Enhanced culture medium for reducing expression of surface polysaccharide of vibrio parahaemolyticus

Abstract

The invention belongs to the field of microbial culture, and particularly relates to an enrichment medium for reducing expression of surface polysaccharide of vibrio parahaemolyticus. According to the formula of the polysaccharide low-expression enrichment culture medium, nutritional ingredients in a specific proportion and specific polysaccharide expression inhibition conditions are selected, so that the expression quantity of surface lipopolysaccharide and capsular polysaccharide after enrichment of vibrio parahaemolyticus is lower than the expression quantity of polysaccharide after enrichment of normally cultured vibrio parahaemolyticus; expression of lipopolysaccharide and capsular polysaccharide on the surface layer can be inhibited to a certain extent, so that outer membrane protein can be better exposed on the surface of a thallus, and immunodetection of vibrio parahaemolyticus is facilitated. The invention provides a technical basis for developing a complete vibrio parahaemolyticus immunodetection method.

Description

Technical Field

[0001] The invention belongs to the field of microbial culture, and in particular relates to a bacterial enrichment culture medium for reducing the expression of surface polysaccharide of Vibrio parahaemolyticus. Background Art

[0002] In recent years, the analysis of national food safety sampling and foodborne disease outbreak detection data show that food safety problems caused by microorganisms are still the main cause of foodborne diseases. Vibrio parahaemolyticus, as a Gram-negative bacterium, can contaminate various foods, mainly seafood, such as fish, shrimp, crab, and shellfish. The number of food safety incidents caused by it has jumped to the top of the number of food poisoning incidents in my country. Vibrio parahaemolyticus is a halophilic, facultative anaerobic Gram-negative bacterium with no spores, flagella, and various morphologies, which can be arc-shaped, rod-shaped, spherical, etc. Vibrio parahaemolyticus is sensitive to acid and has weak resistance to heat. The optimal growth temperature is 30-37℃, and the optimal growth condition of pH value is 7.0-8.5. It is widely distributed in seawater environment and seafood. Vibrio parahaemolyticus can produce a variety of pathogenic factors, which will not only bring huge economic losses to the aquaculture industry, but more seriously, it can cause seafood gastroenteritis, inflammatory diarrhea and other harms to human health.

[0003] Bacterial surface polysaccharides include capsular polysaccharides (CPS) and lipopolysaccharides (LPS), which are important components of the cell wall of Vibrio parahaemolyticus and are essential for its host colonization, invasion, drug resistance, immune escape and pathogenicity. The lipopolysaccharides of most Gram-negative bacteria are composed of lipid A, core polysaccharides and highly variable O side chains (O antigens). The capsule or k antigen is composed of high molecular weight polysaccharides and forms a physical barrier outside the bacterial cell. The expression of surface polysaccharides of Vibrio parahaemolyticus may change under different environmental factors, such as seawater environment, seafood, and animals. At present, immunoassay products for Vibrio parahaemolyticus are very rare at home and abroad. The bottleneck is that the surface specific protein antigens or epitopes have not been revealed, high-quality monoclonal antibodies are difficult to prepare, and the matrix effect of aquatic products may also affect the actual application effect. Outer membrane protein is a commonly used immunoassay antigen, but according to existing literature reports, the difference in the expression of bacterial surface polysaccharides, including lipopolysaccharides and capsular polysaccharides, has a certain impact on the accuracy and sensitivity of immunoassays for Vibrio parahaemolyticus. This is mainly because lipopolysaccharide and capsular polysaccharide are relatively long, forming the outermost physical barrier of bacterial cells. When the expression level is high, it will mask the exposure of bacterial outer membrane protein epitopes, making it difficult for antibodies to recognize the corresponding target proteins in pathogenic bacteria immune detection, resulting in missed detection or poor sensitivity.

[0004] Therefore, it is necessary to study the polysaccharide expression patterns of different strains of Vibrio parahaemolyticus under typical environmental factors, so as to establish the culture medium formula to achieve less surface polysaccharide expression and increased outer membrane protein exposure of Vibrio parahaemolyticus in different environmental and nutritional conditions after enrichment. This can provide a technical basis for further screening of Vibrio parahaemolyticus-specific outer membrane proteins, realizing immunodetection of Vibrio parahaemolyticus in different environmental samples, and developing accurate and reliable immunodetection methods for Vibrio parahaemolyticus. Summary of the invention

[0005] The purpose of the present invention is to provide a culture medium and preparation method for enrichment of Vibrio parahaemolyticus with low expression of surface polysaccharides, in order to screen Vibrio parahaemolyticus outer membrane proteins with better exposure, avoid the influence of surface polysaccharides on antibody recognition in immunoassay after enrichment, solve the problem that differences in polysaccharide expression affect the accuracy and sensitivity of immunoassay of Vibrio parahaemolyticus, and is suitable for pre-treatment and pre-enrichment of immunoassay technology.

[0006] The technical scheme of the present invention is as follows: a bacterial enrichment culture medium for reducing the expression of surface polysaccharide of Vibrio parahaemolyticus comprises peptone, sodium chloride, bile salt, and the balance is water.

[0007] Preferably, the bile salt is selected from bovine bile salt or compound bile salt, and the compound bile salt is composed of 35% glycodeoxycholic acid, 35% sodium iodidecholate and 30% sodium cholate.

[0008] Preferably, the enrichment culture medium comprises 0.25% peptone, 3% sodium chloride, 0.4% to 0.6% bile salt, and the remainder is water, and the pH of the culture medium is 8.0 to 8.5.

[0009] Preferably, the enrichment culture medium consists of: 0.25% peptone, 3% sodium chloride, 0.6% bile salt, and the remainder is water, and the pH of the culture medium is 8.5.

[0010] Preferably, the enrichment culture medium is composed of: 0.25% peptone, 3% sodium chloride, 0.6% ox bile salt, and the remainder is water, and the pH of the culture medium is 8.5.

[0011] Preferably, the enrichment medium is composed of: 2.5 g / L peptone, 30 g / L sodium chloride, 6 g / L ox bile salt, add water to 1000 mL, and adjust the pH to 8.5.

[0012] The bacterial enrichment culture medium provided by the invention is used for reducing the expression of surface polysaccharides of Vibrio parahaemolyticus, wherein the surface polysaccharides are lipopolysaccharides and capsule polysaccharides.

[0013] The invention discloses an application of the enrichment culture medium provided by the invention in preparing a pre-enrichment culture medium for in vitro immunoassay of Vibrio parahaemolyticus.

[0014] The invention provides a method for reducing the expression of polysaccharide on the surface of Vibrio parahaemolyticus. The method comprises the following steps: taking a 1% inoculation amount of a Vibrio parahaemolyticus liquid or inoculating 25 g or 25 mL of a food sample into a bacterial enrichment medium, shaking and culturing for 10-16 hours at 37°C and 180 rpm, wherein the bacterial enrichment medium contains 2.5 g / L of peptone, 30 g / L of sodium chloride and 6 g / L of ox bile salt, adding water to 1000 mL, and adjusting the pH to 8.5.

[0015] The beneficial effects of the present invention are as follows: the formula of the polysaccharide low-expression enrichment culture medium of the present invention selects specific proportions of nutrients and specific conditions for inhibiting polysaccharide expression, so that the amount of surface lipopolysaccharide and capsular polysaccharide expressed after enrichment of Vibrio parahaemolyticus is lower than the amount of polysaccharide expressed after enrichment of normally cultured Vibrio parahaemolyticus, and the expression of surface lipopolysaccharide and capsular polysaccharide can be inhibited to a certain extent, so that the outer membrane protein can be better exposed on the bacterial surface, which is beneficial to the immune detection of Vibrio parahaemolyticus. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 .Silver staining images of lipopolysaccharide expression of Vibrio parahaemolyticus under simulated different environmental factors (A is Vibrio parahaemolyticus CICC 21619, B is CICC 21528, and C is CGMCC 1.1616).

[0017] Figure 2 .Silver staining images of Vibrio parahaemolyticus capsular polysaccharide expression under simulated different environmental factors (A is Vibrio parahaemolyticus CICC 21619, B is CICC 21528, and C is CGMCC 1.1616).

[0018] Figure 3 .Immunoblotting images of lipopolysaccharide expression of Vibrio parahaemolyticus under simulated different environmental factors (A is Vibrio parahaemolyticus CICC 21619, B is CICC 21528, and C is CGMCC 1.1616).

[0019] Figure 4 .Immunoblotting images of the capsular polysaccharide expression of Vibrio parahaemolyticus under simulated different environmental factors (A is Vibrio parahaemolyticus CICC 21619, B is CICC 21528, and C is CGMCC 1.1616).

[0020] in Figure 1-4 M, Lanes 1-34 are:

[0021] M: Marker; 1: pH 5.5; 2: pH 6.5; 3: pH 7.2; 4: pH 8.5; 5: 0.5% NaCl; 6: 1.5% NaCl; 7: 2.5% NaCl; 8: 3.5% NaCl; 9: 25°C; 10: 30°C; 11: 37°C; 12: 0.1% LB; 13: 1% NaCl; 14: 0.25% peptone; 15: 3% peptone; 16: TSB; 17: BHI; 18: Seawater; 19: Aerobic compound bile salt 0 mM; 20: Aerobic compound bile salt 3 mM; 21: aerobic complex bile salts: 7.5mM; 22: aerobic complex bile salts 12mM; 23: anaerobic complex bile salts 0mM; 24: anaerobic complex bile salts 3mM; 25: anaerobic complex bile salts 7.5mM; 26: anaerobic complex bile salts 12mM; 27: aerobic ox bile salts 0%; 28: aerobic ox bile salts 0.05%; 29: aerobic ox bile salts 0.1%; 30: aerobic ox bile salts 0.2%; 31: anaerobic ox bile salts 0%; 32: anaerobic ox bile salts 0.05%; 33: anaerobic ox bile salts 0.1%; 34: anaerobic ox bile salts 0.2%.

[0022] Figure 5 .Growth curve of Vibrio parahaemolyticus in enrichment medium with different concentrations of ox bile salts.

[0023] Figure 6 .Silver staining verification diagram of the amount of ox bile added to the Vibrio parahaemolyticus enrichment medium (A is the lipopolysaccharide verification diagram, and B is the capsular polysaccharide verification diagram). M, the conditions of lanes 1-9 are as follows: M: Marker; 1: CICC 21618 (0.4% ox bile); 2: CICC 21618 (0.6% ox bile); 3: CICC 21619 (0.4% ox bile); 4: CICC 21619 (0.6% ox bile); 5: CICC 21528 (0.4% ox bile); 6: CICC 21528 (0.6% ox bile); 7: CGMCC1.1616 (0.4% ox bile); 8: CGMCC1.1616 (0.6% ox bile); 9: CGMCC1.1616 (BHI)

[0024] Figure 7 .The indirect ELISA method was used to compare the titers of five culture media against five Vibrio parahaemolyticus outer membrane protein immune sera. DETAILED DESCRIPTION

[0025] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0026] If there is no special provision, the conversion relationship of 0.6% of ox bile salt in the present invention is that the concentration of ox bile salt in the culture medium is 6 g / L. This definition is also applicable to other components in the culture medium.

[0027] The main reagent information used in the following examples is shown in Tables 1 and 2; the target strain information is shown in Table 3.

[0028] Table 1 Information of routine reagents for target bacterial culture medium

[0029]

[0030]

[0031] Table 2 Information of main reagents of non-culture medium in the experiment

[0032]

[0033] Table 3 Target bacteria information

[0034]

[0035] The changes in culture conditions are shown in the following table:

[0036] Basic culture conditions: tryptone 10g / L, yeast extract 5g / L, sodium chloride 30g / L, complex bile salt 0mM, ox bile salt 0%, pH adjusted to 8.0, culture temperature 37°C, aerobic culture.

[0037] Sodium chloride alkaline peptone water: peptone 10.0 g, sodium chloride 30.0 g, distilled water 1000.0 mL, adjust the pH to 8.5±0.2.

[0038] TSB medium: trypticase 17.0 g / L, sodium chloride 5.0 g / L, soybean papain hydrolysate 3.0 g / L, potassium dihydrogen phosphate 2.5 g / L, glucose (monohydrate / anhydrous) 2.5 g / L, pH 7.2±0.2.

[0039] BHI medium: peptone 10.0 g / L, dehydrated calf brain powder 12.5 g / L, dehydrated buffalo heart powder 5.0 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L, pH 7.4±0.2.

[0040] The invention uses LB culture medium and sodium chloride alkaline peptone water culture as basic conditions, changes many factors, and screens out culture conditions suitable for normal growth of Vibrio parahaemolyticus and capable of reducing the expression of its surface protein.

[0041] Table 4 Different culture conditions

[0042]

[0043]

[0044] Example 1: Extraction of Surface Lipopolysaccharide and Capsular Polysaccharide of Vibrio parahaemolyticus

[0045] The culture medium of Vibrio parahaemolyticus cultured under different conditions was removed after centrifugation (4°C, 5000 rpm, 30 min), and the wet weight of the bacteria under each condition was adjusted to 0.5 g. The bacteria were resuspended in PBS, centrifuged three times, and resuspended in 6 mL PBS. The bacterial suspension was repeatedly frozen and thawed five times. The phenol was heated to 70°C in a water bath to melt it, and a 90% phenol solution was prepared. It was mixed with an equal volume of the bacterial suspension in a ratio of 1:1, and placed in a 68°C horizontal shaker for vigorous shaking for 30 min. Take it out and put it in an ice water bath to cool it down, centrifuge it (4℃, 5000rpm, 30min), collect the upper aqueous phase, put the collected solution into a dialysis bag with a cutoff molecular weight of 3500, and dialyze it with ultrapure water at 4℃. Change the water every 5-6h, and the dialysis takes a total of 48h (the dialysis is completed when no purple color appears when tested with ferric chloride). After the dialysis is completed, use polyethylene glycol (20000) to concentrate it to 1 / 4 of the original volume, centrifuge and discard the precipitate. The supernatant is the crude LPS.

[0046] The culture medium of Vibrio parahaemolyticus cultured under different conditions was removed by centrifugation (4°C, 5000rpm, 30min), and the wet weight of the bacteria under each condition was adjusted to 0.5g. The cells were washed once in physiological saline, centrifuged, and suspended in 50mL glycine buffer (0.1M, pH9.2), which contained 50mg crystalline salt-free egg white lysozyme. The suspension of Vibrio parahaemolyticus was cultured at 37°C for 6-8h by continuous stirring with a magnetic stirrer. At this time, most of the capsule material, nucleic acid and other components of Vibrio parahaemolyticus were released into the supernatant. After lysozyme lysis and digestion culture, the remaining Vibrio parahaemolyticus residues were removed by centrifugation. Proteinase K was added to 50 mL of the supernatant of the lysate to a concentration of 100 μg / mL and incubated at 55°C for 2 h. CaCl2 was added to the supernatant of the lysate after the addition of proteinase K to obtain a final concentration of 0.1 M and stirred for 1 h. 25% (V / V) anhydrous ethanol was added and the mixture was allowed to stand at 4°C for 2 h. The mixture was centrifuged to precipitate and remove the nucleic acid from the supernatant. Finally, anhydrous ethanol was added to a concentration of 80% (V / V) and the mixture was allowed to stand at 4°C overnight to precipitate and obtain crude capsular polysaccharide.

[0047] Example 2: Silver staining to verify the expression pattern of lipopolysaccharide and capsular polysaccharide

[0048] Vibrio parahaemolyticus was cultured according to the culture conditions in Table 4, and the polysaccharides extracted from the bacteria under different culture conditions (the wet weight of the bacteria under each condition was consistent) were separated by SDS-PAGE gel electrophoresis (the lipopolysaccharide sample was mixed with the loading buffer in a ratio of 1:1, and 10uL was loaded; the capsular polysaccharide was mixed in a ratio of 4:1, and 20uL was loaded), 30% ethanol, 10% acetic acid, 0.85% sodium periodate was added to the lipopolysaccharide, and 4.25% sodium periodate was added to the capsular polysaccharide, and the solution was fixed with ultrapure water for 30 minutes, and the fixation time can be appropriately extended, and then washed 3 times with ultrapure water at room temperature, each time for 5 minutes. Stain with 0.1% AgNO3 for 30 min, precool 3% Na2CO3 colorimetric solution at 4°C, add 40 μL formaldehyde for lipopolysaccharide colorimetry, and add 100 μL formaldehyde for capsular polysaccharide colorimetry. After coloring on the gel for 5-10 min, wash the gel with ultrapure water and image it with GelDoc Go gel imager (Bio-Rad).

[0049] like Figure 1 As shown in the figure, from the silver staining results, different environments have a certain effect on the expression of lipopolysaccharide and capsular polysaccharide of Vibrio parahaemolyticus. The molecular weight of lipopolysaccharide of Vibrio parahaemolyticus accumulates at about 10KDa, and there is an obvious band at 25KDa. The overall trend is: with the increase of pH and salt concentration, there is no significant change in the expression of polysaccharides of the three strains; when the temperature conditions are changed, the expression of polysaccharides is positively correlated with the temperature and the polysaccharide expression is the largest at 37℃; under the nutrient-deficient 0.1% LB condition, there is almost no lipopolysaccharide expression, and the polysaccharide expression in 0.25% peptone water is less than that in 3% peptone water conditions; the richer the nutrition of the culture conditions such as TSB, BHI, and seawater, the greater the increase in lipopolysaccharide expression. The original purpose of adding bile salts in the present invention was to simulate the intestinal environment and make the in vitro culture environment closer to the in vivo growth environment of Vibrio parahaemolyticus. However, it was unexpectedly found that the addition of complex bile salts or ox bile salts could significantly reduce the expression of polysaccharides, regardless of aerobic or anaerobic environment, and that the expression of polysaccharides decreased as the bile salt concentration increased.

[0050] like Figure 2As shown in the figure, under different conditions, the high molecular weight and low molecular weight capsular polysaccharides of the three strains were expressed to varying degrees, and the reaction bands were mainly concentrated above 70KDa; the overall trend was: the expression of capsular polysaccharides increased with the increase of pH value, and there was no significant change in the expression of capsular polysaccharides under different salt concentrations and temperature conditions; under the nutrient-deficient 0.1% LB condition and 0.25% peptone water condition, the expression of high molecular weight and low molecular weight capsular polysaccharides was low, and the expression of other nutrient-rich conditions increased relatively and the difference was not large. The culture conditions with added bile salts were consistent with the expression law of lipopolysaccharides. Whether in aerobic or anaerobic environments, the addition of compound bile salts or ox bile salts could significantly reduce the expression of capsular polysaccharides, and the expression of polysaccharides decreased with the increase of bile salts. In the anaerobic environment, except for 0mM and 3mM, there were shallow reaction bands. Under other conditions, the higher the bile salt concentration, the less polysaccharide expression, and there was almost no polysaccharide expression when the ox bile salt addition reached 0.2%.

[0051] Example 3: Immunoblotting to verify the expression pattern of lipopolysaccharide and capsular polysaccharide

[0052] Follow the steps to perform immunoblotting. After the protein gel electrophoresis, prepare the transfer membrane, pre-cool the transfer buffer, prepare the transfer sandwich, and the order from the negative plate to the positive plate is sponge, filter paper, acrylamide gel, PVDF membrane, filter paper, sponge; use Mini-Trans-Blot Cell ModμLe microtransformer (Bio-Rad) to transfer the membrane, the transfer conditions are 180mA, 80min, and after the transfer, the PVDF membrane is blocked overnight with 5% skim milk powder in 10mM PBS. The monoclonal rabbit secondary antibody is suspended in PBS (containing 1% gelatin) containing 0.05% Tween 20 (PBST) and reacted with the PVDF membrane on an orbital shaker in the dark for 1h at room temperature. After washing, the substrate solution is stained with tetramethylbenzidine (TMB). Once a stable purple-blue band appears, transfer the membrane to a disposable bacterial culture dish, soak it with water, and image it with a gel imager.

[0053] like Figure 3 As shown in the figure, the immunoblotting results of lipopolysaccharide of three strains of Vibrio parahaemolyticus showed that the position and expression pattern of lipopolysaccharide bands were consistent with the silver staining results. The molecular weight of lipopolysaccharide is about 10KDa, and the expression of polysaccharide varies significantly in different environments. The overall trend is: under different pH and temperature conditions, the expression of polysaccharide is not significant; under different temperature conditions, the expression of polysaccharide is relatively large at 37°C; under different nutritional conditions, under the nutrient-deficient 0.1% LB condition, there is almost no lipopolysaccharide expression, and the expression of lipopolysaccharide increases with the increase of nutritional richness; with the increase of the concentration of compound bile salts and ox bile salts, the expression of lipopolysaccharide decreases, and the expression of lipopolysaccharide in anaerobic environment is much lower than that in aerobic conditions.

[0054] like Figure 4 As shown in the figure, the results of immunoblotting of capsular polysaccharide showed that the position and expression pattern of capsular polysaccharide bands were consistent with the results of silver staining. The capsular polysaccharides of the three strains of Vibrio parahaemolyticus had reaction bands at 25KDa to 35KDa, and were also significantly expressed above 70KDa. There were obvious differences in the expression of capsules under different culture environments. The overall trend was: with the increase of pH value, the expression of high molecular weight polysaccharides increased; under the tested salt concentration and temperature conditions, there was no significant change in the expression of capsular polysaccharides; under different nutritional conditions, under the nutrient-deficient 0.1% LB conditions, the capsular polysaccharide imprint bands were shallow, while the capsular polysaccharide imprint bands increased with the increase of nutrient richness; with the increase of the concentration of compound bile salts and ox bile salts, the capsular polysaccharide imprint bands decreased, and the capsular polysaccharide imprint bands in the anaerobic environment were much less than those in the aerobic conditions. This shows that the capsular polysaccharide is similar to the lipopolysaccharide. The addition of compound bile salts or ox bile salts can significantly reduce the expression of polysaccharides, and the expression of polysaccharides decreases with the increase of bile salt concentration.

[0055] Example 4: Determination of the amount of ox bile salt added to the enrichment medium

[0056] Since the anaerobic culture process is relatively cumbersome, solid culture and oxygen-consuming bags are required, the growth is slow, and the bacteria need to be eluted after growth, which is very inconvenient and contrary to the purpose of rapid detection of Vibrio parahaemolyticus; in addition, the composite bile salt is composed of three bile salts, the cost is very high, and it is not universal. Moreover, it has been verified that low nutrients can reduce the expression of polysaccharides, but too low nutrients will cause the bacteria to be unable to reproduce normally and rapidly. Therefore, the present invention uses 2.5 g / L peptone, 30 g / L sodium chloride, the addition of ox bile salts and aerobic culture conditions for subsequent screening.

[0057] Take fresh overnight Vibrio parahaemolyticus liquid and inoculate it into 0.25% peptone water at a 1% inoculum volume, and culture it at 37°C and 180rpm with shaking. Take ox bile salt concentrations of 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, and 2.0% respectively. Take the culture medium without ox bile salt as the growth control, and take the culture medium every 1h to measure its OD 600 .

[0058] According to the OD at each time point 600 The results are plotted as Figure 5 By comparing the growth curves shown, it can be seen that according to the growth of Vibrio parahaemolyticus under different ox bile salt concentrations, 0.6% and 0.4% ox bile salt were selected as the ox bile salt addition amount of the bacterial enrichment medium, which can increase the bacteria normally, while the polysaccharide expression is relatively low.

[0059] Example 5: Identification of the effect of enrichment medium with low expression of surface polysaccharides

[0060] The present embodiment is a culture medium for enriching Vibrio parahaemolyticus with low expression of surface polysaccharide, and its formula composition is: peptone 2.5g / L, sodium chloride 30g / L, ox bile salt 6g / L, add water to 1000mL, adjust pH to 8.5, and culture aerobically. The control group formula is: peptone 2.5g / L, sodium chloride 30g / L, ox bile salt 4g / L, add water to 1000mL, adjust pH to 8.5, and culture aerobically.

[0061] Prepare 0.25% peptone, 30g / L sodium chloride, and 0.6% ox bile salt according to the above ratio, add ultrapure water or distilled water to 1000mL, sonicate until completely dissolved, and adjust the pH value to 8.5. Autoclave at 121℃ for 15min and store at 4℃. Prepare the control group culture medium in the same way.

[0062] Take three kinds of fresh overnight Vibrio parahaemolyticus seed liquids respectively, inoculate them into 0.25% peptone water + 0.4% ox bile salt and 0.25% peptone water + 0.6% ox bile salt medium at a 1% inoculum amount, and culture them at 37°C and 180 rpm with shaking for 10-16 hours;

[0063] According to the extraction method of lipopolysaccharide and capsular polysaccharide in Example 1, lipopolysaccharide and capsular polysaccharide of Vibrio parahaemolyticus cultured at 0.4% and 0.6% ox bile concentration were extracted respectively, and the expression pattern of lipopolysaccharide and capsular polysaccharide was verified by silver staining in Example 2. Figure 6 As shown, when the addition amount of ox bile salt was 0.6%, the overall polysaccharide expression was lower than that when the addition amount of ox bile salt was 0.4%.

[0064] Combining Example 5 and Example 6, it can be seen that in a culture medium for enriching low-expression surface polysaccharides of Vibrio parahaemolyticus, the amount of ox bile salt added can be determined to be 0.6% without affecting the growth of Vibrio parahaemolyticus. The inhibitory effect of this culture medium ratio on surface polysaccharides is better than that of culture media with other concentrations of ox bile salt.

[0065] Example 6: Comparison of the titers of five culture media against five Vibrio parahaemolyticus outer membrane protein immune sera by indirect ELISA

[0066] After overnight culture, the strain was inactivated by boiling in 100°C water for 10 min and cooled to room temperature. The bacterial cell concentration was diluted to 10 with 0.01 mol / L phosphate buffer (PBS). 8 CFU mL -1, 100 μL per well was added to a 96-well ELISA plate, wrapped in a ziplock bag to prevent contamination, and coated in a 37°C oven for 2 hours; after washing 3 times, 200 μL of carbonate buffer (CBS) containing 0.2% gelatin was added to each well to block the ELISA plate, and blocked at 37°C for 2 hours. Mouse serum was diluted 1000 times, 3000 times, 9000 times, and 27000 times with antibody diluent (PBS buffer, 0.1% gelatin, 0.05% Tween-20), and the antibody diluent was used as a negative control. Incubate in a 37°C oven for 35 min; wash the plate three times with PBST; add goat anti-mouse secondary antibody IgG (0.5 ug / mL) diluted 5000 times with antibody diluent, add 100 μL to each well, and incubate in a 37°C oven for 35 min; after washing the plate three times, add 100 μL of color development solution to each well, and incubate in a 37°C oven for 15 min; after incubation, add 50 μL of stop solution (2 mol / L sulfuric acid) to each well, and measure the absorbance at 450 nm with an enzyme reader.

[0067] In this example, mouse immune sera of recombinant proteins OmpAGE002258, OmpAGE003613, OmpAGE003868, BamA and PilQ were used to verify their potency against immune antigens of Vibrio parahaemolyticus cultured in different culture media. Figure 7 , the Y axis represents the absorbance at 450nm, the X axis represents the different culture media tested, and the serum dilution multiple is 1000. The polysaccharide-less enrichment culture medium of the present invention has a higher overall titer than other culture media in terms of reaction with different sera, and has a stronger titer reaction with OmpAGE002258 and OmpAGE003868, indicating that the reduction of polysaccharides on the surface of Vibrio parahaemolyticus will expose more protein sites and more proteins, and the reaction titer with serum will be higher. Therefore, the use of the culture medium of the present invention in actual detection can more sensitively realize the detection of Vibrio parahaemolyticus.

Claims

1. A culture medium for reducing the expression of surface polysaccharides of Vibrio parahaemolyticus, comprising peptone, sodium chloride, bile salts, and the balance being water.

2. The bacterial enrichment medium according to claim 1, characterized in that: The bile salt is selected from bovine bile salt or compound bile salt, and the compound bile salt is composed of 35% glycodeoxycholic acid, 35% bovine sodium iodide lithocholic acid and 30% sodium cholate.

3. The bacterial enrichment medium according to claim 1, characterized in that: The bacterial enrichment culture medium comprises 0.25% peptone, 3% sodium chloride, 0.4% to 0.6% bile salt, and the balance is water. The pH value of the culture medium is 8.0 to 8.

5.

4. The bacterial enrichment medium according to claim 1, characterized in that: The bacterial enrichment culture medium consists of: 0.25% peptone, 3% sodium chloride, 0.6% bile salt, and the remainder is water. The pH value of the culture medium is 8.

5.

5. The bacterial enrichment medium according to claim 1, characterized in that: The bacterial enrichment culture medium consists of: 0.25% peptone, 3% sodium chloride, 0.6% ox bile salt, and the balance is water. The pH value of the culture medium is 8.

5.

6. The bacterial enrichment medium according to claim 1, characterized in that: The enrichment culture medium is composed of: 2.5 g / L peptone, 30 g / L sodium chloride, 6 g / L ox bile salt, water is added to 1000 mL, and the pH is adjusted to 8.

5.

7. The enrichment medium according to any one of claims 1 to 6 is used for reducing the expression of surface polysaccharides of Vibrio parahaemolyticus, wherein the surface polysaccharides are lipopolysaccharides and capsular polysaccharides.

8. Use of the enrichment medium according to any one of claims 1 to 6 in the preparation of a pre-enrichment medium for in vitro immunoassay of Vibrio parahaemolyticus.

9. A method for reducing the expression of surface polysaccharides of Vibrio parahaemolyticus, comprising the following steps: taking a Vibrio parahaemolyticus bacterial solution and inoculating it into a bacterial enrichment medium at a 1% inoculum size, shaking and culturing it at 37°C and 180rpm for 10-16h, wherein the bacterial enrichment medium comprises 2.5g / L peptone, 30g / L sodium chloride, and 6g / L ox bile salt, adding water to 1000mL, and adjusting the pH to 8.5.

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