Vibrio alginolyticus bacteriophage microecological preparation as well as preparation method and application thereof

[0022] The invention provides a preparation method of Vibrio alginolyticus bacteriophage probiotics, comprising the following steps:

[0023] 1) The Vibrio alginolyticus liquid is inserted into the fermentation medium for cultivation to obtain the host fermentation liquid; the initial cell concentration of the Vibrio alginolyticus host in the fermentation medium is 10 10 ~10 16 CFU/ml;

[0024] 2) Set the concentration to 10 8 ~10 16 Vibrio alginolyticus phage liquid of PFU/ml is inoculated into the host fermentation broth for fermentation; the inoculum amount of Vibrio alginolyticus phage liquid is 1% to 10%;

[0025] 3) When the fermentation culture in the step 2) is carried out for 6-12 hours, add Vibrio alginolyticus liquid to the host fermentation liquid in the step 2), continue the fermentation culture, control the entire fermentation period to 18-24 hours, and obtain the fermentation liquid;

[0026] 4) Inactivating the host of Vibrio alginolyticus with the fermented liquid obtained in step 3), to obtain a microecological preparation of Vibrio alginolyticus phage.

[0027] In the present invention, the Vibrio alginolyticus liquid is inserted into the fermentation medium for cultivation to obtain the host fermentation liquid; the initial cell concentration of the Vibrio alginolyticus host in the fermentation medium is 10 10 ~10 16 CFU/ml.

[0028] The present invention has no special limitation on the access method, and the access method known to those skilled in the art can be used.

[0029] In the present invention, the fermentation medium fermentation medium preferably includes the following content components: peptone 1-20g/L, beef extract powder 1-20g/L and sodium chloride 1-20g/L; more preferably peptone 5 ~15g/L, beef extract powder 5~15g/L and sodium chloride 3~10g/L; most preferably peptone 10g/L, beef extract powder 10g/L and sodium chloride 5g/L. The pH value of the fermentation medium is preferably 7.0-9.0, more preferably 7.5-8.5, most preferably 8.0. The present invention has no special limitation on the preparation method of the fermentation medium, and the preparation method of the medium well known to those skilled in the art can be adopted.

[0030] In the present invention, after the Vibrio alginolyticus liquid is inserted into the fermentation medium, the bacterium concentration of the Vibrio alginolyticus host in the fermentation medium is preferably 10 10 ~10 16 CFU/ml, more preferably 10 12 ~10 15 CFU/ml, most preferably 10 14 CFU/ml. In the present invention, there is no special limitation on the source of the Vibrio alginolyticus host, and the strains of Vibrio alginolyticus well known to those skilled in the art can be used. In the embodiment of the present invention, the Vibrio alginolyticus is isolated from the intestinal tract of Penaeus vannamei. Vannamei culture ponds are located in Shandong, Fujian, Guangdong, Hainan and other places, and the collection time is September 2016. The isolated Vibrio alginolyticus is expanded and cultivated by a conventional method to obtain a Vibrio alginolyticus liquid.

[0031] After obtaining the host fermented liquid, the present invention will concentration be 10 8 ~10 16 The PFU/ml Vibrio alginolyticus phage liquid is inoculated into the host fermentation liquid for fermentation; the inoculum amount of the Vibrio alginolyticus phage liquid is 1% to 10%.

[0032] In the present invention, the source of the Vibrio alginolyticus phage is not particularly limited, and the source of the Vibrio alginolyticus phage well known to those skilled in the art can be used. In the embodiment of the present invention, the Vibrio alginolyticus phage is isolated from Vibrio alginolyticus. The present invention has no special limitation on the isolation method, and the phage isolation method well known to those skilled in the art can be used.

[0033] The present invention has no special limitation on the inoculation method, and the access method known to those skilled in the art can be used. In the present invention, the concentration of Vibrio alginolyticus phage liquid is preferably 10 10 ~10 14 PFU/ml, more preferably 10 12PFU/ml. The inoculation amount of Vibrio alginolyticus phage liquid is preferably 3% to 7%, more preferably 5%.

[0034] In the present invention, the conditions for fermenting and cultivating the bacteriophage are independently preferably as follows: the fermentation temperature is 28-35°C, more preferably 30-32°C. The pH value of the host fermentation broth is maintained at 7-9, more preferably 7.5-8.5, most preferably 8.0; the fermentation is carried out under stirring conditions, and the stirring speed is 50-200 rpm, more preferably 100-150 rpm.

[0035] In the present invention, when the fermentation culture is carried out for 6-15 hours, the vibrio alginolyticus liquid is fed into the host fermentation liquid, and the fermentation culture is continued, and the whole fermentation period is controlled to be 18-24 hours to obtain the fermentation liquid.

[0036] In the present invention, the feeding volume of the Vibrio alginolyticus liquid is preferably 1%-10% of the volume of the host fermentation liquid, more preferably 2%-8%, and most preferably 5%. The concentration of the vibrio alginolyticus liquid is 10 10 ~10 14 PFU/ml.

[0037] In the present invention, the volume of Vibrio alginolyticus liquid added per hour is 0.5%-5% of the volume of the host fermentation liquid, more preferably 1%-4%, most preferably 3%.

[0038] In the present invention, the feeding time of the Vibrio alginolyticus liquid is preferably 2-4 hours, more preferably 3 hours.

[0039] In the present invention, the conditions for the continuous fermentation culture are preferably as follows: the fermentation temperature is 28-35°C, more preferably 30-32°C. The pH value of the host fermentation broth is maintained at 6-9, more preferably 7-8; the fermentation is carried out under stirring conditions, and the stirring speed is 50-200 rpm, more preferably 100-150 rpm.

[0040] After the fermented liquid is obtained, the present invention inactivates the Vibrio alginolyticus host to obtain the Vibrio alginolyticus phage probiotics.

[0041] In the present invention, the inactivation method is preferably a water bath; the temperature of the water bath is preferably 45-70°C, more preferably 50°C. The time of the water bath is preferably 2 to 5 hours, more preferably 3 to 4 hours.

[0042] The invention provides a microbial preparation of Vibrio alginolyticus phage, the titer of Vibrio alginolyticus phage is 10 12 ~10 16 PFU/ml.

[0043] In the present invention, the titer determination method of the Vibrio alginolyticus phage is determined by the double-layer plate method; the double-layer plate method: take 1 mL of the bacteriophage fermentation broth, centrifuge at 8000 rpm for 20 min, take 100 μL of the supernatant, add 900 μL Shake well in sterile saline, and perform serial dilutions. take 10 -12～-14 Add 100 μL of dilution gradient solution and 100 μL of host bacteria in the logarithmic growth phase to 8 mL of semi-solid medium cooled to about 50°C, mix well, incubate at 33°C for 12 hours, and count the plaques. The calculation formulas for the number and titer of phage plaques are as follows:

[0044] Cell titer (PFU/mL) = number of plaques on the plate × dilution factor × 10.

[0045] The invention provides the microbial preparation of Vibrio alginolyticus prepared by the method or the application of the microbial preparation of Vibrio alginolyticus phage in identifying or detecting Vibrio alginolyticus in aquaculture.

[0047] Example 1

[0048] Vibrio alginolyticus was isolated from a culture pond of Penaeus vannamei. Penaeus vannamei culture ponds are located in Shandong, Fujian, Guangdong, Hainan and other places. The collection time is September 2016. Vibrio alginolyticus was isolated from the intestinal tract of Penaeus vannamei, and the phage of Vibrio alginolyticus was isolated from cultured water and seawater of Penaeus vannamei.

[0049] The phage expansion culture method of the isolated 3 strains of Vibrio alginolyticus, the specific steps are as follows: first determine the optimal multiplicity of infection of the phage: the multiplicity of infection (MOI) refers to the number of phages and the number of host bacteria when the phage initially infects the host bacteria. The ratio, also known as the infection multiple. Inoculate 100ul of host bacteria into 200ml NB culture medium, and cultivate to logarithmic phase at 150r/min for 12h. Then take the initial valence as 10 9 PFU/mL phage culture medium, according to the ratio of the multiplicity of infection of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, add phage VP11 and PVP11 into the liquid medium in the same volume, 37 ° C Shake, culture at 120r/min for 10h, centrifuge at 4000r/min for 15min, remove the precipitate, filter the supernatant with a 0.22μm membrane, filter 3 times, and measure the titer of the phage with the double-layer plate method. The multiplicity of infection with the highest titer is Optimal multiplicity of infection. The double-layer plate method: take 1 mL of bacteriophage fermentation broth, centrifuge at 8000 rpm for 20 min, take 100 μL of supernatant, add to 900 μL of sterile normal saline, shake well, and perform gradient dilution in sequence. take 10 -11 Add 100 μL of dilution gradient solution and 100 μL of host bacteria in the logarithmic growth phase to 8 mL of semi-solid medium cooled to about 50°C, mix well, incubate at 33°C for 12 hours, and count the plaques. Inoculate according to the optimal multiplicity of infection, each accounting for 2% of the total volume, culture at 33° C., 150 rpm for 18 hours, and obtain phage liquid of 3 strains of Vibrio alginolyticus.

[0050] Example 2

[0051] The method for feeding phage host fermentation to produce phage comprises the following steps:

[0052] 1) Use a 50L fermenter for fermentation. The volume of the fermentation medium in the fermenter is 35L. Insert the host into the fermentation medium so that the number of colonies in the initial medium is 10 10 CFU/ml; the formula of the fermentation medium is as follows: peptone 10g/L; beef extract powder 10gL; sodium chloride 1g/L; pH is 9.0;

[0053] 2) add the 1% 10 of the first strain fermented liquid volume again 8 The Vibrio alginolyticus phage liquid of PFU/ml was cultured in a fermenter, the fermentation temperature was 28° C., the rotation speed was 200 rpm; the pH value was controlled at 9.

[0054] 3) When the fermentation was carried out for 6 hours, the phage host was added to the fermenter, and the concentration was controlled at 10 14 Between CFU/ml, the feeding time is 3h, the flow speed is 2L/h, the feeding volume is 6% of the total fermentation volume, and the fermentation cycle is 18 hours. When the color of the fermentation broth changes from turbid to clear, it can be put into the tank;

[0055] 4) Put the obtained fermentation broth in a water bath at 45° C. for 5 hours to inactivate the host to obtain the bacteriophage probiotics.

[0056] 5) The potency determination method of the phage microecological preparation adopts the double-layer plate method to count the plaques, specifically: take 1 mL of the phage fermentation liquid, centrifuge at 8000 rpm for 20 min, take 100 μL of the supernatant, and add it to 900 μL of sterile saline Shake well, and serially perform serial dilutions. take 10 -11 Add 100 μL of dilution gradient solution and 100 μL of host bacteria in the logarithmic growth phase to 8 mL of semi-solid medium cooled to about 50°C, mix well, incubate at 33°C for 12 hours, and count the plaques.

[0057] The calculation formulas for the number and titer of phage plaques are as follows:

[0058] Cell titer (PFU/mL) = number of plaques on the plate × dilution factor × 10.