Preparation method and application of prawn complete expanded feed rich in vibrio bacteriophage

Abstract

The invention discloses a preparation method and application of a prawn complete expanded feed rich in vibrio bacteriophage. The invention relates to a preparation method and application of a prawn complete expanded feed rich in vibrio bacteriophage. The expanded feed comprises soybean meal, rapeseed meal, cottonseed meal, fish meal, flour, fish meal, monocalcium phosphate, vitamin A + E and various probiotics such as bacillus subtilis, lactic acid bacteria, saccharomyces cerevisiae, enterococcus faecalis and clostridium butyricum. In addition, the compound bacteriophage comprises a vibrio parahaemolyticus bacteriophage, a vibrio alginolyticus bacteriophage, a vibrio harveyi bacteriophage and a The expanded feed is rich in nutrition and probiotics, can effectively promote the growth of prawns and prevent the explosive growth of vibrios, and has broad spectrum, high efficiency, safety and sustainability on the prevention and control of vibrios.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a preparation method and application of a complete puffed shrimp feed rich in Vibrio phages, and in particular to a preparation method and application of a complete puffed shrimp feed rich in multiple Vibrio phages and various probiotics. Background Art

[0002] Bacteriophages are a type of bacterial virus that is widely found in nature. They consist of a nucleic acid head and a tail that specifically recognizes receptors. With the continuous development of the aquaculture industry, intensive, high-density farming practices have also been accompanied by frequent outbreaks of various pathogens, impacting aquaculture safety. Preventing and controlling various bacterial diseases during this period of development is a critical issue that needs to be addressed.

[0003] Vibrio disease is the most common and serious bacterial disease affecting shrimp farming. Common Vibrio species include Vibrio parahaemolyticus Vibriopara haemolyticus , Vibrio alginolyticus V. alginolyticus , Vibrio harveyi V . harveyi , Vibrio vulnificus V. vulnificus In the past, antibiotics were an important means of treating and preventing bacterial diseases. However, due to the excessive use of antibiotics, bacterial resistance can easily occur, residual drugs can pollute the environment, and endanger food safety. Therefore, there is an urgent need for a safe and harmless method to treat and prevent vibriosis. Vibrio phage is a virus of Vibrio bacteria. It has a simple structure, high specificity, and safety. It is safe and non-toxic to animal bodies and probiotics. Studies have shown that adding phages to chick diets can effectively reduce the infection of Salmonella in the chick intestine and the replication of Salmonella in the intestine ( Lim Th 2012) Therefore, there is an urgent need for a method for preparing a complete shrimp feed rich in Vibrio phages to solve the problem of Vibrio outbreaks during shrimp farming. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a preparation method and application of a shrimp puffed feed rich in Vibrio phages. The shrimp complete feed prepared by the preparation method is rich in Vibrio phages and a variety of probiotics, has a strong bactericidal and antibacterial effect, can effectively control the growth of Vibrio in shrimp; and has a wide host range and can lyse Vibrio parahaemolyticus. Vibriopara haemolyticus , Vibrio alginolyticus V. alginolyticus , Vibrio harveyi V. harveyi , Vibrio vulnificus V. vulnificus Compared with other traditional feeds, this expanded feed can promote the healthy growth of shrimp in many aspects, and can also prevent and treat bacterial diseases caused by Vibrio.

[0005] A preparation method and application of a complete puffed shrimp feed rich in Vibrio phages. The first characteristic is that the puffed feed is a complete basic feed for shrimp and has comprehensive nutrition. The second characteristic is that the puffed feed is added with multiple composite probiotics, which can promote the absorption and digestion of nutrients in the shrimp intestine. The third characteristic is that the mixed bacteriophages are added to the puffed feed at the end by spraying, which can effectively prevent and treat bacterial diseases caused by Vibrio.

[0006] The invention discloses a preparation method and application of a complete extruded shrimp feed rich in Vibrio phages. The complete shrimp base feed comprises 30% by weight of soybean meal, 15% of rapeseed meal, 20% of cottonseed meal, 12% of fish meal, 9% of flour, 10% of fish meal, 3% of calcium dihydrogen phosphate, and 1% of vitamin A+E. The complete shrimp base feed is crushed, mixed, and gelatinized, and then granulated at high temperature and cooled for use.

[0007] A preparation method and application of a complete puffed shrimp feed rich in Vibrio phages, wherein the multiple composite probiotics comprise 30% Bacillus subtilis, 20% lactic acid bacteria, 20% Saccharomyces cerevisiae, 20% Enterococcus faecalis, and 10% Clostridium butyricum.

[0008] A preparation method and application of a complete puffed shrimp feed rich in Vibrio phages, wherein the mixed phages contain 25% Vibrio parahaemolyticus phages, 25% Vibrio alginolyticus phages, 25% Vibrio harveyi phages, and 25% Vibrio vulnificus phages, which are mixed uniformly and set aside.

[0009] A preparation method and application of a complete puffed shrimp feed rich in Vibrio phages, the preparation method for the complete puffed shrimp feed rich in phages mainly comprising the following steps: Step S1: Weighing the complete shrimp base material; weighing the soybean meal, rapeseed meal, cottonseed meal, fish meal, flour, fish meal, calcium dihydrogen phosphate, and vitamins A+E in the above proportions respectively for later use.

[0010] Step S2: Mixing the complete shrimp base material: add the weighed raw materials into the mixer in descending order and mix them evenly.

[0011] Step S3: Gelatinization of the complete prawn base feed: The mixed complete prawn base feed is fed into a gelatinization machine for gelatinization.

[0012] Step S4: Granulation of complete shrimp base feed: The gelatinized complete shrimp base feed is transported to a high-temperature granulator for granulation.

[0013] Step S5: Preparation of multiple composite probiotics: Measure various probiotics using a measuring cylinder according to the above ratio, then pour the different strains into a clean and sterilized large stainless steel copper and add 5 times the pure water to mix, use a motor stirrer to stir evenly, stir clockwise for 10 minutes, stir counterclockwise for 10 minutes, seal with plastic wrap after mixing, and set aside.

[0014] Step S6: Preparation of mixed phages: Vibrio parahaemolyticus phages, Vibrio alginolyticus phages, Vibrio harveyi phages, and Vibrio vulnificus phages are weighed and mixed according to the above proportions and set aside.

[0015] Step S7: Probiotic inoculation of complete feed: The pelletized complete shrimp basal feed is transported to a spray inoculator. The motor is turned on and the complete shrimp basal feed is rolled in the inoculator for 10 minutes to fully disperse the particles between the feed. The spray inoculation is turned on and spray inoculation is performed at a ratio of 200:3 of complete basal feed to mixed bacterial solution, so that the mixed bacterial solution is fully attached to each pellet of the complete basal feed.

[0016] Step S8: Preparation of mixed phage oil: Pour the mixed phage powder into a 1:1 mixture of cod liver oil and soybean oil and stir thoroughly. Mix the mixture in a ratio of 1000:1 of mixed oil to mixed phage for more than 10 minutes.

[0017] Step S9: Vacuum spraying: After step S7, the expanded material is quickly transported and loaded into a vacuum sprayer. 3 kg of the bacteriophage oil is sprayed evenly on the surface of the expanded material for every 100 kg of the full-price expanded material.

[0018] Step S10: Packaging: After vacuum spraying the phage oil, quickly package it in a transparent breathing bag.

[0019] It is characterized in that a step S41 is further provided between the step S4 and the step S5. The step S41 is to place the required different wild mushroom liquids into a shaker at 37°C and 180 rpm one day in advance for overnight activation culture.

[0020] A preparation method and application of a complete shrimp puffed feed rich in Vibrio phages, which combines a basic shrimp feed with multiple probiotics and phages of pathogenic bacteria to obtain a functional complete shrimp feed with strong functionality.

[0021] Preferably, the soybean meal used in the above-mentioned base material is cooked soybean meal.

[0022] Preferably, the bacterial content of the Bacillus subtilis used is ≥1.0*10 9 CFU / ml.

[0023] Preferably, the lactic acid bacteria used above contain ≥1.0*10 9 CFU / ml.

[0024] Preferably, the yeast content is ≥1.0*10 9 CFU / ml.

[0025] Preferably, the Enterococcus faecalis used above has a bacterial count of ≥1.0*10 9 CFU / ml.

[0026] Preferably, the Clostridium butyricum used has a bacterial content of ≥1.0*10 8 CFU / ml.

[0027] Preferably, the titer of the Vibrio parahaemolyticus phage used is ≥1.0*10 10 PFU / ml.

[0028] Preferably, the titer of the Vibrio alginolyticus phage used is ≥1.0*10 10 PFU / ml.

[0029] Preferably, the titer of the Vibrio harveyi phage used is ≥1.0*10 10 PFU / ml.

[0030] Preferably, the titer of the Vibrio vulnificus phage used is ≥1.0*10 10 PFU / ml.

[0031] Preferably, in step S8, the phage-mixed oil is prepared by adding the phage mixed powder through a sieve and stirring the mixture during the addition process so that the added phage can be evenly mixed in the oil.

[0032] Preferably, sufficient clockwise and counterclockwise stirring is carried out each time mixing and stirring is required, and the surface of the large barrel for loading materials is covered with transparent preservative film for sealing during the transportation process to prevent foreign matter or reduce the entry of miscellaneous bacteria.

[0033] The advantages of the present invention are that the complete puffed shrimp feed combines the complete shrimp base feed with mixed probiotics and bacteriophages, and not only has comprehensive and rich nutrition, but is also rich in various probiotics and their metabolites, can effectively promote the decomposition of macromolecular substances in the puffed feed, accelerate the absorption and digestion of nutrients in the shrimp intestine, and promote the healthy growth of the shrimp. In addition, the rich variety of Vibrio phages can effectively inhibit the content of Vibrio in the shrimp body and the water environment, and has a broad spectrum, high efficiency, safety and sustainability in the prevention and control of Vibrio. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The effect of shrimp feeding with different expanded feeds on Vibrio prevention and control. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below with reference to the embodiments.

[0035] Example 1 Preparation of 1000 kg of complete expanded shrimp feed rich in bacteriophages The present invention provides a preparation method and application of a complete puffed shrimp feed rich in Vibrio phages, wherein the puffed feed comprises a complete shrimp base material, mixed probiotics, and mixed phage oil. First, the complete shrimp base material comprises 30% soybean meal, 15% rapeseed meal, 20% cottonseed meal, 12% fish meal, 9% flour, 10% fish meal, 3% calcium dihydrogen phosphate, and 1% vitamin A+E in a weight ratio, which are crushed, mixed, gelatinized, and granulated at high temperature to obtain the complete shrimp base material; second, the mixed probiotics are obtained by mixing 30% Bacillus subtilis, 20% lactic acid bacteria, 20% brewer's yeast, 20% Enterococcus faecalis, and 10% Clostridium butyricum in a uniform proportion and then adding 5 times pure water to dilute; third, the mixed phage oil is obtained by mixing cod liver oil and soybean oil in a 1:1 ratio, and adding 1 kg of mixed oil liquid. The mixed phage powder is a uniform mixture of 25% Vibrio parahaemolyticus phage, 25% Vibrio alginolyticus phage, 25% Vibrio harveyi phage, and 25% Vibrio vulnificus phage. A phage-rich complete extruded shrimp feed is a functional shrimp feed enriched with multiple probiotics and phages, obtained by inoculating a complete shrimp base feed with mixed probiotics at a ratio of 10:1 and spraying a 100:1 mixed phage oil solution. This feed is nutritionally comprehensive and rich in various probiotics and their metabolites. It can effectively promote the decomposition of macromolecules in the extruded feed, accelerate nutrient absorption and digestion in the shrimp intestine, significantly reduce diseases caused by Vibrio, and treat various bacterial diseases. The present invention provides a preparation method and application of a complete puffed shrimp feed rich in bacteriophages. The preparation method comprises the following steps according to the standard of 1000 kg of complete basic feed: Step S1: Weighing the complete basic raw materials for shrimp; weigh the soybean meal, rapeseed meal, cottonseed meal, fish meal, flour, fish meal, calcium dihydrogen phosphate, and vitamins A+E in the above proportions respectively and set aside.

[0036] Step S2: Mixing the complete shrimp base material: add the weighed raw materials into the mixer in descending order and mix them evenly.

[0037] Step S3: gelatinization of the complete shrimp base feed; Step S4: pelleting the complete shrimp base feed; Step S40: one day in advance, place the required different probiotic bacterial liquids into a shaker at 37°C and 180 rpm for overnight activation culture.

[0038] Step S5: Preparation of composite probiotics; according to the above proportions, 1.5 kg of Bacillus subtilis, 1 kg of lactic acid bacteria, 1 kg of Saccharomyces cerevisiae, 1 kg of Enterococcus faecalis, and 0.5 kg of Clostridium butyricum were weighed and poured into a clean and sterilized large stainless steel copper for mixing, and 25 kg of pure water was added. The mixture was stirred evenly with a motor stirrer, stirred clockwise for 10 min, stirred counterclockwise for 10 min, and sealed with plastic wrap after mixing for later use.

[0039] Step S6: preparing mixed phages; 8 g of Vibrio parahaemolyticus phage, 8 g of Vibrio alginolyticus phage, 8 g of Vibrio harveyi phage, and 8 g of Vibrio vulnificus phage were weighed separately and mixed into 8 g of phage mixture for later use.

[0040] Step S7: Probiotic inoculation of complete feed: The pelletized complete shrimp basal feed is transported to a spray inoculator, the motor is turned on, and the complete shrimp basal feed is rolled in the inoculator for 10 minutes to fully disperse the feed particles. Simultaneously, three times the volume of purified water is added to the mixed bacterial solution for dilution, and the spray inoculation is turned on to allow the mixed bacterial solution to fully adhere to each pellet of the complete basal feed. Step S8: Preparation of mixed phage oil: 15 kg of cod liver oil and 15 kg of soybean oil were mixed evenly, and then 30 g of mixed phage powder was poured into the mixed oil and stirred thoroughly. The mixture was mixed at a ratio of 1000:1 for mixed phage, and the stirring time was more than 10 minutes.

[0041] Step S9: Vacuum spraying: After step S7, the expanded material is quickly transported and loaded into a vacuum sprayer. 3 kg of the bacteriophage oil is sprayed evenly on the surface of the expanded material for every 100 kg of the full-price expanded material.

[0042] Step S10: Packaging: After vacuum spraying the phage oil, quickly package it in a transparent breathing bag.

[0043] The present invention provides a preparation method of a complete puffed shrimp feed rich in bacteriophages and its application. By preparing the complete puffed shrimp feed rich in bacteriophages according to the method, no less than 1000 kg of the complete puffed shrimp feed rich in bacteriophages can be obtained. Example 2 Effect of Expanded Shrimp Feed Rich in Vibrio Phage on Vibrio Control This experiment aimed to validate the practical application of bacteriophages in preventing pathogenic Vibrio infections. The experiment was conducted in our company's laboratory using aquariums measuring 40 cm × 30 cm × 25 cm in length, width, and height. The aquariums were cultured for 10 days, with a water temperature maintained at 26 ± 1°C and a salinity of 25%. Shrimp seedlings were randomly selected from a client's pond. A total of 270 healthy, active shrimp of similar size were selected, with an average weight of 8 ± 0.5 g. Shrimp were fed daily, one meal in the morning and one in the evening. The aquarium was divided into three groups: an experimental group, a control group, and a blank group, with three replicates per group and 30 shrimp per replicate. Vibrio counts were determined using TCBS plates, with samples collected every 24 hours. The experimental and blank groups were fed a complete extruded shrimp feed enriched with bacteriophages at each meal, while the control group was fed a standard complete extruded feed. On the fifth day of culture, Vibrio lyticus was added to the aquariums of the experimental and control groups. 600 1 ml of bacterial solution with a value of 2.0 was used to simulate the sudden invasion of Vibrio spp., and 1 ml of high-temperature sterilized Vibrio spp. was added to the blank group. Figure 1 As shown in the figure, it can be seen that feeding a complete extruded feed rich in bacteriophages can effectively inhibit the growth of Vibrio; in addition, when foreign Vibrio was added on the fifth day, it can also be seen that compared with the control group, the experimental group fed with a complete extruded feed rich in Vibrio phages and the blank group can effectively prevent the explosive growth of Vibrio.

Claims

1. A method for preparing a complete puffed shrimp feed rich in Vibrio phages and its application, characterized by: Its complete basic feed for shrimp includes 20%-30% soybean meal, 10%-15% rapeseed meal, 10%-20% cottonseed meal, 7%-12% fish meal, 5%-9% flour, 8%-10% fish meal, 1%-3% calcium dihydrogen phosphate, and 0.5-1% vitamin A+E in proportion by weight.

2. A method for preparing a complete expanded shrimp feed rich in Vibrio phages and its application, characterized by: The spray-inoculated composite probiotics include 30% Bacillus subtilis, 20% lactic acid bacteria, 20% Saccharomyces cerevisiae, 20% Enterococcus faecalis, and 10% Clostridium butyricum.

3. A method for preparing a complete expanded shrimp feed rich in Vibrio phages and its application, characterized by: The mixed phages contain 25% Vibrio parahaemolyticus phages, 25% Vibrio alginolyticus phages, 25% Vibrio harveyi phages, and 25% Vibrio vulnificus phages.

4. As described in claim 2, a complete expanded feed for shrimp rich in Vibrio phages, characterized in that: The complete puffed shrimp feed is inoculated with mixed probiotics of Bacillus subtilis, lactic acid bacteria, Saccharomyces cerevisiae, Enterococcus faecalis and Clostridium butyricum.

5. As described in claim 3, a preparation method and application of a complete puffed shrimp feed rich in Vibrio phages, characterized in that The complete expanded shrimp feed is sprayed with mixed phages including Vibrio parahaemolyticus phage, Vibrio alginolyticus phage, Vibrio harveyi phage and Vibrio vulnificus phage.

6. A method for preparing a complete expanded shrimp feed rich in Vibrio phages and its application, characterized by: The ratio of complex phage to spray mixed oil is 1000:

1.

7. A method for preparing a complete expanded shrimp feed rich in Vibrio phages and its application, characterized by: The spray mixture oil used in claim 6 comprises half cod liver oil and half soybean oil.

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