CN114854651B - Micrococcus compositus preparation and preparation method and application thereof - Google Patents
Micrococcus compositus preparation and preparation method and application thereof Download PDFInfo
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- CN114854651B CN114854651B CN202210785196.4A CN202210785196A CN114854651B CN 114854651 B CN114854651 B CN 114854651B CN 202210785196 A CN202210785196 A CN 202210785196A CN 114854651 B CN114854651 B CN 114854651B
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Abstract
The invention belongs to the technical field of microorganism application, and discloses a composite coccus preparation as well as a preparation method and an application thereof, wherein the preparation method comprises the following steps: mixing the composite coccus particles with the filler, stirring, adhering, sieving and extruding, and drying undersize to obtain a composite particle layer; coating the slurry A on the surface of the composite particle layer, and cooling to obtain a water-resisting layer coated composite particle layer; heating and melting the mesh agent, sending the mesh agent to a spinning machine to spray out mesh, and adhering the mesh agent to the surface of the water-resisting layer wrapping the composite particle layer to obtain a reticulate pattern layer; and cooling the reticulate pattern layer, adding the antibacterial powder and the pore-forming agent, mixing, continuously cooling, spraying the slurry B, heating and cooling to obtain the composite sheet coccus preparation. According to the invention, the multi-functional composite coccus preparation is obtained by preparing four hierarchical structures of the composite granular layer, the water-resisting layer, the reticulate pattern layer and the microporous layer, different functions are realized by utilizing different hierarchies, and the hierarchies are tightly combined.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to a pleococcus preparation as well as a preparation method and application thereof.
Background
At present, most of economically and commercially important fish (shrimp) groups in the global range are damaged or overtaken, and face increasing demands on aquatic product consumption, and aquaculture with high yield not only helps to meet the demands on aquatic product consumption, but also can continuously compensate some scarce aquatic product resources, so that the aquaculture industry pays attention to and develops rapidly. Currently, the yield of aquaculture products exceeds the fishing amount of fishery, becomes a main source of aquatic products, and is more and more oriented to the development of intensive aquaculture.
However, in intensive aquaculture, especially large-scale intensive aquaculture, the microbiota in aquaculture have a direct impact on their health and development due to the close interaction of aquatic species with the surrounding environment. For example, when a poor water environment flows into a culture area, fishes can be exposed to various infectious bacterial diseases, are easy to outbreak in intensive aquaculture, cause serious culture economic losses, influence aquaculture progress and limit sustainable development of the industry.
Currently, bacterial fish diseases cause serious culture economic losses in many countries around the world, vibriosis is one of the most common bacterial fish diseases, and the influence range relates to important cultured shrimps, global marine fishes, freshwater fishes and the like. Although the use of antibiotics can achieve control of bacterial pathogens, antibiotics are selected for prophylaxis without restriction, the risk that they will spread to the land through the food chain and eventually enter the human body and accumulate, and in addition, indiscriminate use of antibiotics can hinder the activity of beneficial microorganisms in the fish's intestinal microbiota, alter the microbial system, disrupt the aqueous environment and physiological processes, reduce the immune competence of the fish and the quality of the raw fish.
In the pursuit of high quality, high yield fish farming, a sustainable and environmentally friendly aquaculture mode is one of the main targets of future farming development. In conclusion, on the premise of avoiding the use of antibiotics, the method can be used for inhibiting and controlling vibrio pathogens in aquaculture, in addition, the nutrient quality and the similarity and the relevance between the contact water environment of planktonic microbiota and the animal intestinal microbiota are close, the method is also a determinant factor of the intestinal microbiota in fish, and the method is also an important measure for improving the nutrient quality and improving the water environment for the survival of fish to prevent the outbreak of the vibrio diseases in the nutrient environment. There is therefore an urgent need for a multi-functional type of complex coccus preparation to solve the above problems.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a composite coccus preparation, a preparation method and an application thereof, and the composite coccus preparation has a multilayer structure, is wide in suitable temperature range, simple in process, capable of soaking water for a long time and suitable for farmers to use.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a synechococcus preparation comprises the following steps:
(1) mixing the composite coccus particles with the filler, stirring, adhering, sieving and extruding, and drying undersize to obtain a composite particle layer;
(2) coating the slurry A on the surface of the composite particle layer, and cooling to obtain a water-resisting layer coated composite particle layer;
(3) heating and melting the mesh agent, sending the mesh agent into a spinning machine to spray out mesh, and adhering the mesh agent to the surface of the water-resisting layer wrapping the composite particle layer to obtain a reticulate pattern layer;
(4) reducing the surface temperature of the reticulate pattern layer to 40-50 ℃, adding antibacterial powder and a pore-forming agent, mixing, continuously cooling, spraying slurry B, heating to 50-60 ℃, and cooling to obtain a composite sheet coccus preparation; the slurry A comprises corn starch glue, carboxymethyl cellulose and glycerol; the slurry B comprises xanthan gum, tapioca starch and glycerol.
Preferably, in step (1), the composite particle layer contains at least one composite coccus particle.
Further preferably, the composite particle layer contains two to four particles.
Preferably, in the step (1), the filling material is composed of pig plasma powder, cottonseed protein, sodium alginate and water.
Preferably, in step (1), the process for preparing the synechococcus granule is as follows: the effective viable count is 0.5-8 × 10 8 The effective viable count of the CFU/mL pediococcus is 0.01-2 multiplied by 10 8 The effective viable count of the Clostridium butyricum is 0.01-1 multiplied by 10 8 CFU/mL Bacillus belgii with effective viable count of 0.01-1 × 10 8 And (3) mixing CFU/mL bacillus subtilis and 20-100 kg of matrix material, and performing spray granulation to obtain the bacillus subtilis.
Further preferably, the base material is prepared from pig milk powder, bean dregs, cottonseed protein, calcium dihydrogen phosphate, sodium butyrate, vitamins, sodium carboxymethylcellulose and chicken fat in a mass ratio of (5-20): (5-20): (20-40): (1-3): (0.1-0.8): (0.01-0.03): (0.01-0.05): (1-3).
More preferably, the preparation steps of the swine slurry powder are as follows: (1) crushing pig bones and pig viscera, sieving, adding ethanol, mixing, evaporating at 55-70 ℃, adding de-lipase water, mixing, separating, and concentrating to obtain a concentrated solution; (2) and mixing the concentrated solution with alkaline protease and trypsin, treating at 45-60 ℃ for 3-8 hours to obtain an enzyme decomposition product, concentrating under negative pressure, and drying to obtain the pig pulp powder.
Further preferably, the pediococcus preparation process comprises the following steps: activating lactobacillus with MRS liquid culture medium, and diluting to obtain 2.5 × 10 8 Inoculating the CFU/mL bacterial liquid into an MRS liquid culture medium, culturing for 24 h at 37 ℃, centrifuging and filtering at 4 ℃, taking the supernatant of the lactic acid bacteria, adding the supernatant into an Oxford cup, taking the bacteria A as indicator bacteria, and controlling the inoculation concentration of the indicator bacteria to be 0.1-2 × 10 8 CFU/mL and the volume are both 100 mu L, strains with obvious inhibition zones for each indicator bacterium are screened out, the strains are cultured for 12 hours at 37 ℃, the diameter D of a transparent ring is measured by a cross method, and the strains with the obvious inhibition zones for each indicator bacterium are screened out; the bacteria A is at least one of vibrio, aeromonas, fibrobacter and streptococcus.
More preferably, the MRS liquid medium comprises the following: 10.0g/L of peptone, 8.0g/L of beef extract powder, 4.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0 g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04 g/L of manganese sulfate and 80.0 g/L of tween.
More preferably, after activating pediococcus, clostridium butyricum, bacillus belvesii and bacillus subtilis by using an MRS liquid culture medium, respectively picking strains on each plate lawn into the MRS liquid culture medium, culturing for 40-42h, centrifuging the strains for 10-30 min by 3000-3500r/min, washing and precipitating, then suspending the strain precipitate in a phosphate balanced physiological saline buffer solution, after 50-fold dilution, sucking 0.1mL of diluent, uniformly coating the diluent on a solid culture medium, reversely culturing in a biochemical incubator for 48h, counting, and directly calculating the number of the strains in the strain solution by using an OD600 value according to the linear relation between the optical density value (OD 600 value) of the strain solution and the number of the living strains in the suspension at a wavelength of 600nm of a visible spectrophotometer.
Preferably, in the step (2), the slurry a is prepared from corn starch glue, carboxymethyl cellulose and glycerol according to a mass ratio of (30-50): (10-15): (1-5) mixing, and heating and pasting at 75-85 ℃.
Preferably, in the step (2), the number of times of the wrapping is 3 to 10.
Preferably, in the step (3), the melting temperature is 80-85 ℃.
Preferably, in the step (3), the textured layer is prepared by the following specific steps: heating the mesh agent to 80-85 ℃ to melt to obtain mesh liquid, conveying the mesh liquid to a feeding unit of a spinning machine, extruding the mesh liquid by the feeding unit, conveying the mesh liquid to a metering unit, conveying the metering unit to a melt-blowing unit, spraying the mesh from a spinning jet of the melt-blowing unit, and adhering the mesh to the surface of the rolling composite sheet coccus preparation wrapped by the waterproof layer.
Further preferably, the mesh agent is carrageenan and water according to a mass/volume ratio of (3-10) g: (50-80) ml.
Preferably, the specific steps of step (4) are: and (3) reducing the surface temperature of the water-resisting layer of the composite sheet coccus preparation to 40-50 ℃, adding the antibacterial powder and the pore-forming agent, mixing and adhering, cooling to room temperature, wrapping the slurry B on the surface of the composite sheet coccus preparation by using a slurry wrapping machine, heating to 50-60 ℃, and cooling to room temperature to obtain the composite sheet coccus preparation with the microporous layer coated with the reticulate layer.
The adhesion is to adhere antibacterial powder and pore-forming agent by using the viscosity of the surface of the waterproof layer and the mesh; the above heating to 50-60 deg.C can decompose the pore-forming agent and discharge the generated gas to form a microporous layer.
Preferably, in the step (4), the bacteriostatic powder is composed of candida wilt, streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve and bacillus licheniformis.
Further preferably, said Candida wilt, thermophileThe effective viable count of streptococcus, bifidobacterium longum, bifidobacterium breve and bacillus licheniformis is 0.01-8 multiplied by 10 8 CFU/mL。
Preferably, in the step (4), the addition amount of the antibacterial powder is 0.05-0.2% of the mass of the synechococcus comosum preparation.
Preferably, in the step (4), the pore-forming agent is at least one of ammonium bicarbonate and ammonium carbonate.
Preferably, in the step (4), the addition amount of the pore-forming agent is 0.001-0.1% of the mass of the synechococcus compositus preparation.
Preferably, in the step (4), the slurry B is prepared from xanthan gum, tapioca starch and glycerol according to a mass ratio of (20-50): (30-50): (1-5) mixing, and heating and pasting at 65-75 ℃.
A composite cocci preparation is prepared by the preparation method, and sequentially comprises a composite granular layer, a waterproof layer, a reticulate pattern layer and a microporous layer from inside to outside; the composite particle layer contains at least one particle; the shape of the composite cocci preparation is at least one of irregular sphere, ellipsoid, fusiform, long rod, short rod, sheet and block.
Preferably, the composite particle layer is composed of particle-to-particle bound filler; binding single or multiple particles in the composite particle layer by filler; the particle diameter is 0.2-3 mm.
Further preferably, the diameter of the composite particle layer is 0.35-6.5 mm; more preferably, the diameter of the composite particle layer is 0.50-3.5 mm.
Preferably, the water barrier layer wraps the composite particle layer; the thickness of the water-resisting layer is 0.1-2 mm. The water-resisting layer protects the composite particle layer from being soaked and dispersed in water, ensures the integrity of the composite particle layer and can float on the water surface.
More preferably, the part of the surface of the water-resisting layer, to which the net-shaped structure is attached, forms a plurality of pits, and the pits contain antibacterial powder and residual pore-forming agent; the pores on the microporous layer can release the antibacterial powder into the water body, the microporous layer is gradually decomposed in the water body in the antibacterial powder releasing process, and the exposed reticulate pattern layer is in contact with the water body.
Preferably, the reticulate pattern layer is attached to the surface of the water-resisting layer in a net structure; the thickness of the reticulate pattern layer is 0.15-1 mm. The water-resisting layer is anchored through the reticulate pattern layer, the structural stability is kept, and the water-resisting layer is reduced to break.
Preferably, a plurality of pores are distributed on the microporous layer, and the microporous layer forms a complete composite coccus preparation by covering the reticulate pattern layer; the thickness of the microporous layer is 0.1-1.0 mm.
Preferably, the dissolving time of the microporous layer is 16-48 h.
The invention also provides an application of the composite coccus preparation in fish culture.
Compared with the prior art, the invention has the following beneficial effects:
1. the preparation method comprises the steps of preparing a hierarchical structure of four parts, namely a composite granular layer, a water-resisting layer, a reticulate pattern layer and a microporous layer, realizing different functions by utilizing different levels, and tightly combining the levels to obtain a multifunctional composite pneumococcal preparation; wherein, the pores on the microporous layer are utilized to release the antibacterial powder, and the antibacterial powder contains candida wilt, streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve and bacillus licheniformis which can improve the culture environment: comprises purifying water quality, inhibiting growth of vibrio in water, and simultaneously facilitating population balance propagation of floating microalgae in water; pathogenic bacteria can be indirectly inhibited through interaction with microalgae, and the utilization rate of the feed is improved; moreover, the harmful vibrio is parasitically controlled, so that the vibrio is cracked, and the number of vibrio populations in the environment is reduced, thereby reducing the harm of the vibrio. Secondly, when the microporous layer is gradually dissolved in water, a reticulate pattern layer with a net structure is arranged on the surface of the water-resisting layer, and the water-resisting layer is anchored through the reticulate pattern layer, so that the structural stability is kept, and the cracking of the water-resisting layer is reduced; the water-resisting layer can tightly stick to the composite granular layer, namely the composite granular layer is protected from being soaked and dispersed in water and can float on the water surface, under the condition that interference such as fry feeding does not exist, the long-term integrity of the composite granular layer of the composite coccus preparation is kept by utilizing the function of mutual linkage of the reticulate layer and the water-resisting layer, and the effectiveness of the composite coccus preparation is kept during the whole production period. In addition, the composite granular layer contains pediococcus, clostridium butyricum, bacillus belgii and bacillus subtilis flora, can improve the immunity of the cultured fishes, mainly shows that the macrophage number of the cultured fishes is increased, and the inventor finds that: more phagocytic stimulation results in the release of a large number of biologically active substances, such as enzyme inhibitors, cationic peptides, complement components, and reactive oxygen and nitric oxide-depleting bacteria, which regulate wound healing; can improve the content of immunoglobulin; compete with pathogenic bacteria for nutrition, spatial sites and free iron, and compete for inhibiting pathogenic bacteria; through self secretion or promotion of intestinal flora to secrete various digestive enzymes, the digestive tract enzyme activity of the cultured animals is improved, the conversion of the matrix material and the filling material is promoted, the digestion is promoted, and the utilization rate of the matrix material and the filling material is improved. Finally, because the pig plasma powder is prepared from pig bones and pig internal organs which contain a large amount of bone marrow and collagen, the pig bones and the pig internal organs are used as sources for extracting collagen and chondroitin, and a large amount of small peptides and free amino acids are generated after enzymolysis reaction, and the polypeptides have the characteristics of easy digestion and absorption and low allergy, can promote the absorption of calcium and protein, and promote the growth of fishes.
2. In the preparation process of the reticulate pattern layer, the net wires are sprayed out by the spinning machine to be adhered to the water-resisting layer, the net wires are adhered to the surface of the water-resisting layer in a net structure, the net structure is the reticulate pattern layer, the reticulate pattern layer has good structural stability, a protective layer is formed, the cracking of a composite sheet coccus preparation coated with the water-resisting layer can be prevented, and the functional integrity of the composite sheet coccus preparation is improved; in addition, at 40-50 ℃, the water-resisting layer and the surface mesh can still stick the antibacterial powder and the pore-forming agent, and the water-resisting layer and the surface mesh are not pasted at the temperature and have good shapes.
3. In the preparation process of the microporous layer, the microporous layer is heated at 50-60 ℃, the pore-forming agent is decomposed to release gas, meanwhile, a material layer wrapped by the slurry B is softened at the temperature, the more the gas is, the material layer expands to generate small-pore exhaust gas, after cooling, small pores appear on the material layer, namely the microporous layer, and the antibacterial powder is slowly released from the small pores on the microporous layer.
Drawings
FIG. 1 is a schematic of a planing surface containing 1 Coccocus granulatus preparation;
FIG. 2 is a schematic front view of a plectane layer of a Synechococcus preparation;
FIG. 3 is a schematic front view of a micrococcus formulation overcoated with a microporous layer;
FIG. 4 is an enlarged view of A in the schematic diagram of FIG. 1;
FIG. 5 is a schematic representation of a planar surface containing 2 Micrococcus granulatus preparations;
FIG. 6 is a schematic representation of a planar surface containing 3 of the preparation of Micrococcus granulatus;
FIG. 7 is a schematic representation of a planar surface containing 4 of the preparation of Micrococcus granulatus;
FIG. 8 is a schematic representation of a planar surface containing 5 of the preparation of Micrococcus granulatus;
FIG. 9 is the bacterial flora of the intestinal tract of the first 8 categorical abundances of examples 1-14 and comparative examples 1-5.
In the figure, 1 is a composite particle layer, 11 is particles, and 12 is filler; 2 is a water-resisting layer; 3, a reticulate pattern layer, 31, a mesh and 32, concave points; 4 is a microporous layer, 41 is microporous.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
Examples
The method of making a synechococcus formulation of examples 1-4 comprising the steps of:
the preparation process of pediococcus comprises the following steps: activating lactobacillus with MRS liquid culture medium, and diluting to obtain 2.5 × 10 8 CFU/mL bacterial solution, inoculated to MRS liquid medium (MRS liquid medium: peptone 10.0g/L, beef extract powder 8.0g/L, yeast extract powder 4.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0 g/L, magnesium sulfate 0.2g/L, manganese sulfate 0.04 g/L, Tween)80.0 g/L), culturing at 37 ℃ for 24 h, centrifuging at 4 ℃, filtering, taking 500 muL of lactobacillus supernatant, adding into Oxford cup holes of an upper flat plate, taking vibrio and cellulose as indicator bacteria, and the inoculation concentration of the indicator bacteria is 0.1 multiplied by 10 8 And (3) screening out strains with obvious inhibition zones for each indicator bacterium, culturing for 12 hours in an incubator at 37 ℃, measuring the diameter D of the transparent ring by using a cross method, and screening out strains with obvious inhibition zones for each indicator bacterium to obtain pediococcus.
The preparation process of the composite cocci particles comprises the following steps: the effective viable count is 0.5-8 × 10 8 The effective viable count of the CFU/mL pediococcus is 0.01-2 multiplied by 10 8 The effective viable count of the Clostridium butyricum is 0.01-1 multiplied by 10 8 CFU/mL Bacillus belgii with effective viable count of 0.01-1 × 10 8 And (3) mixing CFU/mL bacillus subtilis and 10-100 kg of matrix material, and performing spray granulation to obtain the bacillus subtilis.
The process of calculating the viable count: activating pediococcus, clostridium butyricum, bacillus belgii and bacillus subtilis, respectively picking strains on each flat lawn of the pediococcus to be cultured in an MRS liquid culture medium for 40h, centrifuging the bacteria liquid for 10 min at 3000 r/min, washing, precipitating, suspending precipitates in a phosphate balanced physiological saline buffer solution, diluting by 50 times, absorbing 0.1mL of diluent to be uniformly coated on a solid culture medium, counting after culturing in a biochemical incubator for 48h in an inverted mode, measuring the optical density value (OD 600 value) of the bacteria solution at a 600nm wavelength of a visible spectrophotometer according to the linear relation of the OD600 value and the number of live bacteria in suspension, and directly calculating the number of bacteria in the bacteria liquid by using the OD600 value in the subsequent culture test.
(1) Adding 20kg of filler into 100kg of composite coccus granules, stirring, adhering the granules by the filler, sieving, extruding, taking undersize, and drying to obtain a composite granule layer (the mixture ratio of the granules, the filler and the matrix materials forming the granules is shown in table 1;
(2) coating the surface of the composite particle layer with slurry A (prepared by mixing corn starch glue, carboxymethyl cellulose and glycerol according to a mass ratio of 40: 10:3 and heating and pasting at 80 ℃) by using a slurry coating machine, and cooling to room temperature to obtain a water-resisting layer coated composite particle layer;
(3) heating a mesh agent (obtained by mixing carrageenan and water according to the mass/volume ratio of 5 g: 60 ml) to 80 ℃ to melt to obtain mesh liquid, sending the mesh liquid to a feeding unit of a spinning machine, extruding the mesh liquid by the feeding unit, sending the mesh liquid to a metering unit, and sending the mesh liquid to a melt-blowing unit, wherein the spinning head of the melt-blowing unit sprays mesh and adheres to the surface of a rolling composite particle layer wrapped by a water-resisting layer;
(4) when the surface temperature of the water-resisting layer is reduced to 40 ℃, adding the antibacterial powder (the effective viable count of each 2mL is 0.15 multiplied by 10) 8 CFU/mL candida wilt, streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve and bacillus licheniformis and 200g ammonium bicarbonate pore-forming agent, and when the mixture is cooled to room temperature, a pulp wrapping machine is used for mixing and adhering the pulp B (xanthan gum, cassava starch and glycerol in a mass ratio of 40: 40: 3 mixing, heating and pasting at 70 ℃) to wrap the surface of the water-resisting layer, heating to 60 ℃ to decompose the pore-forming agent, discharging generated gas, and cooling to room temperature to obtain the composite cocci preparation.
The method of making a synechococcus formulation of examples 5-8 comprising the steps of:
the preparation process of pediococcus comprises the following steps: activating lactobacillus with MRS liquid culture medium, and diluting to obtain 2.5 × 10 8 Inoculating the CFU/mL bacterial solution into an MRS liquid culture medium (MRS liquid culture medium: 10.0g/L peptone, 8.0g/L beef extract, 4.0g/L yeast extract, 20.0g/L glucose, 2.0g/L dipotassium hydrogen phosphate, 2.0g/L diammonium hydrogen citrate, 5.0 g/L sodium acetate, 0.2g/L magnesium sulfate, 0.04 g/L manganese sulfate and 80.0 g/L Tween), culturing at 37 ℃ for 24 h, centrifuging and filtering at 4 ℃, taking 500 muL lactobacillus supernatant, adding into Oxford cupule of an upper plate, taking vibrio and cellulose as indicator bacteria, and inoculating the indicator bacteria at the concentration of 0.5 x 10 8 And (3) screening out strains with obvious inhibition zones for each indicator bacterium, culturing for 12 hours in an incubator at 37 ℃, measuring the diameter D of the transparent ring by using a cross method, and screening out strains with obvious inhibition zones for each indicator bacterium to obtain pediococcus.
And (3) calculating the viable count: activating pediococcus, clostridium butyricum, bacillus belvesii and bacillus subtilis, respectively picking strains on each plate lawn in an MRS liquid culture medium, culturing for 40h, centrifuging the bacteria liquid for 10 min at 3000 r/min, washing, precipitating, suspending the precipitate in a phosphate balanced physiological saline buffer solution, diluting by 50 times, sucking 0.1mL of diluent, uniformly coating the diluent on a solid culture medium, inversely culturing for 48h in a biochemical incubator, counting, measuring the optical density value (OD 600 value) of the bacteria solution at a wavelength of 600nm of a visible spectrophotometer, linearly relating the optical density value (OD 600 value) of the bacteria solution with the number of live bacteria in suspension, and directly calculating the number of bacteria in the bacteria liquid by using the OD600 value in a later culture test.
(1) Adding 20kg of filler into 100kg of composite coccus granules, stirring, adhering the granules by the filler, sieving, extruding, taking undersize, and drying to obtain a composite granule layer (the mixture ratio of the granules, the filler and the matrix materials forming the granules is shown in table 1);
(2) coating the surface of the composite particle layer with slurry A (prepared by mixing corn starch glue, carboxymethyl cellulose and glycerol according to a mass ratio of 40: 10:3 and heating and pasting at 80 ℃) by using a slurry coating machine, and cooling to room temperature to obtain a water-resisting layer coated composite particle layer;
(3) heating a mesh agent (obtained by mixing carrageenan and water according to the mass/volume ratio of 5 g: 60 ml) to 80 ℃ to melt to obtain mesh liquid, sending the mesh liquid to a feeding unit of a spinning machine, extruding the mesh liquid by the feeding unit, sending the mesh liquid to a metering unit, and sending the mesh liquid to a melt-blowing unit, wherein the spinning head of the melt-blowing unit sprays mesh and adheres to the surface of a rolling composite particle layer wrapped by a water-resisting layer;
(4) when the surface temperature of the water-resisting layer is reduced to 40 ℃, adding antibacterial powder (2 mL of each effective viable count is 0.15 multiplied by 10) 8 CFU/mL candida wilt, streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve and bacillus licheniformis and 200g ammonium bicarbonate pore-forming agent, and when the mixture is cooled to room temperature, a pulp wrapping machine is used for mixing and adhering the pulp B (xanthan gum, cassava starch and glycerol in a mass ratio of 40: 40: 3 mixing, heating at 70 deg.C for gelatinization) wrapping on the surface of the water-resisting layer, heating to 60 deg.C for decomposing pore-forming agent, and dischargingCooling the crude gas to room temperature to obtain the Synechococcus preparation.
A method of making a synechococcus formulation according to examples 9-11 comprising the steps of:
the preparation process of pediococcus comprises the following steps: activating lactobacillus with MRS liquid culture medium, and diluting to obtain 2.5 × 10 8 Inoculating the CFU/mL bacterial solution into an MRS liquid culture medium (MRS liquid culture medium: 10.0g/L peptone, 8.0g/L beef extract, 4.0g/L yeast extract, 20.0g/L glucose, 2.0g/L dipotassium hydrogen phosphate, 2.0g/L diammonium hydrogen citrate, 5.0 g/L sodium acetate, 0.2g/L magnesium sulfate, 0.04 g/L manganese sulfate and 80.0 g/L Tween), culturing at 37 ℃ for 24 h, centrifuging and filtering at 4 ℃, taking 500 muL lactobacillus supernatant, adding into Oxford cupule of an upper plate, using vibrio and cellulose as indicator bacteria, and inoculating the indicator bacteria at a concentration of 1.0 x 10 8 And (3) screening out strains with obvious inhibition zones for each indicator bacterium, culturing for 12 hours in an incubator at 37 ℃, measuring the diameter D of the transparent ring by using a cross method, and screening out strains with obvious inhibition zones for each indicator bacterium to obtain pediococcus.
And (3) calculating the viable count: activating pediococcus, clostridium butyricum, bacillus belvesii and bacillus subtilis, respectively picking strains on each plate lawn in an MRS liquid culture medium, culturing for 40h, centrifuging the bacteria liquid for 10 min at 3000 r/min, washing, precipitating, suspending the precipitate in a phosphate balanced physiological saline buffer solution, diluting by 50 times, sucking 0.1mL of diluent, uniformly coating the diluent on a solid culture medium, inversely culturing for 48h in a biochemical incubator, counting, measuring the optical density value (OD 600 value) of the bacteria solution at a wavelength of 600nm of a visible spectrophotometer, linearly relating the optical density value (OD 600 value) of the bacteria solution with the number of live bacteria in suspension, and directly calculating the number of bacteria in the bacteria liquid by using the OD600 value in a later culture test.
(1) Adding 20kg of filler into 100kg of composite cocci granules, stirring, bonding the granules by the filler, then sieving, extruding, taking undersize, and drying to obtain a composite granule layer (the mixture ratio of the granules, the filler and the matrix material forming the granules is shown in table 1);
(2) coating the surface of the composite particle layer with slurry A (prepared by mixing corn starch glue, carboxymethyl cellulose and glycerol according to a mass ratio of 40: 10:3 and heating and pasting at 80 ℃) by using a slurry coating machine, and cooling to room temperature to obtain a water-resisting layer coated composite particle layer;
(3) heating a mesh agent (obtained by mixing carrageenan and water according to the mass/volume ratio of 5 g: 60 ml) to 80 ℃ to melt to obtain mesh liquid, sending the mesh liquid to a feeding unit of a spinning machine, extruding the mesh liquid by the feeding unit, sending the mesh liquid to a metering unit, and sending the mesh liquid to a melt-blowing unit, wherein the spinning head of the melt-blowing unit sprays mesh and adheres to the surface of a rolling composite particle layer wrapped by a water-resisting layer;
(4) when the surface temperature of the water-resisting layer is reduced to 40 ℃, adding antibacterial powder (2 mL of each effective viable count is 0.15 multiplied by 10) 8 CFU/mL candida wilt, streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve and bacillus licheniformis and 200g ammonium bicarbonate pore-forming agent, and when the mixture is cooled to room temperature, a pulp wrapping machine is used for mixing and adhering the pulp B (xanthan gum, cassava starch and glycerol in a mass ratio of 40: 40: 3 mixing, heating and pasting at 70 ℃) to wrap the surface of the water-resisting layer, heating to 60 ℃ to decompose the pore-forming agent, discharging generated gas, and cooling to room temperature to obtain the composite cocci preparation.
A method of making a micrococcus formulation of examples 12-14, comprising the steps of:
the preparation process of pediococcus comprises the following steps: activating lactobacillus with MRS liquid culture medium, and diluting to obtain 2.5 × 10 8 CFU/mL bacterial solution, inoculating in MRS liquid culture medium (MRS liquid culture medium: peptone 10.0g/L, beef extract 8.0g/L, yeast extract 4.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0 g/L, magnesium sulfate 0.2g/L, manganese sulfate 0.04 g/L, Tween 80.0 g/L), culturing at 37 deg.C for 24 h, centrifuging at 4 deg.C, filtering, collecting lactobacillus supernatant 500 μ L, adding into Oxford cup hole of upper plate, using Vibrio and Cellular as indicator bacteria, and the inoculation concentration of indicator bacteria is 1.0 × 10 8 CFU/mL and volume of 100 mu L are respectively selected, strains with obvious inhibition zones for various indicator bacteria are screened out, and the strains are cultured for 12 hours in an incubator at 37 ℃ to benefitAnd measuring the diameter D of the transparent circle by a cross method, and screening strains with obvious inhibition zones of the indicator bacteria to obtain the pediococcus.
And (3) calculating the viable count: activating pediococcus, clostridium butyricum, bacillus belgii and bacillus subtilis, respectively picking strains on each flat lawn of the pediococcus to be cultured in an MRS liquid culture medium for 40h, centrifuging the bacteria liquid for 10 min at 3000 r/min, washing, precipitating, suspending precipitates in a phosphate balanced physiological saline buffer solution, diluting by 50 times, absorbing 0.1mL of diluent to be uniformly coated on a solid culture medium, counting after culturing in a biochemical incubator for 48h in an inverted mode, measuring the optical density value (OD 600 value) of the bacteria solution at a 600nm wavelength of a visible spectrophotometer according to the linear relation of the OD600 value and the number of live bacteria in suspension, and directly calculating the number of bacteria in the bacteria liquid by using the OD600 value in the subsequent culture test.
(1) Adding 20kg of filler into 100kg of composite cocci granules, stirring, bonding the granules by the filler, then sieving, extruding, taking undersize, and drying to obtain a composite granule layer (the mixture ratio of the granules, the filler and the matrix material forming the granules is shown in table 1);
(2) coating the surface of the composite particle layer with slurry A (prepared by mixing corn starch glue, carboxymethyl cellulose and glycerol according to a mass ratio of 40: 10:3 and heating and pasting at 80 ℃) by using a slurry coating machine, and cooling to room temperature to obtain a water-resisting layer coated composite particle layer;
(3) heating a mesh agent (obtained by mixing carrageenan and water according to the mass/volume ratio of 5 g: 60 ml) to 80 ℃ to melt to obtain mesh liquid, sending the mesh liquid to a feeding unit of a spinning machine, extruding the mesh liquid by the feeding unit, sending the mesh liquid to a metering unit, and sending the mesh liquid to a melt-blowing unit, wherein the spinning head of the melt-blowing unit sprays mesh and adheres to the surface of a rolling composite particle layer wrapped by a water-resisting layer;
(4) when the surface temperature of the water-resisting layer is reduced to 40 ℃, adding antibacterial powder (2 mL of each effective viable count is 0.15 multiplied by 10) 8 CFU/mL Candida wilt, Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium breve, Bacillus licheniformis), 200g ammonium bicarbonate pore-forming agent, cooling to room temperature, and coating slurry B (C), (B) with a coating slurry machineXanthan gum, cassava starch and glycerol are mixed according to the mass ratio of 40: 40: 3 mixing, heating and pasting at 70 ℃) to wrap the surface of the water-resisting layer, heating to 60 ℃ to decompose the pore-forming agent, discharging generated gas, and cooling to room temperature to obtain the composite sheet coccus preparation.
Comparative example 1
Comparative example 1 differs from example 3 in that no pediococcus was added to the granules of the synechococcus formulation.
Comparative example 2
This comparative example 2 differs from example 7 in that no pediococcus, clostridium butyricum, bacillus beijerinckii, bacillus subtilis were added to the particles of the synechococcus preparation.
Comparative example 3
Comparative example 3 differs from example 9 in that no antibacterial powder was added to the water barrier of the Synechococcus preparation.
Comparative example 4
This comparative example differs from example 11 in that no pore former was added to the composite coccal formulation and no microporous layer was formed.
Comparative example 5
This comparative example differs from example 11 in that no water barrier layer was provided for the Synechococcus preparation.
TABLE 1 EXAMPLES 1-14 compounding weight ratios
| Base material | Pig milk powder | Bean dregs | Cottonseed protein | Calcium dihydrogen phosphate | Sodium butyrate | Vitamin preparation | Carboxymethyl cellulose Vitamin sodium | Chicken oil |
| Examples 1-4 | 5 | 10 | 20 | 1.2 | 0.4 | 0.01 | 0.01 | 1.5 |
| Examples 5-8 | 10 | 12 | 30 | 1.5 | 0.6 | 0.02 | 0.04 | 2 |
| Examples 9-11 | 15 | 18 | 35 | 2 | 0.7 | 0.03 | 0.05 | 2.5 |
| Examples 12-14 | 20 | 20 | 40 | 3 | 0.8 | 0.03 | 0.05 | 3 |
| Granules | Effective activity of Pediococcus Number of bacteria | Clostridium butyricum has Number of effective viable bacteria | Bacillus belgii Effective number of viable bacteria | Bacillus subtilis Effective viable count | Base material | |||
| Examples 1-4 | 1.2×10 8 CFU/ mL | 0.5× 10 8 CFU/mL | 0.1×10 8 CFU/mL | 0.1×10 8 CFU/ mL | Examples 1 to 4 each independently 40kg of, 50kg、 60kg、 70kg | |||
| Examples 5-8 | 2×10 8 CFU/mL | 1.0× 10 8 CFU/mL | 0.3×10 8 CFU/mL | 0.3×10 8 CFU/ mL | Examples 5 to 8 each 40kg of, 60kg、 80kg、 100kg | |||
| Examples 9-11 | 3.2×10 8 CFU/ mL | 1.2× 10 8 CFU/mL | 0.4×10 8 CFU/mL | 0.4×10 8 CFU/ mL | ||||
| Examples 12-14 | 6×10 8 CFU/mL | 1.5× 10 8 CFU/mL | 1×10 8 CFU/mL | 1×10 8 CFU/mL | ||||
| Filler material | Pig milk powder | Cottonseed protein | Sodium alginate | water/L | ||||
| Examples 1-4 | 8 | 25 | 3 | 20 | ||||
| Examples 5-8 | 15 | 40 | 6 | 25 | ||||
| Examples 9-11 | 15 | 50 | 8 | 35 | ||||
| Examples 12-14 | 18 | 60 | 8 | 40 |
TABLE 2 examples 1-14 Comptococcus preparations diameter or thickness in each structure
| Examples 1 to 4 | Examples 5 to 8 | Examples 9 to 11 | Examples 12 to 14 | |
| Particle diameter | 0.27~1.3mm | 0.31~17mm | 0.26~1.3mm | 0.25~1.69mm |
| Diameter of composite particle layer | 0.63~3.7mm | 0.64~3.9mm | 0.66~3.8mm | 0.71~4.3mm |
| Thickness of water barrier layer | 0.36~0.98mm | 0.46~1.28mm | 0.32~1.75mm | 0.40~1.64mm |
| Thickness of the cross-hatch layer | 0.15~0.3mm | 0.12~0.49mm | 0.27~0.84mm | 0.32~0.73mm |
| Microporous layer | 0.34~0.66mm | 0.33~0.70mm | 0.38~0.76mm | 0.33~0.78mm |
And (3) data analysis:
1.1 feeding treatment of cultured weever
The water for culturing weever is culture water filtered by sand surface, and is aerated for one day, and has 19 culture tanks, each culture tank is a water tank with length, width and depth of 2m, and the water amount of each culture tank is 6m 3 Half of the water in the culture tank is updated every 15 days; the size of the screened weever is 14-15 cm/tail, the mass of the weever is 185-195 g/tail, all the weever is domesticated for 7 days and then is put into culture tanks, 30 weever is put into each culture tank (the average mass of the weever in each tank is recorded), and 5mL of vibrio parahaemolyticus bacterial liquid is put into the culture tanks (the viable count of the vibrio parahaemolyticus diluted by the sterile physiological saline solution is 1.0-1.2 multiplied by 10) 6 CFU/mL). The average weight of the weever per day is calculated by randomly taking 3 weever in each culture tank and calculating the total weight/3 of the 3 weever per day, the preparations prepared in examples 1-14 and comparative examples 1-5 are respectively fed into 19 water tanks, the composite sheet coccus preparations prepared in examples 1-14 are fed into the corresponding examples 1-14 water tanks, the preparations prepared in comparative examples 1-5 are fed into the corresponding comparative examples 1-5 water tanks, and the mass of the preparations fed by each culture tank per day is as follows: the average weight of the weever is multiplied by 30 multiplied by 3.5 percent, and the weever is fed once at 8, 12 and 16 points every day for 30 consecutive days.
1.2 index determination
Randomly taking 5 fish from the culture tanks of examples 1-14 and comparative examples 1-5, putting the fish into 3L of water, anesthetizing the weever by using alkyl sulfonate, adding 0.2mL of alkyl sulfonate into each L of water, anesthetizing the weever, treating the surface of the weever by using alcohol disinfectant, taking out the intestinal tract of the fish, extruding the intestinal contents, putting the intestinal contents into a sterile centrifuge tube, quickly freezing by using liquid nitrogen, and freezing and storing at ultralow temperature (minus 40 ℃) for analysis of intestinal flora change. Total DNA of the microorganisms in the intestinal contents was extracted using the Mag-Bind Soil DNA Kit. Scraping secretion and mucous membrane in the middle intestinal tract by using a knife, placing in a low-temperature refrigerator for standing for 4 h, centrifuging at 4 ℃, collecting supernatant, and measuring trypsin (trypsin) activity, Amylase (AMS) activity and Lipase (LPS) activity by using an ultraviolet colorimetric method, a starch-iodine colorimetric method and a nanogold colorimetric method by using an ultraviolet colorimetric method.
Randomly taking 5 fish from each culture tank corresponding to the control group and the test group, taking blood from veins, collecting blood sample of the weever, placing the blood sample in a low-temperature refrigerator for standing for 4 h, centrifuging at 4 ℃, and collecting the upper serum for detecting serum immunity indexes. Measuring macrophage inflammatory protein-1 beta (MIP-1 beta), Total Protein (TP) and Globulin (GLB) in serum by adopting an enzyme-linked immunosorbent assay method; glutathione peroxidase is measured by a glutathione peroxidase (GSH-PX) measuring kit (a colorimetric method), Catalase (CAT) is measured by a catalase kit (an ultraviolet light method), and superoxide dismutase (SOD) is measured by a superoxide dismutase measuring kit (a hydroxylamine method).
Randomly taking 5 weever from each culture tank corresponding to the control group and the test group, and measuring the length (cm) of the weever, the mass (g) of the weever, the growth rate and the survival rate in each culture tank, wherein the length (g) of the weever, the mass (g) of the weever and the growth rate are averaged, the survival rate = the number of the weever fed on the 30 th day/the number of the weever fed on the 1 st day 100%, and the growth rate = (the average mass of the weever fed on the 30 th day-the average mass of the weever fed on the 1 st day)/30; and observing and recording the scaly skin and spleen of the weever.
1.3 determination of the Water in suspension of the preparation
10 of each of the preparations prepared in examples 1 to 14 and comparative examples 1 to 5 were placed in a water tank containing water for cultivation, the water tank was placed near a cultivation tank, the water tanks were kept at the same humidity and temperature, the time required for the preparations prepared in examples 1 to 14 and comparative examples 1 to 5 to sink water was recorded, and the time for the microporous layer of the preparations of examples 1 to 14 to first dissolve was recorded. Wherein 50% of the preparation is settled for 50% of the time required for settling, and 100% of the preparation is settled for the total time required for settling.
1.4 Water quality determination in culture tanks
The sampling time of the culture water sample is 10: 30-11: 30 in the morning on the 31 st day after the preparation is put in, the water sample is collected at a position 50cm below the water surface in the center of the culture tank, total nitrogen, ammonia nitrogen and COD are measured by a portable water quality multi-parameter detector, dissolved oxygen is measured by a dissolved oxygen analyzer, and pH is measured by a pH value and pH detector.
Serum was centrifuged at 2000 g (centrifuge radius 8.5 cm). The serum MIP-1 alpha, MIP-1 beta and MCP-1 index levels are measured by adopting an enzyme-linked immunosorbent assay, an experimental kit is provided by Beijing kang peptide biotechnology limited, and the experimental steps are strictly carried out according to the kit specification.
And (3) chart analysis:
in fig. 1 and 4, the particles are wrapped by the filler to obtain a particle layer, a water-proof layer is arranged outside the particle layer, and a reticulate pattern layer wraps the water-proof layer, wherein in fig. 2, the reticulate pattern layer contains mesh concave points, and in fig. 2, a microporous layer is wrapped outside the reticulate pattern layer and is provided with a plurality of pores; fig. 5-8 show the filler coating 2, 3, 4, 5 particles of the composite cocci preparation. The intestinal flora provides a habitat, and the intestinal microorganisms have irreplaceable influence on the nutrition, health, immunity and the like of a host. In table 1, compared with comparative examples 1 to 5, the intestinal microorganisms of the preparations prepared in examples 1 to 14 have increased abundance of firmicutes, proteobacteria, bacteroidetes and gibberella, and are all dominant microorganisms, so that intestinal microorganisms of the dominant microorganisms of fusobacteria, actinomycetes and firmicutes, proteobacteria, bacteroidetes and gibberella have irreplaceable effects on nutrition, immunity and the like of hosts, and have certain correlation with the health of the hosts. In contrast, in the preparation of comparative example 2, pediococcus, clostridium butyricum, bacillus belgii and bacillus subtilis are not added, and the abundance of each firmicutes, proteobacteria, bacteroidetes and gibberella in intestinal tracts is lowest, which indicates that the addition of pediococcus, clostridium butyricum, bacillus belgii and bacillus subtilis has the greatest change to the intestinal microorganisms, and is beneficial to the growth of dominant microorganisms; compared with comparative examples 1 to 3, the micrococcus preparation prepared in comparative example 4 has only no microporous layer, the micrococcus preparation prepared in comparative example 5 has only no water-stop layer, the firmicutes phylum, proteobacteria, bacteroidetes and gibberella are slightly increased, and the fusobacteria and actinomycetes are slightly decreased, so that the micrococcus preparation has a certain effect on intestinal microorganisms due to the removal of the microporous layer and the water-stop layer.
TABLE 3 Perch intestinal enzyme Activity
| Trypsin (U/mg prot) | Amylase (U/mg prot) | Lipase (U/mg prot) | |
| Example 1 | 145.7 | 4.70 | 68.4 |
| Example 2 | 137.8 | 4.20 | 60.5 |
| Example 3 | 136.4 | 4.40 | 64.7 |
| Example 4 | 143.2 | 4.62 | 67.0 |
| Example 5 | 146.8 | 4.88 | 67.7 |
| Example 6 | 148.0 | 4.68 | 66.1 |
| Example 7 | 148.8 | 4.76 | 66.9 |
| Example 8 | 150.5 | 4.81 | 71.5 |
| Example 9 | 145.7 | 4.72 | 68.2 |
| Example 10 | 146.8 | 4.67 | 67.7 |
| Example 11 | 141.1 | 4.52 | 63.5 |
| Example 12 | 146.8 | 4.88 | 68.7 |
| Example 13 | 144.7 | 4.62 | 68.2 |
| Example 14 | 145.7 | 4.70 | 67.7 |
| Comparative example 1 | 126.0 | 4.19 | 60.7 |
| Comparative example 2 | 130.4 | 4.21 | 60.3 |
| Comparative example 3 | 133.7 | 4.17 | 62.6 |
| Comparative example 4 | 136.5 | 4.39 | 62.9 |
| Comparative example 5 | 129.9 | 4.46 | 63.3 |
TABLE 4 Perch serum immune index data
| Macrophage inflammatory protein-1 beta (ng/L) | Total protein (g/L) | Globulin (g/L) | Glutathione peroxidase ((U/mL)) | Catalase (U/mL) | Superoxide dismutase (U/mL) | |
| Example 1 | 47.9 | 36.13 | 26.14 | 342 | 3.78 | 26.3 |
| Example 2 | 38.1 | 35.86 | 25.78 | 336 | 3.18 | 25.4 |
| Example 3 | 41.7 | 35.94 | 26.48 | 337 | 3.63 | 24.5 |
| Example 4 | 44.4 | 36.40 | 26.84 | 360 | 3.62 | 25.9 |
| Example 5 | 41.7 | 36.05 | 26.25 | 339 | 3.91 | 23.1 |
| Example 6 | 48.0 | 36.13 | 25.92 | 342 | 3.79 | 25.5 |
| Example 7 | 46.8 | 35.86 | 26.42 | 351 | 3.58 | 25.4 |
| Example 8 | 49.8 | 36.07 | 26.39 | 352 | 4.01 | 26.2 |
| Example 9 | 45.0 | 35.72 | 26.64 | 347 | 3.86 | 24.9 |
| Example 10 | 44.2 | 35.80 | 25.76 | 345 | 3.69 | 25.3 |
| Example 11 | 42.5 | 35.80 | 26.38 | 353 | 3.76 | 24.7 |
| Example 12 | 46.0 | 36.03 | 26.86 | 334 | 3.65 | 25.2 |
| Example 13 | 41.7 | 35.68 | 26.30 | 336 | 3.68 | 24.6 |
| Example 14 | 47.8 | 35.25 | 25.64 | 321 | 3.69 | 25.7 |
| Comparative example 1 | 34.5 | 29.98 | 23.48 | 307 | 3.25 | 22.3 |
| Comparative example 2 | 34.3 | 28.23 | 23.24 | 294 | 3.32 | 21.5 |
| Comparative example 3 | 37.3 | 32.69 | 25.16 | 297 | 3.08 | 22.8 |
| Comparative example 4 | 33.8 | 33.72 | 24.86 | 309 | 3.26 | 21.1 |
| Comparative example 5 | 36.7 | 33.53 | 24.75 | 300 | 2.92 | 21.3 |
Table 3 shows the intestinal enzyme activity of the weever cultured by the preparations prepared in examples 1-14 and comparative examples 1-5, Table 4 shows the serum immunity index data of the weever cultured by the preparations prepared in examples 1-14 and comparative examples 1-5, and the intestinal trypsin, amylase, lipase and macrophage inflammatory protein, total protein, globulin, glutathione peroxidase, catalase and superoxide dismutase in the weever intestinal tract of the preparations prepared in examples 1-14 are all increased compared with the intestinal enzyme activity and serum immunity index of the weever cultured by the preparations prepared in comparative examples 1-5, which shows that the pleococcus preparation prepared in examples 1-14 of the invention enhances the humoral immunity and cellular immunity of the weever, and is poorer than the intestinal enzyme activity and serum immunity index of the weever prepared in comparative examples 1-2, this is similar to the situation in fig. 9.
TABLE 5 growth of weever
| Length (cm) | Quality (g) | Growth rate (g/d) | Survival rate (%) | Scaly skin and spleen | |
| Example 1 | 30.1 | 389 | 6.49 | 96.7 | Scaly skin and spleen are intact |
| Example 2 | 28.9 | 346 | 5.09 | 100.0 | The scaly skin and spleen are intact |
| Example 3 | 29.5 | 367 | 5.76 | 100.0 | The scaly skin and spleen are intact |
| Example 4 | 29.9 | 382 | 6.12 | 96.7 | The scaly skin and spleen are intact |
| Example 5 | 30.1 | 389 | 6.49 | 100.0 | The scaly skin and spleen are intact |
| Example 6 | 30.1 | 390 | 6.51 | 96.7 | The scaly skin and spleen are intact |
| Example 7 | 30.3 | 397 | 6.97 | 100.0 | The scaly skin and spleen are intact |
| Example 8 | 30.4 | 405 | 6.97 | 100.0 | The scaly skin and spleen are intact |
| Example 9 | 30.1 | 389 | 6.62 | 96.7 | Scaly skin and spleen are intact |
| Example 10 | 30.2 | 397 | 6.70 | 100.0 | The scaly skin and spleen are intact |
| Example 11 | 29.7 | 374 | 6.02 | 100.0 | The scaly skin and spleen are intact |
| Example 12 | 29.0 | 349 | 5.14 | 100.0 | 1 small part of the tail with discolored scaly skin and intact other spleens |
| Example 13 | 29.9 | 382 | 6.33 | 96.7 | Scaly skin and spleen are intact |
| Example 14 | 29.4 | 363 | 5.48 | 96.7 | The scaly skin and spleen are intact |
| Comparative example 1 | 28.6 | 334 | 4.65 | 86.7 | Discoloration of 7 small parts of scales, 1-tail scale falling off and 3-tail spleen slight bleeding |
| Comparative example 2 | 29.0 | 325 | 4.31 | 83.3 | 9 small parts of scaly skin fade, 2 tail scales fall off, and 6 spleen slight bleeding |
| Comparative example 3 | 28.3 | 351 | 5.43 | 86.7 | With 6 small parts of scale discolored and spleen intact |
| Comparative example 4 | 28.1 | 320 | 5.32 | 93.3 | With 3 small parts of the scale discolored and the spleen intact |
| Comparative example 5 | 28.1 | 318 | 5.20 | 90.0 | 2 small part of the tail is discolored and the spleen is intact |
Table 5 shows the growth of the weever cultured by the preparations prepared in examples 1-14 and comparative examples 1-5, after the weever is fed for 30 days, the growth and survival rate of the weever cultured by the preparations prepared in examples 1-14 are better, the growth rate of the weever cultured by the preparations prepared in comparative examples 1 and 2 is lower, and the survival rate of the weever treated by the preparation prepared in comparative example 2 is the lowest. From observation of the scales and spleens of the weever, only 1 small part of the scales of the weever cultured by the preparation prepared in example 12 faded, and the small parts of the scales of the multi-tail weever, which are faded, exfoliated and slightly bleeding from the spleens, of the weever cultured by the preparations prepared in comparative examples 1 to 5. The discoloration of the scaly skin, the flaking off and the slight bleeding of the spleen are the manifestations of the symptom of the parahemolytic vibrio of the weever, which indicates that the preparation prepared in the examples 1 to 14 has better effect on inhibiting the parahemolytic vibrio.
TABLE 6 time for the formulation to settle and the microporous layer to dissolve
| Sink 100% (d) | Sink 50% (d) | First dissolution time (h) | |
| Example 1 | 15 | 6 | 26 |
| Example 2 | 15 | 7 | 25 |
| Example 3 | 14 | 7 | 23 |
| Example 4 | 13 | 6 | 30 |
| Example 5 | 16 | 6 | 26 |
| Example 6 | 13 | 6 | 31 |
| Example 7 | 15 | 5 | 26 |
| Example 8 | 14 | 8 | 28 |
| Example 9 | 15 | 5 | 26 |
| Example 10 | 16 | 8 | 27 |
| Example 11 | 16 | 5 | 24 |
| Example 12 | 15 | 5 | 23 |
| Example 13 | 14 | 5 | 28 |
| Example 14 | 15 | 7 | 24 |
| Comparative example 1 | 15 | 7 | 26 |
| Comparative example 2 | 15 | 6 | 25 |
| Comparative example 3 | 13 | 6 | 28 |
| Comparative example 4 | 23 | 14 | 37 |
| Comparative example 5 | 9 | 3 | 14 |
Table 6 shows the sinking and dissolution times of the microporous layers of the formulations prepared in examples 1 to 14 and comparative examples 1 to 5, 50% and 100% of the formulations prepared in examples 1 to 14, 50% and 100% of the first dissolution times of 5 to 8 days, 13 to 16 days and 23 to 31 hours, respectively, 50% and 100% of the formulations prepared in comparative examples 1 to 3, and 50% and 100% of the first dissolution times of 6 to 7 days, 13 to 15 days and 26 to 28 hours, respectively, are not different from the 50% and 100% of the formulations prepared in examples 1 to 14, and the first dissolution time is not different from the first dissolution time of the formulations prepared in examples 1 to 14, so that the floating state is better, while the formulations prepared in comparative example 4, which has no microporous layer, has 50% and 100% of the first dissolution time, and the composite coccus preparation prepared in comparative example 5, has no water barrier, resulting in 50% and 100% of the shortest first dissolution time and fastest water of the formulations, is not beneficial to the floating of the preparation.
Table 7 water quality data of each water tank
| pH | Dissolved oxygen (mg/L) | Total nitrogen (mg/L) | Ammonia nitrogen (mg/L) | COD（mg/L） | |
| Example 1 | 7.41 | 6.37 | 2.66 | 0.86 | 6.36 |
| Example 2 | 7.37 | 6.58 | 2.72 | 0.87 | 6.60 |
| Example 3 | 7.66 | 6.64 | 2.72 | 0.85 | 6.05 |
| Example 4 | 7.57 | 6.51 | 2.75 | 0.78 | 6.78 |
| Example 5 | 7.60 | 6.38 | 2.76 | 0.85 | 6.06 |
| Example 6 | 7.42 | 6.52 | 2.78 | 0.82 | 6.87 |
| Example 7 | 7.16 | 6.49 | 2.72 | 0.85 | 6.21 |
| Example 8 | 7.46 | 6.97 | 2.86 | 0.81 | 6.10 |
| Example 9 | 7.63 | 6.42 | 2.83 | 0.88 | 6.53 |
| Example 10 | 7.47 | 6.67 | 2.69 | 0.83 | 5.74 |
| Example 11 | 7.60 | 6.43 | 2.79 | 0.87 | 6.46 |
| Example 12 | 7.45 | 6.52 | 2.83 | 0.73 | 6.78 |
| Example 13 | 7.34 | 6.57 | 2.88 | 0.82 | 7.15 |
| Example 14 | 7.58 | 6.72 | 2.80 | 0.87 | 6.74 |
| Comparative example 1 | 7.76 | 6.64 | 3.26 | 0.98 | 7.40 |
| Comparative example 2 | 7.71 | 6.56 | 3.19 | 0.96 | 7.99 |
| Comparative example 3 | 7.81 | 5.68 | 3.53 | 0.98 | 7.73 |
| Comparative example 4 | 8.32 | 5.35 | 3.83 | 1.06 | 6.62 |
| Comparative example 5 | 7.37 | 5.72 | 2.85 | 0.94 | 6.93 |
Table 7 shows the cases of the tanks into which the formulations prepared in examples 1 to 14 and comparative examples 1 to 5 were fed, compared to comparative examples 1 to 5, the water quality of each tank was visible in pH, dissolved oxygen, total nitrogen, ammonia nitrogen, and COD, the cases of examples 1 to 14 were better, and the water quality of the tank into which the formulation prepared in comparative example 4 was fed was worse, and it was likely that the formulation prepared in comparative example 4 was most rapidly dissolved in water, resulting in poor water quality.
The present invention is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.
Claims (3)
1. A method for preparing a complex coccus preparation is characterized by comprising the following steps:
(1) mixing the composite coccus particles with the filler, stirring, adhering, sieving and extruding, and drying undersize to obtain a composite particle layer;
(2) coating the slurry A on the surface of the composite particle layer, and cooling to obtain a water-resisting layer coated composite particle layer;
(3) heating and melting the mesh agent, sending the mesh agent into a spinning machine to spray out mesh, and adhering the mesh agent to the surface of the water-resisting layer wrapping the composite particle layer to obtain a reticulate pattern layer;
(4) reducing the surface temperature of the reticulate pattern layer to 40-50 ℃, adding antibacterial powder and a pore-forming agent, mixing, continuously cooling, spraying slurry B, heating to 50-60 ℃, and cooling to obtain a composite sheet coccus preparation; the slurry A comprises corn starch glue, carboxymethyl cellulose and glycerol; the slurry B comprises xanthan gum, tapioca starch and glycerol; in the step (1), the preparation process of the composite coccus particles comprises the following steps: the effective viable count is 0.5-8 × 10 8 The effective viable count of the CFU/mL pediococcus is 0.01-2 multiplied by 10 8 The effective viable count of the Clostridium butyricum is 0.01-1 multiplied by 10 8 CFU/mL Bacillus belgii with effective viable count of 0.01-1 × 10 8 Mixing CFU/mL bacillus subtilis and 20-100 kg of matrix material, and performing spray granulation to obtain the bacillus subtilis preparation; in the step (1), the filling material consists of pig plasma powder, cottonseed protein, sodium alginate and water; in the step (3), the mesh agent is carrageenan, and the mass/volume ratio of water is (3-10) g: (50-80) ml; in the step (4), the antibacterial powder consists of candida wilt, streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve and bacillus licheniformis; the base material is prepared from pig milk powder, bean dregs, cottonseed protein, monocalcium phosphate, sodium butyrate, vitamins, sodium carboxymethylcellulose and chicken fat in a mass ratio of (5-20): (5-20): (20-40): (1-3): (0.1-0.8): (0.01-0.03): (0.01-0.05): (1-3); in the step (4), the pore-forming agent is at least one of ammonium bicarbonate and ammonium carbonate.
2. The preparation method of claim 1, wherein the preparation steps of the pig starch are as follows: (1) crushing pig bones and pig viscera, sieving, adding ethanol, mixing, evaporating at 55-70 ℃, adding de-lipase water, mixing, separating, and concentrating to obtain a concentrated solution; (2) and mixing the concentrated solution with alkaline protease and trypsin, treating at 45-60 ℃ for 3-8 hours to obtain an enzymolysis product, concentrating under negative pressure, and drying to obtain the pig pulp powder.
3. A complex coccus preparation prepared by the preparation method of any one of claims 1-2, wherein the complex coccus preparation comprises a complex particle layer, a water-resisting layer, a reticulate pattern layer and a microporous layer in sequence from inside to outside; the composite granular layer contains at least one composite coccus granule; the shape of the composite coccal preparation is at least one of irregular sphere, ellipsoid, fusiform, long rod shape, short rod shape, sheet shape or block shape.
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