A rich culture medium for reducing expression of surface polysaccharide of vibrio parahaemolyticus

Abstract

The present application belongs to the field of microbial culture, and particularly relates to a culture medium for reducing expression of surface polysaccharide of Vibrio parahaemolyticus. The formula of the polysaccharide low-expression culture medium is selected from specific proportion of nutritional components and specific conditions for inhibiting polysaccharide expression, so that the expression amount of surface lipopolysaccharide and capsule polysaccharide of Vibrio parahaemolyticus after the culture medium is increased is lower than the expression amount of polysaccharide of Vibrio parahaemolyticus after normal culture, the expression of surface lipopolysaccharide and capsule polysaccharide can be inhibited to a certain extent, the surface of the bacterial body can better expose outer membrane protein, and the immunodetection of Vibrio parahaemolyticus is facilitated. The present application provides a technical basis for developing a complete immunodetection method of Vibrio parahaemolyticus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial culture, and particularly relates to a culture medium for increasing Vibrio parahaemolyticus expression of surface polysaccharide. BACKGROUND

[0002] In recent years, the analysis of national food safety sampling and the detection data of foodborne disease outbreaks show that the problem of food safety unqualified caused by microorganisms is still the main reason for foodborne diseases. Vibrio parahaemolyticus, as a gram-negative bacterium, can contaminate various foods such as fish, shrimp, crab, and shellfish, and the number of food safety incidents caused by Vibrio parahaemolyticus has jumped to the top of food poisoning incidents in China. Vibrio parahaemolyticus is a salt-tolerant, facultative anaerobic gram-negative bacterium without spores and with flagella. It has various morphologies, such as arc-shaped, rod-shaped, and spherical. Vibrio parahaemolyticus is sensitive to acid and weak to heat, with an optimal growth temperature of 30-37℃ and an optimal pH value of 7.0-8.5. It is widely distributed in seawater environment and seafood. Vibrio parahaemolyticus can produce various pathogenic factors, which not only brings huge economic losses to aquaculture industry, but also causes seafood gastroenteritis, inflammatory diarrhea and other hazards to human health.

[0003] Bacterial surface polysaccharides, including capsular polysaccharide (CPS) and lipopolysaccharide (LPS), are important components of Vibrio parahaemolyticus cell wall, which are crucial for its host colonization, invasion, drug resistance, immune escape and pathogenicity. The lipopolysaccharide of most gram-negative bacteria is composed of lipid A, core polysaccharide and highly variable O side chain (O antigen). The capsule or k antigen is composed of high molecular weight polysaccharide and forms a physical barrier outside the bacterial cell. Under different environmental factors such as seawater environment, seafood and animal body, the expression of Vibrio parahaemolyticus surface polysaccharide may change. At present, there are very few Vibrio parahaemolyticus immune detection products at home and abroad, and the bottleneck is that the surface specific protein antigen or epitope has 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 immunodetection antigen, but according to the existing literature reports, the expression difference of bacterial surface polysaccharide including lipopolysaccharide and capsular polysaccharide has a certain influence on the accuracy and sensitivity of Vibrio parahaemolyticus immune detection. This is mainly because the length of lipopolysaccharide and capsular polysaccharide is relatively long, which constitutes the outermost physical barrier of bacterial cells, and when the expression amount is high, it will cover the epitope exposure of bacterial outer membrane protein, causing the antibody to be difficult to recognize the corresponding target protein in the pathogenic bacteria immune detection, resulting in missed detection or poor sensitivity.

[0004] Therefore, the polysaccharide expression rules of different strains of Vibrio parahaemolyticus under typical environmental factors are studied, and a culture medium formula is established to realize that Vibrio parahaemolyticus under different environmental and nutritional conditions has less surface polysaccharide expression and improved exposure of outer membrane protein after enrichment, which can provide a technical basis for further screening of Vibrio parahaemolyticus specific outer membrane protein, and for developing an accurate and reliable Vibrio parahaemolyticus immunodetection method. SUMMARY

[0005] The purpose of the present application is to provide a culture medium for reducing the expression of surface polysaccharide of Vibrio parahaemolyticus and a preparation method, which is suitable for pre-enrichment of immunodetection technology.

[0006] The technical solution of the present application is as follows: a culture medium for reducing the expression of surface polysaccharide of Vibrio parahaemolyticus, which comprises proteose peptone, sodium chloride, bile salt, and the rest is water.

[0007] Preferably, the bile salt is selected from bovine bile salt or complex bile salt, and the composition of the complex bile salt is 35% glycol deoxycholic acid, 35% sodium bovine iodine stone cholic acid, and 30% sodium cholate.

[0008] Preferably, the composition of the culture medium comprises 0.25% proteose peptone, 3% sodium chloride, 0.4%-0.6% bile salt, and the rest is water, and the pH of the culture medium is 8.0-8.5.

[0009] Preferably, the composition of the culture medium is 0.25% proteose peptone, 3% sodium chloride, 0.6% bile salt, and the rest is water, and the pH of the culture medium is 8.5.

[0010] Preferably, the composition of the culture medium is 0.25% proteose peptone, 3% sodium chloride, 0.6% bovine bile salt, and the rest is water, and the pH of the culture medium is 8.5.

[0011] Preferably, the composition of the culture medium is 0.25% proteose peptone, 3% sodium chloride, 0.6% bovine bile salt, and the rest is water, and the pH of the culture medium is 8.5.

[0012] The culture medium provided by the present application is used for reducing the expression of surface polysaccharide of Vibrio parahaemolyticus, and the surface polysaccharide is lipopolysaccharide and capsular polysaccharide.

[0013] The culture medium provided by the present application is used for reducing the expression of surface polysaccharide of Vibrio parahaemolyticus, and the surface polysaccharide is lipopolysaccharide and capsular polysaccharide.

[0014] The application provides a method for reducing expression of surface polysaccharide of Vibrio parahaemolyticus, comprising the following steps: inoculating Vibrio parahaemolyticus liquid culture at a 1% inoculation amount or a food sample at 25 g or 25 mL into a enrichment culture medium, and culturing at 37 DEG C and 180 rpm for 10-16 h, wherein the enrichment culture medium is composed of 2.5 g / L of proteose peptone, 30 g / L of sodium chloride, 6 g / L of butyric acid salt, water added to 1000 mL, and pH adjusted to 8.5.

[0015] The application has the following beneficial effects: the formula of the polysaccharide low-expression enrichment culture medium of the application selects specific proportions of nutritional components and specific polysaccharide expression inhibition conditions, so that the expression amount of surface lipopolysaccharide and capsule polysaccharide of Vibrio parahaemolyticus after enrichment is lower than that of Vibrio parahaemolyticus cultured normally, the expression of surface lipopolysaccharide and capsule polysaccharide can be inhibited to a certain extent, so that the outer membrane protein on the surface of the bacterial body can be better exposed, and the immune detection of Vibrio parahaemolyticus is facilitated. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0019] Figure 4 . Immunoblot images of Vibrio parahaemolyticus capsule polysaccharide expression under simulation of different environmental factors (wherein A is Vibrio parahaemolyticus CICC 21619, B is CICC 21528, and C is CGMCC 1.1616).

[0020] M, lanes 1-34 of Figures 1-4

[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 complexed bile salt 0 mM; 20: aerobic complexed bile salt 3 mM; 21: aerobic complexed bile salt: 7.5 mM; 22: aerobic complexed bile salt 12 mM; 23: anaerobic complexed bile salt 0 mM; 24: anaerobic complexed bile salt 3 mM; 25: anaerobic complexed bile salt 7.5 mM; 26: anaerobic complexed bile salt 12 mM; 27: aerobic oxgall 0%; 28: aerobic oxgall 0.05%; 29: aerobic oxgall 0.1%; 30: aerobic oxgall 0.2%; 31: anaerobic oxgall 0%; 32: anaerobic oxgall 0.05%; 33: anaerobic oxgall 0.1%; 34: anaerobic oxgall 0.2%.

[0022] Figure 5 . Growth curve of Vibrio parahaemolyticus in different concentrations of oxgall in the enrichment medium.

[0023] Figure 6 . Silver staining verification of Vibrio parahaemolyticus in the enrichment medium with different concentrations of oxgall (A: verification of lipopolysaccharide; B: verification of capsular polysaccharide). M, conditions of lanes 1-9 are as follows: M: Marker; 1: CICC 21618 (0.4% oxgall); 2: CICC 21618 (0.6% oxgall); 3: CICC 21619 (0.4% oxgall); 4: CICC 21619 (0.6% oxgall); 5: CICC 21528 (0.4% oxgall); 6: CICC 21528 (0.6% oxgall); 7: CGMCC 1.1616 (0.4% oxgall); 8: CGMCC 1.1616 (0.6% oxgall); 9: CGMCC 1.1616 (BHI)

[0024] Figure 7 . Comparison of the titers of the immune serum against the outer membrane proteins of five Vibrio parahaemolyticus strains in five media by indirect ELISA. DETAILED DESCRIPTION

[0025] The following examples can make the person skilled in the art more fully understand the present application, but the present application is not limited in the scope of the examples.

[0026] Unless otherwise specified, 0.6% of the oxgall salt in the present application is converted to a concentration of 6 g / L of oxgall salt in the medium. The same definition applies to other components in the 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 table of common reagents for target bacteria culture medium

[0029]

[0030]

[0031] Table 2. Information table of main reagents for test non-culture medium

[0032]

[0033] Table 3. Information table of target bacteria

[0034]

[0035] The change of culture conditions is shown in the following table:

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

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

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

[0039] BHI medium: tryptone 10.0 g / L, dehydrated calf brain extract powder 12.5 g / L, dehydrated bovine heart extract powder 5.0 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, sodium phosphate dibasic 2.5 g / L, pH 7.4±0.2.

[0040] The present application takes LB medium and sodium chloride alkaline tryptone water culture as the basic condition, and changes many factors to screen out the culture conditions suitable for the normal growth of Vibrio parahaemolyticus and at the same time can reduce the expression of surface protein.

[0041] Table 4. Different culture conditions

[0042]

[0043]

[0044] Example 1: Extraction of surface lipopolysaccharide and capsule polysaccharide of Vibrio parahaemolyticus

[0045] The Vibrio parahaemolyticus cultured under different conditions was centrifuged (4°C, 5000 rpm, 30 min) to remove the culture medium, and the wet weight of the bacterial body of each condition was adjusted to 0.5 g. The bacterial body was resuspended in PBS, centrifuged repeatedly three times, resuspended in 6 mL of PBS, and 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. The bacterial suspension was mixed with the phenol solution at a ratio of 1:1, and the mixture was placed in a 68°C horizontal shaker for vigorous shaking for 30 min. The mixture was then placed in an ice water bath for cooling, centrifuged (4°C, 5000 rpm, 30 min), and the upper aqueous phase was collected. The collected solution was placed in a dialysis bag with a molecular weight cutoff of 3500, and ultra-pure water was used for flow dialysis at 4°C. The water was changed every 5-6 h, and a total of 48 h of dialysis was required (dialysis was completed when no purple color appeared in the detection of ferric chloride). After dialysis, polyethylene glycol (20000) was used to concentrate the solution to 1 / 4 of the original volume. The precipitate was removed by centrifugation, and the supernatant was the crude LPS product.

[0046] The Vibrio parahaemolyticus cultured under different conditions was centrifuged (4°C, 5000 rpm, 30 min) to remove the culture medium, and the wet weight of the bacterial body of each condition was adjusted to 0.5 g. The bacterial body was washed once in physiological saline, centrifuged, and suspended in 50 mL of glycine buffer (0.1 M, pH 9.2) containing 50 mg of crystalline salt-free egg white lysozyme. The suspension was incubated at 37°C for 6-8 h with constant stirring by a magnetic stirrer. At this time, most of the capsule material, along with nucleic acids and other components of Vibrio parahaemolyticus, was released into the supernatant. After lysozyme digestion, the remaining Vibrio parahaemolyticus residues were removed by centrifugation. Proteinase K was added to the supernatant to a final concentration of 100 μg / mL, and the mixture was incubated at 55°C for 2 h. After the addition of proteinase K, CaCl2 was added to the supernatant to a final concentration of 0.1 M, and the mixture was stirred for 1 h. Then, 25% (V / V) of anhydrous ethanol was added, and the mixture was incubated at 4°C for 2 h. The nucleic acids were precipitated and removed from the supernatant by centrifugation. Finally, anhydrous ethanol was added to a concentration of 80% (V / V), and the mixture was incubated at 4°C overnight to precipitate the capsule polysaccharide.

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

[0048] The culture conditions according to Table 4 were used for the culture of Vibrio parahaemolyticus, and polysaccharides were extracted from bacteria in different culture conditions (the wet weight of bacteria in each condition was consistent), and separated by SDS-PAGE gel electrophoresis (lipopolysaccharide samples were mixed with loading buffer at a ratio of 1:1, and 10 uL was loaded; capsular polysaccharide was mixed at a ratio of 4:1, and 20 uL was loaded), 30% ethanol, 10% acetic acid, 0.85% sodium periodate was added to lipopolysaccharide, 4.25% sodium periodate was added to capsular polysaccharide, and ultrapure water was used for dissolution and fixation for 30 min, and the fixation time can be appropriately extended, then washed with ultrapure water at room temperature for 3 times, each time for 5 min. 0.1% AgNO3 staining for 30 min, 3% Na2CO3 color developing solution was pre-cooled at 4°C, 40 uL of formaldehyde was added for lipopolysaccharide color development, 100 uL of formaldehyde was added for capsular polysaccharide color development, and the gel was colored for 5-10 min, then the gel was washed with ultrapure water, and imaged using a GelDoc Go gel imager (Bio-Rad Company).

[0049] As shown in Figure 1 From the results of silver staining, different environments have a certain effect on the expression of Vibrio parahaemolyticus lipopolysaccharide and capsular polysaccharide, the molecular weight of Vibrio parahaemolyticus lipopolysaccharide is about 10 KDa, and there is a clear band at 25 KDa; the overall trend is that as the pH increases and the salt concentration increases, the polysaccharide expression of the three strains has no significant change rule; changing the temperature conditions, the polysaccharide expression rule is positively correlated with the temperature and the polysaccharide expression is the largest at 37°C; under the 0.1% LB condition of nutrient deficiency, there is almost no lipopolysaccharide expression, and the polysaccharide expression under the 0.25% proteose peptone water condition is less than that under the 3% proteose peptone water condition; the more nutrient-rich the culture conditions such as TSB, BHI and seawater, the more the lipopolysaccharide expression increases. The purpose of initially adding bile salt in the present application is to simulate the intestinal environment, so that the in vitro culture environment is closer to the in vivo growth environment of Vibrio parahaemolyticus, but it is accidentally found that adding compound bile salt or bovine bile salt can significantly reduce the polysaccharide expression under aerobic or anaerobic environment, and the polysaccharide expression decreases with the increase of bile salt concentration.

[0050] As shown in Figure 2As shown, under different conditions, both high-molecular-weight and low-molecular-weight capsular polysaccharides of the three strains were expressed to varying degrees, with reaction bands mainly concentrated above 70 kDa. The overall trend was that capsular polysaccharide expression increased with increasing pH, while no significant variation was observed under different salt concentrations and temperatures. In nutrient-deficient conditions (0.1% LB and 0.25% peptone water), both high-molecular-weight and low-molecular-weight capsular polysaccharides were expressed at relatively low levels, while under other nutrient-rich conditions, expression levels increased relatively but with little difference. The expression pattern of lipopolysaccharide (LPS) was consistent with that of bile salts in culture conditions. Regardless of whether the culture was aerobic or anaerobic, the addition of compound bile salts or ox bile salts significantly reduced capsular polysaccharide expression, and the expression decreased with increasing bile salt concentration. Under anaerobic conditions, only 0 mM and 3 mM showed relatively faint reaction bands; under other conditions, higher bile salt concentrations resulted in lower polysaccharide expression, with almost no polysaccharide expression observed at 0.2% ox bile salt concentration.

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

[0052] Perform the immunoblotting procedure according to the steps. After protein gel electrophoresis, prepare for membrane transfer. Pre-cool the transfer buffer and prepare the transfer sandwich clamp. The order from negative to positive electrode is sponge, filter paper, acrylamide gel, PVDF membrane, filter paper, sponge. Use a Mini-Trans-Blot Cell ModμLe (Bio-Rad) for transfer at 180mA for 80 min. After transfer, block the PVDF membrane overnight in 10mM PBS with 5% skim milk powder. The monoclonal antibody and rabbit secondary antibody are suspended in PBS containing 0.05% Tween 20 (PBST) (containing 1% gelatin) and reacted with the PVDF membrane on a rocker in the dark for 1 h at room temperature. After washing, develop the membrane with tetramethylbenzidine (TMB) blotting substrate solution. Once a stable purple-blue band appears, transfer the membrane to a disposable bacterial culture dish, soak it in water, and image it using a gel imaging system.

[0053] like Figure 3 As shown, the immunoblotting results of lipopolysaccharide (LPS) in three Vibrio parahaemolyticus strains were consistent with the silver staining results. The molecular weight of LPS was approximately 10 kDa. LPS expression levels varied significantly under different environments, with the following general trends: LPS expression did not change significantly under different pH and temperature conditions; at 37℃, LPS expression was relatively high; under different nutritional conditions, LPS expression was almost nonexistent under nutrient-deficient 0.1% LB conditions, while LPS expression increased with increasing nutrient richness; LPS expression decreased with increasing concentrations of complex bile salts and ox bile salts, with LPS expression levels significantly lower under anaerobic conditions than under aerobic conditions.

[0054] likeFigure 4 As shown in Fig. 6, the results of the capsule polysaccharide immunoblotting show that the position and expression regularity of the capsule polysaccharide bands are consistent with the silver staining results. The capsule polysaccharides of the three strains of Vibrio parahaemolyticus have reaction bands at 25KDa-35KDa, and have obvious expression above 70KDa. The expression of the capsule is obviously different under different culture environments. The overall trend is that, as the pH value increases, the expression of the high molecular weight polysaccharide increases; under the tested salt concentration and temperature conditions, there is no significant change in the expression of the capsule polysaccharide; under different nutritional conditions, the capsule polysaccharide immunoblotting band is shallower under the 0.1% LB condition of nutritional deficiency, and the capsule polysaccharide immunoblotting band increases with the increase of the richness of the nutrition; as the concentration of the compound cholate and the bovine cholate increases, the capsule polysaccharide immunoblotting band decreases, and the capsule polysaccharide immunoblotting band under the anaerobic environment is much lower than that under the aerobic condition. It is shown that the capsule polysaccharide is similar to the lipopolysaccharide, and the addition of the compound cholate or the bovine cholate can significantly reduce the expression of the polysaccharide, and the expression amount of the polysaccharide decreases with the increase of the concentration of the cholate.

[0055] Example 4: Determination of the addition amount of bovine cholate in the enrichment culture medium

[0056] Since the anaerobic culture process is complicated, 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 is contrary to the purpose of rapid detection of Vibrio parahaemolyticus. In addition, the compound cholate is composed of three cholates, and the cost is very high, which is not universal. Furthermore, it has been verified that low nutritional ingredients can reduce the expression of polysaccharide, but too low nutritional ingredients can cause the bacteria to be unable to grow normally and rapidly, therefore, in the present application, 2.5g / L of proteose peptone, 30g / L of sodium chloride, the addition of bovine cholate and aerobic culture conditions are used for subsequent screening.

[0057] Fresh Vibrio parahaemolyticus liquid after overnight culture was inoculated into 0.25% proteose peptone water at an inoculation amount of 1%, and was cultured under the condition of 37°C and 180rpm vibration, and bovine cholate concentrations of 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8% and 2.0% were taken, and the culture medium without the addition of bovine cholate was taken as the growth condition control, and the culture was taken every 1h to measure the OD 600 .

[0058] The growth curves were drawn according to the OD 600 results at each time point, as shown in Fig. 4, and it can be seen from the comparison that, according to the growth conditions of Vibrio parahaemolyticus under different bovine cholate concentrations, 0.6% and 0.4% bovine cholate are selected as the addition amount of bovine cholate in the enrichment culture medium, which can normally enrich the bacteria, and the expression of polysaccharide is less. Figure 5 Example 5: Effect identification of the enrichment culture medium with low expression of surface polysaccharide

[0059]

[0060] ​The culture medium for low expression of surface polysaccharide of Vibrio parahaemolyticus in the embodiment comprises 2.5 g / L of proteose peptone, 30 g / L of sodium chloride, and 6 g / L of bovine bile salt, and is added with water to 1000 mL and adjusted to pH 8.5 for aerobic culture.

[0061] The 0.25% proteose peptone, 30 g / L of sodium chloride, and 0.6% bovine bile salt are prepared according to the above proportions, added with ultrapure water or distilled water to 1000 mL, ultrasonically dissolved, and adjusted to pH 8.5. The solution is sterilized at 121°C for 15 min and stored at 4°C. The control medium is prepared in the same way.

[0062] The three kinds of fresh overnight Vibrio parahaemolyticus seed liquids are respectively inoculated into 0.25% proteose peptone water + 0.4% bovine bile salt and 0.25% proteose peptone water + 0.6% bovine bile salt culture media at an inoculation amount of 1%, and cultured at 37°C and 180 rpm for 10-16 h.

[0063] According to the extraction method of lipopolysaccharide and capsular polysaccharide in Example 1, the lipopolysaccharide and capsular polysaccharide of Vibrio parahaemolyticus cultured at 0.4% and 0.6% bovine bile salt concentrations are respectively extracted, and the expression rules of lipopolysaccharide and capsular polysaccharide are verified by silver staining in Example 2, as shown in Figure 6 As shown in the figure, the total polysaccharide expression at the bovine bile salt addition amount of 0.6% is lower than that at the bovine bile salt addition amount of 0.4%.

[0064] It can be known from the combination of Example 5 and Example 6 that the culture medium for low expression of surface polysaccharide of Vibrio parahaemolyticus can be determined to be added with 0.6% bovine bile salt without affecting the growth of Vibrio parahaemolyticus, and the inhibition effect of the culture medium ratio on surface polysaccharide is better than that of other concentrations of bovine bile salt culture medium.

[0065] Example 6: Comparison of the titers of five kinds of Vibrio parahaemolyticus outer membrane protein immune serum in five kinds of culture media by indirect ELISA method

[0066] After the strain is cultured overnight, it is boiled in 100°C boiling water for 10 min to inactivate, and cooled to room temperature. The bacterial concentration is diluted to 10 8 CFU / mL -1, 100 μL per well was added to a 96-well enzyme plate, and a self-sealing bag was used to prevent contamination, and the plate was coated in an oven at 37°C for 2 h; after washing 3 times, 200 μL of carbonate buffer solution (CBS) containing 0.2% gelatin was added to each well to block the enzyme plate, and the plate was blocked in an oven at 37°C for 2 h. Mouse serum was diluted 1000-fold, 3000-fold, 9000-fold, and 27000-fold with an antibody diluent (PBS buffer, 0.1% gelatin, 0.05% Tween-20), and the antibody diluent was used as a negative control. The plate was incubated in an oven at 37°C for 35 min; the plate was washed 3 times with PBST; 100 μL of goat anti-mouse secondary antibody IgG (0.5 ug / mL) diluted 5000-fold with the antibody diluent was added to each well, and the plate was incubated in an oven at 37°C for 35 min; after washing the plate 3 times, 100 μL of color developing solution was added to each well, and the plate was incubated in an oven at 37°C for 15 min; after the incubation was completed, 50 μL of stop solution (2 mol / L sulfuric acid) was added to each well, and the absorbance at 450 nm was measured using an enzyme marker.

[0067] In this example, mouse immune serum of recombinant proteins OmpAGE002258, OmpAGE003613, OmpAGE003868, BamA and PilQ was used to verify the titer of the immune antigen of Vibrio parahaemolyticus cultured in different media. The results are shown in Figure 7 , the Y-axis represents the absorbance at 450 nm, and the X-axis represents the different media tested, and the serum dilution factor is 1000. The reaction titer of the polysaccharide-depleted enrichment medium of the present application with different serum is higher than that of other media as a whole, and has a strong titer reaction with OmpAGE002258 and OmpAGE003868, indicating that the reduction of polysaccharide on the surface of Vibrio parahaemolyticus exposes more protein sites, and exposes more proteins, and the reaction titer with serum will be higher. Therefore, the use of the medium of the present application in actual detection can more sensitively achieve the detection of Vibrio parahaemolyticus.

Claims

1. A bacterial enrichment medium for reducing the expression of surface polysaccharides of Vibrio parahaemolyticus, wherein the surface polysaccharides are lipopolysaccharides and capsular polysaccharides; the bacterial enrichment medium comprises 0.25% of proteose peptone, 3% of sodium chloride, 0.4%-0.6% of bile salts, and the rest is water, and the pH of the medium is 8.0-8.5; and the bile salts are selected from bovine bile salts or composite bile salts, and the composite bile salts comprise 35% of glycol deoxycholic acid, 35% of sodium cholate and 30% of sodium cholate.

2. Use of a bacterial enrichment medium in the preparation of a pre-enrichment medium for the in vitro immunological detection of Vibrio parahaemolyticus, wherein the bacterial enrichment medium comprises 0.25% of proteose peptone, 3% of sodium chloride, 0.4%-0.6% of bile salts, and the rest is water, and the pH of the medium is 8.0-8.5; and the bile salts are selected from bovine bile salts or composite bile salts, and the composite bile salts comprise 35% of glycol deoxycholic acid, 35% of sodium cholate and 30% of sodium cholate.

3. A method for reducing the expression of surface polysaccharides of Vibrio parahaemolyticus, comprising the following steps: inoculating Vibrio parahaemolyticus liquid culture into a bacterial enrichment medium at an inoculation amount of 1%, and culturing at 37°C and 180 rpm for 10-16 hours, wherein the bacterial enrichment medium comprises 2.5 g / L of proteose peptone, 30 g / L of sodium chloride, 6 g / L of bovine bile salts, and the rest is water, and the pH of the medium is adjusted to 8.5.

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