Pig feed, feed additive and preparation method thereof

Abstract

The invention discloses a pig feed. The pig feed comprises the following components in percentage by mass: (1) 2% of fish collagen hydrolysate; (2) 1.5% of whey protein hydrolysate; (3) 1% of lactic acid bacteria for expressing the PEDV monoclonal antibody; (4) 1% of bifidobacterium; (5) 1% of beta-glucan; (6) 2% of a wolfberry extract; (7) 45% of corn flour; (8) 35% of soybean meal; and (9) 0.1% of vitamin E. According to the present invention, various functional components such as the small molecule polypeptide, the lactic acid bacteria expressing the PEDV monoclonal antibody, the probiotics and the like are combined together, such that the immune system of the pig can be comprehensively enhanced, the intestinal health can be improved, and the growth performance of the pig can be improved. Compared with the prior art, the formula provided by the invention not only solves the problem that the traditional feed only depends on a single component, but also can provide directional immune protection for a specific virus (PEDV), and has remarkable market competitiveness.

Description

Technical Field

[0001] The invention relates to the technical field of animal feed, in particular to a pig feed and a feed additive, and specifically to a feed additive capable of improving the immunity of pigs and promoting their growth and a preparation method thereof. Background Art

[0002] With the rapid development of modern pig farming, the nutritional content and additives of feed have gradually become key factors in improving pig health and production efficiency. Traditional pig feed mainly provides basic energy and protein sources. However, in terms of improving pig immunity, disease resistance and promoting growth, existing feed and feed additives often have certain limitations. In particular, when dealing with problems such as decreased immunity, poor disease resistance and frequent epidemics of pigs, the effect of traditional feed is not ideal.

[0003] In recent years, the health of the pig immune system has received more and more attention, especially in the breeding environment. The threat of pathogens such as viruses and bacteria puts the pig immune system in a state of long-term stress, which directly affects its growth and disease resistance. Improving pig immunity and enhancing its disease resistance has become a difficult problem that needs to be solved in the current pig farming industry. At the same time, with the expansion of breeding scale and the increase in production costs, the pig farming industry has higher and higher requirements for feed conversion rate. How to reduce the occurrence of diseases and improve the health level of pigs while ensuring the growth performance of pigs has become the focus of research.

[0004] At present, there are some immune-enhancing feeds and additives on the market, such as feed additives containing immunomodulatory ingredients (such as β-glucan, antioxidants, etc.) and Chinese herbal medicine ingredients, which can improve the immunity and disease resistance of pigs to a certain extent, but there are still problems such as insufficient formulation and limited effect. Therefore, it is urgent to develop a new feed and feed additive that can improve the immunity of pigs, promote healthy growth, and improve feed utilization efficiency and breeding benefits. Summary of the invention

[0005] The invention provides a pig feed and a feed additive, which can effectively improve the immunity of pigs, promote their healthy growth, and improve feed conversion rate.

[0006] Therefore, the present invention discloses a pig feed in one aspect, wherein the composition of the pig feed is as follows in terms of mass percentage:

[0007] (1) Fish collagen hydrolysate: 2%;

[0008] (2) Whey protein hydrolysate: 1.5%;

[0009] (3) Lactic acid bacteria expressing PEDV monoclonal antibodies: 1%;

[0010] (4) Bifidobacterium: 1%;

[0011] (5) β-glucan: 1%;

[0012] (6) Lycium barbarum extract: 2%;

[0013] (7) Corn flour: 45%;

[0014] (8) Soybean meal: 35%;

[0015] (9) Vitamin E: 0.1%.

[0016] Preferably, the pig feed of the present invention comprises the following features:

[0017] (1) Moisture content: 13.2%;

[0018] (2) Particle hardness: 4.5N;

[0019] (3) Particle diameter: 5 mm;

[0020] (4) Lycium barbarum polysaccharide content: 2.05%;

[0021] (5) Probiotic activity: 1.2×10 8 CFU / g;

[0022] (6) Total number of microorganisms: Not detected.

[0023] Preferably, the fish collagen hydrolysate of the present invention is rich in glycine, proline, leucine and phenylalanine; the purity of the fish collagen hydrolysate is 85%; the protein content in every 100g of fish collagen hydrolysate is 65g; and the molecular weight of 80% of the fish collagen hydrolysate is between 300-1000Da.

[0024] Preferably, the whey protein hydrolysate of the present invention is rich in glycine and proline, wherein the glycine content in every 100g of whey protein hydrolysate is 28.5g, and the proline content in every 100g of whey protein hydrolysate is 16.2g; each 100g of whey protein hydrolysate contains 60-65g of total protein, and the molecular weight distribution of 80% of the whey protein hydrolysate is between 500-800Da.

[0025] Preferably, the viable bacterial count of the lactic acid bacteria expressing the PEDV monoclonal antibody of the present invention after freeze-drying is 1.2×10^10 CFU / g, and the viable bacterial count is maintained at 8.5×10^9 CFU / g after storage for 3 months, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the PEDV monoclonal antibody are as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0026] Preferably, the number of viable bifidobacteria after freeze-drying of the present invention is 1×10^10 CFU / g, and the survival rate of viable bacteria after storage for 3 months is >85%.

[0027] Preferably, the wolfberry extract of the present invention is rich in polysaccharides and flavonoids, wherein the content of polysaccharides in every 100g of wolfberry extract is 45±2g, and the content of flavonoid components in every 100g of wolfberry extract is 12±1g.

[0028] In one aspect, the present invention also discloses a use of the fish collagen hydrolyzate in preparing pig feed.

[0029] In one aspect, the present invention also discloses a use of the whey protein hydrolyzate in preparing pig feed.

[0030] In one aspect, the present invention also discloses a use of the lactic acid bacteria expressing PEDV monoclonal antibodies in preparing pig feed.

[0031] The present invention combines a variety of functional ingredients such as small molecule peptides, lactic acid bacteria expressing PEDV monoclonal antibodies, and probiotics, which can comprehensively enhance the immune system of pigs, improve intestinal health, and enhance the growth performance of pigs. Compared with the prior art, the formula of the present invention not only solves the problem that traditional feed relies on only a single ingredient, but also provides targeted immune protection against specific viruses (PEDV), and has significant market competitiveness.

[0032] Fish collagen hydrolysate: Through hydrolysis, the small molecule peptides generated are easily absorbed by the pig intestine, and have the functions of enhancing immunity, anti-oxidation and promoting growth. Whey protein hydrolysate: As a high-quality protein source, it can provide rich amino acids to support the muscle development and immune response of pigs. Lactic acid bacteria expressing PEDV monoclonal antibodies: Specific antibodies against porcine epidemic diarrhea virus (PEDV) can enhance the immune protection of pigs against PEDV and effectively reduce PEDV infection. Probiotics (Bifidobacteria): Improve intestinal microecology, promote digestion and absorption, increase feed utilization, and enhance the intestinal health of pigs. β-Glucan: Activates the immune system in pigs and enhances defense against external pathogens. Lycium barbarum extract: Rich in antioxidants, enhances the immune system, and has anti-fatigue and anti-stress effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Figure 1. Pig feed prepared by the present invention. DETAILED DESCRIPTION

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0035] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0036] Example 1: Pig feed formula

[0037] 1. The raw material composition is as follows in terms of mass percentage:

[0038] (1) Fish collagen hydrolysate: 2%;

[0039] (2) Whey protein hydrolysate: 1.5%;

[0040] (3) Lactic acid bacteria expressing PEDV monoclonal antibodies: 1%;

[0041] (4) Bifidobacterium: 1%;

[0042] (5) β-glucan: 1%;

[0043] (6) Lycium barbarum extract: 2%;

[0044] (7) Corn flour: 45%;

[0045] (8) Soybean meal: 35%;

[0046] (9) Vitamin E: 0.1%.

[0047] Example 2: Pig feed preparation method

[0048] 1. Preparation of fish collagen hydrolysate

[0049] 1.1 Raw material preparation

[0050] Fish collagen raw material selection: Collagen is extracted from fish skin, fish scales or fish bones. Common choices include salmon skin, cod skin, etc. The freshness of the raw materials must be ensured to avoid excessive oxidation or deterioration. Fish collagen should be cut into small pieces to facilitate subsequent extraction and hydrolysis.

[0051] Collagen extraction: Place fish skin or scales in distilled water at a ratio of 1:3 (fish skin: water). Treat the fish skin with an acidic solution (1% acetic acid) and adjust the pH to 4-5. Let stand at 4°C for 1-2 hours to remove impurities. Extract collagen by high-temperature steaming (about 80°C, 30 minutes), filter the extract and store.

[0052] 1.2 Collagen hydrolysis

[0053] Enzymatic hydrolysis: Take an appropriate amount of fish collagen extract and add an appropriate amount of enzyme (such as pepsin or trypsin). The amount of enzyme added is usually 1-2% of collagen (enzyme: collagen mass ratio). Adjust the solution pH to 4-6 (by adding an appropriate amount of acetic acid) to make it suitable for the optimal action range of the selected enzyme. Put the hydrolyzate into a water bath, set the temperature to 50℃-60℃, and perform the hydrolysis reaction. The reaction time is usually 2-4 hours, and the degree of hydrolysis is controlled according to actual needs.

[0054] Reaction termination: When the hydrolyzate reaches the predetermined time, the reaction is terminated in a water bath heated to 80°C to kill the enzyme and stop the hydrolysis process. The reaction solution can be further neutralized by adjusting the pH to 6-7 to ensure the stability of the hydrolyzate.

[0055] 1.3 Hydrolyzate separation and purification

[0056] Centrifugation and separation: Centrifuge the hydrolyzate (4000rpm, 10-15 minutes) to remove macromolecular residues and impurities. The supernatant is the initial hydrolyzed collagen peptide.

[0057] Ultrafiltration: Use an ultrafiltration membrane (molecular weight cutoff of 300-1000Da) to filter the hydrolyzate to remove the unhydrolyzed high molecular weight protein and obtain small molecular weight peptides. Separate the small molecular weight polypeptides (molecular weight 300-1000Da) with biological activity and retain the supernatant.

[0058] 1.4 Drying and finished product preparation

[0059] Drying: The ultrafiltered hydrolyzate liquid is spray dried, and the drying temperature is controlled below 60°C to prevent degradation of the active substance. During the spray drying process, the atomization particle size should be appropriate to ensure fine powder quality and good fluidity.

[0060] Finished product storage: The dried fish collagen hydrolysate powder can be packaged and stored in a cool, dry, dark environment. Finished collagen hydrolysate needs to be tested for quality control to ensure its stability and activity.

[0061] 1.5 Detection

[0062] (1) Protein quantification: The protein content of collagen hydrolysate was quantified by the BCA method. The specific results are shown in Table 1.

[0063] (2) Molecular weight analysis: The molecular weight distribution of the hydrolyzate was analyzed by SDS-PAGE electrophoresis. The specific results are shown in Table 1.

[0064] (3) Amino acid composition analysis: The amino acid composition of the hydrolyzate was analyzed using an amino acid analyzer to ensure that it contained a high proportion of essential amino acids and met the requirements of animal feed. The specific results are shown in Table 1.

[0065] (4) Antioxidant activity test: The antioxidant activity of the hydrolyzate was evaluated using the DPPH free radical scavenging method or the ABTS method. The specific results are shown in Table 1.

[0066] Table 1 Summary of test results

[0067]

[0068] 2. Preparation of whey protein hydrolysate

[0069] 2.1 Raw material preparation

[0070] Whey powder selection: Choose high-quality skim whey powder and ensure that its protein content reaches more than 80%. Make sure the whey powder is fresh and avoid moisture and contamination.

[0071] Whey protein extraction: Dissolve whey powder in distilled water, with the ratio of whey powder to water being 1:4. Let stand for 15 minutes, stir evenly, and filter with filter paper to remove impurities.

[0072] 2.2 Whey protein hydrolysis

[0073] Enzymatic reaction: Place the whey protein solution in a reaction vessel, add appropriate amounts of pepsin and trypsin, and the mass ratio of enzyme to protein is 1:2. The reaction temperature is 50-55°C, the pH value is adjusted to 4.5, and a pH meter is used for real-time monitoring to ensure maximum enzyme activity. The reaction time is 2-3 hours.

[0074] End and termination of hydrolysis: After the hydrolysis is completed, the reaction is terminated by heating to 80°C to prevent excessive hydrolysis. Cool the liquid to room temperature using cooling water.

[0075] 2.3 Hydrolyzate separation and purification

[0076] Centrifugal separation: Place the hydrolyzate in a centrifuge (4000 rpm, 15 minutes) to remove macromolecular proteins and impurities that are not completely hydrolyzed during the hydrolysis reaction.

[0077] Ultrafiltration purification: Use an ultrafiltration membrane with a molecular weight cutoff of 300-1000Da to further purify the hydrolyzate, remove unhydrolyzed high-molecular-weight proteins, and obtain a hydrolyzate rich in small-molecule peptides.

[0078] 2.4 Drying and finished product preparation

[0079] Spray drying: Using spray drying technology, the purified hydrolyzate is dried through a spray dryer, and the temperature is controlled below 50°C to maintain the biological activity of the peptide substances.

[0080] Finished product storage: Package and store the dried whey protein hydrolysate powder in a cool, dry environment, avoiding exposure to light and high temperature.

[0081] 2.5. Detection

[0082] (1) Protein quantification and analysis: The protein content of the hydrolysate was quantified using the BCA method to ensure the standardization of each batch of products. Results: Each 100 g of hydrolysate contained 60-65 g of total protein.

[0083] (2) Molecular weight distribution analysis: The molecular weight distribution of the hydrolyzate was analyzed by SDS-PAGE. The results showed that 80% of the hydrolyzate had a molecular weight distribution between 500-800 Da, which met the requirements for biological activity.

[0084] (3) Amino acid composition analysis: The amino acid composition of the hydrolyzate was analyzed using an amino acid analyzer. The results are shown in Table 2. The hydrolyzate is rich in glycine and proline, which is consistent with the characteristics of whey protein and can provide the basic amino acids required for intestinal health and immune regulation.

[0085] Table 2 Amino acid composition analysis results

[0086]

[0087] (4) Antioxidant activity: The antioxidant capacity of the hydrolysate was tested using the DPPH method. Results: The DPPH free radical scavenging rate of the 50 mg / mL hydrolysate solution was 45%.

[0088] 3. Preparation of lactic acid bacteria expressing PEDV monoclonal antibodies

[0089] (1) A monoclonal antibody against PEDV virus was prepared using conventional mouse hybridoma cell technology (as described in the specifications of CN118684771A, CN118325846A, etc.). The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody were detected as shown in SEQ ID NO.1 and SEQ ID NO.2. The monoclonal antibody was subjected to a neutralization experiment on PEDV virus of different titers, and it was found that the monoclonal antibody could inhibit 105.0 TCID 50 / ml of PEDV virus, while the commercial monoclonal antibody (Shanghai Yuduo Biotechnology Co., Ltd.) can only inhibit 10 3.5 TCID 50 / ml of PEDV virus, which shows that the monoclonal antibody of the present invention has a better inhibitory effect on PEDV virus.

[0090] (2) Using the gene sequences of the monoclonal antibody light chain and heavy chain against PEDV virus, the codons were optimized to adapt to the expression of lactic acid bacteria. The synthesized gene was inserted into the plasmid pNZ8148, and the ligase reaction temperature was 16°C overnight. Transformed into DH5α, positive clones were screened, and the recombinant plasmid was extracted and verified. PCR amplification, double enzyme digestion (NcoI and HindIII) and sequencing were used to ensure that the gene was correctly inserted.

[0091] (3) Lactococcus lactis NZ3900 was cultured in GM17 medium until the logarithmic growth phase (OD600 = 0.6). Washed with sterile cold PBS three times, resuspended in 10% glycerol solution, and prepared electrocompetent cells. The recombinant plasmid was used to transform lactic acid bacteria. The electrotransformation conditions were: 2.5 kV, 25 μF, 200 Ω. Positive clones were selected on GM17 solid medium containing chloramphenicol, and PCR was used to verify the correct insertion of the vector.

[0092] (4) Positive strains were inoculated into GM17 medium and pre-cultured at 37°C overnight. Diluted to OD600 = 0.2, and continued to culture until OD600 = 0.6. 0.5% lactose was added as an inducer and cultured at 37°C for 12 hours. The cells were collected and the expression level was determined.

[0093] (5) Collect the cells by centrifugation (6,000 g, 4°C, 10 minutes) and wash twice with sterile PBS. Resuspend the cells in a freeze-drying protective agent (10% skim milk powder, 5% sucrose, 1% antioxidant). Divide the bacterial suspension into freeze-drying bottles and freeze at -80°C overnight. Use a freeze dryer to dry, and control the temperature to rise gradually from -40°C to 25°C. After freeze-drying, detect the number of viable cells, package in sealed bags, and store at room temperature.

[0094] (6) Test the viable bacterial count (the viable bacterial count after freeze-drying is 1.2×10^10 CFU / g, and the viable bacterial count remains at 8.5×10^9 CFU / g after storage for 3 months) to ensure that each gram of freeze-dried powder contains ≥10^9 CFU.

[0095] 4. Cultivation and freeze-drying of Bifidobacterium

[0096] (1) Inoculate the strain from the storage tube onto MRS agar medium and culture anaerobically at 37°C for 24 hours. Pick a single colony and inoculate it into MRS liquid medium and culture anaerobically at 37°C until the logarithmic growth phase (16-18 hours). Centrifuge at 6,000g for 10 minutes at 4°C and discard the supernatant. Wash the cells twice with sterile PBS solution to remove the culture medium residue.

[0097] (2) Resuspend the collected bacteria in the freeze-drying protective agent solution, and adjust the concentration of the bacterial suspension to 10^9 CFU / mL. Divide the bacterial suspension into freeze-drying bottles and freeze at -80°C for 12 hours. Use a freeze dryer (initial temperature -40°C), gradually increase the temperature to 25°C, and freeze-dry for 24 hours. Divide the freeze-dried bacterial powder into sealed bags and store at 4°C or -20°C. After testing, the number of live bacteria after freeze-drying is 1×10^10 CFU / g, and the survival rate of live bacteria is >85% after storage for 3 months.

[0098] 5. Preparation of Lycium barbarum Extract

[0099] 5.1 Pretreatment of wolfberry

[0100] Washing and drying wolfberries: Put the dried wolfberries into a sieve and gently rinse them with running water to remove surface impurities. Dry the washed wolfberries at 60°C until the moisture content is less than 10% (which can be measured with a moisture meter).

[0101] Crushing: The dried wolfberries are processed using a crusher or grinder to obtain powder with smaller particles (particle size is about 100 mesh).

[0102] 5.2 Solvent extraction

[0103] Selection of extraction solvent: This experiment selected 80% ethanol solution as the extraction solvent. Ethanol can better extract the polysaccharides and flavonoids in wolfberry.

[0104] Extraction process: Mix wolfberry powder with 80% ethanol at a ratio of 1:10 (i.e. 10g wolfberry powder plus 100mL ethanol). Place in an ultrasonic cleaner and extract at 50°C for 30 minutes. Keep the solution slightly stirred during the extraction process to ensure uniform extraction. Filter the extract to remove undissolved solid impurities.

[0105] Second extraction: Add 80% ethanol to the wolfberry residue after the first extraction for a second extraction, and the operation method is the same as the first extraction.

[0106] Combine the two extracts: Combine the two extracts and filter to remove impurities.

[0107] 5.3 Concentration and purification

[0108] Solvent recovery: Use a rotary evaporator to concentrate the combined extracts, evaporate most of the ethanol under reduced pressure, and the remaining solution volume is about 1 / 3 of the original solution. Note that the evaporation temperature should be controlled below 50°C to avoid degradation of the active ingredients due to high temperature.

[0109] Removal of fat-soluble impurities: Add an equal volume of chloroform to the concentrate, mix well, place in a separatory funnel to separate the aqueous phase and the chloroform phase. Remove the chloroform phase and retain the aqueous phase.

[0110] Removal of impurities and further concentration: The aqueous phase is further purified by ultrafiltration membrane, and the filtrate is concentrated to the desired concentration. The concentrate is further dried in a vacuum drying oven to obtain wolfberry extract powder.

[0111] 5.4 Drying and storage of extracts

[0112] Drying: The concentrate is vacuum dried, and the temperature is maintained below 40°C until the extract is completely dry, and a powdery finished product is obtained.

[0113] Storage: Pack the dried wolfberry extract powder in an airtight container and store in a cool and dry place away from direct sunlight and high temperature.

[0114] 5.5. Detection

[0115] (1) Detection of polysaccharide content of wolfberry extract: The polysaccharide content in wolfberry extract was quantified using the phenol-sulfuric acid method. The results showed that the polysaccharide content in every 100g of wolfberry extract was 45±2g.

[0116] (2) Detection of flavonoid content in wolfberry: The flavonoid content in wolfberry extract was determined by UV-visible spectrophotometry, mainly detecting the biologically active flavonoid compounds in wolfberry (such as wolfberry flavonoids). The results showed that the content of flavonoid components in every 100g of wolfberry extract was 12±1g.

[0117] (3) Antioxidant activity detection: The antioxidant activity of wolfberry extract was detected using the DPPH method. The results showed that the DPPH free radical scavenging rate of 50 mg / mL wolfberry extract solution was 65%.

[0118] (4) Microbial limit test: The aseptic culture method was used to test the microbial contamination in the extract. The results showed that it met the standards and no harmful microorganisms were detected.

[0119] Table 3 Summary of test results

[0120]

[0121] 6. Feed mixing

[0122] 6.1 Mixing process

[0123] Preliminary mixing of dry materials: Add corn flour, soybean meal, β-glucan, antioxidant (vitamin E) and other dry materials into the mixer. Set the mixer speed to 20-30rpm and perform preliminary mixing for 5-10 minutes to ensure that these dry materials are fully mixed and evenly mixed.

[0124] Add wet raw materials: Add dissolved fish collagen hydrolysate, whey protein hydrolysate, wolfberry extract, lactic acid bacteria expressing PEDV monoclonal antibodies, and bifidobacteria to the mixer in proportion. Use a spray device to evenly spray the liquid raw materials onto the mixture to ensure that each particle is evenly covered with the liquid ingredients.

[0125] Evenly mix: After adding the liquid raw materials, continue to mix, adjust the speed to 40-60rpm, and mix for 10-15 minutes to ensure that the liquid and dry materials are evenly combined. Check the mixing uniformity by sampling to ensure that the liquid ingredients (such as wolfberry extract, fish collagen hydrolysate, etc.) are evenly distributed.

[0126] Humidity control: Check the humidity of the mixture and ensure it is within the range of 12%-15%. If the humidity is too high, it can be adjusted by adding an appropriate amount of dry powder (such as corn flour). Humidity measurement can be monitored in real time using a dedicated moisture meter.

[0127] 6.2 Granulation and Cooling

[0128] Granulation: After mixing, the feed enters the granulation equipment. The sieve aperture of the granulator is set to 3-5mm to ensure that the size of the particles meets the standard. The gap between the pressing wheel and the die is adjusted to control the density and hardness of the particles.

[0129] Pellet cooling: The pelletized feed needs to be cooled by the cooling system. The cooling process uses air circulation cooling, and the temperature is controlled within the range of 25-30℃. The cooling time is 20-30 minutes to ensure the stability of the pellets.

[0130] 6.3 Detection

[0131] (1) Finished granule quality inspection (such as Figure 1 (shown)

[0132] Hardness test: Use a feed pellet hardness tester to ensure that the pellet hardness meets the standard (≥4N).

[0133] Particle size: Check the particle diameter by sieve screening method to ensure that the particle diameter is between 3-8mm.

[0134] (2) Component analysis

[0135] Moisture content: Use a moisture meter to test the final feed to ensure that the moisture content is between 12%-15%.

[0136] Lycium barbarum polysaccharide content: The mixed feed was analyzed by HPLC to ensure that the polysaccharide content of the wolfberry extract reached the predetermined standard (2%).

[0137] (3) Microbiological testing

[0138] Conduct microbial testing on feed to ensure that the activity and quantity of probiotics (lactic acid bacteria and bifidobacteria expressing PEDV monoclonal antibodies) meet the standard requirements.

[0139] 6.4 Storage and packaging: Package qualified feed and store it in sealed containers to avoid the influence of moisture and air. The storage environment temperature should be controlled at 20-25℃ and the humidity should be maintained at 50%-60%.

[0140] Table 4 Summary of test results

[0141]

[0142] Example 3: Application of pig feed

[0143] 1. Experimental grouping: Feed the prepared feed to weaned piglets for 60 days. The number of pigs in the experimental group and the control group is 20, the experimental group is fed with the above functional feed, and the control group is fed with conventional feed.

[0144] Experimental group: fish collagen hydrolysate, whey protein hydrolysate, lactic acid bacteria expressing PEDV monoclonal antibodies, and bifidobacteria were added to the feed.

[0145] Control group: The above functional ingredients were not added to the feed, and only conventional feed was used.

[0146] 2. Sampling and data collection: The weight and daily weight gain of the pig group were measured weekly, and the feed consumption was recorded. Blood samples were collected every 10 days to test the immune indicators (such as IgG, IgA, IgM) and physiological indicators of the pigs. At the end of the experiment, the pigs were challenged with PEDV virus, and intestinal and blood samples were collected from each group of pigs to test the viral load and immune response.

[0147] 3. PEDV challenge test: The pigs at the end of the experiment were challenged with PEDV virus by oral administration of PEDV virus solution (dose: 1×10^5TCID 50 / ml, 2ml / head) and observe the clinical symptoms and immune response after virus infection.

[0148] 4. Test results: The specific tracking results are shown in Table 5.

[0149] Growth performance: The daily weight gain of the experimental group (411g) was significantly higher than that of the control group (326g), indicating that the addition of small molecule peptides and lactic acid bacteria expressing PEDV monoclonal antibodies can promote the growth of pigs.

[0150] Immune response: The IgG, IgA and IgM levels of the experimental group pigs were significantly higher than those of the control group, indicating that the feed formula can significantly enhance the immunity of the pig herd.

[0151] PEDV protection: In the PEDV challenge experiment, the viral load of the experimental group was significantly lower than that of the control group, indicating that the added lactic acid bacteria expressing PEDV monoclonal antibodies can effectively improve the pig herd's protection against PEDV.

[0152] Intestinal health: The number of intestinal probiotics in the experimental group was significantly higher than that in the control group, indicating that the feed can promote the balance of intestinal microflora and improve intestinal health.

[0153] Table 5 Summary of test tracking results

[0154]

[0155] The pig feed formula provided by the present invention significantly improves the immunity of pigs, promotes growth and development, and shows excellent protective effects in PEDV challenge experiments by comprehensively applying functional ingredients such as fish collagen hydrolysate, whey protein hydrolysate, lactic acid bacteria expressing PEDV monoclonal antibodies, and bifidobacteria.

[0156] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A pig feed, characterized in that: The composition of the pig feed is as follows in terms of mass percentage: (1) Fish collagen hydrolysate: 2%; (2) Whey protein hydrolysate: 1.5%; (3) Lactic acid bacteria expressing PEDV monoclonal antibodies: 1%; (4) Bifidobacterium: 1%; (5) β-glucan: 1%; (6) Lycium barbarum extract: 2%; (7) Corn flour: 45%; (8) Soybean meal: 35%; (9) Vitamin E: 0.1%.

2. The pig feed according to claim 1, characterized in that The pig feed includes the following features: (1) Moisture content: 13.2%; (2) Particle hardness: 4.5N; (3) Particle diameter: 5 mm; (4) Lycium barbarum polysaccharide content: 2.05%; (5) Probiotic activity: 1.2×10 8 CFU / g; (6) Total number of microorganisms: Not detected.

3. The pig feed according to claim 1, characterized in that The fish collagen hydrolysate is rich in glycine, proline, leucine and phenylalanine; the purity of the fish collagen hydrolysate is 85%; the protein content in every 100g of the fish collagen hydrolysate is 65g; and the molecular weight of 80% of the fish collagen hydrolysate is between 300-1000Da.

4. The pig feed according to claim 1, characterized in that The whey protein hydrolysate is rich in glycine and proline, wherein the glycine content in each 100g of whey protein hydrolysate is 28.5g, and the proline content in each 100g of whey protein hydrolysate is 16.2g; each 100g of whey protein hydrolysate contains 60-65g of total protein, and the molecular weight distribution of 80% of the whey protein hydrolysate is between 500-800Da.

5. The pig feed according to claim 1, characterized in that: The viable bacterial count of the lactic acid bacteria expressing the PEDV monoclonal antibody after freeze-drying is 1.2×10^10 CFU / g, and the viable bacterial count is maintained at 8.5×10^9 CFU / g after storage for 3 months, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the PEDV monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.

2.

6. The pig feed according to claim 1, characterized in that: The number of viable bifidobacteria after freeze-drying is 1×10^10 CFU / g, and the survival rate of viable bacteria after storage for 3 months is >85%.

7. The pig feed according to claim 1, characterized in that The wolfberry extract is rich in polysaccharides and flavonoids, wherein the polysaccharide content in every 100g of wolfberry extract is 45±2g, and the flavonoid content in every 100g of wolfberry extract is 12±1g.

8. Use of the fish collagen hydrolyzate according to claim 3 in preparing pig feed.

9. Use of the whey protein hydrolyzate according to claim 4 in preparing pig feed.

10. Use of the lactic acid bacteria expressing PEDV monoclonal antibody according to claim 5 in preparing pig feed.

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