Culture medium formula for improving vibrio inhibition capability of bacillus

Abstract

The invention provides a culture medium formula capable of effectively improving vibrio inhibition capability of bacillus. The culture medium formula comprises 10-20 g of corn starch, 2-10 g of yeast powder, 2-10 g of malt extract, 5-20 g of glycerin, 0.5-1 g of L-aspartic acid, 1 g of dipotassium phosphate, 0.3 g of monopotassium phosphate, 0.5 g of magnesium sulfate, 1 g of calcium carbonate and 1000 mL of distilled water. When the culture medium is used for culturing bacillus capable of naturally inhibiting vibrio, the obtained culture has a better vibrio inhibiting effect.

Description

Technical Field

[0001] The invention relates to the field of microbial culture, in particular to a culture medium formula for improving the ability of bacillus to inhibit vibrio. Background Art

[0002] Bacillus belongs to the phylum Firmicutes, class Bacillus, orders Bacilales, family Bacilaceae, and genus Bacillus. It is a type of Gram-positive bacteria that can form spores and is strictly aerobic or facultative anaerobic. Bacillus grows fast, has rich metabolites, and can produce antibacterial substances with broad-spectrum bactericidal activity. Therefore, it is used in many fields such as agricultural production and food processing.

[0003] Vibrio belongs to the phylum Proteobacteria, class γ-Proteobacteria, order Vibrioles, family Vibrio, and genus Vibrio. It is a type of Gram-negative bacteria with a curved body and a flagellum at the tail. It mostly lives in various aquatic environments in nature. Many Vibrio are conditionally pathogenic bacteria, such as Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio alginolyticus. In aquaculture, an outbreak of Vibrio can cause serious diseases in farmed species such as fish and shrimp, posing a great threat to aquaculture.

[0004] Many Bacillus species have natural resistance to Vibrio, but when cultured using common bacterial culture media, the growth of Bacillus species and the amount of metabolites are relatively average, resulting in their ability to inhibit Vibrio being less than outstanding. Summary of the invention

[0005] The present invention provides a culture medium formula for improving the ability of bacillus to inhibit vibrio. The culture medium used to culture bacillus has a better effect of inhibiting vibrio than the culture obtained by culturing with a common bacterial culture medium.

[0006] A culture medium formula for improving the ability of Bacillus to inhibit Vibrio comprises: 10-20 g corn starch, 2-10 g yeast powder, 2-10 g malt extract, 5-20 g glycerol, 0.5-1 g L-aspartic acid, 1 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 1 g calcium carbonate, and 1000 mL distilled water.

[0007] Preferably, the ingredients of the culture medium formula include: 15 g corn starch, 5 g yeast powder, 5 g malt extract, 10 g glycerol, 0.5 g L-aspartic acid, 1 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 1 g calcium carbonate, and 1000 mL distilled water.

[0008] Preferably, the pH of the culture medium is 7.0-7.2.

[0009] Preferably, the culture medium is sterilized at a temperature of 115° C. for 30 min.

[0010] The beneficial effect of the present invention is that the present invention provides a culture medium formula that can improve the ability of Bacillus to inhibit Vibrio. Compared with the common bacterial culture medium formula, the Bacillus culture cultured with the formula has stronger activity in inhibiting Vibrio and better effect. DETAILED DESCRIPTION

[0012] Example 1 A culture medium formula for improving the ability of Bacillus to inhibit Vibrio comprises the following ingredients: 20 g corn starch, 5 g yeast powder, 2 g malt extract, 20 g glycerol, 1 g L-aspartic acid, 1 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 1 g calcium carbonate, and 1000 mL distilled water.

[0013] Place 50 mL of the culture medium in a 500 mL Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus licheniformis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 mL of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h, and obtain Bacillus licheniformis culture liquid.

[0014] Example 2 A culture medium formula for improving the ability of Bacillus to inhibit Vibrio comprises the following ingredients: 10 g corn starch, 5 g yeast powder, 10 g malt extract, 5 g glycerol, 1 g L-aspartic acid, 1 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 1 g calcium carbonate, and 1000 mL distilled water.

[0015] Place 50 mL of the culture medium in a 500 mL Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus licheniformis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 mL of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h, and obtain Bacillus licheniformis culture liquid.

[0016] Example 3 A culture medium formula for improving the ability of Bacillus to inhibit Vibrio comprises the following ingredients: 15 g corn starch, 10 g yeast powder, 5 g malt extract, 20 g glycerol, 0.5 g L-aspartic acid, 1 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 1 g calcium carbonate, and 1000 mL distilled water.

[0017] Place 50 mL of the culture medium in a 500 mL Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus licheniformis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 mL of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h, and obtain Bacillus licheniformis culture liquid.

[0018] Example 4 A culture medium formula for improving the ability of Bacillus to inhibit Vibrio comprises the following ingredients: 15 g corn starch, 5 g yeast powder, 5 g malt extract, 10 g glycerol, 0.5 g L-aspartic acid, 1 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 1 g calcium carbonate, and 1000 mL distilled water.

[0019] Place 50 mL of the culture medium in a 500 mL Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus licheniformis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 mL of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h, and obtain Bacillus licheniformis culture liquid.

[0020] Comparative Example 1 Common bacterial culture medium - LB medium formula: tryptone 10 g, yeast extract 5 g, NaCl 10 g, distilled water 1000 mL.

[0021] Place 50 ml of the culture medium in a 500 mL Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus licheniformis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 mL of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h, and obtain Bacillus licheniformis culture liquid.

[0022] Comparative Example 2 Common bacterial culture medium - nutrient broth culture medium formula: peptone 10 g, beef extract powder 3 g, sodium chloride 5 g, distilled water 1000 mL.

[0023] Place 50 mL of the culture medium in a 500 mL Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus licheniformis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 ml of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h, and obtain Bacillus licheniformis culture liquid.

[0024] Example 5 A culture medium formula for improving the ability of Bacillus to inhibit Vibrio comprises the following ingredients: 20 g corn starch, 5 g yeast powder, 2 g malt extract, 20 g glycerol, 1 g L-aspartic acid, 1 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 1 g calcium carbonate, and 1000 mL distilled water.

[0025] Place 50 ml of the culture medium in a 500 ml Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus subtilis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 ml of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h to obtain Bacillus subtilis culture liquid.

[0026] Example 6 A culture medium formula for improving the ability of Bacillus to inhibit Vibrio comprises the following ingredients: 10 g corn starch, 5 g yeast powder, 10 g malt extract, 5 g glycerol, 1 g L-aspartic acid, 1 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 1 g calcium carbonate, and 1000 mL distilled water.

[0027] Place 50 ml of the culture medium in a 500 ml Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus subtilis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 ml of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h to obtain Bacillus subtilis culture liquid.

[0028] Example 7 A culture medium formula for improving the ability of Bacillus to inhibit Vibrio comprises: 15 g corn starch, 10 g yeast powder, 5 g malt extract, 20 g glycerol, 0.5 g L-aspartic acid, 1 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 1 g calcium carbonate, and 1000 mL distilled water.

[0029] Place 50 ml of the culture medium in a 500 ml Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus subtilis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 ml of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h to obtain Bacillus subtilis culture liquid.

[0030] Example 8 A culture medium formula for improving the ability of Bacillus to inhibit Vibrio comprises the following ingredients: 15 g corn starch, 5 g yeast powder, 5 g malt extract, 10 g glycerol, 0.5 g L-aspartic acid, 1 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 1 g calcium carbonate, and 1000 mL distilled water.

[0031] Place 50 ml of the culture medium in a 500-mL Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus subtilis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 mL of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h, and obtain Bacillus subtilis culture liquid.

[0032] Comparative Example 3 Common bacterial culture medium - LB medium formula: tryptone 10 g, yeast extract 5 g, NaCl 10 g, distilled water 1000 mL.

[0033] Place 50 ml of the culture medium in a 500-mL Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus subtilis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 mL of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h, and obtain Bacillus subtilis culture liquid.

[0034] Comparative Example 4 Common bacterial culture medium - nutrient broth culture medium formula: peptone 10 g, beef extract powder 3 g, sodium chloride 5 g, distilled water 1000 mL.

[0035] Place 50 mL of the culture medium in a 500 mL Erlenmeyer flask, adjust the pH to 7.2, and sterilize at high temperature; inoculate a strain of Bacillus subtilis known to have inhibitory effects on Vibrio parahaemolyticus and Vibrio vulnificus, with an inoculation volume of 1 mL of bacterial liquid; place in a shaker, culture at 35°C, 180 rpm for 16 h, and obtain Bacillus subtilis culture liquid.

[0036] Evaluation test The bacillus culture solutions of Examples 1 to 8 and Comparative Examples 1 to 4 were used to conduct antibacterial tests; indicator plates containing Vibrio parahaemolyticus and Vibrio vulnificus were prepared by the plate pouring method, and after solidification, an Oxford cup with a diameter of 7 mm was carefully placed; 200 μl of the bacillus culture solution was carefully injected into the Oxford cup, and cultured upright at 37°C for 24 h, and the size of the inhibition zone was observed and recorded; the results are as shown in the following table: Bacillus species Vibrio parahaemolyticus inhibition zone radius (mm) Radius of inhibition zone of Vibrio vulnificus (mm) Example 1 Bacillus licheniformis 15.79 16.25 Example 2 Bacillus licheniformis 16.17 15.89 Example 3 Bacillus licheniformis 15.88 16.20 Example 4 Bacillus licheniformis 16.86 17.11 Comparative Example 1 Bacillus licheniformis 10.23 10.84 Comparative Example 2 Bacillus licheniformis 9.57 11.06 Example 5 Bacillus subtilis 17.10 16.70 Example 6 Bacillus subtilis 16.87 16.43 Example 7 Bacillus subtilis 16.85 16.65 Example 8 Bacillus subtilis 17.45 17.00 Comparative Example 3 Bacillus subtilis 10.45 10.32 Comparative Example 4 Bacillus subtilis 10.33 9.98

[0037] In summary, the Bacillus culture fluid cultured using the culture medium formula of the present invention has a significantly better inhibitory effect on Vibrio parahaemolyticus and Vibrio vulnificus than the Bacillus culture fluid cultured using the common bacterial culture medium formula.

[0038] Although the implementation scheme of the present invention has been described above in conjunction with the embodiments, it should be noted that the described embodiments are only a part of the specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent replacements, improvements, etc. made within the scope of the technical concept and technical method of the present invention belong to the protection scope and disclosure scope of the present invention.

Claims

1. A method for improving the ability of Bacillus to inhibit Vibrio and its formulation, It is characterized in that The ingredients include: 10-20 g corn starch, 2-10 g yeast powder, 2-10 g malt extract, 5-20 g glycerol, 0.5-1 g L-aspartic acid, 1 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 1 g calcium carbonate, and 1000 mL distilled water.

2. The culture medium formulation as claimed in claim 1, It is characterized in that The method for preparing the culture and formulation comprises the following steps: Step A: Weigh each ingredient according to the recipe; Step B: Add the ingredients weighed in step A into the container in sequence and stir evenly; Step C: adjusting the pH of the culture medium obtained in step B; Step D: sterilize the culture medium obtained in step C under high temperature and high pressure.

3. The culture medium formulation as claimed in claim 1, Features: The pH of the culture medium is 7.0-7.

2.

4. The culture medium formulation as claimed in claim 1, Features: The culture medium is sterilized at 115°C for 30 min.