Culture medium for prolonging preservation period of vibrio seeds for phage fermentation production as well as preparation method and application of culture medium

Abstract

The invention belongs to the technical field of microorganisms, and discloses a culture medium for prolonging the preservation period of vibrio seeds for bacteriophage fermentation production as well as a preparation method and application of the culture medium. The culture medium is prepared from the following components in parts by mass: 5 to 10 parts of peptone, 1 to 3 parts of yeast extract powder, 0.1 to 0.3 part of ferric citrate, 19 to 20 parts of sodium chloride, 5 to 6.5 parts of magnesium chloride, 3 to 5 parts of sodium sulfate, 0.3 to 0.5 part of calcium chloride, 0.5 to 0.7 part of potassium chloride, 0.15 to 0.25 part of sodium carbonate, 0.05 to 0.1 part of potassium bromide, 0.01 to 0.03 part of boric acid, 0.002 to 0.005 part of sodium silicate, 0.002 to 0.005 part of sodium fluoride, 0.005 to 0.01 part of disodium hydrogen phosphate and 980 to 1100 parts of water. The composition further comprises one or more of the following three components in parts by mass: 0.1 to 0.2 part of vitamin C, 0.06 to 0.16 part of gelatin and 0.05 to 0.15 part of glycine. The culture medium can be applied to preservation of vibrio seeds for bacteriophage production, is simple to prepare, convenient to use and wide in vibrio application range, and can improve vibrio activity in the seed preparation process in the bacteriophage fermentation production process, prolong the seed preservation period and greatly improve the production efficiency.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a culture medium for improving the preservation period of Vibrio seeds used in bacteriophage fermentation production, its preparation method, and its application. Background Technology

[0002] Vibrio infection is a group of diseases caused by Vibrio species. Vibrio can infect shrimp, oysters, shellfish, and other aquaculture, causing significant economic losses to the global aquaculture industry and hindering its stable development. Currently, the traditional methods of Vibrio control in aquaculture involve the use of antibiotics and chemical disinfectants. However, prolonged use of antibiotics can lead to antibiotic resistance, with some bacteria even developing multidrug resistance, making the treatment of aquatic diseases more difficult. Furthermore, drug residues can cause food safety issues. Therefore, finding green and healthy biocontrol agents to inhibit Vibrio pathogens in aquaculture environments is crucial for maintaining the health and food safety of aquatic products.

[0003] Bacteriophages are a type of virus that specifically targets bacteria, invading and lysing host bacteria. Due to their high specificity and lack of toxic side effects, bacteriophages may gradually replace antibiotics as an effective means of preventing and treating bacterial diseases. Bacteriophages have unique advantages in treating aquatic animals and disinfecting aquatic environments. Especially since the national promotion of "antibiotic-free aquaculture," bacteriophages have begun to attract attention as an alternative to antibacterial drugs. Currently, a large number of Vibrio bacteriophage resources have been screened.

[0004] However, in the fermentation production of Vibrio phage, the host bacterial seed culture suffers from a short shelf life and rapid decline in activity. This necessitates frequent activation of the host bacterial seed used in Vibrio phage fermentation, leading to a cumbersome and inefficient workflow. Therefore, researching the shelf life and activity of Vibrio phage fermentation host bacterial seeds, and obtaining a culture medium that can extend the usage period and maintain the activity of Vibrio seeds used in phage fermentation production, will significantly improve the production efficiency of Vibrio phage.

[0005] Therefore, existing technologies need further improvement. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a culture medium for improving the preservation period of Vibrio seeds used in phage fermentation production, its preparation method, and its application. The culture medium, through formulation optimization, significantly extends the preservation period and viability of Vibrio used in phage fermentation production, thereby improving the production efficiency of Vibrio phage fermentation.

[0007] To address the above problems, this application provides the following technical solution: In a first aspect, this application provides a culture medium for improving the preservation period of Vibrio seeds used in bacteriophage fermentation production, comprising the following components in parts by weight: 5-10 parts peptone, 1-3 parts yeast extract, 0.1-0.3 parts ferric citrate, 19-20 parts sodium chloride, 5-6.5 parts magnesium chloride, 3-5 parts sodium sulfate, 0.3-0.5 parts calcium chloride, 0.5-0.7 parts potassium chloride, 0.15-0.25 parts sodium carbonate, 0.05-0.1 parts potassium bromide, 0.01-0.03 parts boric acid, 0.002-0.005 parts sodium silicate, 0.002-0.005 parts sodium fluoride, 0.005-0.01 parts disodium hydrogen phosphate, and 980-1100 parts water; The culture medium also includes one or more of the following three components in parts by weight: 0.1-0.2 parts vitamin C, 0.06-0.16 parts gelatin, and 0.05-0.15 parts glycine.

[0008] The culture medium mentioned above can be either liquid or solid, depending on the specific circumstances.

[0009] Optionally, the culture medium may further include the following components in parts by weight: 10-20 parts agar powder. Adding agar powder prepares a solid culture medium, which can be used for subsequent preservation of Vibrio via slant inoculation.

[0010] Based on the nutritional characteristics of Vibrio and the conditions required to maintain its activity, this invention optimizes the culture conditions of Vibrio seeds by adding vitamin C, gelatin, glycine, and other components to the basic culture medium and further optimizing the content of these components, thereby significantly extending the service life and activity of Vibrio for phage fermentation production.

[0011] Preferably, the culture medium comprises the following components in parts by weight: 5 parts peptone, 1 part yeast extract, 0.1 parts ferric citrate, 19.45 parts sodium chloride, 5.98 parts magnesium chloride, 3.24 parts sodium sulfate, 0.3 parts calcium chloride, 0.55 parts potassium chloride, 0.16 parts sodium carbonate, 0.08 parts potassium bromide, 0.022 parts boric acid, 0.004 parts sodium silicate, 0.0024 parts sodium fluoride, 0.008 parts disodium hydrogen phosphate, 15 parts agar powder, 0.1-0.2 parts vitamin C, 0.06-0.16 parts gelatin, and 0.05-0.15 parts glycine, and 1000 parts water.

[0012] In the aforementioned culture medium, added vitamin C inhibits bacterial growth by acting as an oxygen-inducing agent, thus extending the bacterial growth cycle. Gelatin, as a heterogeneous mixture of polypeptides, possesses unique biocompatibility, biodegradability, and properties that promote cell differentiation and proliferation. Amino acids, used as protective monomers, are zwitterions with amino and carboxyl groups, exhibiting strong buffering capacity and serving as buffer solutions. Through the synergistic effect of these three components with other components in the culture, the preservation period of Vibrio is extended, maintaining its seed viability.

[0013] Preferably, in the culture medium, the gelatin is first prepared to a mass concentration of 2%, and then 3-8 mL is added.

[0014] Optionally, the pH of the culture medium is 7.60 ± 0.2.

[0015] Preferably, the culture medium contains 0.2 g / L of vitamin C, 0.1 g / L of gelatin, and 0.1 g / L of glycine.

[0016] Optionally, the culture medium is a slant culture medium. This medium allows for longer preservation of Vibrio inoculated at 25°C.

[0017] Secondly, this application also provides a method for preparing the culture medium for improving the preservation period of Vibrio seeds used in bacteriophage fermentation production, which includes the following steps: S1. Weigh all ingredients except vitamins and add them to the corresponding amount of water. Mix well and adjust the pH to 7.60. Sterilize to obtain a sterile mixture. S2. After cooling the obtained sterile mixture, add sterile water-soluble vitamin C and mix well. S3. Dispense the mixture prepared in the previous step into containers.

[0018] Optionally, in the preparation method, the container in step S3 is a test tube or a petri dish, and the slant is prepared by tilting.

[0019] Thirdly, this application also provides the application of the above-mentioned culture medium for improving the preservation period of Vibrio seeds for phage fermentation production in the preservation of Vibrio seeds. The application method is as follows: after inoculating Vibrio into the aforementioned culture medium, incubate it in a 37°C incubator and then store it in a 25°C incubator.

[0020] Optionally, the Vibrio species include common aquatic Vibrio species such as Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, Vibrio cholerae, Vibrio campeum, and Vibrio mermaidii.

[0021] The present invention has the following beneficial effects: This invention provides a culture medium for extending the storage period of Vibrio seeds used in phage fermentation. This medium is simple to prepare, convenient to use, and applicable to a wide range of Vibrio species. It not only significantly extends the storage period of Vibrio seeds used in phage fermentation but also maintains the host bacterial activity of the Vibrio seeds, ensuring that they do not affect the phage fermentation titer during the storage period. This solves the problems of short storage period and poor viability of Vibrio seeds used in phage fermentation and also improves production efficiency. This culture medium is of great significance for the industrial preservation of Vibrio colonies. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0023] Example 1: A culture medium for improving the preservation period of Vibrio seeds used in bacteriophage fermentation production. 1. This embodiment provides a culture medium for improving the preservation period of Vibrio seeds used in bacteriophage fermentation production. Each liter of this culture medium comprises the following components: 5.0g peptone, 1.0g yeast extract, 0.1g ferric citrate, 19.45g sodium chloride, 5.98g magnesium chloride, 3.24g sodium sulfate, 0.3g calcium chloride, 0.55g potassium chloride, 0.16g sodium carbonate, 0.08g potassium bromide, 0.022g boric acid, 0.004g sodium silicate, 0.0024g sodium fluoride, 0.008g disodium hydrogen phosphate, 0.2g vitamin C, 0.1g gelatin, 0.1g glycine, 15g agar powder, and the remainder diluted with water to 1L.

[0024] 2. This embodiment also provides a method for preparing the above-mentioned culture medium, which includes the following steps: S1. Weigh out the corresponding masses of peptone, yeast extract, ferric citrate, sodium chloride, magnesium chloride, sodium sulfate, calcium chloride, potassium chloride, sodium carbonate, potassium bromide, boric acid, sodium silicate, sodium fluoride, disodium hydrogen phosphate, gelatin, glycine, and agar powder according to the above formula, and add them to the corresponding amounts of water and mix well. Adjust the pH value to 7.60; sterilize at 121℃ for 20 minutes to obtain a sterile mixture. S2. Add sterile water-soluble vitamin C to the sterile mixture cooled to 60°C and mix well. S3. Dispense the mixture prepared in the previous step into individual test tubes at a rate of 5 mL / tube to prepare slant test tube culture medium.

[0025] 3. This embodiment also provides a method for preserving Vibrio seeds, which is as follows: Vibrio is streaked onto the above-mentioned slant culture medium, incubated at 37°C for 16-24 hours, and then stored at 25°C.

[0026] When using it, the Vibrio colonies on the slant culture tube are directly inoculated into the production culture medium for fermentation and used for the production of bacteriophages.

[0027] Example 2: Optimization of Culture Medium Formulation This embodiment optimizes the content of the three core components in the culture medium: vitamin C, gelatin, and glycine.

[0028] 1. Experimental Methods (1) Set up an orthogonal experiment with vitamin C (0, 0.1, 0.2, 0.3) g / L, gelatin (0, 0.06, 0.1, 0.14) g / L and glycine (0, 0.05, 0.1, 0.15) g / L, with 3 factors and 4 levels. The specific formulas for each group are shown in Table 1.

[0029] (2) The preparation method of the culture medium for each group is as follows: Add the following to every 1000 mL of water: 5.0 g peptone, 1.0 g yeast extract, 0.1 g ferric citrate, 19.45 g sodium chloride, 5.98 g magnesium chloride, 3.24 g sodium sulfate, 0.3 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium carbonate, 0.08 g potassium bromide, 0.022 g boric acid, 0.004 g sodium silicate, 0.0024 g sodium fluoride, 0.008 g disodium hydrogen phosphate, 15 g agar powder, gelatin, and glycine. Prepare nine liquid culture media, adjust the pH to 7.6, dispense into 5 mL test tubes, autoclave at 121 °C for 30 min, and then add the corresponding amount of water-soluble vitamin C according to the orthogonal array to form solid slant agar. Each experiment is conducted in triplicate. The amounts of gelatin, glycine, and vitamin C used in each group are added according to the amounts in Table 1.

[0030] (3) Pick Vibrio DYW34-1 and streak it in a zigzag pattern on the prepared solid slant culture medium. After culturing in a 37°C incubator for 16-24 hours, transfer it to a 25°C incubator and store it for 7 days. Then, determine the number of bacteria on the solid slant culture medium.

[0031] (4) To verify the actual optimality of the theoretical optimal combination in the orthogonal experiment, a verification experiment was carried out by setting up the theoretical optimal combination group, the highest value group measured in the orthogonal experiment group, the second highest value group, and the blank control group without additives. All operating procedures and culture conditions were consistent with those of the orthogonal experiment.

[0032] 2. Experimental Results and Analysis The results of the orthogonal experiment are shown in Table 1. Based on range calculations at various levels, the combination of 0.2 g / L vitamin C, 0.1 g / L gelatin, and 0.1 g / L glycine in the above-mentioned culture medium is the optimal one. The culture medium using this combination is the preferred culture medium.

[0033] Table 1. Orthogonal Experiment for Optimization of Vitamin C, Gelatin, and Glycine Components

[0034] The theoretically optimal combination was validated, and the results are shown in Table 2. The combination of 0.2 g / L vitamin C, 0.1 g / L gelatin, and 0.1 g / L glycine was the actual optimal culture medium combination for the preservation of Vibrio DYW34-1 slant agar. This combination significantly improved the bacterial survival rate of Vibrio slant agar after 7 days of preservation, with the bacterial count reaching 10-1. 8.65 The cfu / mL concentration was superior to all other combinations in Table 1, proving it to be the preferred culture medium for the preservation of Vibrio DYW34-1 slant culture.

[0035] Table 2 Results of the validation experiment for the optimal combination of Vibrio DYW34-1 slant culture medium

[0036] Example 3: Application of the culture medium of the present invention in the preservation of Vibrio harveyi seeds. 1. Test method: (1) Prepare solid slant culture medium, 2216E solid slant culture medium, TCBS culture medium and TSA solid slant culture medium of Example 1 respectively. The preparation method is the same as in Example 1.

[0037] Formula for 2216E solid culture medium: Each liter of medium contains 5.0g peptone, 1.0g yeast extract, 0.1g ferric citrate, 19.45g sodium chloride, 8.8g magnesium chloride (MgCl2·6H2O), 3.24g sodium sulfate, 1.8g calcium chloride, 0.55g potassium chloride, 0.16g sodium bicarbonate, 0.08g potassium bromide, 0.034g strontium chloride, 0.022g boric acid, 0.004g sodium silicate, 0.0024g sodium fluoride, 0.0016g ammonium nitrate, 0.008g disodium hydrogen phosphate, and 15.0g agar. pH: 7.6±0.2. Preparation method: Weigh approximately 55.2g of all the above ingredients in dry powder form, add 1000mL of distilled water, heat and stir until completely dissolved, then autoclave at 121℃ for 20 minutes.

[0038] The TCBS medium formula is as follows: per liter, it contains 5.0g yeast extract, 10.0g peptone, 10.0g sodium thiosulfate, 10.0g sodium citrate, 5.0g ox bile powder, 3.0g sodium tauronate, 20.0g sucrose, 10.0g sodium chloride, 1.0g ferric citrate, 0.04g bromothymol blue, 0.04g thymol blue, and 15.0g agar. The pH is 8.6±0.1. Weigh approximately 89g of all the above dry powders, add them to 1000mL of distilled water, boil and stir until completely dissolved. Autoclaving is not required. Once the medium has cooled to 50–60℃, it is ready for use in plate culture.

[0039] The formula for TSA solid slant culture medium is as follows: 15.0 g tryptone, 5.0 g soybean peptone, 5.0 g sodium chloride, and 15.0 g agar per liter of medium. pH 7.1–7.5. Weigh approximately 40 g of the dry powder of all the above ingredients, add 1000 mL of distilled water, and autoclave at 121°C for 15 min.

[0040] (2) Vibrio harveyi was inoculated onto the four types of solid slant agar plates mentioned above and cultured in a 37°C incubator for 16 hours. After being transferred to a 25°C incubator, the bacterial count and phage fermentation titer were measured daily. The titer of the proliferated phages was measured, and the culture effects were compared.

[0041] The method for detecting bacterial count A is as follows: resuspend the colonies on a slant in 10 mL of physiological saline and then measure the bacterial count.

[0042] Experimental steps for propagating bacteriophages: Inoculate the host bacteria (bacterial suspension washed from slants preserved for different days) and the stock phage solution into 2216E liquid medium at a volume ratio of 1:1. Incubate at 37℃ and 180 rpm in a constant temperature shaker, observing the state of the culture medium every 2-3 hours. Stop incubation when the culture medium changes from turbid to completely clear (usually 3-5 hours).

[0043] B. Phage titer detection method: The proliferated phages were diluted 10-fold with PBS (e.g., 10-fold). -1 Up to 10 -8 Melt 2216E solid medium and cool to 50-60℃. Pour approximately 5 mL into each dish as the bottom plate. Take 5 mL of 45-50℃ semi-solid medium, add 0.1 mL of host bacteria and mix well. Add 0.1 mL of phage dilutions of different gradients and mix quickly. Pour quickly onto the bottom plate, shake well and spread. Each experiment should have 2 replicates. Incubate upside down at 37℃ for 12-18 hours. Select plates with 30-300 plaques and count the average number.

[0044] The potency is calculated as follows: Potency (PFU / mL) = Average number of plaques × Dilution factor ÷ Inoculation volume (0.1 mL) Storage time: The storage time point is defined as when the number of colonies decreases by 10 times relative to the initial amount, and the titer of the proliferating phage is not less than one order of magnitude relative to the initial amount.

[0045] 2. Experimental Results and Analysis The test results are shown in Tables 3, 4, and 5. The existing three commonly used solid slant culture media, 2216E, TCBS, and TSA, all have a storage time of 7 days for Vibrio harveyi inoculated on them. However, the storage time of Vibrio harveyi on the optimized culture medium of this invention can be as high as 10 days, which is 4 days longer than the existing level. Moreover, its phage production activity remains unchanged during the extended storage time.

[0046] Table 3 Comparison of optimized solid inclined plane with 2216E solid inclined plane

[0047] Table 4 Comparison of optimized solid slope with TCBS solid slope

[0048] Table 5 Comparison of optimized solid slope with TSA solid slope

[0049] Example 4: Application of the culture medium of the present invention in the preservation of Vibrio alginolyticus seeds 1. Experimental Methods (1) Prepare solid slant culture medium, 2216E solid slant culture medium, TCBS culture medium and TSA solid slant culture medium of Example 1 respectively. The preparation method is the same as in Example 1.

[0050] (2) Inoculate Vibrio alginolyticus onto the four types of solid slant culture media mentioned above, incubate at 37°C for 16 hours, then transfer to a 25°C incubator, and test the bacterial count and phage fermentation titer daily. The test method is the same as in Example 3.

[0051] 2. Experimental Results and Analysis The test results are shown in Tables 6, 7 and 8. For Vibrio alginolyticus seeds, the storage time of 2216E solid slant was only 7 days, while the storage time of TCBS solid slant and TSA solid slant was only 6 days. However, the optimized culture medium provided in this application can be stored for up to 9 days, which significantly extends the storage time of Vibrio alginolyticus seeds.

[0052] Table 6 Comparison of optimized solid inclined plane with 2216E solid inclined plane

[0053] Table 7 Comparison of optimized solid inclined plane with TCBS solid inclined plane

[0054] Table 8 Comparison of optimized solid slope with TSA solid slope

[0055] Example 5: Application of the culture medium of the present invention in the preservation of Vibrio parahaemolyticus seeds. 1. Experimental Methods (1) Prepare solid slant culture medium, 2216E solid slant culture medium, TCBS culture medium and TSA solid slant culture medium of Example 1 respectively. The preparation method is the same as in Example 1.

[0056] (2) Inoculate Vibrio alginolyticus onto the four types of solid slant culture media mentioned above, incubate at 37°C for 16 hours, then transfer to a 25°C incubator, and test the bacterial count and phage fermentation titer daily. The test method is the same as in Example 3.

[0057] 2. Experimental Results and Analysis The test results are shown in Tables 9, 10 and 11. For Vibrio alginolyticus seeds, the preservation time of TSA solid slant was only 7 days, while the preservation time of 2216E solid slant and TCBS solid slant was only 8 days. However, the optimized culture medium of this application preserved Vibrio alginolyticus for up to 12 days, which extended the preservation time by 4 to 5 days, showing outstanding results.

[0058] Table 9 Comparison of the optimized solid inclined plane with the 2216E solid inclined plane

[0059] Table 10 Comparison of optimized solid inclined plane with TCBS solid inclined plane

[0060] Table 11 Comparison of optimized solid inclined plane with TSA solid inclined plane

[0061] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A culture medium for improving the preservation period of Vibrio seeds used in bacteriophage fermentation production, characterized in that, The product comprises the following components in parts by weight: 5-10 parts peptone, 1-3 parts yeast extract, 0.1-0.3 parts ferric citrate, 19-20 parts sodium chloride, 5-6.5 parts magnesium chloride, 3-5 parts sodium sulfate, 0.3-0.5 parts calcium chloride, 0.5-0.7 parts potassium chloride, 0.15-0.25 parts sodium carbonate, 0.05-0.1 parts potassium bromide, 0.01-0.03 parts boric acid, 0.002-0.005 parts sodium silicate, 0.002-0.005 parts sodium fluoride, 0.005-0.01 parts disodium hydrogen phosphate, and 980-1100 parts water. It also includes one or more of the following three ingredients in parts by weight: 0.1-0.2 parts vitamin C, 0.06-0.16 parts gelatin, and 0.05-0.15 parts glycine.

2. The culture medium according to claim 1, characterized in that, It also includes the following other ingredients in parts by weight: 10-20 parts agar powder.

3. The culture medium according to claim 2, characterized in that, The product comprises the following components in parts by weight: 5 parts peptone, 1 part yeast extract, 0.1 part ferric citrate, 19.45 parts sodium chloride, 5.98 parts magnesium chloride, 3.24 parts sodium sulfate, 0.3 parts calcium chloride, 0.55 parts potassium chloride, 0.16 parts sodium carbonate, 0.08 parts potassium bromide, 0.022 parts boric acid, 0.004 parts sodium silicate, 0.0024 parts sodium fluoride, 0.008 parts disodium hydrogen phosphate, 15 parts agar powder, 0.1-0.2 parts vitamin C, 0.06-0.16 parts gelatin, and 0.05-0.15 parts glycine, and 1000 parts water.

4. The culture medium according to claim 1, characterized in that, The gelatin was first prepared to a mass concentration of 2%, and then 3-8 mL was added; the pH of the culture medium was 7.60±0.

2.

5. The culture medium according to claim 1, characterized in that, The vitamin C content is 0.2 g / L, the gelatin content is 0.1 g / L, and the glycine content is 0.1 g / L.

6. The culture medium according to claim 1, characterized in that, The culture medium is a slant culture medium.

7. The method for preparing the culture medium for improving the preservation period of Vibrio seeds used in bacteriophage fermentation production as described in claim 1, characterized in that, Includes the following steps: S1. Weigh all components of the culture medium formula as described in claim 1 except for vitamins, add them to the corresponding amount of water and mix well, adjust the pH value to 7.60; perform sterilization treatment to obtain a sterile mixture; S2. Add sterile water-soluble vitamin C to the cooled sterile mixture and mix well; S3. Dispense the mixture prepared in the previous step into containers.

8. The preparation method according to claim 7, characterized in that, In step S3, the container is a test tube or a petri dish, and an inclined plane is prepared by tilting it.

9. The application of the culture medium for improving the preservation period of Vibrio seeds used in bacteriophage fermentation production as described in claim 1 in the preservation of Vibrio seeds, characterized in that, The application method is as follows: Vibrio is inoculated into the culture medium as described in any one of claims 1-6, cultured in an incubator at 37°C, and then stored in an incubator at 25°C.

10. The application according to claim 9, characterized in that, The Vibrio species include: Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, Vibrio cholerae, Vibrio campeum, and Vibrio mermaidii.

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