CN115074288B - Anti-diarrhea egg yolk antibody coated lactobacillus preparation and preparation method and application thereof - Google Patents
Anti-diarrhea egg yolk antibody coated lactobacillus preparation and preparation method and application thereof Download PDFInfo
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- CN115074288B CN115074288B CN202210780368.9A CN202210780368A CN115074288B CN 115074288 B CN115074288 B CN 115074288B CN 202210780368 A CN202210780368 A CN 202210780368A CN 115074288 B CN115074288 B CN 115074288B
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- egg yolk
- yolk antibody
- lactobacillus
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- diarrhea virus
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a preparation of anti-diarrhea egg yolk antibody coated lactobacillus, a preparation method and application thereof, and relates to the technical field of feeds. The preparation method comprises the following steps: adding lactobacillus into a solution containing epidemic diarrhea virus egg yolk antibody, stirring and standing, and separating a first coating; mixing the first coating with vegetable oil, stirring, standing, and separating the second coating; mixing the second coating with the glue solution, stirring, standing for solidification, separating and drying. The egg yolk antibody coats the lactobacillus, so that the survival rate of the lactobacillus in gastric acid is improved, the lactobacillus is more beneficial to colonization in intestinal tracts, harmful flora is inhibited, and normal microecological balance is maintained. In addition, the anti-diarrhea egg yolk antibody coated lactobacillus preparation has stable performance, is suitable for reducing and forbidding cultivation, can improve the palatability of daily ration, enhance the immunity of piglet organisms, improve the feed intake and improve the digestion utilization rate of nutrient substances.
Description
Technical Field
The invention relates to the technical field of feeds, in particular to a preparation of anti-diarrhea egg yolk antibody coated lactobacillus, a preparation method and application thereof.
Background
The abuse of antibiotics has caused a series of problems to society, bacterial resistance problems caused by the use of large-scale antibiotics, food safety problems, and in order to avoid tragedy occurrence where no drugs are available, it is necessary to limit the use of antibiotics in large quantities in feeds. According to No. 194 document issued by the national agricultural rural department, the addition of antibiotics is completely forbidden in the feed of China from 1 month and 1 day in 2020, so that the harm caused by abuse of antibiotics is reduced, and the animal source food safety and public health safety are maintained.
Porcine epidemic diarrhea is a hyperacute, infectious enterovirus disease caused by porcine epidemic diarrhea virus. The clinical symptoms of the piglet feed are severe diarrhea, vomiting and dehydration, the death rate of the piglet is 50%, and if the piglet feed is not timely supplied with water, the death rate can be up to 100%. There is no particularly effective treatment for outbreak of epidemic diarrhea virus, and the antibacterial treatment is ineffective. The main control method at present is to vaccinate epidemic diarrhea virus, and the vaccinations comprise: inactivated vaccine, attenuated vaccine, transgenic plant vaccine, lactobacillus vaccine, subunit vaccine, stomach-flow combined vaccine, stomach-flow-round combined vaccine and the like, because the vaccine has slow effect, antigens in the vaccine stimulate the organism to generate fewer antibodies, and the mesentery and lymph nodes thereof can not be stimulated to generate antibodies locally to generate high-efficiency immune protection, the pig group still has high probability of infecting epidemic diarrhea virus after being immunized by the multi-linked vaccine, and the survival rate of piglets in a delivery room is seriously influenced.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide an anti-diarrhea egg yolk antibody coated lactobacillus preparation, a preparation method and application.
The invention is realized in the following way:
In a first aspect, the invention provides a method for preparing an anti-diarrhea egg yolk antibody coated lactobacillus preparation, comprising the following steps: adding lactobacillus into a solution containing epidemic diarrhea virus egg yolk antibody, stirring and standing, and separating a first coating; mixing the first coating with vegetable oil, stirring and standing, and separating the second coating; mixing the second coating with the glue solution, stirring, standing for solidification, separating and drying.
In a second aspect, the present invention provides an anti-diarrhea egg yolk antibody coated lactobacillus preparation prepared by the method for preparing an anti-diarrhea egg yolk antibody coated lactobacillus preparation according to any of the previous embodiments; preferably, the anti-diarrhea egg yolk antibody coated lactobacillus preparation is granular, and the grain size of the preparation is 6-600 mu m.
In a third aspect, the present invention provides the use of the anti-diarrhea egg yolk antibody coated lactobacillus preparation according to the previous embodiments for the preparation of a feed additive or feed.
The invention has the following beneficial effects:
The preparation method of the anti-diarrhea egg yolk antibody coated lactobacillus preparation provided by the application uses an epidemic diarrhea virus egg yolk antibody to carry out coating treatment fusion technology on lactobacillus, and is matched with vegetable oil and glue solution to form a multilayer structure, wherein the first layer is formed by coating the egg yolk antibody on the lactobacillus, the outer surface of the first layer is coated by the vegetable oil, and the outer surface of the vegetable oil is coated by the glue layer. The multilayer coating structure ensures that the lactobacillus is fully protected, the stability is better, the survival rate of the lactobacillus in gastric acid is further improved, the field planting of the lactobacillus in intestinal tracts is further facilitated, the harmful flora is inhibited, and the normal microecological balance is maintained. The preparation method provided by the application is simple, has good condition controllability, low production cost and stable product performance, is suitable for cultivation with resistance reduction and resistance inhibition, has good application prospect in the aspects of medicine residue reduction action and food safety, and can be especially applied to preparation of feed additives or feeds. In addition, the anti-diarrhea egg yolk antibody coated lactobacillus preparation provided by the application can improve the palatability of daily ration, enhance the immunity of piglets, improve the feed intake and improve the digestion utilization rate of nutrient substances.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention provides a preparation method of an anti-diarrhea egg yolk antibody coated lactobacillus preparation, which comprises the following steps:
s1, preparing an epidemic diarrhea virus egg yolk antibody.
Selecting a farrowing house piglet with infection onset at 2-3 days old, aseptically collecting small intestine, shearing, fully grinding, diluting with sterile physiological saline, centrifuging at 7000-9000r/min for 10-20 min, collecting supernatant, adding 0.1-0.2% formaldehyde, and oscillating at 35-40deg.C for 24-28 hr.
Inactivating the collected supernatant, diluting to obtain diluent for inactivating the epidemic diarrhea virus, and mixing the diluent for inactivating the epidemic diarrhea virus with tween to obtain mixed solution as water phase, wherein the tween accounts for 3-5% of the mass of the mixed solution; the oil phase comprises 92-94% of white oil, 5-7% of span 85 and 1-2% of aluminum stearate by mass percent; mixing the water phase and the oil phase according to the proportion of 1:1.4-1.6, and stirring to obtain the epidemic diarrhea virus oil emulsion immune antigen.
Injecting 0.4-0.6mL of epidemic diarrhea virus oil emulsion immune antigen into chicken breast muscle of laying hen as first immunization; injecting 0.8-1.2mL of epidemic diarrhea virus oil emulsion immunization antigen into chicken breast muscle of the laying hen at the 2 nd week and 4 th week respectively for the second immunization and the third immunization, and injecting 1.6-2.4mL of epidemic diarrhea virus oil emulsion immunization antigen into chicken breast muscle and leg muscle of the laying hen at the 6 th week for the fourth immunization; detecting antibody titer, when the antibody titer is more than 128, starting to collect eggs, aseptically collecting yolk, adding pullulan polysaccharide and procyanidine, and uniformly stirring to obtain epidemic diarrhea virus yolk antibody; the mass ratio of the yolk to the pullulan to the procyanidine is 100-120:8-10:0.3-0.5.
By four immunizations, the application stimulates the laying hens to produce more antibodies, thereby producing high-efficiency immune protection. Meanwhile, the addition of the pullulan polysaccharide can ensure that the epidemic diarrhea virus egg yolk antibody has water solubility and viscosity, is easier to form a film, and is easy to coat the lactobacillus subsequently. Furthermore, the addition of the antigen Huang Qingsu enables the epidemic diarrhea virus egg yolk antibody to have the functions of resisting oxidization and eliminating free radicals.
The egg yolk antibody of the epidemic diarrhea virus has similar structure and function as the antibody in the colostrum of the sow, and the capability of adhering antigen and neutralizing virus is far stronger than that of the antibody in sow and hyperimmune serum. Because the digestive system development of the newborn piglets is not complete, the yolk antibody fed by the feed can quickly and effectively permeate the intestinal wall of the newborn piglets, and maintain long-term activity in the intestinal tract. After piglets eat the feed containing the yolk antibody, the long-term passive immunity can be obtained, and the yolk antibody has the characteristics of high yield, strong stability, low cost, safety, no toxicity and the like. The mechanism of action of egg yolk antibodies mainly comprises four aspects: firstly, specific adhesion factors which can specifically bind to pathogens are inactivated, so that the pathogenicity is reduced; secondly, the biological agent can be actively adhered to the cell wall of a specific pathogen, and the structure and the shape of the pathogen are changed, so that the normal biological activity of the pathogen is inhibited; thirdly, the microbial inoculum can be adhered to pili of specific pathogenic bacteria, so that the pathogenic bacteria cannot be adhered to intestinal wall epithelial cells, and the microbial inoculum plays a role in inhibiting the colonization of the pathogenic bacteria; fourthly, the yolk antibody part which is eaten orally is decomposed into small peptide fragments by digestive enzymes, and the small peptide fragments are easily absorbed by organisms, enter a blood circulation system through intestinal walls, are transported to a specific area to be combined with specific pathogen adhesion factors, so that pathogens lose pathogenicity, and the organisms are protected from being damaged by external pathogens.
S2, preparing a first coating.
Adding lactobacillus into a solution containing epidemic diarrhea virus egg yolk antibody, stirring, standing, and separating the first coating.
Specifically, adding the epidemic diarrhea virus egg yolk antibody into the water solution of the skimmed milk powder, adding the transglutaminase, and uniformly stirring to obtain a solution containing the epidemic diarrhea virus egg yolk antibody; adding lactobacillus into a solution containing epidemic diarrhea virus egg yolk antibody, and then adding CaCl 2; stirring at 150-170r/min for 3-5 min min, standing for 30-50min, cleaning, and filtering to obtain the first coating.
Wherein, the skim milk powder is selected as a carrier, so that the epidemic diarrhea virus egg yolk antibody can be better loaded, and the mass volume ratio of the epidemic diarrhea virus egg yolk antibody to the water solution of the skim milk powder is (5-6) g: (18-22) ml. The transglutaminase can modify protein, improve functionality and increase nutritional value, and in the application, the mass ratio of the epidemic diarrhea virus egg yolk antibody to the transglutaminase is 100:1-2; the mass ratio of the epidemic diarrhea virus egg yolk antibody to the lactobacillus is (5-6): (2-3); the CaCl 2 in the epidemic diarrhea virus egg yolk antibody is used at a concentration of 2-5wt%, and the CaCl 2 can be added to serve as a flocculating agent, so that the epidemic diarrhea virus egg yolk antibody and the lactobacillus are beneficial to being mutually close, and the aggregation property of CaCl 2 is utilized to concentrate and separate the epidemic diarrhea virus egg yolk antibody and the lactobacillus. The application discovers that better coating effect can be obtained by adding the components according to the proportion. It should be appreciated that the flocculant may also be selected from a variety of materials including, but not limited to, at least one of aluminum chloride, ferric chloride, and alum.
Preferably, in the present application, the bacterial content of the lactic acid bacillus is 1.0X10 8-3.0×108 cfu/g; the lactobacillus is pig-origin lactobacillus, and it is understood that the lactobacillus may also be chicken-origin lactobacillus or duck-origin lactobacillus. More preferably, the Bacillus subtilis of porcine origin is Bacillus subtilis Q-21 of porcine origin.
The application is notable that the specific selection application number is CN201711312542.2, and the application is named as the swine lactobacillus which is the swine lactobacillus Q-21 recorded in the swine lactobacillus, the microecological preparation, the preparation method and the application. The swine lactobacillus Q-21 is a lactobacillus containing spores, has special physiological functions, and can resist the influence of adverse environmental factors such as high temperature, high humidity, high pressure, strong acid and the like. The lactobacillus is also a living bacteria preparation, can colonize in intestinal tracts, inhibit harmful bacteria, maintain normal microecological balance and activate the immunity of immune cells of organisms. Lactic acid bacillus can produce a large number of metabolites including lactic acid, acetic acid, hydrogen peroxide, bacteriocins, etc., and secreted hydrogen peroxide can activate a catalase system and inhibit many pathogenic bacteria. The lactobacillus can secrete a bacteriocin substance, the bacteriocin can inhibit pathogenic bacteria inside and outside the organism, main nisin in the bacteriocin has stronger bactericidal activity, and the membrane potential change is caused by destroying the cell membrane of harmful bacteria, so that the permeability of the cell membrane is improved, and a large amount of ATP and a large amount of ions in the cell membrane are lost, so that the death of the harmful bacteria is caused.
S3, preparing a second coating.
Mixing the first coating with vegetable oil, stirring, standing, and separating the second coating. The vegetable oil comprises one or more of moringa seed oil, kiwi seed oil, avocado oil, perilla seed oil and merry-go-round oil. Stirring and standing for 10-20min at stirring speed of 150-170r/min, and standing for 20-30min. Separating comprises centrifuging at 4000-6000r/min at 3-5deg.C for 10-20min, layering, and removing residual vegetable oil to obtain second coating.
S4, preparing a third coating.
Mixing the second coating with the glue solution, stirring, standing for solidification, and separating. Wherein the glue solution comprises one or more of sodium alginate, flaxseed gum and carboxymethyl tamarind gum; preferably, the gum solution comprises sodium alginate, flaxseed gum and carboxymethyl tamarind gum; more preferably, the weight parts ratio of the sodium alginate, the flaxseed gum and the carboxymethyl tamarind gum is (8-10): (2-3): (2-3).
S5, drying.
And drying the separated third coating. There are various ways of drying including, but not limited to: oven drying, atmospheric drying, microwave drying, spray drying, and the like. In the present application, the third coating is preferably dried by spray drying.
The anti-diarrhea egg yolk antibody coated lactobacillus preparation prepared by the preparation method of the anti-diarrhea egg yolk antibody coated lactobacillus preparation is granular, and the grain diameter of the anti-diarrhea egg yolk antibody coated lactobacillus preparation is 6-600 mu m. According to the preparation method, the anti-diarrhea egg yolk antibody coated lactobacillus preparation has a multilayer structure, wherein the egg yolk antibody coated lactobacillus is used as the first layer, the outer surface of the first layer is coated by vegetable oil, and the outer surface of the vegetable oil is coated by a glue layer. The multilayer coating structure ensures that the lactobacillus is fully protected, the stability is better, the survival rate of the lactobacillus in gastric acid is further improved, the field planting of the lactobacillus in intestinal tracts is further facilitated, the harmful flora is inhibited, and the normal microecological balance is maintained. In addition, the anti-diarrhea egg yolk antibody coated lactobacillus preparation provided by the application can improve the palatability of daily ration, enhance the immunity of piglets, improve the feed intake and improve the digestion utilization rate of nutrient substances.
According to the application, modern biotechnology is firstly used for putting epidemic diarrhea virus into the small intestine of a sterile SPF pig for proliferation, and the virus is used for inoculating and producing antibodies in the egg laying period of a healthy egg laying hen, so that the titer of the yolk antibody of the epidemic diarrhea virus can be improved; the method has the advantages that the epidemic diarrhea virus egg yolk antibody is firstly used for coating, processing and fusing the lactobacillus, so that the stability of the egg yolk antibody and the microecological preparation is improved; the preparation method provided by the application is simple, has good condition controllability, low production cost and stable product performance, is suitable for cultivation with resistance reduction and resistance inhibition, has good application prospect in the aspects of medicine residue reduction action and food safety, and can be especially applied to preparation of feed additives or feeds.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a preparation of anti-diarrhea egg yolk antibody coated lactobacillus, and the preparation method comprises the following steps:
s1, preparing an epidemic diarrhea virus egg yolk antibody.
Selecting a bearing house piglet with infection onset of 2-3 days old, aseptically collecting small intestine, shearing, fully grinding, diluting with sterile physiological saline, centrifuging at 8000r/min for 15min, collecting supernatant, adding formaldehyde (the addition amount is 0.15% of the volume of the supernatant), and oscillating at 37 ℃ for 24h at constant temperature.
Inactivating the collected supernatant, diluting to obtain diluent for inactivating the epidemic diarrhea virus, mixing the diluent for inactivating the epidemic diarrhea virus with 4% Tween to obtain mixed solution as water phase, wherein the oil phase comprises 92% white oil, 6% span 85 and 2% aluminum stearate by mass percent; sterilizing at 115 deg.C for 30min, and cooling to room temperature. Mixing and stirring the water phase and the oil phase according to the proportion of 1:1.5 to obtain the epidemic diarrhea virus oil emulsion immune antigen.
Injecting 0.5mL of epidemic diarrhea virus oil emulsion immune antigen into chicken breast muscle of the laying hen as first immunization; injecting 1mL of epidemic diarrhea virus oil emulsion immunization antigen into the breast muscle of the laying hen at the 2 nd week and the 4 th week for the second immunization and the third immunization, and injecting 3mL of epidemic diarrhea virus oil emulsion immunization antigen into the breast muscle and the leg muscle of the laying hen at the 6 th week for the fourth immunization; detecting antibody titer, when the antibody titer is more than 128, starting to collect eggs, aseptically collecting yolk, adding pullulan polysaccharide and procyanidine, and uniformly stirring to obtain epidemic diarrhea virus yolk antibody;
The mass ratio of the yolk to the pullulan to the procyanidine is 105:9:0.5.
S2, preparing a first coating.
60G of epidemic diarrhea virus egg yolk antibody is added into 200ml of defatted milk powder water solution with the concentration of 20wt%, then transglutaminase (the addition amount is 1% of the egg yolk antibody) is added, and the solution containing the epidemic diarrhea virus egg yolk antibody is prepared after uniform stirring; 20g of Bacillus subtilis Q-21 of porcine origin (bacterial content 2.0X10 8) was added, followed by CaCl 2 having a concentration of 1 ml of 3% by weight; stirring at 160r/min for 5 min min, standing for 40min, cleaning, and filtering to obtain the first coating.
S3, preparing a second coating.
Mixing the first coating with vegetable oil (including moringa seed oil, perilla seed oil and merry go round machine oil in the ratio of 5:2:2), stirring at 160r/min for 15min, standing for 30min, centrifuging at 4deg.C for 15min at 5000r/min, layering, and removing residual vegetable oil to obtain the second coating.
S4, preparing a third coating.
Mixing the second coating with gelatin solution (sodium alginate 10g, flaxseed gum 2g, carboxymethyl tamarind gum 2 g), stirring, standing, solidifying for 2-3 hr, and separating.
S5, drying.
And (3) carrying out spray drying on the third coating obtained by separation to obtain the anti-diarrhea egg yolk antibody coated lactobacillus preparation with the particle size of 6-600 mu m.
Example 2
This embodiment is substantially the same as embodiment 1, except that: the amounts of the partial components used in step S1 and step S2 are different.
In step S1 of this example, the mass ratio of yolk, pullulan and procyanidins is 110:8:0.4; the dosage of the epidemic diarrhea virus egg yolk antibody in the step S2 is 52g, and the dosage of the swine lactic acid bacillus Q-21 is 25g.
Example 3
This embodiment is substantially the same as embodiment 1, except that: the amounts of the partial components used in step S1, step S2 and step S4 are different.
In step S1 of this example, the mass ratio of yolk, pullulan and procyanidins is 110:10:0.5; the dosage of the epidemic diarrhea virus egg yolk antibody in the step S2 is 58g, and the dosage of the swine lactic acid bacillus Q-21 is 26g; the amount of sodium alginate used in step S4 was 10g.
Example 4
The embodiment provides a preparation of anti-diarrhea egg yolk antibody coated lactobacillus, and the preparation method comprises the following steps:
s1, preparing an epidemic diarrhea virus egg yolk antibody.
Selecting a bearing house piglet with infection onset of 2-3 days old, aseptically collecting small intestine, shearing, fully grinding, diluting with sterile physiological saline, centrifuging at 7000r/min for 15 min, collecting supernatant, adding formaldehyde (the addition amount is 0.2% of the volume of the supernatant), and oscillating at constant temperature of 35 ℃ for 28h.
Inactivating the collected supernatant, diluting to obtain diluent for inactivating the epidemic diarrhea virus, mixing the diluent for inactivating the epidemic diarrhea virus with 4% Tween to obtain mixed solution as water phase, wherein the oil phase comprises 92% white oil, 6% span 85 and 2% aluminum stearate by mass percent; sterilizing at 115 deg.C for 30min, and cooling to room temperature. Mixing and stirring the water phase and the oil phase according to the proportion of 1:1.5 to obtain the epidemic diarrhea virus oil emulsion immune antigen.
Injecting 0.5mL of epidemic diarrhea virus oil emulsion immune antigen into chicken breast muscle of the laying hen as first immunization; injecting 1mL of epidemic diarrhea virus oil emulsion immunization antigen into the breast muscle of the laying hen at the 2 nd week and the 4 th week for the second immunization and the third immunization, and injecting 3mL of epidemic diarrhea virus oil emulsion immunization antigen into the breast muscle and the leg muscle of the laying hen at the 6 th week for the fourth immunization; detecting antibody titer, when the antibody titer is more than 128, starting to collect eggs, aseptically collecting yolk, adding pullulan polysaccharide and procyanidine, and uniformly stirring to obtain epidemic diarrhea virus yolk antibody;
the mass ratio of the yolk to the pullulan to the procyanidine is 105:9:0.3.
S2, preparing a first coating.
Adding 55g of epidemic diarrhea virus egg yolk antibody into 200ml of defatted milk powder water solution with concentration of 20wt%, adding transglutaminase (the addition amount is 1% of the egg yolk antibody), and stirring to obtain solution containing epidemic diarrhea virus egg yolk antibody; 25g of Bacillus subtilis Q-21 of porcine origin (bacterial content 2.0X10 8) was added, followed by CaCl 2 having a concentration of 1 ml of 2% by weight; stirring at 160r/min for 4 min min, standing for 40min, cleaning, and filtering to obtain the first coating.
S3, preparing a second coating.
Mixing the first coating with vegetable oil (the vegetable oil is kiwi seed oil and avocado oil in a ratio of 1:1), stirring at 160r/min for 15min, standing for 30min, centrifuging at 4deg.C for 15min at 5000r/min, layering, and removing residual vegetable oil to obtain the second coating.
S4, preparing a third coating.
Mixing the second coating with glue solution (sodium alginate 10 parts, flaxseed gum 3 parts, carboxymethyl tamarind gum 2 parts), stirring, standing, solidifying for 3 hr, and separating.
S5, drying.
And (3) carrying out spray drying on the third coating obtained by separation to obtain the anti-diarrhea egg yolk antibody coated lactobacillus preparation with the particle size of 6-600 mu m.
Example 5
The embodiment provides a preparation of anti-diarrhea egg yolk antibody coated lactobacillus, and the preparation method comprises the following steps:
s1, preparing an epidemic diarrhea virus egg yolk antibody.
Selecting a bearing house piglet with infection onset of 2-3 days old, aseptically collecting small intestine, shearing, fully grinding, diluting with sterile physiological saline, centrifuging at 8000r/min for 15min, collecting supernatant, adding formaldehyde (the addition amount is 0.15% of the volume of the supernatant), and oscillating at 37 ℃ for 24h at constant temperature.
Inactivating the collected supernatant, diluting to obtain diluent for inactivating the epidemic diarrhea virus, mixing the diluent for inactivating the epidemic diarrhea virus with 4% Tween to obtain mixed solution as water phase, wherein the oil phase comprises 92% white oil, 6% span 85 and 2% aluminum stearate by mass percent; sterilizing at 115 deg.C for 30min, and cooling to room temperature. Mixing and stirring the water phase and the oil phase according to the proportion of 1:1.5 to obtain the epidemic diarrhea virus oil emulsion immune antigen.
Injecting 0.5mL of epidemic diarrhea virus oil emulsion immune antigen into chicken breast muscle of the laying hen as first immunization; injecting 1mL of epidemic diarrhea virus oil emulsion immunization antigen into the breast muscle of the laying hen at the 2 nd week and the 4 th week for the second immunization and the third immunization, and injecting 3mL of epidemic diarrhea virus oil emulsion immunization antigen into the breast muscle and the leg muscle of the laying hen at the 6 th week for the fourth immunization; detecting antibody titer, when the antibody titer is more than 128, starting to collect eggs, aseptically collecting yolk, adding pullulan polysaccharide and procyanidine, and uniformly stirring to obtain epidemic diarrhea virus yolk antibody;
The mass ratio of the yolk to the pullulan to the procyanidine is 108:9:0.3.
S2, preparing a first coating.
58G of epidemic diarrhea virus egg yolk antibody is added into 200ml of defatted milk powder water solution with concentration of 20wt%, then transglutaminase (the addition amount is 1% of the egg yolk antibody) is added, and the solution containing the epidemic diarrhea virus egg yolk antibody is prepared after uniform stirring; 22g of Bacillus subtilis Q-21 of porcine origin (bacterial content 2.0X10 8) was added followed by CaCl 2 having a concentration of 1 ml of 5% by weight; stirring at 160r/min for 5 min min, standing for 40min, cleaning, and filtering to obtain the first coating.
S3, preparing a second coating.
Mixing the first coating with vegetable oil (the vegetable oil is kiwi seed oil and avocado oil in a ratio of 1:1), stirring at 160r/min for 15min, standing for 30min, centrifuging at 4deg.C for 15min at 5000r/min, layering, and removing residual vegetable oil to obtain the second coating.
S4, preparing a third coating.
Mixing the second coating with glue solution (sodium alginate 8 parts, flaxseed gum 2 parts, carboxymethyl tamarind gum 2 parts), stirring, standing, solidifying for 2-3 hr, and separating.
S5, drying.
And (3) carrying out spray drying on the third coating obtained by separation to obtain the anti-diarrhea egg yolk antibody coated lactobacillus preparation with the particle size of 6-600 mu m.
Example 6
This embodiment is substantially the same as embodiment 1, except that: the parameters of step S2 are different. In this embodiment, step S2 includes: adding 50g of epidemic diarrhea virus egg yolk antibody into 180ml of defatted milk powder water solution with concentration of 20wt%, adding transglutaminase (the addition amount is 2% of the egg yolk antibody), and stirring uniformly to obtain a solution containing epidemic diarrhea virus egg yolk antibody; 20g of Bacillus subtilis Q-21 of porcine origin (bacterial content: 1.8X10 8) was added, followed by CaCl 2 having a concentration of 1ml of 3% by weight; stirring at 160r/min for 5 min min, standing for 40min, cleaning, and filtering to obtain the first coating.
Example 7
This embodiment is substantially the same as embodiment 1, except that: the parameters of step S2 are different. In this embodiment, step S2 includes: 60g of epidemic diarrhea virus egg yolk antibody is added into 220ml of defatted milk powder water solution with the concentration of 20wt%, then transglutaminase (the addition amount is 1.5 percent of the egg yolk antibody) is added, and the solution containing the epidemic diarrhea virus egg yolk antibody is prepared after uniform stirring; 30g of Bacillus subtilis Q-21 of porcine origin (bacterial content: 2.2X10 8) was added, followed by CaCl 2 having a concentration of 1 ml of 5% by weight; stirring at 160r/min for 5min, standing for 40min, cleaning, and filtering to obtain the first coating.
Example 8
This embodiment is substantially the same as embodiment 1, except that: in this example, bacillus licheniformis Q-21 was not used, and Bacillus licheniformis from Henan New Yangshao biotechnology Co. Otherwise, the same as in example 1 was conducted.
Comparative example 1
This comparative example is substantially identical to example 1, except that: procyanidins were not added in this comparative example. Otherwise, the same as in example 1 was conducted.
Comparative example 2
This comparative example is substantially identical to example 1, except that: pullulan was not added in this comparative example. Otherwise, the same as in example 1 was conducted.
Comparative example 3
This comparative example is substantially identical to example 1, except that: in this comparative example, the coating glue solution of step S4 was directly applied to the first coating obtained in step S2 without adding vegetable oil, and at this time, the two-layer coating was obtained in this comparative example. Otherwise, the same as in example 1 was conducted.
Comparative example 4
This comparative example is substantially identical to example 1, except that: in the comparative example, no linseed gum and carboxymethyl tamarind gum were added, and only sodium alginate was used. Otherwise, the same as in example 1 was conducted.
Comparative example 5
This comparative example is substantially identical to example 1, except that: step S3 and step S4 are omitted, namely vegetable oil and glue solution are omitted.
Comparative example 6
This comparative example is substantially identical to example 1, except that: in this comparative example, transglutaminase was omitted.
Comparative example 7
This comparative example is substantially identical to example 1, except that: caCl 2 was omitted from this comparative example.
Comparative example 8
This comparative example is substantially identical to example 1, except that: in the step S1, the mass ratio of the yolk to the pullulan to the procyanidine is 130:7:0.2.
Comparative example 9
This comparative example is substantially identical to example 1, except that: in the step S2, the dosage of the egg yolk antibody of the epidemic diarrhea virus is 65g, and the dosage of the porcine lactobacillus Q-21 is 15g.
Test example 1
In the conical flask of 250 mI, 200 mL artificial gastric acid solution (ph=2.1) is placed, numbered, and bacillus lactis is sequentially put into the conical flask, and the number of viable bacteria is measured after 2 hours, specifically: a certain amount of solution is taken for sample dilution, colony count (cfu/mL) is calculated by a dilution plate colony counting method, and the solution is cultured for 48 hours in a culture box at 37 ℃ to calculate the survival rate.
TABLE 1 Effect of gastric acid on the Activity of embedded Lactobacillus
The results show that the survival rate of the egg yolk antibody coated lactobacillus in the present embodiment is more than 72.22% after 2 hours in gastric juice, and the survival rate of the comparative example group is less than 57.5%, wherein the effect of example 3 is optimal.
Test example two
The test adopts a single factor test design, healthy 14-day-old piglets with similar birth date and weight are selected, 20 piglets are treated each, and the test period is 21 days. The addition amount of the egg yolk antibody coated lactobacillus is 2%.
Daily weight gain and feed conversion ratio of piglets are calculated, the diarrhea number of each group of piglets is counted daily, and the diarrhea rate = the total accumulated diarrhea number of each group of piglets in the test period/(the total number of each group in the test period x the number of test days) x 100%.
TABLE 2 Effect of egg yolk antibody coated lactic acid bacteria on daily weight gain and feed/meat ratio of piglets
The experiment shows that the average daily gain of the weaned piglets of the example group is higher than 310.95g, and the average daily gain of the weaned piglets of the comparative example group is lower than 308.57g, wherein the effect of the example 3 is optimal. The weaned piglets of the test group have a lower feed to meat ratio than the control group. The egg yolk antibody coated lactobacillus improves the palatability of daily ration to a certain extent, enhances the immunity of piglet organisms, improves the feed intake and improves the digestion utilization rate of nutrient substances.
TABLE 3 Effect of egg yolk antibody coated lactic acid bacteria on diarrhea Rate of piglets
The test shows that the diarrhea rate of the comparative example group is higher than that of the example group by observing piglets, wherein the effect of example 3 is the best. The clinical application effect of the egg yolk antibody coated lactobacillus preparation is good.
In summary, the preparation method of the anti-diarrhea egg yolk antibody coated lactobacillus preparation provided by the application uses the technology of coating treatment and fusion of epidemic diarrhea virus egg yolk antibody on lactobacillus, and is matched with vegetable oil and glue solution to form a multi-layer structure, wherein the first layer is formed by coating the egg yolk antibody on the lactobacillus, the outer surface of the first layer is coated by vegetable oil, and the outer surface of the vegetable oil is coated by a glue layer. The multilayer coating structure ensures that the lactobacillus is fully protected, the stability is better, the survival rate of the lactobacillus in gastric acid is further improved, the field planting of the lactobacillus in intestinal tracts is further facilitated, the harmful flora is inhibited, and the normal microecological balance is maintained. The preparation method provided by the application is simple, has good condition controllability, low production cost and stable product performance, is suitable for cultivation with resistance reduction and resistance inhibition, has good application prospect in the aspects of medicine residue reduction action and food safety, and can be especially applied to preparation of feed additives or feeds. In addition, the anti-diarrhea egg yolk antibody coated lactobacillus preparation provided by the application can improve the palatability of daily ration, enhance the immunity of piglets, improve the feed intake and improve the digestion utilization rate of nutrient substances.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A method for preparing an anti-diarrhea egg yolk antibody coated lactobacillus preparation, which is characterized by comprising the following steps:
Adding lactobacillus into a solution containing epidemic diarrhea virus egg yolk antibody, adding CaCl 2 into the mixed solution, stirring and standing to separate a first coating, wherein the mass ratio of the epidemic diarrhea virus egg yolk antibody to the lactobacillus is (5-6): (2-3), wherein the mass ratio of the epidemic diarrhea virus egg yolk antibody to the CaCl 2 is 100:2-5, the bacterial content of the lactobacillus is 1.8X10- 8-2.2×108 cfu/g, and the lactobacillus is swine lactobacillus Q-21; the solution containing the epidemic diarrhea virus egg yolk antibody is prepared by adding the epidemic diarrhea virus egg yolk antibody into a skimmed milk powder aqueous solution, adding transglutaminase, and stirring uniformly; the mass volume ratio of the epidemic diarrhea virus egg yolk antibody to the skimmed milk powder aqueous solution is (5-6) g: (18-22) ml; the mass ratio of the epidemic diarrhea virus egg yolk antibody to the transglutaminase is 100:1-2; the preparation method of the epidemic diarrhea virus egg yolk antibody comprises the following steps: injecting epidemic diarrhea virus oil emulsion immune antigen into laying hens for multiple immunity, detecting antibody titer, collecting eggs when the antibody titer is more than 128, aseptically collecting yolk, adding pullulan polysaccharide and procyanidine, and stirring uniformly to obtain the epidemic diarrhea virus yolk antibody; the mass ratio of the yolk to the pullulan to the procyanidine is 100-120:8-10:0.3-0.5; stirring at a stirring speed of 150-170r/min for 3-5 min while preparing the first coating, standing for 30-50min, cleaning, and filtering;
Mixing the first coating with vegetable oil, stirring and standing, and separating the second coating; stirring for 10-20min at a stirring speed of 150-170r/min, standing for 20-30min, centrifuging for 10-20min at a centrifuging speed of 4000-6000r/min at 3-5deg.C, layering, and removing residual vegetable oil to obtain the second coating;
Mixing the second coating with a glue solution, stirring, standing, solidifying, separating and drying, wherein the glue solution comprises sodium alginate, flaxseed gum and carboxymethyl tamarind gum; the weight parts ratio of the sodium alginate to the linseed gum to the carboxymethyl tamarind gum is (8-10): (2-3): (2-3).
2. The method of preparing an anti-diarrhea egg yolk antibody coated lactobacillus preparation according to claim 1, wherein the multiple immunization comprises: injecting 0.4-0.6mL of epidemic diarrhea virus oil emulsion immune antigen into chicken breast muscle of laying hen as first immunization; injecting 0.8-1.2mL of the epidemic diarrhea virus oil emulsion immunization antigen into the chicken breast muscle of the laying hen at the 2 nd week and the 4 th week respectively for the second immunization and the third immunization, and injecting 1.6-2.4mL of the epidemic diarrhea virus oil emulsion immunization antigen into the chicken breast muscle and leg muscle of the laying hen at the 6 th week for the fourth immunization.
3. The preparation method of the anti-diarrhea egg yolk antibody coated lactobacillus preparation according to claim 1, wherein the epidemic diarrhea virus oil emulsion immune antigen is a mixed solution of an aqueous phase and an oil phase, wherein the aqueous phase comprises a mixed solution of a diluent for inactivating the epidemic diarrhea virus and tween, and the tween accounts for 3-5% of the mass of the mixed solution; the oil phase comprises 92-94% of white oil, 5-7% of span 85 and 1-2% of aluminum stearate by mass percent; mixing and stirring the water phase and the oil phase according to the proportion of 1:1.4-1.6.
4. The method for preparing the anti-diarrhea egg yolk antibody coated lactobacillus preparation according to claim 3, wherein the method for preparing the diluent for inactivating the epidemic diarrhea virus comprises the following steps: selecting a farrowing house piglet with infection onset at 2-3 days old, aseptically collecting small intestine, shearing, fully grinding, diluting with sterile physiological saline, centrifuging at 7000-9000r/min for 10-20 min, collecting supernatant, adding 0.1-0.2% formaldehyde, and oscillating at 35-40deg.C for 24-28 hr.
5. The method for preparing the anti-diarrhea egg yolk antibody coated lactobacillus preparation according to any one of claims 1 to 4, wherein the vegetable oil comprises one or more of moringa seed oil, kiwi seed oil, avocado oil, perilla seed oil and mero oil.
6. The method of preparing an anti-diarrhea egg yolk antibody coated lactobacillus preparation according to any of claims 1-4 wherein the drying is spray drying.
7. An anti-diarrhea egg yolk antibody coated lactobacillus preparation, which is prepared by the preparation method of the anti-diarrhea egg yolk antibody coated lactobacillus preparation according to any one of claims 1 to 6.
8. The anti-diarrhea egg yolk antibody coated lactobacillus preparation of claim 7, wherein the anti-diarrhea egg yolk antibody coated lactobacillus preparation is in the form of particles with a particle size of 6-600 μm.
9. The use of the anti-diarrhea egg yolk antibody coated lactic acid bacillus formulation of claim 7 in the preparation of a feed additive or feed.
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