CN111109473A - Compound probiotic preparation for preventing and treating gibelism hemorrhagic disease of carassius auratus gibelio and preparation method thereof - Google Patents
Compound probiotic preparation for preventing and treating gibelism hemorrhagic disease of carassius auratus gibelio and preparation method thereof Download PDFInfo
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- CN111109473A CN111109473A CN202010112498.6A CN202010112498A CN111109473A CN 111109473 A CN111109473 A CN 111109473A CN 202010112498 A CN202010112498 A CN 202010112498A CN 111109473 A CN111109473 A CN 111109473A
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Abstract
The application discloses a compound probiotic preparation for preventing and treating gibelism disease of carassius auratus gibelio and a preparation method thereof, and the compound probiotic preparation is composed of 12-16 parts of rapeseed meal, 10-14 parts of fish meal, 2-6 parts of soybean oil, 3-7 parts of corn flour, 1-3 parts of premix, 1-3 parts of monocalcium phosphate, 6-8 parts of microcrystalline cellulose, 15-19 parts of wheat flour, 2-6 parts of fish oil, 26-30 parts of soybean meal and 0.07-0.12 part of bacillus; the disease resistance of the carassius auratus gibelio high-quality breeding variety to CyHV-2 virus is improved, and the immunity of the carassius auratus gibelio is enhanced by the probiotics and the immunopotentiator, so that the disease resistance of the carassius auratus gibelio is improved.
Description
Technical Field
The invention belongs to the technical field of fishing biological products in animal and maintenance industries, and particularly relates to a compound probiotic preparation for preventing and treating gibelism of carassius auratus gibelio and a preparation method thereof.
Background
Carassius auratus gibelio is a new Carassius auratus breeding variety which is developed by inducing eggs of triploid Carassius auratus gibelio to perform gynogenesis by utilizing triploid fang zheng silver crucian (female parent) and diploid Xingguo red carp (male parent) through an artificial hybridization method. The variety has the characteristics of strong adaptability, high survival rate, high growth speed, short feeding period and the like, and particularly achieves great success in popularization and cultivation in Subei areas of Jiangsu province. However, in recent years, the carassius auratus gibelio breeding industry is severely restricted by diseases (such as carassius auratus herpesvirus disease, bacterial septicemia and myxosporidiosis) frequently, and the economic loss caused by the diseases is about 30 billion yuan each year. In order to prevent and control the spread of diseases, farmers add a large amount of chemical drugs such as antibiotics and the like into the feed, so that the drug resistance of the fish body is increased, and the drug residue of the finished fish is serious. Therefore, the development of the green, environment-friendly and safe immunity-enhanced compound feed for improving the self immunity of the carassius auratus gibelio has great significance for improving the disease resistance of the carassius auratus gibelio.
The Poly β -hydroxybutyrate (Poly- β -hydroxybutyate, PHB) is a short-chain fatty acid polymer which is generated by a bacterium in order to maintain the intracellular and extracellular osmotic pressure and accumulated in a large amount when the proportion of carbon and nitrogen in the environment is unbalanced, the short-chain fatty acid polymer generally exists in a particle state, can be degraded into β -hydroxybutyrate monomer in the intestinal tract of an animal in a chemical hydrolysis or enzymolysis mode, and is firstly found to be in bacillus megaterium.
In some researches on cultivation of fin fishes and shellfishes and the like, the results show that the probiotics are a feed additive beneficial to the growth performance, intestinal microbiota, immunity and disease resistance of aquatic animals. As with similar effects of other immunopotentiators in aquaculture, researchers have demonstrated the use of probiotics to enhance immune responses, resist disease and reduce malformations in carp species. When the fish is fed with a bait rich in Aeromonas veronii, Vibrio and Flavobacterium, both humoral immunity and cellular immune response are improved. It has been demonstrated that bacillus circulans PB7 enhances the innate immune response of carp and increases phagocytic activity after challenge with the fish pathogen aeromonas hydrophila.
Chinese patent CN 201811609176.1 discloses an immunopotentiation type compound feed for carassius auratus gibelio, which comprises the following components in parts by weight: 5-10% of fish meal, 10-20% of cottonseed meal, 20-30% of rapeseed meal, 20-25% of soybean meal, 10-15% of peanut meal, 3-5% of soybean oil, 15-20% of wheat middling, 0.2-0.5% of choline chloride, 1.5-2% of monocalcium phosphate, 1.5-2.0% of premix, 0.10-0.20% of yucca extract and 0.50-1.00% of purslane powder. The invention also comprises a preparation method of the carassius auratus gibelio immune enhancement type compound feed. The invention is helpful for enhancing the disease resistance of carassius auratus gibelio. The invention has obvious synergistic effect, the weight gain rate of fish bodies is obviously improved after the use, the indexes of ammonia nitrogen content and the like in water bodies are obviously reduced, but the purslane has the effect of preventing and treating diseases in the invention, but the preparation cost of the purslane powder is high and the efficiency is low, a vacuum microwave drying method is required, fresh purslane is intermittently dried to constant weight in a microwave vacuum low-temperature heating furnace under the conditions of 1000W and 0.08MPa of vacuum degree, the purslane is taken out and crushed and passes through a 80-mesh screen for standby, and the Yucca and the purslane are used as Chinese herbal medicines, so the invention has the defect of higher cost, and the prior art is slightly insufficient.
Disclosure of Invention
The technical problem to be solved is as follows: the application mainly provides a compound probiotic preparation for preventing and treating the gill hemorrhage of carassius auratus gibelio and a preparation method thereof, and solves the technical problems that purslane has the effect of preventing and treating diseases in the prior art, but the preparation cost of purslane powder is high and the efficiency is low, a vacuum microwave drying method is adopted, fresh purslane is dried to constant weight intermittently in a microwave vacuum low-temperature heating furnace under the conditions of 1000W and 0.08MPa of vacuum degree, the purslane is taken out and crushed and then passes through a 80-mesh screen for standby, the costs of the yucca and the purslane as Chinese herbal medicines are high, and the like, so that the disease resistance of the carassius auratus gibelio as a high-quality breeding variety to CyHV 2 virus is improved, and the immunity of the carassius auratus gibelio is enhanced from the perspective.
The technical scheme is as follows:
the compound probiotic preparation for preventing and treating the carassius auratus gibelio hemorrhagic disease comprises the following raw materials in parts by weight: 12-16 parts of rapeseed meal, 10-14 parts of fish meal, 2-6 parts of soybean oil, 3-7 parts of corn flour, 1-3 parts of premix, 1-3 parts of monocalcium phosphate, 6-8 parts of microcrystalline cellulose, 15-19 parts of wheat flour, 2-6 parts of fish oil, 26-30 parts of soybean meal and 0.07-0.12 part of bacillus.
As a preferred technical scheme of the invention: the compound probiotic preparation for preventing and treating the gill hemorrhage of the carassius auratus gibelio is prepared from the following raw materials in parts by mass: 14 parts of rapeseed meal, 12 parts of fish meal, 4 parts of soybean oil, 5 parts of corn flour, 2 parts of premix, 2 parts of monocalcium phosphate, 8 parts of microcrystalline cellulose, 17 parts of wheat flour, 4 parts of fish oil, 28 parts of soybean meal and 0.1 part of bacillus.
As a preferred technical scheme of the invention: the premix is a mineral and vitamin premix, wherein 5500 IU of magnesium sulfate, 4 g of cobalt chloride, 3g of manganese sulfate, 8 g of aluminum chloride, 7 g of potassium iodide, 140 g of zinc sulfate, 150g of ferric citrate, 0.6 g of sodium selenite, 15000IU of calcium dihydrogen phosphate, 4000IU of potassium chloride, 8 g of copper sulfate, 5 mg of biotin, 200mg of inositol, 150 mg of vitamin B, 220 mg of vitamin B, 650 mg of vitamin B, 120.1 mg of vitamin B, 250 IU of nicotinic acid, 50mg of pantothenic acid, 15 mg of folic acid, 5000IU of vitamin A, 400 mg of vitamin C, 32000 IU of vitamin D, 400 mg of vitamin E and 340 mg of vitamin K.
As a preferred technical scheme of the invention: the final concentration of the bacillus is 107cfu/g。
As a preferred technical scheme of the invention: the bacillus was stored in a 4 ℃ refrigerator.
A preparation method of a compound probiotic preparation for preventing and treating carassius auratus gibelio hemorrhagic disease comprises the following steps:
firstly, preparing basic feed raw materials: crushing rapeseed meal, fish meal, corn flour, wheat flour and bean pulp, sieving with a 80-mesh sieve, and uniformly mixing to obtain a basic feed raw material;
step two, preparing a basic feed powder mixture: uniformly mixing monocalcium phosphate, microcrystalline cellulose and the premix, and uniformly mixing the mixture with the basic feed raw material mixture again to obtain a basic feed powder mixture;
the third step: slowly adding soybean oil and fish oil into the basic feed powder mixture, then adding 10-20% of distilled water, uniformly stirring, processing into granules on a small granulator, drying at 70 ℃, and standing at-20 ℃ for later use to obtain hard pellet feed, namely the basic feed;
the fourth step: will have a final concentration of 107counting the number of the coated plates of cfu/g of bacillus every week to ensure the consistency of the number of the added bacillus during the experiment;
the fifth step: according to the mass ratio of 96: 4 weighing PHB powder, dissolving the PHB powder in chloroform to prepare 4-6% saturated chloroform solution;
and a sixth step: diluting bacillus by 100 times of pbs, and spraying the diluted bacillus solution on the surface of the basic feed;
the seventh step: spraying the saturated chloroform solution obtained in the fifth step on the surface of the basic feed;
eighth step: rubbing, drying in the shade, volatilizing chloroform, and standing at-20 deg.C for use;
the ninth step: and coating a layer of sodium alginate adhesive on the surface of the crucian carp, and storing the crucian carp and sodium alginate adhesive in a refrigerator at 4 ℃ after the preparation is finished to obtain the compound probiotic preparation for preventing and treating the gibelism of the carassius auratus gibelio.
Has the advantages that: compared with the prior art, the composite probiotic preparation for preventing and treating the gill hemorrhage of the carassius auratus gibelio and the preparation method thereof adopt the technical scheme, and have the following technical effects:
1. the artificial infection experiment and the immunity-related gene expression comparison are carried out on the same batch of fish fed with different proportions of probiotic preparations for 30 days, the experimental result is as follows, the conclusion can be drawn from the accumulated mortality, and the composite probiotic preparation prepared by mixing PHB and bacillus has obvious effect on preventing and treating gill hemorrhage of carassius auratus gibelio.
2. The difference multiple of the immune related genes of the crucian control group and the obvious increase of the expression quantity of the immune related genes relative to the control group indicate that the resistance of the compound probiotic preparation to gill hemorrhage is greatly increased by enhancing the immunity of the carassius auratus gibelio.
3. The disease resistance of the carassius auratus gibelio as a high-quality breeding variety to the CyHV-2 virus is improved.
4. The probiotic bacteria and immunopotentiator enhance immunity and thus disease resistance.
5. The product has low cost and simple preparation method, and is suitable for various crucian varieties.
Description of the drawings: fig. 1 is a graph of cumulative mortality.
FIG. 2 is a graph showing the difference in the fold number of the immune-related genes.
Detailed Description
Comparative example 1:
the compound probiotic preparation for preventing and treating the hemorrhagic disease of the gill of the carassius auratus gibelio comprises the following raw materials in parts by mass: 12 parts of rapeseed meal, 10 parts of fish meal, 2 parts of soybean oil, 3 parts of corn flour, 1 part of premix, 1 part of monocalcium phosphate, 6 parts of microcrystalline cellulose, 15 parts of wheat flour, 2 parts of fish oil and 26 parts of soybean meal.
The premix is a mineral and vitamin premix, wherein 5500 IU of magnesium sulfate, 4 g of cobalt chloride, 3g of manganese sulfate, 8 g of aluminum chloride, 7 g of potassium iodide, 140 g of zinc sulfate, 150g of ferric citrate, 0.6 g of sodium selenite, 15000IU of calcium dihydrogen phosphate, 4000IU of potassium chloride, 8 g of copper sulfate, 5 mg of biotin, 200mg of inositol, 150 mg of vitamin B, 220 mg of vitamin B, 650 mg of vitamin B, 120.1 mg of vitamin B, 250 IU of nicotinic acid, 50mg of pantothenic acid, 15 mg of folic acid, 5000IU of vitamin A, 400 mg of vitamin C, 32000 IU of vitamin D, 400 mg of vitamin E and 340 mg of vitamin K.
A preparation method of a compound probiotic preparation for preventing and treating carassius auratus gibelio hemorrhagic disease comprises the following steps:
the first step is as follows: preparing basic feed raw materials: crushing rapeseed meal, fish meal, corn flour, wheat flour and bean pulp, sieving with a 80-mesh sieve, and uniformly mixing to obtain a basic feed raw material;
the second step is that: preparing a base feed powder mixture: uniformly mixing monocalcium phosphate, microcrystalline cellulose and the premix, and uniformly mixing the mixture with the basic feed raw material mixture again to obtain a basic feed powder mixture;
the third step: slowly adding soybean oil and fish oil into the base feed powder mixture, adding 10-20% of distilled water, uniformly stirring, processing into granules on a small granulator, drying at 70 ℃, and standing at-20 ℃ for later use to obtain the hard pellet feed, namely the base feed
Comparative example 2:
the compound probiotic preparation for preventing and treating the hemorrhagic disease of the gill of the carassius auratus gibelio comprises the following raw materials in parts by mass: 12 parts of rapeseed meal, 10 parts of fish meal, 2 parts of soybean oil, 3 parts of corn flour, 1 part of premix, 1 part of monocalcium phosphate, 6 parts of microcrystalline cellulose, 15 parts of wheat flour, 2 parts of fish oil and 26 parts of soybean meal.
The premix is a mineral and vitamin premix, wherein 5500 IU of magnesium sulfate, 4 g of cobalt chloride, 3g of manganese sulfate, 8 g of aluminum chloride, 7 g of potassium iodide, 140 g of zinc sulfate, 150g of ferric citrate, 0.6 g of sodium selenite, 15000IU of calcium dihydrogen phosphate, 4000IU of potassium chloride, 8 g of copper sulfate, 5 mg of biotin, 200mg of inositol, 150 mg of vitamin B, 220 mg of vitamin B, 650 mg of vitamin B, 120.1 mg of vitamin B, 250 IU of nicotinic acid, 50mg of pantothenic acid, 15 mg of folic acid, 5000IU of vitamin A, 400 mg of vitamin C, 32000 IU of vitamin D, 400 mg of vitamin E and 340 mg of vitamin K.
A preparation method of a compound probiotic preparation for preventing and treating carassius auratus gibelio hemorrhagic disease comprises the following steps:
the first step is as follows: preparing basic feed raw materials: crushing rapeseed meal, fish meal, corn flour, wheat flour and bean pulp, sieving with a 80-mesh sieve, and uniformly mixing to obtain a basic feed raw material;
the second step is that: preparing a base feed powder mixture: uniformly mixing monocalcium phosphate, microcrystalline cellulose and the premix, and uniformly mixing the mixture with the basic feed raw material mixture again to obtain a basic feed powder mixture;
the third step: slowly adding soybean oil and fish oil into the basic feed powder mixture, then adding 10-20% of distilled water, uniformly stirring, processing into granules on a small granulator, drying at 70 ℃, and standing at-20 ℃ for later use to obtain hard pellet feed, namely the basic feed;
the fourth step: according to the mass ratio of 96: 4 weighing PHB powder, dissolving the PHB powder in chloroform to prepare 4-6% saturated chloroform solution;
the fifth step: spraying the saturated chloroform solution obtained in the fifth step on the surface of the basic feed;
and a sixth step: rubbing, drying in the shade, volatilizing chloroform, and standing at-20 deg.C for use;
the seventh step: and coating a layer of sodium alginate adhesive on the surface of the crucian carp, and storing the crucian carp and sodium alginate adhesive in a refrigerator at 4 ℃ after the preparation is finished to obtain the compound probiotic preparation for preventing and treating the gibelism of the carassius auratus gibelio.
Example 1:
the compound probiotic preparation for preventing and treating the hemorrhagic disease of the gill of the carassius auratus gibelio comprises the following raw materials in parts by mass: 12 parts of rapeseed meal, 10 parts of fish meal, 2 parts of soybean oil, 3 parts of corn flour, 1 part of premix, 1 part of monocalcium phosphate, 6 parts of microcrystalline cellulose, 15 parts of wheat flour, 2 parts of fish oil, 26 parts of soybean meal and 0.07 part of bacillus.
The premix is a mineral and vitamin premix, wherein 5500 IU of magnesium sulfate, 4 g of cobalt chloride, 3g of manganese sulfate, 8 g of aluminum chloride, 7 g of potassium iodide, 140 g of zinc sulfate, 150g of ferric citrate, 0.6 g of sodium selenite, 15000IU of calcium dihydrogen phosphate, 4000IU of potassium chloride, 8 g of copper sulfate, 5 mg of biotin, 200mg of inositol, 150 mg of vitamin B, 220 mg of vitamin B, 650 mg of vitamin B, 120.1 mg of vitamin B, 250 IU of nicotinic acid, 50mg of pantothenic acid, 15 mg of folic acid, 5000IU of vitamin A, 400 mg of vitamin C, 32000 IU of vitamin D, 400 mg of vitamin E and 340 mg of vitamin K.
The final concentration of the bacillus is 107cfu/g, the Bacillus was stored in a refrigerator at 4 ℃.
A preparation method of a compound probiotic preparation for preventing and treating carassius auratus gibelio hemorrhagic disease comprises the following steps:
the first step is as follows: preparing basic feed raw materials: crushing rapeseed meal, fish meal, corn flour, wheat flour and bean pulp, sieving with a 80-mesh sieve, and uniformly mixing to obtain a basic feed raw material;
the second step is that: preparing a base feed powder mixture: uniformly mixing monocalcium phosphate, microcrystalline cellulose and the premix, and uniformly mixing the mixture with the basic feed raw material mixture again to obtain a basic feed powder mixture;
the third step: slowly adding soybean oil and fish oil into the basic feed powder mixture, then adding 10-20% of distilled water, uniformly stirring, processing into granules on a small granulator, drying at 70 ℃, and standing at-20 ℃ for later use to obtain hard pellet feed, namely the basic feed;
the fourth step: will have a final concentration of 107counting the number of the coated plates of cfu/g of bacillus every week to ensure the consistency of the number of the added bacillus during the experiment;
the fifth step: according to the mass ratio of 96: 4 weighing PHB powder, dissolving the PHB powder in chloroform to prepare 4-6% saturated chloroform solution;
and a sixth step: diluting bacillus by 100 times of pbs, and spraying the diluted bacillus solution on the surface of the basic feed;
the seventh step: spraying the saturated chloroform solution obtained in the fifth step on the surface of the basic feed;
eighth step: rubbing, drying in the shade, volatilizing chloroform, and standing at-20 deg.C for use;
the ninth step: and coating a layer of sodium alginate adhesive on the surface of the crucian carp, and storing the crucian carp and sodium alginate adhesive in a refrigerator at 4 ℃ after the preparation is finished to obtain the compound probiotic preparation for preventing and treating the gibelism of the carassius auratus gibelio.
Example 2
The compound probiotic preparation for preventing and treating the hemorrhagic disease of the gill of the carassius auratus gibelio comprises the following raw materials in parts by mass: 16 parts of rapeseed meal, 14 parts of fish meal, 6 parts of soybean oil, 7 parts of corn flour, 3 parts of premix, 3 parts of monocalcium phosphate, 8 parts of microcrystalline cellulose, 19 parts of wheat flour, 6 parts of fish oil, 30 parts of soybean meal and 0.12 part of bacillus.
The premix is a mineral and vitamin premix, wherein the premix comprises 5500 IU of magnesium sulfate, 4 g of cobalt chloride, 3g of manganese sulfate, 8 g of aluminum chloride, 7 g of potassium iodide, 140 g of zinc sulfate, 150g of ferric citrate, 0.6 g of sodium selenite, 15000IU of monocalcium phosphate, 4000IU of potassium chloride, 8 g of copper sulfate, 5 mg of biotin, 200mg of inositol, 150 mg of vitamin B, 220 mg of vitamin B, 650 mg of vitamin B, 120.1 mg of vitamin B, 250 IU of nicotinic acid, 50mg of pantothenic acid, 15 mg of folic acid, 5000IU of vitamin A, 400 mg of vitamin C, 32000 IU of vitamin D, 400 mg of vitamin E and 340 mg of vitamin K340.
The final concentration of the bacillus is 107cfu/g, the Bacillus was stored in a refrigerator at 4 ℃.
A preparation method of a compound probiotic preparation for preventing and treating carassius auratus gibelio hemorrhagic disease comprises the following steps:
the first step is as follows: preparing basic feed raw materials: crushing rapeseed meal, fish meal, corn flour, wheat flour and bean pulp, sieving with a 80-mesh sieve, and uniformly mixing to obtain a basic feed raw material;
the second step is that: preparing a base feed powder mixture: uniformly mixing monocalcium phosphate, microcrystalline cellulose and the premix, and uniformly mixing the mixture with the basic feed raw material mixture again to obtain a basic feed powder mixture;
the third step: slowly adding soybean oil and fish oil into the basic feed powder mixture, then adding 10-20% of distilled water, uniformly stirring, processing into granules on a small granulator, drying at 70 ℃, and standing at-20 ℃ for later use to obtain hard pellet feed, namely the basic feed;
the fourth step: will have a final concentration of 107counting the number of the coated plates of cfu/g of bacillus every week to ensure the consistency of the number of the added bacillus during the experiment;
the fifth step: according to the mass ratio of 96: 4 weighing PHB powder, dissolving the PHB powder in chloroform to prepare 4-6% saturated chloroform solution;
and a sixth step: diluting bacillus by 100 times of pbs, and spraying the diluted bacillus solution on the surface of the basic feed;
the seventh step: spraying the saturated chloroform solution obtained in the fifth step on the surface of the basic feed;
eighth step: rubbing, drying in the shade, volatilizing chloroform, and standing at-20 deg.C for use;
the ninth step: and coating a layer of sodium alginate adhesive on the surface of the crucian carp, and storing the crucian carp and sodium alginate adhesive in a refrigerator at 4 ℃ after the preparation is finished to obtain the compound probiotic preparation for preventing and treating the gibelism of the carassius auratus gibelio.
Example 3
The compound probiotic preparation for preventing and treating the hemorrhagic disease of the gill of the carassius auratus gibelio comprises the following raw materials in parts by mass: 14 parts of rapeseed meal, 12 parts of fish meal, 4 parts of soybean oil, 5 parts of corn flour, 2 parts of premix, 2 parts of monocalcium phosphate, 8 parts of microcrystalline cellulose, 17 parts of wheat flour, 4 parts of fish oil, 28 parts of soybean meal and 0.1 part of bacillus.
The premix is a mineral and vitamin premix, wherein 5500 IU of magnesium sulfate, 4 g of cobalt chloride, 3g of manganese sulfate, 8 g of aluminum chloride, 7 g of potassium iodide, 140 g of zinc sulfate, 150g of ferric citrate, 0.6 g of sodium selenite, 15000IU of calcium dihydrogen phosphate, 4000IU of potassium chloride, 8 g of copper sulfate, 5 mg of biotin, 200mg of inositol, 150 mg of vitamin B, 220 mg of vitamin B, 650 mg of vitamin B, 120.1 mg of vitamin B, 250 IU of nicotinic acid, 50mg of pantothenic acid, 15 mg of folic acid, 5000IU of vitamin A, 400 mg of vitamin C, 32000 IU of vitamin D, 400 mg of vitamin E and 340 mg of vitamin K.
The final concentration of the bacillus is 107cfu/g, the Bacillus was stored in a refrigerator at 4 ℃.
A preparation method of a compound probiotic preparation for preventing and treating carassius auratus gibelio hemorrhagic disease comprises the following steps:
the first step is as follows: preparing basic feed raw materials: crushing rapeseed meal, fish meal, corn flour, wheat flour and bean pulp, sieving with a 80-mesh sieve, and uniformly mixing to obtain a basic feed raw material;
the second step is that: preparing a base feed powder mixture: uniformly mixing monocalcium phosphate, microcrystalline cellulose and the premix, and uniformly mixing the mixture with the basic feed raw material mixture again to obtain a basic feed powder mixture;
the third step: slowly adding soybean oil and fish oil into the basic feed powder mixture, then adding 10-20% of distilled water, uniformly stirring, processing into granules on a small granulator, drying at 70 ℃, and standing at-20 ℃ for later use to obtain hard pellet feed, namely the basic feed;
the fourth step: will have a final concentration of 107counting the number of the coated plates of cfu/g of bacillus every week to ensure the consistency of the number of the added bacillus during the experiment;
the fifth step: according to the mass ratio of 96: 4 weighing PHB powder, dissolving the PHB powder in chloroform to prepare 4-6% saturated chloroform solution;
and a sixth step: diluting bacillus by 100 times of pbs, and spraying the diluted bacillus solution on the surface of the basic feed;
the seventh step: spraying the saturated chloroform solution obtained in the fifth step on the surface of the basic feed;
eighth step: rubbing, drying in the shade, volatilizing chloroform, and standing at-20 deg.C for use;
the ninth step: and coating a layer of sodium alginate adhesive on the surface of the crucian carp, and storing the crucian carp and sodium alginate adhesive in a refrigerator at 4 ℃ after the preparation is finished to obtain the compound probiotic preparation for preventing and treating the gibelism of the carassius auratus gibelio.
As shown in fig. 1 and fig. 2, artificial infection experiments and immunity-related gene expression comparisons were performed on the same batch of fish fed with different ratios of probiotic preparations for 30 days, and it can be concluded from the cumulative mortality that the composite probiotic preparation prepared by mixing PHB and bacillus has a significant effect on prevention and treatment of gill hemorrhagic disease of carassius auratus gibelio, and the fold difference of the immunity-related gene crucian control group and the significant increase of the expression level of the immunity-related gene relative to the control group indicate that the resistance of the composite probiotic preparation to gill hemorrhagic disease is greatly increased by enhancing the immunity of carassius auratus gibelio.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. The compound probiotic preparation for preventing and treating carassius auratus gibelio hemorrhagic disease is characterized by comprising the following components in parts by weight: the compound probiotic preparation for preventing and treating the gill hemorrhage of the carassius auratus gibelio is prepared from the following raw materials in parts by mass: 12-16 parts of rapeseed meal, 10-14 parts of fish meal, 2-6 parts of soybean oil, 3-7 parts of corn flour, 1-3 parts of premix, 1-3 parts of monocalcium phosphate, 6-8 parts of microcrystalline cellulose, 15-19 parts of wheat flour, 2-6 parts of fish oil, 26-30 parts of soybean meal and 0.07-0.12 part of bacillus.
2. The compound probiotic preparation for preventing and treating allogynogenetic crucian carp gill hemorrhage according to claim 1, is characterized in that: the compound probiotic preparation for preventing and treating the gill hemorrhage of the carassius auratus gibelio is prepared from the following raw materials in parts by mass: 14 parts of rapeseed meal, 12 parts of fish meal, 4 parts of soybean oil, 5 parts of corn flour, 2 parts of premix, 2 parts of monocalcium phosphate, 8 parts of microcrystalline cellulose, 17 parts of wheat flour, 4 parts of fish oil, 28 parts of soybean meal and 0.1 part of bacillus.
3. The compound probiotic preparation for preventing and treating allogynogenetic crucian carp gill hemorrhage according to claim 1, is characterized in that: the premix is a mineral and vitamin premix, wherein 5500 IU of magnesium sulfate, 4 g of cobalt chloride, 3g of manganese sulfate, 8 g of aluminum chloride, 7 g of potassium iodide, 140 g of zinc sulfate, 150g of ferric citrate, 0.6 g of sodium selenite, 15000IU of calcium dihydrogen phosphate, 4000IU of potassium chloride, 8 g of copper sulfate, 5 mg of biotin, 200mg of inositol, 150 mg of vitamin B, 220 mg of vitamin B, 650 mg mg of vitamin B, 120.1 mg of vitamin B, 250 IU of nicotinic acid, 50mg of pantothenic acid, 15 mg of folic acid, 5000IU of vitamin A, 400 mg of vitamin C, 32000 IU of vitamin D, 400 mg of vitamin E and 340 mg of vitamin K.
4. The compound probiotic preparation for preventing and treating allogynogenetic crucian carp gill hemorrhage according to claim 1, is characterized in that: the final concentration of the bacillus is 107cfu/g。
5. The compound probiotic preparation for preventing and treating crucian carp gill hemorrhage according to claim 4, is characterized in that: the bacillus was stored in a 4 ℃ refrigerator.
6. A method for preparing a compound probiotic preparation for preventing and treating the hemorrhagic disease of gill of carassius auratus gibelio according to any one of claims 1 to 5, which comprises the following steps:
firstly, preparing basic feed raw materials: crushing rapeseed meal, fish meal, corn flour, wheat flour and bean pulp, sieving with a 80-mesh sieve, and uniformly mixing to obtain a basic feed raw material;
step two, preparing a basic feed powder mixture: uniformly mixing monocalcium phosphate, microcrystalline cellulose and the premix, and uniformly mixing the mixture with the basic feed raw material mixture again to obtain a basic feed powder mixture;
the third step: slowly adding soybean oil and fish oil into the basic feed powder mixture, then adding 10-20% of distilled water, uniformly stirring, processing into granules on a small granulator, drying at 70 ℃, and standing at-20 ℃ for later use to obtain hard pellet feed, namely the basic feed;
the fourth step: will have a final concentration of 107counting the number of the coated plates of cfu/g of bacillus every week to ensure the consistency of the number of the added bacillus during the experiment;
the fifth step: according to the mass ratio of 96: 4 weighing PHB powder, dissolving the PHB powder in chloroform to prepare 4-6% saturated chloroform solution;
and a sixth step: diluting bacillus by 100 times of pbs, and spraying the diluted bacillus solution on the surface of the basic feed;
the seventh step: spraying the saturated chloroform solution obtained in the fifth step on the surface of the basic feed;
eighth step: rubbing, drying in the shade, volatilizing chloroform, and standing at-20 deg.C for use;
the ninth step: and coating a layer of sodium alginate adhesive on the surface of the crucian carp, and storing the crucian carp and sodium alginate adhesive in a refrigerator at 4 ℃ after the preparation is finished to obtain the compound probiotic preparation for preventing and treating the gibelism of the carassius auratus gibelio.
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