Pig feed, feed additive and preparation method thereof

Abstract

The present invention discloses a pig feed, wherein the composition of the pig feed is as follows, by mass percentage: (1) fish collagen hydrolysate: 2%; (2) whey protein hydrolysate: 1.5%; (3) lactic acid bacteria expressing PEDV monoclonal antibodies: 1%; (4) bifidobacteria: 1%; (5) β-glucan: 1%; (6) wolfberry extract: 2%; (7) corn flour: 45%; (8) soybean meal: 35%; and (9) vitamin E: 0.1%. The present invention combines multiple functional ingredients such as small molecule polypeptides, 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 existing technology, the formula of the present invention not only solves the problem of traditional feed relying on only a single ingredient, but also provides targeted immune protection against a specific virus (PEDV), and has significant market competitiveness.

Description

Technical Field

[0001] The present 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 become key factors in improving pig health and production efficiency. Traditional pig feeds primarily provide basic energy and protein sources. However, existing feeds and feed additives often have limitations when it comes to improving pig immunity, disease resistance, and growth. This is particularly true when addressing issues such as decreased immunity, poor disease resistance, and frequent epidemics.

[0003] In recent years, the health of pig immune systems has garnered increasing attention. This is particularly true in livestock farming environments, where threats from pathogens such as viruses and bacteria place pigs' immune systems under chronic stress, directly impacting their growth, development, and disease resistance. Improving pig immunity and enhancing their disease resistance has become a pressing challenge for the pig farming industry. Furthermore, with the expansion of livestock farming and rising production costs, the pig farming industry is placing increasingly stringent demands on feed conversion rates. Therefore, ensuring pig growth performance while reducing disease and improving pig health has become a key research priority.

[0004] Currently, there are some immune-enhancing feeds and additives on the market, such as those containing immunomodulatory ingredients (such as β-glucan and antioxidants) and Chinese herbal ingredients. These can improve pigs' immunity and disease resistance to a certain extent, but they still suffer from issues such as inadequate formulations and limited effectiveness. Therefore, there is an urgent need to develop new feeds and feed additives that can enhance pig immunity and promote healthy growth while also improving feed utilization efficiency and breeding profitability. Summary of the Invention

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

[0006] Therefore, the present invention discloses a pig feed, which comprises the following components in percentage by mass:

[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 includes 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 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 80% of the whey protein hydrolysate has a molecular weight distribution between 500-800Da.

[0025] Preferably, the lactic acid bacteria expressing PEDV monoclonal antibodies of the present invention have a viable cell count of 1.2×10^10 CFU / g after freeze-drying, and the viable cell 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 as SEQ ID NO.1 and SEQ ID NO.2.

[0026] Preferably, the viable bacterial count of the bifidobacterium after freeze-drying of the present invention is 1×10^10 CFU / g, and the viable bacterial survival rate after storage for 3 months is greater than 85%.

[0027] Preferably, the wolfberry extract of the present invention 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.

[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] This invention combines multiple functional ingredients, including small molecule peptides, lactic acid bacteria expressing PEDV monoclonal antibodies, and probiotics, to comprehensively enhance the pig's immune system, improve intestinal health, and enhance its growth performance. Compared with existing feeds, this formula not only overcomes the problem of traditional feeds relying on a single ingredient but also provides targeted immune protection against a specific virus (PEDV), making it significantly more competitive in the market.

[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), which 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 shows the 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 commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated 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 composition of raw materials 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, scales, or bones. Common choices include salmon skin and cod skin. Ensure the freshness of the raw material 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 (skin:water). Treat the 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. Steam at high temperature (approximately 80°C for 30 minutes) to extract the collagen. 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 enzyme dosage is typically 1-2% of the collagen (enzyme:collagen mass ratio). Adjust the pH of the solution to 4-6 (by adding an appropriate amount of acetic acid) to the optimal range for the selected enzyme. Place the hydrolyzate in a water bath set to 50°C-60°C for hydrolysis. The reaction typically lasts 2-4 hours, but the degree of hydrolysis can be adjusted based on actual needs.

[0054] Reaction Termination: When the hydrolyzate reaches the predetermined time, terminate the reaction 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 (4000 rpm, 10-15 minutes) to remove large molecular residues and impurities. The supernatant is the initial hydrolyzed collagen peptides.

[0057] Ultrafiltration: Filter the hydrolyzate using an ultrafiltration membrane (molecular weight cutoff of 300-1000Da) to remove unhydrolyzed high-molecular-weight proteins and obtain small-molecule peptides. Isolate small-molecule peptides (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 at a temperature below 60°C to prevent degradation of the active ingredient. During the spray-drying process, the atomized particle size should be appropriate to ensure a fine powder with good flowability.

[0060] Finished Product Storage: After drying, the fish collagen hydrolysate powder can be packaged and stored in a cool, dry, dark environment. Finished collagen hydrolysate undergoes quality control testing 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 hydrolyzates 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 with a protein content of more than 80%. Ensure the whey powder is fresh and avoid moisture and contamination.

[0071] Whey protein extraction: Dissolve whey powder in distilled water at a ratio of 1:4. Let stand for 15 minutes, stir thoroughly, and filter through filter paper to remove impurities.

[0072] 2.2 Whey protein hydrolysis

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

[0074] End of hydrolysis and termination: 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 standardization of each batch of product. 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 hydrolyzate was tested using the DPPH method. Results: The DPPH free radical scavenging rate of the 50 mg / mL hydrolyzate solution was 45%.

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

[0089] (1) Conventional mouse hybridoma cell technology (as described in the specifications of CN118684771A, CN118325846A, etc.) was used to prepare monoclonal antibodies against PEDV virus. The amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody were shown in SEQ ID NO.1 and SEQ ID NO.2. The monoclonal antibody was subjected to neutralization experiments on PEDV virus of different titers. It was found that the monoclonal antibody could inhibit 10 μg / ml of PEDV virus at a concentration of 10 μg / ml.5.0 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 and heavy chains against PEDV virus, the codons were optimized to adapt to expression in lactic acid bacteria. The synthesized gene was inserted into the plasmid pNZ8148, and the ligase reaction temperature was 16°C overnight. Transformation was performed 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). The cells were washed three times with sterile cold PBS and resuspended in 10% glycerol to prepare electrocompetent cells. The recombinant plasmid was used to transform the lactic acid bacteria using electroporation conditions: 2.5 kV, 25 μF, 200 Ω. Positive clones were selected on GM17 solid medium containing chloramphenicol, and correct vector insertion was verified by PCR.

[0092] (4) Positive strains were inoculated into GM17 medium and pre-cultured at 37°C overnight. Dilute to an OD600 of 0.2 and continue culturing until an OD600 of 0.6 was reached. 0.5% lactose was added as an inducer and cultured at 37°C for 12 hours. The cells were harvested and expression levels were 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) Detect 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) Streak the strain from the storage tube onto MRS agar medium and incubate anaerobically at 37°C for 24 hours. Pick a single colony and inoculate it onto MRS liquid medium and incubate 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 any residual culture medium.

[0097] (2) Resuspend the collected bacteria in a 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℃ for 12 hours. Use a freeze dryer (initial temperature -40℃), gradually increase the temperature to 25℃, and freeze-dry for 24 hours. Divide the freeze-dried bacterial powder into sealed bags and store at 4℃ or -20℃. After testing, the number of viable bacteria after freeze-drying was 1×10^10 CFU / g, and the survival rate of viable bacteria was >85% after storage for 3 months.

[0098] 5. Preparation of wolfberry extract

[0099] 5.1 Pretreatment of wolfberry

[0100] Washing and drying wolfberries: Place the dried wolfberries in a sieve and gently rinse with running water to remove surface impurities. Dry the washed wolfberries at 60°C until the moisture content is less than 10% (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 goji berry powder with 80% ethanol in a 1:10 ratio (i.e., 10g goji berry powder to 100mL ethanol). Place in an ultrasonic cleaner and extract at 50°C for 30 minutes. Gently stir the solution during extraction 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. The operation method is the same as the first extraction.

[0106] 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 and remove most of the ethanol under reduced pressure. The remaining solution volume should be approximately 1 / 3 of the original solution. Be sure to keep the evaporation temperature below 50°C to avoid degradation of the active ingredients due to high temperatures.

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

[0110] Impurity Removal and Further Concentration: The aqueous phase is further purified by ultrafiltration, and the filtrate is concentrated to the desired concentration. The concentrate is then 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, maintaining the temperature below 40°C, until the extract is completely dry, and a powdery finished product is obtained.

[0113] Storage: Store the dried wolfberry extract powder in a sealed container in a cool, dry place away from direct sunlight and high temperatures.

[0114] 5.5. Detection

[0115] (1) Detection of polysaccharide content in 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 100 g of wolfberry extract was 45 ± 2 g.

[0116] (2) Detection of flavonoid content in wolfberry: The flavonoid content in wolfberry extract was determined using UV-visible spectrophotometry, primarily to detect bioactive flavonoids (e.g., wolfberry flavonoids). The results showed that the flavonoid content in every 100g of wolfberry extract was 12±1g.

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

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

[0119] Table 3 Summary of test results

[0120]

[0121]

[0122] 6. Feed mixing

[0123] 6.1 Mixing process

[0124] (1) Preliminary mixing of dry materials: Add corn flour, soybean meal, β-glucan, antioxidant (vitamin E) and other dry ingredients into a mixer. Set the mixer speed to 20-30 rpm and perform preliminary mixing for 5-10 minutes to ensure that the dry ingredients are fully mixed.

[0125] (2) Adding wet materials: Add the dissolved fish collagen hydrolysate, whey protein hydrolysate, wolfberry extract, lactic acid bacteria expressing PEDV monoclonal antibodies, and bifidobacteria to a mixer in proportion and mix thoroughly. Use a spray device to evenly spray the mixed wet materials onto the mixture (the dry materials mixed in step (1)), ensuring that each particle is evenly covered with the wet materials.

[0126] (3) Even mixing: After adding the wet ingredients, continue mixing at a speed of 40-60 rpm for 10-15 minutes to ensure that the wet ingredients are evenly combined with the dry ingredients. Check the mixing uniformity by sampling to ensure that the wet ingredients (such as wolfberry extract, fish collagen hydrolyzate, etc.) are evenly distributed.

[0127] (4) 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.

[0128] 6.2 Granulation and Cooling

[0129] Granulation: After mixing, the feed enters the granulation equipment. The sieve aperture of the granulator is set to 3-5mm to ensure that the particle size meets the standard. The gap between the pressing wheel and the die is adjusted to control the density and hardness of the particles.

[0130] Pellet cooling: Pelletized feed needs to be cooled through a cooling system. Air circulation is used during the cooling process, and the temperature is controlled within the range of 25-30°C. The cooling time is 20-30 minutes to ensure pellet stability.

[0131] 6.3 Detection

[0132] (1) Finished product granule quality inspection (such as Figure 1 shown)

[0133] Hardness test: Use a feed pellet hardness tester to ensure that the hardness of the pellets meets the standard (≥4N).

[0134] Particle size: Check the particle diameter through sieve screening method to ensure that the particle diameter is between 3-8mm.

[0135] (2) Component analysis

[0136] Moisture content: Use a moisture meter to test the final feed to ensure that the moisture content is between 12% and 15%.

[0137] Lycium barbarum polysaccharide content: The mixed feed was analyzed by HPLC to ensure that the polysaccharide content of the Lycium barbarum extract reached the predetermined standard (2%).

[0138] (3) Microbial testing

[0139] 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.

[0140] 6.4 Storage and Packaging: Qualified feeds should be packaged and stored 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%.

[0141] Table 4 Summary of test results

[0142]

[0143] Example 3: Application of pig feed

[0144] 1. Experimental Grouping: The prepared feed was fed to weaned piglets for 60 days. The experimental group and control group consisted of 20 pigs each. The experimental group was fed the functional feed described above, while the control group was fed conventional feed.

[0145] Experimental group: fish collagen hydrolysate, whey protein hydrolysate, lactic acid bacteria expressing PEDV monoclonal antibodies, and bifidobacteria were added to the feed.

[0146] Control group: The above functional ingredients were not added to the feed, and only conventional feed was used.

[0147] 2. Sampling and Data Collection: Pig body weight and daily weight gain were measured weekly, and feed consumption was recorded. Blood samples were collected every 10 days to test immune markers (such as IgG, IgA, and IgM) and physiological indicators. At the end of the experiment, the pigs were challenged with PEDV. Intestinal and blood samples were collected from each group to test for viral load and immune response.

[0148] 3. PEDV challenge test: The pigs were challenged with PEDV virus at the end of the experiment. The PEDV virus solution (dose: 1×10^5TCID 50 / ml, 2ml / head), and observe the clinical symptoms and immune response after virus infection.

[0149] 4. Test results: The specific tracking results are shown in Table 5.

[0150] 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.

[0151] Immune response: The IgG, IgA and IgM levels of the pigs in the experimental group were significantly higher than those in the control group, indicating that the feed formula can significantly enhance the immunity of the pig herd.

[0152] 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.

[0153] 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 microbial communities and improve intestinal health.

[0154] Table 5 Summary of test tracking results

[0155]

[0156] The pig feed formula provided by the present invention significantly improves the immunity of pigs, promotes growth and development, and demonstrates 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.

[0157] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A pig feed, characterized in that: The pig feed is made from the following raw materials in percentage by mass: (1) Fish collagen hydrolysate: 2%; wherein the fish collagen hydrolysate is rich in glycine, proline, leucine and phenylalanine; wherein the purity of the fish collagen hydrolysate is 85%; the protein content in each 100g of fish collagen hydrolysate is 65g; and the molecular weight of 80% of the fish collagen hydrolysate is between 300-1000 Da; (2) Whey protein hydrolysate: 1.5%; wherein 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 80% of the whey protein hydrolysate has a molecular weight distribution between 500-800 Da; (3) Lactic acid bacteria expressing PEDV monoclonal antibodies: 1%; wherein the viable bacterial count of the lactic acid bacteria expressing PEDV monoclonal antibodies 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, 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; (4) Bifidobacterium: 1%; wherein the number of viable bacteria of the bifidobacterium after freeze-drying is 1×10^10 CFU / g, and the survival rate of the viable bacteria after storage for 3 months is greater than 85%; (5) β-glucan: 1%; (6) Lycium barbarum extract: 2%; wherein the Lycium barbarum extract is rich in polysaccharides and flavonoids, wherein the polysaccharide content in each 100g of Lycium barbarum extract is 45±2g, and the flavonoid content in each 100g of Lycium barbarum extract is 12±1g; (7) Corn flour: 45%; (8) Soybean meal: 35%; (9) Vitamin E: 0.1%.

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