Oligopeptide for improving immune performance of laying hens and preparation method of oligopeptide

Abstract

The invention relates to an oligopeptide for improving the immune performance of laying hens and a preparation method thereof, and belongs to the technical field of functional oligopeptides. The preparation method of the oligopeptide comprises the following steps: performing fractional enzymolysis on sunflower discs and vital gluten by using hydrolase I and hydrolase II obtained by fermenting bacillus licheniformis and aspergillus oryzae, and purifying to obtain the target oligopeptide. The prepared oligopeptide can effectively improve the immune performance of laying hens, improve the oxidation resistance of the laying hens and prevent the laying hens from oxidative damage in a high-temperature environment.

Description

Technical Field

[0001] The invention belongs to the technical field of functional short peptides and relates to a short peptide for improving the immune performance of laying hens and a preparation method thereof. Background Art

[0002] Layers are poultry belonging to the genus Gallus in the family Phasianidae. Unlike broiler chickens, laying hens are raised specifically for egg production. Scientific breeding practices are crucial to improving egg quality and maintaining or increasing egg production, not improving the quality of the chicken itself.

[0003] As one of the most common poultry in the farming industry, the immune health of laying hens is extremely important. During the growth of laying hens, the stability and health of their immune systems are directly related to their production performance and production costs. Therefore, scientific immune support in laying hen farming is crucial to the health and profitability of laying hen operations.

[0004] It has been disclosed in the prior art that adding antimicrobial peptides to feed can improve the production performance of caged laying ducks during the peak laying period without affecting egg quality. It can also improve serum biochemical indicators, enhance antioxidant capacity, and enhance immune function. Summary of the Invention

[0005] The main purpose of the present invention is to provide a short peptide for improving the immune performance of laying hens and a preparation method thereof. The short peptide can prevent laying hens from being damaged by oxidation in a high-temperature environment.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned purpose: A method for preparing a short peptide for improving the immune performance of laying hens, specifically comprising the following steps: Step 1, preparation of hydrolase I: after activating Bacillus licheniformis, inoculate it into seed culture medium I containing 5 g / L tryptone, 5 g / L yeast extract, 10 g / L glucose, and 18 g / L dipotassium hydrogen phosphate, and place it in a shaking incubator for shaking culture for 24 hours to obtain seed solution I; inoculate seed solution I into fermentation medium I containing 10 g peptone, 5 g beef extract, 10 g sucrose, 2 g disodium hydrogen phosphate, and 1 L water, with an initial pH of 7.5, shake culture at 35°C and 180 r / min for 4 hours, add Chlorella powder, stop fermentation after 36 hours, filter, concentrate the filtrate, and dry to obtain hydrolase I; Step 2, preparation of hydrolase II: After activating Aspergillus oryzae, inoculate it into a seed culture medium II containing 200g of potato, 20g of glucose, 15-20g of agar, and 1L of water, and culture it at 30°C for 48h. Scrape the spores, shake them to break them up, filter out the mycelium, and dilute them with sterile saline to obtain a spore suspension; inoculate the spore suspension into a fermentation medium II containing 30% bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. After culture at 30°C for 72h, stop the fermentation, filter, and dry to obtain hydrolase II; Step 3: drying and crushing the sunflower disk to obtain sunflower disk powder for later use; adding water to the sunflower disk powder and mixing evenly, adding Bacillus subtilis, fermenting at 35° C. for 6 hours, then heating and boiling for 20 minutes, cooling to room temperature, adding gluten and mixing evenly, adding hydrolase I, enzymatically hydrolyzing at 40-50° C. for 2-3 hours, and centrifuging to obtain hydrolyzate I for later use; Step 4: Filter hydrolyzate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolyzate fractions with molecular weights of 3-5 kDa and >10 kDa. After combining the fractions, add hydrolase II and aminopeptidase, perform enzymatic hydrolysis at 40-50°C for 1-1.5 hours, heat to 80°C to inactivate the enzyme, and centrifuge to obtain hydrolyzate II for later use. Step 5, the hydrolyzate II is filtered using a 5 kDa ultrafiltration membrane to obtain a hydrolyzate with a molecular weight of less than 5 kDa, which is concentrated and dried to obtain a primary product; after the primary product is dissolved in water, it is loaded onto a Sephadex G25 gel column, eluted with water as the elution solvent, the eluate is collected, and the eluate is tested. The eluates containing short peptides with a molecular weight of 500-5000Da are combined, concentrated, and dried to obtain the target short peptide.

[0007] Furthermore, the inoculation amount of seed solution I in step 1 is 10%.

[0008] Furthermore, the amount of chlorella powder added in step 1 is 20 g / L.

[0009] Furthermore, the protease activity of the hydrolase I in step 1 is 1503 U / g.

[0010] Furthermore, the amount of spores in the spore suspension in step 2 is 1×10 6 pieces / mL.

[0011] Furthermore, the inoculation amount of the spore suspension in step 2 is 5%.

[0012] Furthermore, the protease activity of the hydrolase II in step 2 is 2128 U / g.

[0013] Furthermore, the amount of Bacillus subtilis added in step 3 is 1% of the weight of the sunflower disc powder.

[0014] Furthermore, the amount of gluten added in step 3 is 30% of the weight of the sunflower disc powder.

[0015] Furthermore, the amount of hydrolase I added in step 3 is 10 g / L.

[0016] Furthermore, in step 4, the amount of hydrolase II added is 15 g / L, and the amount of aminopeptidase added is 8 g / L.

[0017] The present invention provides a short peptide prepared by the above preparation method.

[0018] The present invention provides a specific application of the short peptide prepared by the preparation method, that is, the short peptide can improve the immune performance of laying hens.

[0019] The present invention has the following beneficial effects: 1. The short peptide prepared in this invention has the ability to improve the immune function of laying hens and reduce the oxidative stress response caused by heat stress. Test results show that the short peptide of this invention can significantly increase the immunoglobulin level in the serum of laying hens under high temperature conditions, enhancing their immune function; at the same time, it can also improve their antioxidant capacity and reduce oxidative damage.

[0020] 2. In the short peptide preparation method of the present invention, sunflower discs and gluten are creatively selected as raw materials, and short peptides of different fragments are obtained by preliminary enzymatic hydrolysis. After filtering through a filter membrane, the peptide of the target fragment is subjected to secondary enzymatic hydrolysis to obtain small molecule peptides, and then purified by a filter to obtain the short peptide of the target fragment; in the enzymatic hydrolysis process, the enzymatic hydrolysis effect of simply using traditional protease is not ideal, and the short peptide obtained has poor activity. Therefore, the present invention further screens the hydrolases used for enzymatic hydrolysis, selects a complex enzyme system produced by fermentation of Bacillus licheniformis and Aspergillus oryzae, and adopts a partial enzymatic hydrolysis method to obtain short peptides. The short peptides obtained by this method have high physiological activity, can significantly improve the immune performance of laying hens, and improve their antioxidant capacity. DETAILED DESCRIPTION

[0021] The present invention is further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art all fall within the scope of protection of the claims of this application.

[0022] Example 1 Step 1, preparation of hydrolase I: Bacillus licheniformis CCTCC AB 2016311 purchased from the China Center for Type Culture Collection was activated according to the instructions and inoculated into a seed culture medium I containing 5 g / L tryptone, 5 g / L yeast extract, 10 g / L glucose, and 18 g / L dipotassium hydrogen phosphate. The culture was placed in a shaking incubator (30°C, 150 r / min) and cultured for 24 hours to obtain seed solution I; the seed solution I was inoculated at a 10% (v / v) inoculum amount into a fermentation medium I containing 10 g peptone, 5 g beef extract, 10 g sucrose, 2 g disodium hydrogen phosphate, and 1 L water, with an initial pH of 7.5, and cultured at 35°C, 180 r / min, and 20 g / L Chlorella powder was added. The fermentation was stopped after 36 hours, filtered, and the filtrate was concentrated and dried to obtain hydrolase I, wherein the protease activity was 1503 U / g; Step 2, preparation of hydrolase II: Aspergillus oryzae CCTCC AF 2016006 purchased from China Center for Type Culture Collection was activated according to the instructions and inoculated into seed culture medium II containing 200 g potato, 20 g glucose, 20 g agar, and 1 L water. The culture was statically incubated at 30°C for 48 h, the spores were scraped off, shaken to disperse, the mycelium was filtered to remove, and the spores were diluted with sterile saline to a spore volume of 1×10 6 / mL to obtain a spore suspension; the spore suspension was inoculated at a 5% (v / v) inoculum size into a fermentation medium II containing 30% bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. The culture was carried out at 30°C for 72 hours, and then the fermentation was stopped. The culture was filtered and dried to obtain hydrolase II, of which the protease activity was 2128 U / g. Step 3: drying and crushing the sunflower disk to obtain sunflower disk powder for later use; taking 1 kg of the sunflower disk powder, adding 5 L of water and mixing evenly, adding 10 g of Bacillus subtilis with an effective viable count of 20 billion cfu / g, fermenting at 35° C. for 6 hours, then heating and boiling for 20 minutes, cooling to room temperature, adding 0.3 kg of gluten and mixing evenly, adding 10 g / L of hydrolase I, enzymatically hydrolyzing at 50° C. for 2 hours, and centrifuging to obtain hydrolyzate I for later use; Step 4: Filter hydrolyzate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolyzate fractions with molecular weights of 3-5 kDa and >10 kDa. After combining the two fractions, add 15 g / L hydrolase II and 8 g / L aminopeptidase (enzyme activity of 5000 U / g), perform enzymatic hydrolysis at 50°C for 1 hour, heat to 80°C to inactivate the enzyme, and centrifuge to obtain hydrolyzate II for later use. Step 5: Filter the hydrolyzate II using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight of less than 5 kDa, concentrate and dry to obtain the initial product; after dissolving the initial product in water, load it onto a Sephadex G25 gel column, elute with water as the elution solvent, collect the eluate, detect the eluate, combine the eluates containing short peptides with a molecular weight of 500-5000Da, concentrate and dry to obtain the target short peptide.

[0023] Example 2 Step 1, preparation of hydrolase I: Bacillus licheniformis CCTCC AB 2016311 purchased from the China Center for Type Culture Collection was activated according to the instructions and inoculated into a seed culture medium I containing 5 g / L tryptone, 5 g / L yeast extract, 10 g / L glucose, and 18 g / L dipotassium hydrogen phosphate. The culture was placed in a shaking incubator (30°C, 150 r / min) and cultured for 24 hours to obtain seed solution I; the seed solution I was inoculated at a 10% (v / v) inoculum amount into a fermentation medium I containing 10 g peptone, 5 g beef extract, 10 g sucrose, 2 g disodium hydrogen phosphate, and 1 L water, with an initial pH of 7.5, and cultured at 35°C, 180 r / min, and 20 g / L Chlorella powder was added. The fermentation was stopped after 36 hours, filtered, and the filtrate was concentrated and dried to obtain hydrolase I, wherein the protease activity was 1503 U / g; Step 2, preparation of hydrolase II: Aspergillus oryzae CCTCC AF 2016006 purchased from China Center for Type Culture Collection was activated according to the instructions and inoculated into seed culture medium II containing 200 g potato, 20 g glucose, 20 g agar, and 1 L water. The culture was statically incubated at 30°C for 48 h, the spores were scraped off, shaken to disperse, the mycelium was filtered to remove, and the spores were diluted with sterile saline to a spore volume of 1×10 6 / mL to obtain a spore suspension; the spore suspension was inoculated at a 5% (v / v) inoculum size into a fermentation medium II containing 30% bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. The culture was carried out at 30°C for 72 hours, and then the fermentation was stopped. The culture was filtered and dried to obtain hydrolase II, of which the protease activity was 2128 U / g. Step 3: drying and crushing the sunflower disk to obtain sunflower disk powder for later use; taking 1 kg of the sunflower disk powder, adding 5 L of water and mixing evenly, adding 10 g of Bacillus subtilis with an effective viable count of 20 billion cfu / g, fermenting at 35° C. for 6 hours, then heating and boiling for 20 minutes, cooling to room temperature, adding 0.3 kg of gluten and mixing evenly, adding 10 g / L of hydrolase I, enzymatically hydrolyzing at 40° C. for 3 hours, and centrifuging to obtain hydrolyzate I for later use; Step 4: Filter hydrolyzate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolyzate fractions with molecular weights of 3-5 kDa and >10 kDa. After combining the two fractions, add 15 g / L hydrolase II and 8 g / L aminopeptidase (enzyme activity of 5000 U / g), perform enzymatic hydrolysis at 40°C for 1.5 h, heat to 80°C to inactivate the enzyme, and centrifuge to obtain hydrolyzate II for later use. Step 5: Filter the hydrolyzate II using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight of less than 5 kDa, concentrate and dry to obtain the initial product; after dissolving the initial product in water, load it onto a Sephadex G25 gel column, elute with water as the elution solvent, collect the eluate, detect the eluate, combine the eluates containing short peptides with a molecular weight of 500-5000Da, concentrate and dry to obtain the target short peptide.

[0024] Example 3 Step 1, preparation of hydrolase I: Bacillus licheniformis CCTCC AB 2016311 purchased from the China Center for Type Culture Collection was activated according to the instructions and inoculated into a seed culture medium I containing 5 g / L tryptone, 5 g / L yeast extract, 10 g / L glucose, and 18 g / L dipotassium hydrogen phosphate. The culture was placed in a shaking incubator (30°C, 150 r / min) and cultured for 24 hours to obtain seed solution I; the seed solution I was inoculated at a 10% (v / v) inoculum amount into a fermentation medium I containing 10 g peptone, 5 g beef extract, 10 g sucrose, 2 g disodium hydrogen phosphate, and 1 L water, with an initial pH of 7.5, and cultured at 35°C, 180 r / min, and 20 g / L Chlorella powder was added. The fermentation was stopped after 36 hours, filtered, and the filtrate was concentrated and dried to obtain hydrolase I, wherein the protease activity was 1503 U / g; Step 2, preparation of hydrolase II: Aspergillus oryzae CCTCC AF 2016006 purchased from China Center for Type Culture Collection was activated according to the instructions and inoculated into seed culture medium II containing 200 g potato, 20 g glucose, 20 g agar, and 1 L water. The culture was statically incubated at 30°C for 48 h, the spores were scraped off, shaken to disperse, the mycelium was filtered to remove, and the spores were diluted with sterile saline to a spore volume of 1×10 6 / mL to obtain a spore suspension; the spore suspension was inoculated at a 5% (v / v) inoculum size into a fermentation medium II containing 30% bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. The culture was carried out at 30°C for 72 hours, and then the fermentation was stopped. The culture was filtered and dried to obtain hydrolase II, of which the protease activity was 2128 U / g. Step 3: drying and crushing the sunflower disk to obtain sunflower disk powder for later use; taking 1 kg of the sunflower disk powder, adding 5 L of water and mixing evenly, adding 10 g of Bacillus subtilis with an effective viable count of 20 billion cfu / g, fermenting at 35° C. for 6 hours, then heating and boiling for 20 minutes, cooling to room temperature, adding 0.3 kg of gluten and mixing evenly, adding 10 g / L of hydrolase I, enzymatically hydrolyzing at 50° C. for 2 hours, and centrifuging to obtain hydrolyzate I for later use; Step 4: Filter hydrolyzate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolyzate fractions with molecular weights of 3-5 kDa and >10 kDa. After combining the two fractions, add 15 g / L hydrolase II and 8 g / L aminopeptidase (enzyme activity of 5000 U / g), perform enzymatic hydrolysis at 40°C for 1.5 h, heat to 80°C to inactivate the enzyme, and centrifuge to obtain hydrolyzate II for later use. Step 5: Filter the hydrolyzate II using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight of less than 5 kDa, concentrate and dry to obtain the initial product; after dissolving the initial product in water, load it onto a Sephadex G25 gel column, elute with water as the elution solvent, collect the eluate, detect the eluate, combine the eluates containing short peptides with a molecular weight of 500-5000Da, concentrate and dry to obtain the target short peptide.

[0025] Comparative Example 1 Step 1, preparation of hydrolase: Bacillus licheniformis CCTCC AB 2016311 purchased from the China Center for Type Culture Collection was activated according to the instructions and inoculated into a seed culture medium I containing 5 g / L tryptone, 5 g / L yeast extract, 10 g / L glucose, and 18 g / L dipotassium hydrogen phosphate. The culture was placed in a shaking incubator (30°C, 150 r / min) and cultured for 24 hours to obtain seed solution I; the seed solution I was inoculated at a 10% (v / v) inoculum amount into a fermentation medium I containing 10 g peptone, 5 g beef extract, 10 g sucrose, 2 g disodium hydrogen phosphate, and 1 L water, with an initial pH of 7.5, and cultured at 35°C, 180 r / min, and 20 g / L Chlorella powder was added. The fermentation was stopped after 36 hours, filtered, and the filtrate was concentrated and dried to obtain a hydrolase, wherein the protease activity was 1503 U / g; Step 2: drying and crushing the sunflower disk to obtain sunflower disk powder for later use; taking 1 kg of the sunflower disk powder, adding 5 L of water and mixing evenly, adding 10 g of Bacillus subtilis with an effective viable count of 20 billion cfu / g, fermenting at 35° C. for 6 hours, then heating and boiling for 20 minutes, cooling to room temperature, adding 0.3 kg of gluten and mixing evenly, adding 50 g of hydrolase, and enzymatically hydrolyzing at 40° C. for 3 hours, centrifuging, and obtaining a hydrolyzate for later use; Step 3: Filter hydrolyzate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolyzate fractions with molecular weights of 3-5 kDa and >10 kDa. After combining the two fractions, add 5 g / L papain (100,000 U / g) and 8 g / L aminopeptidase (enzyme activity of 5000 U / g), perform enzymatic hydrolysis at 40°C for 1.5 h, heat to 80°C to inactivate the enzyme, and centrifuge to obtain hydrolyzate II for later use. Step 4: Filter the hydrolyzate II using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight of less than 5 kDa, concentrate and dry to obtain the initial product; after dissolving the initial product in water, load it onto a Sephadex G25 gel column, elute with water as the elution solvent, collect the eluate, detect the eluate, combine the eluates containing short peptides with a molecular weight of 500-5000Da, concentrate and dry to obtain the target short peptide.

[0026] Comparative Example 2 Step 1, preparation of hydrolase: Aspergillus oryzae CCTCC AF 2016006 purchased from China Center for Type Culture Collection was activated according to the instructions and inoculated into seed culture medium II containing 200 g potato, 20 g glucose, 20 g agar, and 1 L water. The culture was statically incubated at 30°C for 48 h, the spores were scraped off, shaken to disperse, the mycelium was filtered to remove, and the spores were diluted with sterile saline to a spore volume of 1×10 6 / mL to obtain a spore suspension; the spore suspension was inoculated at a 5% (v / v) inoculum size into a fermentation medium containing 30% bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. The culture was carried out at 30°C for 72 hours, and then the fermentation was stopped. The hydrolase was filtered and dried to obtain a protease activity of 2128 U / g. Step 2: drying and crushing the sunflower disk to obtain sunflower disk powder, which is set aside; taking 1 kg of the sunflower disk powder, adding 5 L of water and mixing evenly, adding 10 g of Bacillus subtilis with an effective viable bacterial count of 20 billion cfu / g, fermenting at 35°C for 6-8 hours, then heating and boiling for 20-30 minutes, cooling to room temperature, adding 0.3 kg of gluten and mixing evenly, adding 50 g of hydrolase and 20 g / L of aminopeptidase (enzyme activity of 5000 U / g), enzymatically hydrolyzing at 40-50°C for 1-1.5 hours, heating to 80°C to inactivate the enzyme, and centrifuging to obtain a hydrolyzate for set aside; Step 3: Filter the hydrolyzate using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight of less than 5 kDa, concentrate and dry to obtain the initial product; after dissolving the initial product in water, load it onto a Sephadex G25 gel column, elute with water as the elution solvent, collect the eluate, detect the eluate, combine the eluates containing short peptides with a molecular weight of 500-5000Da, concentrate and dry to obtain the target short peptide.

[0027] Comparative Example 3 Step 1, preparation of hydrolase I: Bacillus licheniformis CCTCC AB 2016311 purchased from the China Center for Type Culture Collection was activated according to the instructions and inoculated into a seed culture medium I containing 5 g / L tryptone, 5 g / L yeast extract, 10 g / L glucose, and 18 g / L dipotassium hydrogen phosphate. The culture was placed in a shaking incubator (30°C, 150 r / min) and cultured for 24 hours to obtain seed solution I; the seed solution I was inoculated at a 10% (v / v) inoculum amount into a fermentation medium I containing 10 g peptone, 5 g beef extract, 10 g sucrose, 2 g disodium hydrogen phosphate, and 1 L water, with an initial pH of 7.5. The culture was shaken at 35°C and 180 r / min for 36 hours, and then the fermentation was stopped. The filtrate was filtered, concentrated, and dried to obtain hydrolase I, wherein the protease activity was 366 U / g; Step 2, preparation of hydrolase II: Aspergillus oryzae CCTCC AF 2016006 purchased from China Center for Type Culture Collection was activated according to the instructions and inoculated into seed culture medium II containing 200 g potato, 20 g glucose, 20 g agar, and 1 L water. The culture was statically incubated at 30°C for 48 h, the spores were scraped off, shaken to disperse, the mycelium was filtered to remove, and the spores were diluted with sterile saline to a spore volume of 1×10 6 / mL to obtain a spore suspension; the spore suspension was inoculated at a 5% (v / v) inoculum size into a fermentation medium II containing 30% bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. The culture was carried out at 30°C for 72 hours, and then the fermentation was stopped. The culture was filtered and dried to obtain hydrolase II, of which the protease activity was 2128 U / g. Step 3: drying and crushing the sunflower disk to obtain sunflower disk powder for later use; taking 1 kg of the sunflower disk powder, adding 5 L of water and mixing evenly, adding 10 g of Bacillus subtilis with an effective viable count of 20 billion cfu / g, fermenting at 35° C. for 6-8 hours, then heating and boiling for 20-30 minutes, cooling to room temperature, adding 10 g / L of hydrolase I, enzymatically hydrolyzing at 40-50° C. for 2-3 hours, and centrifuging to obtain hydrolyzate I for later use; Step 4: Filter hydrolyzate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolyzate fractions with molecular weights of 3-5 kDa and >10 kDa. After combining the two fractions, add 15 g / L hydrolase II and perform enzymatic hydrolysis at 40-50°C for 1-1.5 hours. Heat to 80°C to inactivate the enzyme, centrifuge, and obtain hydrolyzate II for later use. Step 5, the hydrolyzate II is filtered using a 5 kDa ultrafiltration membrane to obtain a hydrolyzate with a molecular weight of less than 5 kDa, which is concentrated and dried to obtain a primary product; after the primary product is dissolved in water, it is loaded onto a Sephadex G25 gel column, eluted with water as the elution solvent, the eluate is collected, and the eluate is tested. The eluates containing short peptides with a molecular weight of 500-5000Da are combined, concentrated, and dried to obtain the target short peptide.

[0028] Comparative Example 4 Step 1, preparation of hydrolase I: Bacillus licheniformis CCTCC AB 2016311 purchased from the China Center for Type Culture Collection was activated according to the instructions and inoculated into a seed culture medium I containing 5 g / L tryptone, 5 g / L yeast extract, 10 g / L glucose, and 18 g / L dipotassium hydrogen phosphate. The culture was placed in a shaking incubator (30°C, 150 r / min) and cultured for 24 hours to obtain seed solution I; the seed solution I was inoculated at a 10% (v / v) inoculum amount into a fermentation medium I containing 10 g peptone, 5 g beef extract, 10 g sucrose, 2 g disodium hydrogen phosphate, and 1 L water, with an initial pH of 7.5, and cultured at 35°C, 180 r / min, and 20 g / L Chlorella powder was added. The fermentation was stopped after 36 hours, filtered, and the filtrate was concentrated and dried to obtain hydrolase I, wherein the protease activity was 1503 U / g; Step 2, preparation of hydrolase II: Aspergillus oryzae CCTCC AF 2016006 purchased from China Center for Type Culture Collection was activated according to the instructions and inoculated into seed culture medium II containing 200 g potato, 20 g glucose, 20 g agar, and 1 L water. The culture was statically incubated at 30°C for 48 h, the spores were scraped off, shaken to disperse, the mycelium was filtered to remove, and the spores were diluted with sterile saline to a spore volume of 1×10 6 / mL to obtain a spore suspension; the spore suspension was inoculated at a 5% (v / v) inoculum size into a fermentation medium II containing 30% bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. The culture was carried out at 30°C for 72 hours, and then the fermentation was stopped. The culture was filtered and dried to obtain hydrolase II, of which the protease activity was 2128 U / g. Step 3: drying and crushing the sunflower disk to obtain sunflower disk powder for later use; taking 1 kg of the sunflower disk powder, adding 5 L of water and mixing evenly, adding 10 g of Bacillus subtilis with an effective viable count of 20 billion cfu / g, fermenting at 35° C. for 6 hours, then heating and boiling for 20 minutes, cooling to room temperature, adding 0.3 kg of gluten and mixing evenly, adding 10 g / L of hydrolase I, enzymatically hydrolyzing at 50° C. for 2 hours, and centrifuging to obtain hydrolyzate I for later use; Step 4: Filter hydrolyzate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolyzate fractions with molecular weights <3 kDa and 5-10 kDa. After combining the two fractions, add 15 g / L hydrolase II and 8 g / L aminopeptidase (enzyme activity 5000 U / g), perform enzymatic hydrolysis at 40°C for 1.5 h, heat to 80°C to inactivate the enzyme, and centrifuge to obtain hydrolyzate II for later use. Step 5: Filter the hydrolyzate II using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight of less than 5 kDa, concentrate and dry to obtain the initial product; after dissolving the initial product in water, load it onto a Sephadex G25 gel column, elute with water as the elution solvent, collect the eluate, detect the eluate, combine the eluates containing short peptides with a molecular weight of 500-5000Da, concentrate and dry to obtain the target short peptide.

[0029] Determination of types and contents of short peptide amino acids The target short peptide prepared in Example 1 was measured for amino acid types and contents using an amino acid analyzer. The results are shown in Table 1.

[0030] Table 1 Types and contents of amino acids in short peptides

[0031] Performance test: Improving effect of short peptides on immune performance of laying hens 1. Materials and Methods The experiment was conducted at our company's farm. Thirty-week-old laying hens of similar weight and condition were randomly divided into eight groups, each containing 30 chickens. One group served as a blank control group, fed a basic diet and maintained at a henhouse temperature of approximately 20°C. Another served as a control group, fed a basic diet and maintained at a henhouse temperature of approximately 30°C. The remaining groups received a basic diet supplemented with different short peptides (at a 1% additive level) and maintained at a henhouse temperature of approximately 30°C. The pre-trial period lasted one week, and the final trial period lasted four weeks.

[0032] Basic feed formula: corn 57.9%, wheat bran 4%, soybean meal 21.5%, fish meal 2%, rapeseed meal 4%, calcium hydrogen phosphate 1.3%, stone powder 8%, and salt 0.3%.

[0033] 2. Detection indicators On the last day of the experiment, 10 laying hens were randomly selected from each group, and blood was collected from the wing vein on an empty stomach to obtain serum. According to the instructions of the kit, the activities of immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), tumor necrosis factor-α (TNF-α), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and malondialdehyde (MDA) in the serum were detected.

[0034] Five laying hens were randomly selected from each group, weighed and then slaughtered, the spleens were removed, the spleen weight was measured, and the spleen index was calculated.

[0035]

[0036] The above results are expressed as "mean ± standard deviation". The data were analyzed by one-way analysis of variance using SPSS22.0, and P < 0.05 indicated significant differences between the groups.

[0037] 3. Results and Analysis Under high temperature conditions, the immune function of laying hens decreases and the mortality rate increases. As can be seen from Table 1 below, the IgA, IgG, and IgM levels of the laying hens in the control group fed normally decreased significantly under high temperature conditions, and the spleen index decreased. This result also verifies that the immune performance of laying hens decreases under high temperature conditions. As can be seen from the results in Table 1, on the basis of feeding the basic feed, adding the short peptides prepared in Examples 1-3 of the present invention can significantly increase the IgA, IgG, and IgM levels in the laying hens' serum and increase their spleen index, indicating that the short peptides prepared by the present invention have the effect of enhancing the immune function of laying hens, and their effect is significantly better than the short peptides prepared in Comparative Examples 1-4.

[0038] Table 2 Comparison of immune performance of laying hens

[0039] Note: * indicates P < 0.05 compared with the control group Under high temperature heat stress conditions, the metabolism of laying hens will be abnormal, the body's oxidative metabolism will be stronger, and peroxides will increase, causing problems such as oxidative damage or oxidative stress. The results in Table 2 show that the T-AOC of the laying hens in the control group decreased, the activities of SOD, GSH-Px, and CAT decreased significantly, and the MDA content increased significantly, indicating that high temperature environments can cause oxidative stress in laying hens. This is consistent with the prior art. Adding the short peptides prepared by Examples 1-3 of the present invention during feeding can significantly reduce their oxidative stress response, increase T-AOC, increase the activities of SOD, GSH-Px, and CAT, and reduce MDA content. The effect is significantly better than the short peptides prepared by Comparative Examples 1-4.

[0040] Table 3 Comparison of antioxidant capacity of laying hens

[0041] Note: * indicates P < 0.05 compared with the control group.

Claims

1. A method for preparing a short peptide for improving the immune performance of laying hens, characterized in that: The following steps are involved: Step 1, preparation of hydrolase I: after activating Bacillus licheniformis, inoculate it into seed culture medium I containing 5 g / L tryptone, 5 g / L yeast extract, 10 g / L glucose, and 18 g / L dipotassium hydrogen phosphate, and place it in a shaking incubator for shaking culture for 24 hours to obtain seed solution I; inoculate seed solution I into fermentation medium I containing 10 g peptone, 5 g beef extract, 10 g sucrose, 2 g disodium hydrogen phosphate, and 1 L water, with an initial pH of 7.5, shake culture at 35°C and 180 r / min for 4 hours, add Chlorella powder, stop fermentation after 36 hours, filter, concentrate the filtrate, and dry to obtain hydrolase I; Step 2, preparation of hydrolase II: After activating Aspergillus oryzae, inoculate it into a seed culture medium II containing 200g of potato, 20g of glucose, 15-20g of agar, and 1L of water, and culture it at 30°C for 48h. Scrape the spores, shake them to break them up, filter out the mycelium, and dilute them with sterile saline to obtain a spore suspension; inoculate the spore suspension into a fermentation medium II containing 30% bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. After culture at 30°C for 72h, stop the fermentation, filter, and dry to obtain hydrolase II; Step 3: drying and crushing the sunflower disk to obtain sunflower disk powder for later use; adding water to the sunflower disk powder and mixing evenly, adding Bacillus subtilis, fermenting at 35° C. for 6 hours, then heating and boiling for 20 minutes, cooling to room temperature, adding gluten and mixing evenly, adding hydrolase I, enzymatically hydrolyzing at 40-50° C. for 2-3 hours, and centrifuging to obtain hydrolyzate I for later use; Step 4: Filter hydrolyzate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolyzate fractions with molecular weights of 3-5 kDa and >10 kDa. After combining the fractions, add hydrolase II and aminopeptidase, perform enzymatic hydrolysis at 40-50°C for 1-1.5 hours, heat to 80°C to inactivate the enzyme, and centrifuge to obtain hydrolyzate II for later use. Step 5, the hydrolyzate II is filtered using a 5 kDa ultrafiltration membrane to obtain a hydrolyzate with a molecular weight of less than 5 kDa, which is concentrated and dried to obtain a primary product; after the primary product is dissolved in water, it is loaded onto a Sephadex G25 gel column, eluted with water as the elution solvent, the eluate is collected, and the eluate is tested. The eluates containing short peptides with a molecular weight of 500-5000Da are combined, concentrated, and dried to obtain the target short peptide.

2. The preparation method according to claim 1, wherein The inoculation amount of the seed solution I in step 1 is 10%; the addition amount of the Chlorella powder is 20 g / L.

3. The preparation method according to claim 1, wherein The protease activity of the hydrolase I in step 1 is 1503 U / g.

4. The preparation method according to claim 1, wherein The amount of spores in the spore suspension in step 2 is 1×10 6 The inoculation amount of the spore suspension in step 2 is 5%.

5. The preparation method according to claim 1, wherein The protease activity of the hydrolase II in step 2 is 2128 U / g.

6. The preparation method according to claim 1, wherein The amount of Bacillus subtilis added in step 3 is 1% of the weight of the sunflower disk powder; the amount of gluten added in step 3 is 30% of the weight of the sunflower disk powder; and the amount of hydrolase I added in step 3 is 10 g / L.

7. The preparation method according to claim 1, wherein In step 4, the amount of hydrolase II added is 15 g / L, and the amount of aminopeptidase added is 8 g / L.

8. A short peptide for improving the immune performance of laying hens, characterized in that: The short peptide is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of a short peptide prepared by the preparation method according to any one of claims 1 to 7 in improving the immune performance of laying hens.

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