freshwater aquaculture, Vibrio freshwaterii are important pathogens threatening the health of farmed organisms and the development of the aquaculture industry, among which Vibrio mimicry (Vibrio spp.) V. mimic ) and Vibrio cholerae ( V. cholera Due to their strong pathogenicity, these bacteria pose a significant threat to the stability of freshwater aquaculture environments. Studies have shown that the motility of these two types of freshwater Vibrio bacteria, mainly including flagellated swimming and swarming, is closely related to their adhesion and colonization processes in the host or aquaculture environment, as well as the spread of infection. The strength of their motility directly affects their pathogenicity and the degree of harm. Therefore, research on the motility of freshwater Vibrio bacteria and related influencing factors has become one of the key directions for controlling the diseases they cause.

[0003] However, current research on the motility of Vibrio freshwater still faces many unresolved challenges, which severely restrict in-depth exploration of its pathogenic mechanisms and the development of effective control measures. Specifically, these challenges are mainly reflected in the following three aspects: First, traditional culture media suffer from significant nutritional deficiencies. While TCBS medium is widely used in the isolation and culture of Vibrio brevis in freshwater Vibrio culture research, its nutrient composition cannot meet the needs of flagellar synthesis, particularly lacking crucial precursors such as tyrosine and magnesium ions. This deficiency results in significantly reduced motility of Vibrio brevis cultured in TCBS medium, with a migration radius typically ≤15 mm. This falls far short of mimicking the normal pathogenic state of Vibrio brevis in natural aquaculture environments, negatively impacting the accuracy of subsequent research on Vibrio pathogenic mechanisms.

[0004] Secondly, there is an issue of insufficient expression of movement-related genes. Flagellin (FliC) in Vibrio freshwater is a core component of its flagellum structure, ensuring its motility. The synthesis of this flagellin is highly dependent on exogenous purine and pyrimidine metabolites. However, commonly used traditional culture media are extremely deficient in nucleotides, failing to provide Vibrio freshwater with sufficient purine and pyrimidine metabolites. This directly leads to inhibited flagellin synthesis in Vibrio freshwater, resulting in a significant reduction in the number of flagella and a marked decrease in flagellar beating frequency. Ultimately, this weakens Vibrio motility, making it difficult to meet the needs of research on its movement-related pathogenic mechanisms.

[0005] Finally, there is the problem of an imbalance between culture efficiency and activity. In the cultivation of Vibrio freshwaterii, some studies, in pursuit of higher cell density, choose media such as LB broth, which promotes rapid cell proliferation. However, in LB broth, as the cells multiply rapidly, metabolic waste accumulates in the medium. This accumulated metabolic waste significantly inhibits the flagellar function of Vibrio freshwaterii, resulting in increased cell density but a substantial decrease in motility. This imbalance between culture efficiency and cell activity makes it impossible to simultaneously meet the research requirements of high cell density and high motility, further limiting in-depth research on the characteristics and pathogenic mechanisms of Vibrio freshwaterii.

[0006] Based on the above problems, there is an urgent need to develop a novel culture technology or optimized culture medium formulation that can simultaneously meet the nutritional requirements for flagellar synthesis in Vibrio freshwater, promote the efficient expression of motility-related genes, and achieve a balance between high-density bacterial culture and high motility activity. Existing culture media (such as TCBS and LB) all have significant shortcomings, especially the lack of composite culture media specifically designed to balance high-density growth and high motility activity of Vibrio freshwater. The limitations of current traditional culture media have prevented research from accurately simulating the natural pathogenic state of Vibrio, hindering in-depth analysis of the correlation mechanisms between its motility, adhesion, colonization, and infection spread. This, in turn, restricts the development of targeted control technologies against the pathogenic pathways of Vibrio freshwater and the establishment of efficient detection methods. Therefore, breaking through the bottlenecks of existing culture technologies and filling this technological gap has become a key requirement for promoting the research on the pathogenic mechanisms of Vibrio freshwater and the prevention and control of aquatic diseases. Summary of the Invention

[0007] The present invention aims to provide a high-efficiency culture medium for Vibrio freshwater containing nucleotide complexes, in order to solve the technical problem that the existing technology lacks a culture method that can effectively promote the growth of Vibrio freshwater and ensure its motility.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A high-efficiency culture medium for freshwater Vibrio containing nucleotide complexes, comprising the following active ingredients by weight: 2000-5000 parts carbon source, 1500-3000 parts nitrogen source, 225-405 parts precursor substances, 65-125 parts amino acids and amino acid analogs, 55-110 parts energy system, 650-1300 parts ion regulator, 35-85 parts membrane stabilizer, and 10-20 parts antioxidant.

[0009] Furthermore, the carbon source includes D-glucose; the nitrogen source includes 1000-2000 parts of ammonium sulfate and 500-1000 parts of yeast extract.

[0010] Furthermore, the precursor substances include 100-150 parts of hypoxanthine, 20-50 parts of deoxyguanosine, 50-100 parts of uridine, 40-80 parts of cytidine, and 15-25 parts of deoxycytidine acid.

[0011] Furthermore, the amino acids and amino acid analogues include 30-70 parts of L-tryptophan and 35-55 parts of cycloserine.

[0012] Furthermore, the energy system includes NAD + 5-10 parts and 50-100 parts of ribose-1-phosphate.

[0013] Furthermore, the ion regulator includes 150-300 parts of magnesium chloride hexahydrate and 0.5-1 parts of sodium chloride.

[0014] Furthermore, the membrane stabilizer includes 5-15 parts of ganglioside GM1 and 30-70 parts of β-cyclodextrin; the antioxidant includes taurine.

[0015] Furthermore, each liter of the compound culture medium contains: D-glucose 2-5g, ammonium sulfate 1-2g, yeast extract 0.5-1g, hypoxanthine 100-150mg, deoxyguanosine 20-50mg, uridine 50-100mg, cytidine 40-80mg, deoxycytidine acid 15-25mg, L-tryptophan 30-70mg, cyclic serine 35-55mg, and NAD+. + 5-10mg, ribose-1-phosphate 50-100mg, magnesium chloride hexahydrate 150-300mg, sodium chloride 0.5-1g, ganglioside GM1 5-15mg, β-cyclodextrin 30-70mg, taurine 10-20mg.

[0016] This technical solution also provides a method for preparing a high-efficiency culture medium for freshwater Vibrio containing nucleotide complexes: Sodium chloride and magnesium chloride hexahydrate are dissolved in water sequentially, and sterilized to obtain a sterile base solution; D-glucose, ammonium sulfate, and yeast extract are dissolved in water, filtered and sterilized to obtain a carbon and nitrogen source nutrient solution; hypoxanthine, deoxyguanosine, uridine, cytidine, deoxycytidine acid, and NAD+ are added... + Ribose-1-phosphate was dissolved in water, and the nucleotide filtrate was obtained after filtration and sterilization. Ganglioside GM1 was mixed with β-cyclodextrin, and after sonication, an inclusion complex was formed. After filtration and sterilization, the GM1-β-cyclodextrin complex was obtained. The following were added sequentially to the sterilized base solution: carbon and nitrogen source nutrient solution, nucleotide filtrate, GM1-β-cyclodextrin complex, L-tryptophan, cycloserine, and taurine. The solution was brought to a final volume, stirred, and the pH was adjusted to 7.2±0.1 to obtain the composite culture medium.

[0017] Furthermore, ganglioside GM1 was mixed with β-cyclodextrin and sonicated at 40 kHz, 80-120 W for 30 min. The resulting composite culture medium was then dispensed into culture flasks with 0.2 μm breathable membranes.

[0018] In summary, the technical principle of this invention is as follows: This invention is based on the core physiological needs of freshwater Vibrio (Vibrio mimicry, Vibrio cholerae) regarding "flagellate synthesis-motility-metabolic balance." Through multi-component synergistic regulation and precise process assurance, it overcomes the technical bottlenecks of traditional culture media. Its core technical principles are as follows: To address the problem of insufficient expression of FliC (flagellate protein), flhD (flagellate regulatory factor), and motA (flagellate motor protein) genes due to the lack of purine / pyrimidine precursors in traditional culture media, this invention designs a nucleotide complex composed of hypoxanthine, deoxyguanosine, uridine, cytidine, and deoxycytidine acid, which effectively improves the growth rate and migratory diffusion radius of Vibrio freshwater.

[0019] To address the weakened flagellar function caused by insufficient energy supply and the absence of key ions in traditional culture media, this invention constructs a synergistic system of "energy system + ion regulator". In the energy system, NAD+... + Synergistically acting with ribose-1-phosphate, it enhances the migration diffusion radius and transcription levels of movement-related genes. In the ion regulator, the addition of magnesium chloride hexahydrate maintains matrix structural stability, while sodium chloride maintains intracellular osmotic pressure stability. Addressing the decreased motility due to metabolic waste accumulation and cell membrane damage during high-density culture, this invention designs a "membrane stabilizer + antioxidant" protection system. Ganglioside GM1 and β-cyclodextrin form an inclusion complex through ultrasonic treatment, solving the problem of poor water solubility of GM1 and enabling targeted embedding into the Vibrio cell membrane, enhancing membrane fluidity and osmotic pressure resistance, and reducing the damage of metabolic waste to the flagellar base binding site. The antioxidant taurine scavenges reactive oxygen species (ROS) generated during culture, preventing ROS-induced oxidative damage to microorganisms while maintaining intracellular redox balance and ensuring normal microbial growth. Through differentiated sterilization processes (autoclaving base solution, filtration to remove heat-sensitive components, and ultrasonic inclusion membrane stabilizer), it avoids nucleotide degradation and NAD+ loss caused by high temperatures. + Deactivation etc.

[0020] The beneficial effects of this technical solution are as follows: (1) Overcoming nutritional deficiencies and significantly improving the motility of freshwater Vibrio. Compared to TCBS medium, this invention provides sufficient precursors and cofactors for flagellar synthesis through the synergistic supply of nucleotide complexes and ion regulators, significantly increasing the migration and diffusion radius of Vibrio mimicus and Vibrio cholerae. It can accurately simulate the pathogenic state of Vibrio in natural breeding environments, providing a realistic bacterial model for the study of its adhesion, colonization, infection, and diffusion mechanisms.

[0021] (2) Promote the efficient expression of movement-related genes and ensure the integrity of flagellar function. The culture medium of this protocol can significantly upregulate the relative expression levels of FliC (flagellate protein), flhD (regulatory factor), and motA (motor protein) genes, ensuring the integrity of Vibrio motility at the molecular level and providing reliable samples for research on the correlation between motility and pathogenicity.

[0022] (3) Achieving a balance between high-density culture and high motility. This invention optimizes the energy system and protects the membrane with a stabilizer-antioxidant, thereby improving the retention rate of motility activity while achieving a high cell density. This solves the problem of "density and activity imbalance" and meets the dual needs of large-scale cell preparation and high-activity functional research.

[0023] In summary, this invention, through the technical principle of multi-component synergy and precise process design, breaks through the bottleneck of existing freshwater Vibrio culture technology, providing key support for the study of pathogenic mechanisms of aquatic pathogens, the development of targeted prevention and control technologies, and the establishment of efficient detection methods, and has significant scientific research value and application prospects. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0025] Example 1 Table 1 shows the component ratios of the high-efficiency culture medium for Vibrio freshwater containing nucleotide complexes.

[0026] Table 1: Culture medium composition and ratio (each L of culture medium is prepared using sterile water as the solvent)

[0027] Prepare the culture medium as follows (using the preparation of 1L of culture medium as an example): (1) Sterilization of base solution: Take 800 mL of deionized water, dissolve sodium chloride and magnesium chloride hexahydrate in sequence, stir magnetically until completely dissolved, autoclave at 121℃ for 20 min, cool to 40℃ for later use, and obtain sterilized base solution.

[0028] (2) Preparation of nutrient solution: Heat-stable components: D-glucose, ammonium sulfate, and yeast extract were dissolved in 50 mL of sterile water and filtered through a 0.22 μm filter membrane to obtain a carbon and nitrogen source nutrient solution.

[0029] Heat-sensitive components: hypoxanthine, deoxyguanosine, uridine, cytidine, deoxycytidine acid, NAD + Ribose-1-phosphate was dissolved in 30 mL of sterile water and filtered through a 0.22 μm filter membrane to obtain a nucleotide filtrate.

[0030] Lipid complex: Ganglioside GM1 was premixed with β-cyclodextrin and dissolved in 20 mL of sterile water. The solution was sonicated (40 kHz, 100 W, 30 min) to form an inclusion complex. The solution was filtered through a 0.45 μm filter membrane to obtain the GM1-β-cyclodextrin complex.

[0031] (3) Mixing and Filling: Place the sterile base solution in a sterile mixing tank, and add in sequence: carbon and nitrogen source nutrient solution, nucleotide filtrate, GM1-β cyclodextrin complex, L-tryptophan, cyclic serine, and taurine (L-tryptophan, cyclic serine, and taurine need to be prepared into solutions and then sterilized by routine 0.22μm filtration before being added to the sterile mixing tank). Then, bring the volume to 1L with sterile water, maintain a stirring speed of 200rpm for 15min to ensure uniformity, and adjust the pH to 7.2±0.1. Dispense into culture flasks with 0.2μm breathable membranes and store at 4℃ protected from light (shelf life 7 days).

[0032] Example 2 (1) Culture medium formulation and preparation The following formulation and preparation method of Example 1 were used to prepare a high-efficiency culture medium for Vibrio freshwater containing nucleotide complexes: D-glucose 3g, ammonium sulfate 1.5g, yeast extract 0.8g, hypoxanthine 120mg, deoxyguanosine 35mg, uridine 70mg, cytidine 60mg, deoxycytidine acid 20mg, L-tryptophan 50mg, cyclic serine 45mg, NAD+ 8mg, ribose-1-phosphate 80mg, magnesium chloride hexahydrate 200mg, sodium chloride 0.8g, ganglioside GM1 10mg, β-cyclodextrin 50mg, taurine 15mg.

[0033] (2) Cultivation of freshwater Vibrio Take the bacterial culture in the logarithmic growth phase (Vibrio mimicus or Vibrio cholerae, OD...) 600 =0.5) Inoculate the culture medium at a 1% inoculation rate (inoculation rate = volume of inoculated bacterial solution / volume of fresh culture medium × 100%) and culture at 28℃ with shaking at 180-220 rpm for 12-16 hours.

[0034] Subsequent experiments (experimental group) used Vibrio mimicry ( Vibrio mimic The study used an aquatic isolate (ATCC33653, a commercially available model strain of Vibrio mimicry) for experimental research. Vibrio mimicry was inoculated into conventional TCBS medium and cultured until the logarithmic growth phase (OD2). 600 =0.5). Then, inoculate 1% into the culture medium of this protocol and incubate at 28°C with shaking at 200 rpm for 14 hours. After incubation, perform various tests on the Vibrio mimicus.

[0035] As a control group, Vibrio mimicry in the logarithmic growth phase was inoculated into TCBS medium at a 1% inoculum and cultured with shaking in the same manner. After the culture was completed, various tests were performed on Vibrio mimicry.

[0036] TCBS medium, short for Thiosulfate-Citrate-Bile Salts-Sucrose Medium, is a selective culture medium commonly used for the isolation and culture of Vibrio bacteria and is commercially available. The TCBS liquid medium formulation contains the following components and their corresponding amounts: peptone 10g, yeast extract 5g, sodium thiosulfate 10g, sodium citrate 10g, sodium taurocholate 3g, sucrose 20g, sodium chloride 10g, bromothymol blue 0.04g, thymol blue 0.04g, and distilled water is used as the solvent, with a final volume of 1000mL.

[0037] (3) Indicator detection Detecting bacterial cell density (OD) after 14 hours of culture 600 Dilute the bacterial solution appropriately with sterile physiological saline (dilution ratio 10-100 times, ensuring OD). 600 The value is within the linear detection range of 0.1-1.0); the diluted bacterial solution is added to a quartz cuvette, with uninoculated corresponding culture medium (the same dilution factor as the sample) as a blank control, and the absorbance value (OD) is measured at a wavelength of 600 nm using a spectrophotometer. 600 Multiplying the measured absorbance value by the dilution factor yields the bacterial cell density (OD) after 14 hours of culture. 600 Through the standard curve (in OD) 600 The values ​​are plotted on the x-axis, and the corresponding viable cell concentration is plotted on the y-axis (a linear regression equation is fitted). The measured absorbance values ​​are converted into the cell density (cells / mL) of the bacterial culture after 14 hours of incubation. Each sample is measured in triplicate, and the average value is taken as the final result. The result is expressed as "mean ± standard deviation (Mean ± SD)".

[0038] Motion diffusion radius detection: Prepare 0.3% agar semi-solid medium (LB), sterilize it, and pour it into sterile petri dishes (90 mm in diameter) while still hot, 20 mL per dish, and place them horizontally to solidify; take the bacterial suspension after 14 h of culture and adjust the density to a concentration of 1×10⁻⁶. 8 Prepare a bacterial suspension of CFU / mL, then take 5 μL of the bacterial suspension and vertically inoculate it into the center of the semi-solid culture medium (to a depth of about 0.5 cm). Incubate at 28°C for 12 h. Remove the culture dish and use a vernier caliper (accuracy of 0.01 mm) to measure the diameter of the circular migration zone formed by the colony spreading from the inoculation point. Calculate the radius. Set up 3 replicates for each sample and take the average value. The results are expressed as "mean ± standard deviation (Mean ± SD)".

[0039] Methods for detecting adhesion and colonization ability: Healthy yellow catfish (50±5g) were selected, and the intestines were dissected and removed under aseptic conditions. The intestinal mucosa was rinsed with pre-cooled sterile phosphate-buffered saline (PBS, pH 7.2) until no contents remained. The intestinal mucosa was cut into 1cm×1cm tissue blocks and placed in 24-well cell culture plates. 1mL of a 1×10⁻⁶ solution was added to each well. 8 A bacterial suspension of CFU / mL (obtained by adjusting the density of the bacterial suspension cultured for 14 hours) was incubated at 28°C for 1 hour. After incubation, the tissue block was gently rinsed three times with PBS to remove unattached bacteria. The tissue block was then transferred to a centrifuge tube containing 1 mL of sterile PBS and vortexed for 1 minute to detach the attached bacteria. 100 μL of the bacterial suspension was then subjected to 10... 3 -10 6 The samples were serially diluted and spread onto LB agar plates. After incubation at 37°C for 18 hours, the colony count (CFU) of plates with a colony count of 30-300 was counted and converted into the number of adhering bacteria per piece of intestinal mucosa tissue (1cm×1cm). Each sample was tested in triplicate, and the average value was taken. The results are expressed as mean ± standard deviation (Mean±SD).

[0040] Gene expression level detection: Total RNA was extracted from bacterial cells cultured for 14 hours, and quantitative PCR was performed to detect the FliC gene (flagellate protein), flhD gene, and motA gene. -ΔΔCt The relative gene expression level (transcriptional level) was calculated using a method. Each sample was set up in triplicate, and the average value was taken.

[0041] The FliC gene (flagellin gene) encodes flagellin, a major structural protein of bacterial flagellar filaments. The flhD gene (flagellation regulation gene) encodes the FlhD protein, which typically forms a complex with the FlhC protein and is a primary regulator (early regulator) of flagellar synthesis. The motA gene (flagellate motor protein gene) encodes the MotA protein, a core component of the flagellar motor. If the motA gene is missing or malfunctions, the flagellar motor cannot obtain energy, and the bacteria lose their ability to move (manifesting as "non-motorized"). flhD is related to the initiation of flagellar synthesis, FliC is related to the integrity of the flagellar structure, and motA is related to the ability of the flagella to generate movement. Detecting the expression levels of these three genes allows for analysis of the bacterial flagellar system status from three levels: regulation, structure, and function. This is commonly used to study bacterial motility, environmental adaptation mechanisms, and pathogenicity (e.g., the host colonization ability of pathogenic bacteria often depends on flagellar movement).

[0042] (4) Experimental results Using the nucleotide complex-containing high-efficiency culture medium for freshwater Vibrio culturing according to this protocol, after 14 hours of culture, the cell density (OD) of *Vibrio mimicus* was determined. 600 The value was 2.8 ± 0.2, and the cell density calculated from the standard curve was 3.2 × 10⁻⁶. 9 Cell density (cells / mL); after 14 h of culture of Vibrio mimicry on TCBS medium, the cell density (OD) 600 The value was 0.9 ± 0.1. Using the culture medium of this scheme increased the cell density of Vibrio mimicus by 211%.

[0043] Using the nucleotide complex-containing high-efficiency culture medium for freshwater Vibrio mimicry in this protocol, after 14 hours of culture, the mimicry diffusion radius of Vibrio mimicry was 13.5 ± 1.8 mm; after 14 hours of culture in TCBS medium, the mimicry diffusion radius was 10.2 ± 1.2 mm. Using the culture medium in this protocol can improve the motility of Vibrio mimicry by 32.4%.

[0044] Using the nucleotide complex-containing high-efficiency culture medium for freshwater Vibrio culturing according to this protocol, after 14 hours of cultivation, the adhesion and colonization capacity of *Vibrio mimicus* was 8.5 × 10⁻⁶. 6 CFU / intestinal mucosa; after 14 h of culture in TCBS medium, the adhesion and colonization capacity of *Vibrio mimicus* was 3.2 × 10⁻⁶. 6 CFU / intestinal mucosa. The culture medium used in this protocol can increase the intestinal mucosal colonization capacity of Vibrio mimicus by 166%.

[0045] After quantitative PCR detection of FliC, flhD, and motA genes, it was found that the transcriptional levels (mRNA) of FliC, flhD, and motA genes in the experimental group were upregulated by 180%, 130%, and 120% respectively compared with the control group (calculation method: (relative gene expression level in experimental group - relative gene expression level in control group) / relative gene expression level in control group × 100%).

[0046] Comparative Example Extensive research was conducted on the formulation composition before determining the optimal formulation, as detailed in Tables 2 and 3. Apart from the formulation, the experimental methods for each test group were the same as those for the test group in Example 2 (i.e., test group 1 in Table 2). In test group 10, β-cyclodextrin was not added, so no lipid complex was prepared; ganglioside GM1 was directly added during the filling process after filtration and sterilization.

[0047] Table 2: Formulation composition of each test group (prepared with sterile water as solvent per L of culture medium).

[0048] Table 3: Formulation composition of each test group (prepared with sterile water as solvent per L of culture medium).

[0049] The experimental results are shown in Table 4.

[0050] Table 4: Experimental results of test groups 1-13 (mean ± standard deviation)

[0051] The analysis of the experimental results is as follows: (1) Regarding nucleic acid precursors, the formulation of test group 2 did not contain deoxyguanosine, which resulted in a lower cell density (OD) compared to test group 1. 600 The migration diffusion radius and adhesion and colonization ability were significantly reduced, at 1.9±0.1 mm (a decrease of 32.1%) vs. 2.8±0.2 mm and 9.8±1.1 mm (a decrease of 27.4%), respectively, compared to 13.5±1.8 mm and 5.2×10⁻⁶ mm. 6 CFU / intestinal mucosa (decreased by 38.8%) vs 8.5×10 6CFU / intestinal mucosa analysis indicated that deoxyguanosine, a key precursor in nucleic acid synthesis, inhibited bacterial growth, motility, and intestinal colonization due to its deficiency. In test group 3, uric acid replaced hypoxanthine; uric acid is a commonly used additive in existing technologies. Compared to test group 1, this also resulted in decreased bacterial density, migration radius, and adhesion / colonization ability, specifically 2.0±0.2 (a decrease of 28.6%) vs 2.8±0.2, 10.1±1.3 mm (a decrease of 25.2%) vs 13.5±1.8 mm, and 5.5×10⁻⁶ mm, respectively. 6 CFU / intestinal mucosa (decreased by 35.3%) vs 8.5×10 6 The CFU / intestinal mucosa ratio indicates that uric acid is less effective as a substitute than hypoxanthine, which is more important for maintaining bacterial metabolic activity. Furthermore, the percentage decrease is calculated using the formula: (Test Group 1 index value - Test Group index value) / Test Group 1 index value × 100%.

[0052] (2) Regarding amino acids and analogues, test group 4 used 95 mg L-tryptophan (used alone, without cyclic serine), and test group 5 used 95 mg cyclic serine (used alone, without L-tryptophan). Compared to test group 1 (50 mg L-tryptophan + 45 mg cyclic serine, with the same total amino acid addition of 95 mg), the technical indicators of test groups 4 and 5 showed a significant decrease compared to test group 1. Bacterial cell density (OD) 600 ): Test group 4 decreased to 1.7±0.1 (a decrease of 39.3% compared to test group 1), and test group 5 decreased to 1.6±0.1 (a decrease of 42.9%). Swimming diffusion radius: Test group 4 decreased to 8.6±1.0 mm (a decrease of 36.3%), and test group 5 decreased to 8.3±0.9 mm (a decrease of 38.5%). Adhesion and colonization ability: Test group 4 decreased to 4.8×10 6 CFU / intestinal mucosa (decreased by 43.5%), test group 5 decreased to 4.5×10 6 CFU / intestinal mucosa (decreased by 47.1%).

[0053] The data shows that even when the total dose of amino acids used individually was exactly the same as when used in combination (95 mg), the effect of using them individually was far inferior to the combination. This confirms that the synergistic effect of L-tryptophan and cyclic serine is not a simple dose additive effect, but rather the result of functional complementarity and synergistic mechanism. If the effect of using L-tryptophan and cyclic serine in combination were simply a dose additive effect, then the technical indicators of test group 1 should be between those of test groups 4 and 5. Currently, the experimental results show that the technical indicators of test group 1 are far superior to those of test groups 4 and 5, indicating the existence of a synergistic effect.

[0054] (3) For the energy system, test group 6 used 80 mg of ribose-1-phosphate (without added NAD). + Test group 7 used 8mg NAD + (Without added ribose-1-phosphate), test group 8 did not include an energy system (without added ribose-1-phosphate and NAD). + Compared to test group 1 (80mg ribose-1-phosphate + 8mg NAD⁺), the technical indicators of test groups 6-8 decreased, and the effect of using energy substances alone was significantly different from that of using them in combination. Bacterial cell density (OD) 600 Test group 6 decreased to 1.5±0.1 (a decrease of 46.4%), test group 7 decreased to 1.4±0.1 (a decrease of 50.0%), and test group 8 decreased to 1.0±0.1 (a decrease of 64.3%). Swimming diffusion radius: Test group 6 decreased to 7.9±0.8 mm (a decrease of 41.5%), test group 7 decreased to 7.5±0.7 mm (a decrease of 44.4%), and test group 8 decreased to 6.2±0.6 mm (a decrease of 54.1%). Adhesion and colonization ability: Test group 6 decreased to 4.2×10 6 CFU / intestinal mucosa (decreased by 50.6%), in test group 7, it decreased to 4.0×10. 6 CFU / intestinal mucosa (decreased by 52.9%), in test group 8, it decreased to 3.1×10 6 CFU / intestinal mucosa (decreased by 63.5%).

[0055] The data shows that the energy supply efficiency of using ribose-1-phosphate or NAD⁺ alone is only slightly better than that of test group 8, which has no energy system at all, but is far lower than that of the combination of the two.

[0056] The Bliss independent model was used to illustrate the synergistic effect of ribose-1-phosphate and NAD⁺. The Bliss independent model is a model used to evaluate the combined effects of two or more bioactive substances, primarily to determine whether the combination exhibits a synergistic, additive, or antagonistic effect. The Bliss independent model is calculated using the following formula: EAB = EA + EB - EAEB; where EA and EB represent substance A (ribose-1-phosphate) and substance B (NAD⁺), respectively. + The relative effect value when a substance acts alone is expressed as the "ratio of the substance's effect to the control group" (ranging from 0 to 1, reflecting the proportion of the effect intensity of the single substance on the control group). Here, the "control group" refers to the bacterial culture system lacking ribose-1-phosphate and NAD. +Test group 8; EAB represents the expected effect when the two are used in combination. This model is often used for the optimization of bioactive substance combinations. By comparing the actual combined effect with the expected effect, it is determined whether a synergistic effect exists. If the actual effect exceeds the expectation, a synergistic effect is considered to exist. The specific calculation is as follows: Control group (Test group 8: without ribose-1-phosphate and NAD) + The molecular diffusion radius of the bacterial culture group was 6.2 mm (as the baseline effect); the increase in molecular diffusion radius of ribose-1-phosphate alone (test group 6) was 7.9 - 6.2 = 1.7 mm (the increase in molecular diffusion radius of ribose-1-phosphate relative to the control group); the increase in molecular diffusion radius of NAD⁺ alone (test group 7) was 7.5 - 6.2 = 1.3 mm (the increase in molecular diffusion radius of NAD⁺ relative to the control group); the actual increase in molecular diffusion radius of ribose-1-phosphate + NAD⁺ combined (test group 1) was 13.5 - 6.2 = 7.3 mm (the actual increase in molecular diffusion radius of ribose-1-phosphate relative to the control group after the two substances were used together).

[0057] EA (Relative effect of ribose-1-phosphate) = 1.7 / 6.2 ≈ 0.274 (i.e., ribose-1-phosphate increases the migratory diffusion radius by 27.4% relative to the control group); EB (Relative effect of NAD⁺) = 1.3 / 6.2 ≈ 0.210 (i.e., NAD⁺ increases the migratory diffusion radius by 21.0% relative to the control group); EAB = 0.274 + 0.210 - 0.274 × 0.210 ≈ 0.427 (theoretically, the combined use of ribose-1-phosphate and NAD⁺ can increase the migratory diffusion radius by 42.7% relative to the control group).

[0058] The actual improvement effect of the combined use of ribose-1-phosphate and NAD⁺ on the migratory diffusion radius was: (13.5-6.2) / 6.2×100%≈117.7%>42.7%. The actual value is much higher than the expected value, indicating a significant synergistic effect. Since the actual relative effect (117.7%) is significantly higher than the model's expected relative effect (42.7%), it can be determined that the combined use of ribose-1-phosphate and NAD⁺ has a synergistic enhancing effect on the migratory diffusion ability of bacteria.

[0059] In addition to the migration diffusion radius index, the combined use of ribose-1-phosphate and NAD⁺ also showed a synergistic effect in terms of cell density and adhesion and colonization ability.

[0060] (4) Regarding the membrane stabilizer, ganglioside GM1 was not added to test group 9. Compared with test group 1, its cell density, migration radius, and adhesion and colonization ability all decreased. The specific data and changes are as follows: Cell density: 2.4±0.1 (a decrease of about 14.3% compared with 2.8±0.2 in test group 1); Migration radius: 12.5±1.0 mm (a decrease of about 7.4% compared with 13.5±1.8 mm in test group 1); Adhesion and colonization ability: 5.0×10 6 CFU / intestinal mucosa (compared to 8.5×10 in test group 1) 6 CFU / intestinal mucosa decreased by approximately 41.2%. This indicates that ganglioside GM1 plays a role in maintaining bacterial membrane stability, promoting growth, and colonization. In test group 10, the added ganglioside GM1 was not encapsulated with β-cyclodextrin, and its performance indicators were as follows: bacterial density 2.5±0.1 (a decrease of approximately 10.7% compared to test group 1, slightly higher than test group 9); migratory diffusion radius 12.1±0.9 mm (a decrease of approximately 10.4% compared to test group 1, close to test group 9); adhesion and colonization capacity 4.3×10⁻⁶. 6 CFU / intestinal mucosa (decreased by approximately 49.4% compared to test group 1, and lower than test group 9). This indicates that the inclusion of β-cyclodextrin is crucial for ganglioside GM1, affecting its supporting role in bacterial adhesion and colonization. β-cyclodextrin inclusion is essential for fully leveraging the role of ganglioside GM1 in enhancing bacterial adhesion and colonization; unencapsulated GM1 may have poor stability or produce degradation products, resulting in an adhesion-promoting effect that is even less than that of GM1 without any addition. The inventors analyzed that the reason is that unencapsulated ganglioside GM1 is prone to degradation, producing short-chain glycolipid fragments. These fragments have certain surface activity, which can further negatively impact the flagellar structure and adhesion proteins of bacteria, thereby weakening their adhesion and colonization ability. Therefore, β-cyclodextrin-encapsulated ganglioside GM1 can significantly promote bacterial adhesion and colonization, and the inclusion form of β-cyclodextrin must be used.

[0061] (5) Regarding antioxidants, the inventors attempted to replace taurine with vitamin C (test group 11), ethoxyquin (test group 12), and 2-hydroxycinnamic acid (test group 13). Compared with test group 1, the results showed that the effects of the three antioxidants were inferior to taurine, and all indicators showed a significant decrease: Bacterial cell density (OD) 600 The scores for test groups 11, 12, and 13 decreased to 2.0±0.1 (a decrease of 28.6%), all of which were lower than the 2.8±0.2 of test group 1. The swimming diffusion radius: Test group 11 decreased to 10.3±1.2mm (a decrease of 23.7%), test group 12 decreased to 10.5±1.2mm (a decrease of 22.2%), and test group 13 decreased to 10.0±1.1mm (a decrease of 25.9%), all of which were lower than the 13.5±1.8mm of test group 1; Adhesion and colonization ability: Test group 11 decreased to 5.8 × 10⁻⁶. 6 CFU / intestinal mucosa (decreased by 31.8%), test group 12 decreased to 6.0×10 6 CFU / intestinal mucosa (decreased by 29.4%), test group 13 decreased to 5.6×10 6 CFU / intestinal mucosa (decreased by 34.1%) was lower than that of test group 1 (8.5 × 10⁻⁶). 6 CFU / intestinal mucosa.

[0062] This demonstrates that using taurine as an antioxidant is particularly effective compared to other conventional antioxidants, achieving unexpected technological results.

[0063] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A high-efficiency culture medium for freshwater Vibrio containing nucleotide complexes, characterized in that: By weight, its active ingredients include: 2000-5000 parts carbon source, 1500-3000 parts nitrogen source, 225-405 parts precursor substances, 65-125 parts amino acids and amino acid analogs, 55-110 parts energy system, 650-1300 parts ion regulator, 35-85 parts membrane stabilizer, and 10-20 parts antioxidant. The carbon source includes D-glucose; the nitrogen source includes 1000-2000 parts of ammonium sulfate and 500-1000 parts of yeast extract. The precursor substances include 100-150 parts of inosine, 20-50 parts of deoxyguanosine, 50-100 parts of uridine, 40-80 parts of cytidine, and 15-25 parts of deoxycytidine acid. Amino acids and amino acid analogues include 30-70 parts of L-tryptophan and 35-55 parts of cyclic serine; Energy systems include NAD + 5-10 parts and 50-100 parts of ribose-1-phosphate; The ion regulator consists of 150-300 parts magnesium chloride hexahydrate and 500-1000 parts sodium chloride; Membrane stabilizers include 5-15 parts of ganglioside GM1 and 30-70 parts of β-cyclodextrin; antioxidants include taurine.

2. The high-efficiency culture medium for freshwater Vibrio containing nucleotide complexes according to claim 1, characterized in that: Each liter of the compound culture medium contains: D-glucose 2-5g, ammonium sulfate 1-2g, yeast extract 0.5-1g, hypoxanthine 100-150mg, deoxyguanosine 20-50mg, uridine 50-100mg, cytidine 40-80mg, deoxycytidine acid 15-25mg, L-tryptophan 30-70mg, cyclic serine 35-55mg, NAD+. + 5-10mg, ribose-1-phosphate 50-100mg, magnesium chloride hexahydrate 150-300mg, sodium chloride 0.5-1g, ganglioside GM1 5-15mg, β-cyclodextrin 30-70mg, taurine 10-20mg.

3. The method for preparing a high-efficiency culture medium for freshwater Vibrio containing nucleotide complexes according to claim 2, characterized in that: Sodium chloride and magnesium chloride hexahydrate were dissolved in water sequentially, and sterilized to obtain a sterile base solution; D-glucose, ammonium sulfate, and yeast extract were dissolved in water, and filtered to obtain a carbon and nitrogen source nutrient solution; hypoxanthine, deoxyguanosine, uridine, cytidine, deoxycytidine acid, and NAD were added. + Ribose-1-phosphate was dissolved in water, and the nucleotide filtrate was obtained after filtration and sterilization. Ganglioside GM1 was mixed with β-cyclodextrin, and after sonication, an inclusion complex was formed. After filtration and sterilization, the GM1-β-cyclodextrin complex was obtained. The following were added sequentially to the sterilized base solution: carbon and nitrogen source nutrient solution, nucleotide filtrate, GM1-β-cyclodextrin complex, L-tryptophan, cycloserine, and taurine. The solution was brought to a final volume, stirred, and the pH was adjusted to 7.2±0.1 to obtain the composite culture medium.

4. The method for preparing a high-efficiency culture medium for freshwater Vibrio containing nucleotide complexes according to claim 3, characterized in that: Ganglioside GM1 was mixed with β-cyclodextrin and then sonicated at 40 kHz, 80-120 W for 30 min. The obtained composite culture medium was dispensed into culture flasks equipped with 0.2 μm breathable membranes.

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