A short peptide for enhancing the immune function of laying hens and its preparation method

Abstract

This invention relates to a short peptide for improving the immune performance of laying hens and its preparation method, belonging to the field of functional short peptide technology. The preparation method of the short peptide of this invention includes using hydrolytic enzymes I and II obtained from the fermentation of Bacillus licheniformis and Aspergillus oryzae to enzymatically hydrolyze sunflower flower heads and wheat gluten in stages, followed by purification to obtain the target short peptide. The short peptide prepared by this invention can effectively improve the immune performance of laying hens, enhance their antioxidant capacity, and prevent oxidative damage to laying hens in high-temperature environments.

Description

Technical Field

[0001] This invention belongs to the field of functional short peptide technology, and relates to a short peptide that improves the immune performance of laying hens and its preparation method. Background Technology

[0002] Egg-laying hens are domesticated birds belonging to the genus *Gallus* in the family Phasianidae, order Galliformes. Unlike broiler chickens, egg-laying hens are raised specifically to produce eggs. Scientific breeding methods are needed to improve egg quality and maintain or increase egg production, not to improve the quality of chicken meat.

[0003] As one of the most common poultry in the livestock industry, the immunization and health care of laying hens are extremely important. During the growth process of laying hens, the stability and health of their immune system directly affect their production performance and costs. Therefore, scientific immunization support for laying hens is of great significance to the health and profitability of laying hen farming.

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

[0005] The main objective of this invention is to provide a short peptide that improves the immune performance of laying hens and its preparation method, which can prevent laying hens from suffering oxidative damage in high-temperature environments.

[0006] The present invention employs the following technical solutions to achieve the above objectives: A method for preparing a short peptide to enhance the immune function of laying hens, specifically including the following steps: Step 1, Preparation of hydrolase I: After activating Bacillus licheniformis, it was inoculated 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. The medium was then placed in a shaker incubator and cultured for 24 h to obtain seed solution I. Seed solution I was then inoculated into fermentation culture 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 medium was cultured at 35 °C and 180 r / min for 4 h with shaking. Chlorella powder was added, and fermentation was stopped after 36 h. The medium was then filtered, and the filtrate was concentrated and dried to obtain hydrolase I. Step 2, preparation of hydrolase II: After activating Aspergillus oryzae, it was inoculated into seed culture medium II containing 200g potato, 20g glucose, 15-20g agar and 1L water. It was incubated at 30℃ for 48h. The spores were scraped off, shaken to disperse, filtered to remove mycelium, and diluted with sterile physiological saline to obtain a spore suspension. The spore suspension was inoculated into fermentation culture medium II containing 30% wheat bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride and 50% water. Fermentation was stopped after incubation at 30℃ for 72h. The mixture was then filtered and dried to obtain hydrolase II. Step 3: Dry and pulverize the sunflower head to obtain sunflower head powder for later use; mix the sunflower head powder with water evenly, add Bacillus subtilis, ferment at 35℃ for 6 hours, then heat to boiling for 20 minutes, cool to room temperature, add gluten powder and mix well, add hydrolytic enzyme I, and enzymatically hydrolyze at 40-50℃ for 2-3 hours, centrifuge to obtain hydrolysate I for later use. Step 4: Filter hydrolysate I using 3 kDa, 5 kDa and 10 kDa ultrafiltration membranes to obtain hydrolysate fractions with molecular weights of 3-5 kDa and >10 kDa. After combining the fractions, add hydrolase II and aminopeptidase, and enzymatically hydrolyze at 40-50℃ for 1-1.5 h. Heat to 80℃ to inactivate the enzymes, centrifuge, and obtain hydrolysate II for later use. Step 5: Filter hydrolysate II using a 5 kDa ultrafiltration membrane to obtain hydrolysate with a molecular weight <5 kDa. Concentrate and dry to obtain the initial product. Dissolve the initial product in water and load it onto a Sephadex G25 gel column. Use water as the elution solvent for elution. Collect the eluent and analyze it. Combine the eluents containing short peptides with a molecular weight of 500-5000 Da, concentrate and dry to obtain the target short peptide.

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

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

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

[0010] Furthermore, in step 2, the spore quantity in the spore suspension is 1×10⁻⁶. 6 per mL.

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

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

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

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

[0015] Furthermore, in step 3, the amount of hydrolase I added 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] This invention provides short peptides prepared by the above-described preparation method.

[0018] This invention provides a specific use for the short peptides prepared by the above preparation method, namely, the short peptides can improve the immune performance of laying hens.

[0019] The present invention has the following beneficial effects: 1. The short peptides prepared in this invention can improve the immune function of laying hens and reduce oxidative stress caused by heat stress. Experimental results show that the short peptides of this invention can significantly increase the level of immunoglobulins in the serum of laying hens under high-temperature conditions, thereby enhancing immune function; at the same time, they can improve their antioxidant capacity and reduce oxidative damage.

[0020] 2. In the short peptide preparation method of this invention, sunflower flower heads and gluten powder are creatively selected as raw materials. First, preliminary enzymatic hydrolysis is performed to obtain short peptides of different fragments. After filtration through a filter membrane, the target fragment peptide is subjected to secondary enzymatic hydrolysis to obtain small molecule peptides. After purification by a filter, the target fragment short peptide is obtained. During the enzymatic hydrolysis process, the effect of simply using traditional proteases is not ideal, and the short peptides obtained have poor activity. Therefore, this invention further screens the hydrolytic enzymes used for enzymatic hydrolysis, selecting a complex enzyme system produced by fermentation of Bacillus licheniformis and Aspergillus oryzae, and adopts a fractional enzymatic hydrolysis method to obtain short peptides. The short peptides obtained by this method have high physiological activity and can significantly improve the immune performance and antioxidant capacity of laying hens. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope of protection of the claims of this application.

[0022] Example 1 Step 1, Preparation of hydrolase I: Bacillus licheniformis AB 2016311 (purchased from the China Center for Type Culture Collection) was activated according to the instructions and inoculated 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. The medium was then cultured in a shaker (30℃, 150 rpm) for 24 h to obtain seed solution I. Seed solution I was then inoculated at 10% (v / v) 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. The medium was cultured at 35℃ and 180 rpm for 4 h with shaking. 20 g / L Chlorella powder was added, and fermentation was stopped after 36 h. The mixture was filtered, and the filtrate was concentrated and dried to obtain hydrolase I, with a protease activity of 1503 U / g. Step 2, Preparation of hydrolase II: The *Aspergillus oryzae* strain CCTCC AF 2016006 purchased from the China Center for Type Culture Collection was activated according to the instructions and inoculated into seed culture medium II containing 200g potato, 20g glucose, 20g agar, and 1L water. The medium was incubated statically at 30℃ for 48 hours. The spores were scraped off, shaken to disperse, filtered to remove mycelium, and diluted with sterile physiological saline to a spore count of 1×10⁻⁶. 6 The spores were collected at a concentration of 100 spores / mL to obtain a spore suspension. The spore suspension was inoculated at a concentration of 5% (v / v) into fermentation medium II containing 30% wheat bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. After culturing at 30°C for 72 h, fermentation was stopped, and the mixture was filtered and dried to obtain hydrolytic enzyme II, in which the protease activity was 2128 U / g. Step 3: Dry and pulverize the sunflower head to obtain sunflower head powder for later use; take 1 kg of sunflower head powder, add 5 L of water and mix well, add 10 g of Bacillus subtilis with an effective viable count of 20 billion CFU / g, ferment at 35℃ for 6 h, then heat to boiling for 20 min, cool to room temperature, add 0.3 kg of gluten powder and mix well, add 10 g / L hydrolytic enzyme I, enzymatically hydrolyze at 50℃ for 2 h, centrifuge to obtain hydrolysate I for later use; Step 4: Filter hydrolysate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolysate fractions with molecular weights of 3-5 kDa and >10 kDa. Combine the two fractions, add 15 g / L hydrolase II and 8 g / L aminopeptidase (enzyme activity of 5000 U / g), and enzymatically hydrolyze at 50°C for 1 h. Heat to 80°C to inactivate the enzymes, centrifuge, and obtain hydrolysate II for later use. Step 5: Filter hydrolysate II using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight <5 kDa. Concentrate and dry the fraction to obtain the initial product. Dissolve the initial product in water and load it onto a Sephadex G25 gel column. Use water as the elution solvent for elution. Collect the eluent and analyze it. Combine the eluents containing short peptides with a molecular weight of 500-5000 Da, concentrate and dry the eluent to obtain the target short peptide.

[0023] Example 2 Step 1, Preparation of hydrolase I: Bacillus licheniformis AB 2016311 (purchased from the China Center for Type Culture Collection) was activated according to the instructions and inoculated 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. The medium was then cultured in a shaker (30℃, 150 rpm) for 24 h to obtain seed solution I. Seed solution I was then inoculated at 10% (v / v) 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. The medium was cultured at 35℃ and 180 rpm for 4 h with shaking. 20 g / L Chlorella powder was added, and fermentation was stopped after 36 h. The mixture was filtered, and the filtrate was concentrated and dried to obtain hydrolase I, with a protease activity of 1503 U / g. Step 2, Preparation of hydrolase II: The *Aspergillus oryzae* strain CCTCC AF 2016006 purchased from the China Center for Type Culture Collection was activated according to the instructions and inoculated into seed culture medium II containing 200g potato, 20g glucose, 20g agar, and 1L water. The medium was incubated statically at 30℃ for 48 hours. The spores were scraped off, shaken to disperse, filtered to remove mycelium, and diluted with sterile physiological saline to a spore count of 1×10⁻⁶. 6 The spores were collected at a concentration of 100 spores / mL to obtain a spore suspension. The spore suspension was inoculated at a concentration of 5% (v / v) into fermentation medium II containing 30% wheat bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. After culturing at 30°C for 72 h, fermentation was stopped, and the mixture was filtered and dried to obtain hydrolytic enzyme II, in which the protease activity was 2128 U / g. Step 3: Dry and pulverize the sunflower head to obtain sunflower head powder for later use; take 1 kg of sunflower head powder, add 5 L of water and mix well, add 10 g of Bacillus subtilis with an effective viable count of 20 billion CFU / g, ferment at 35℃ for 6 h, then heat to boiling for 20 min, cool to room temperature, add 0.3 kg of gluten powder and mix well, add 10 g / L hydrolytic enzyme I, enzymatically hydrolyze at 40℃ for 3 h, centrifuge to obtain hydrolysate I for later use; Step 4: Filter hydrolysate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolysate fractions with molecular weights of 3-5 kDa and >10 kDa. Combine the two fractions, add 15 g / L hydrolase II and 8 g / L aminopeptidase (enzyme activity of 5000 U / g), and enzymatically hydrolyze at 40℃ for 1.5 h. Heat to 80℃ to inactivate the enzymes, centrifuge, and obtain hydrolysate II for later use. Step 5: Filter hydrolysate II using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight <5 kDa. Concentrate and dry the fraction to obtain the initial product. Dissolve the initial product in water and load it onto a Sephadex G25 gel column. Use water as the elution solvent for elution. Collect the eluent and analyze it. Combine the eluents containing short peptides with a molecular weight of 500-5000 Da, concentrate and dry the eluent to obtain the target short peptide.

[0024] Example 3 Step 1, Preparation of hydrolase I: Bacillus licheniformis AB 2016311 (purchased from the China Center for Type Culture Collection) was activated according to the instructions and inoculated 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. The medium was then cultured in a shaker (30℃, 150 rpm) for 24 h to obtain seed solution I. Seed solution I was then inoculated at 10% (v / v) 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. The medium was cultured at 35℃ and 180 rpm for 4 h with shaking. 20 g / L Chlorella powder was added, and fermentation was stopped after 36 h. The mixture was filtered, and the filtrate was concentrated and dried to obtain hydrolase I, with a protease activity of 1503 U / g. Step 2, Preparation of hydrolase II: The *Aspergillus oryzae* strain CCTCC AF 2016006 purchased from the China Center for Type Culture Collection was activated according to the instructions and inoculated into seed culture medium II containing 200g potato, 20g glucose, 20g agar, and 1L water. The medium was incubated statically at 30℃ for 48 hours. The spores were scraped off, shaken to disperse, filtered to remove mycelium, and diluted with sterile physiological saline to a spore count of 1×10⁻⁶. 6 The spores were collected at a concentration of 100 spores / mL to obtain a spore suspension. The spore suspension was inoculated at a concentration of 5% (v / v) into fermentation medium II containing 30% wheat bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. After culturing at 30°C for 72 h, fermentation was stopped, and the mixture was filtered and dried to obtain hydrolytic enzyme II, in which the protease activity was 2128 U / g. Step 3: Dry and pulverize the sunflower head to obtain sunflower head powder for later use; take 1 kg of sunflower head powder, add 5 L of water and mix well, add 10 g of Bacillus subtilis with an effective viable count of 20 billion CFU / g, ferment at 35℃ for 6 h, then heat to boiling for 20 min, cool to room temperature, add 0.3 kg of gluten powder and mix well, add 10 g / L hydrolytic enzyme I, enzymatically hydrolyze at 50℃ for 2 h, centrifuge to obtain hydrolysate I for later use; Step 4: Filter hydrolysate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolysate fractions with molecular weights of 3-5 kDa and >10 kDa. Combine the two fractions, add 15 g / L hydrolase II and 8 g / L aminopeptidase (enzyme activity of 5000 U / g), and enzymatically hydrolyze at 40℃ for 1.5 h. Heat to 80℃ to inactivate the enzymes, centrifuge, and obtain hydrolysate II for later use. Step 5: Filter hydrolysate II using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight <5 kDa. Concentrate and dry the fraction to obtain the initial product. Dissolve the initial product in water and load it onto a Sephadex G25 gel column. Use water as the elution solvent for elution. Collect the eluent and analyze it. Combine the eluents containing short peptides with a molecular weight of 500-5000 Da, concentrate and dry the eluent to obtain the target short peptide.

[0025] Comparative Example 1 Step 1, preparation of hydrolytic enzyme: CCTCC AB 2016311 Bacillus licheniformis (purchased from the China Center for Type Culture Collection) was activated according to the instructions and inoculated 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. The medium was then cultured in a shaker (30℃, 150 rpm) for 24 h to obtain seed solution I. Seed solution I was inoculated at 10% (v / v) 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. The medium was cultured at 35℃ and 180 rpm for 4 h with shaking. 20 g / L Chlorella powder was added, and fermentation was stopped after 36 h. The mixture was filtered, and the filtrate was concentrated and dried to obtain the hydrolytic enzyme, with a protease activity of 1503 U / g. Step 2: Dry and pulverize the sunflower head to obtain sunflower head powder for later use; take 1 kg of sunflower head powder, add 5 L of water and mix well, add 10 g of Bacillus subtilis with an effective viable count of 20 billion CFU / g, ferment at 35℃ for 6 hours, then heat to boiling for 20 minutes, cool to room temperature, add 0.3 kg of gluten powder and mix well, add 50 g of hydrolytic enzyme, enzymatically hydrolyze at 40℃ for 3 hours, centrifuge to obtain hydrolysate for later use; Step 3: Filter hydrolysate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolysate fractions with molecular weights of 3-5 kDa and >10 kDa. Combine the two fractions, add 5 g / L papain (100,000 U / g) and 8 g / L aminopeptidase (enzyme activity of 5000 U / g), and enzymatically hydrolyze at 40°C for 1.5 h. Heat to 80°C to inactivate the enzymes, centrifuge, and obtain hydrolysate II for later use. Step 4: Filter hydrolysate II using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight <5 kDa. Concentrate and dry the fraction to obtain the initial product. Dissolve the initial product in water and load it onto a Sephadex G25 gel column. Use water as the elution solvent for elution. Collect the eluent and analyze it. Combine the eluents containing short peptides with a molecular weight of 500-5000 Da, concentrate and dry the eluent to obtain the target short peptide.

[0026] Comparative Example 2 Step 1, preparation of hydrolytic enzyme: CCTCC AF 2016006 Aspergillus oryzae (purchased from the China Center for Type Culture Collection) was activated according to the instructions and inoculated into seed culture medium II containing 200g potato, 20g glucose, 20g agar, and 1L water. The medium was incubated at 30℃ for 48 hours. Spores were scraped off, shaken to disperse, and filtered to remove mycelium. The spores were then diluted with sterile physiological saline to a concentration of 1×10⁻⁶ spores. 6 The spores were collected at a concentration of 1 spore per mL to obtain a spore suspension. The spore suspension was inoculated at a concentration of 5% (v / v) into a fermentation medium containing 30% wheat bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. After culturing at 30°C for 72 h, the fermentation was stopped, filtered, and dried to obtain a hydrolytic enzyme with a protease activity of 2128 U / g. Step 2: Dry and pulverize the sunflower heads to obtain sunflower head powder for later use; take 1 kg of sunflower head powder, add 5 L of water and mix well, add 10 g of Bacillus subtilis with an effective viable count of 20 billion CFU / g, ferment at 35℃ for 6-8 h, then heat to boiling for 20-30 min, cool to room temperature, add 0.3 kg of gluten powder and mix well, then add 50 g of hydrolytic enzyme and 20 g / L aminopeptidase (enzyme activity of 5000 U / g), enzymatically hydrolyze at 40-50℃ for 1-1.5 h, heat to 80℃ to inactivate the enzyme, centrifuge to obtain hydrolysate for later use; Step 3: Filter the hydrolysate using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight <5 kDa. Concentrate and dry the fraction to obtain the initial product. Dissolve the initial product in water and load it onto a Sephadex G25 gel column. Use water as the elution solvent for elution. Collect the eluent and analyze it. Combine the eluents containing short peptides with a molecular weight of 500-5000 Da, concentrate and dry the eluent to obtain the target short peptide.

[0027] Comparative Example 3 Step 1, Preparation of hydrolase I: Bacillus licheniformis AB 2016311 (purchased from the China Center for Type Culture Collection) was activated according to the instructions and inoculated 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. The medium was then cultured in a shaker (30℃, 150 rpm) for 24 h to obtain seed solution I. Seed solution I was then inoculated at 10% (v / v) 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. The medium was cultured at 35℃ and 180 rpm for 36 h, after which fermentation was stopped. The mixture was filtered, concentrated, and dried to obtain hydrolase I, with a protease activity of 366 U / g. Step 2, Preparation of hydrolase II: The *Aspergillus oryzae* strain CCTCC AF 2016006 purchased from the China Center for Type Culture Collection was activated according to the instructions and inoculated into seed culture medium II containing 200g potato, 20g glucose, 20g agar, and 1L water. The medium was incubated statically at 30℃ for 48 hours. The spores were scraped off, shaken to disperse, filtered to remove mycelium, and diluted with sterile physiological saline to a spore count of 1×10⁻⁶. 6 The spores were collected at a concentration of 100 spores / mL to obtain a spore suspension. The spore suspension was inoculated at a concentration of 5% (v / v) into fermentation medium II containing 30% wheat bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. After culturing at 30°C for 72 h, fermentation was stopped, and the mixture was filtered and dried to obtain hydrolytic enzyme II, in which the protease activity was 2128 U / g. Step 3: Dry and pulverize the sunflower head to obtain sunflower head powder for later use; take 1 kg of sunflower head powder, add 5 L of water and mix evenly, add 10 g of Bacillus subtilis with an effective viable count of 20 billion CFU / g, ferment at 35℃ for 6-8 h, then heat to boiling for 20-30 min, cool to room temperature, add 10 g / L of hydrolytic enzyme I, and enzymatically hydrolyze at 40-50℃ for 2-3 h, centrifuge to obtain hydrolysate I for later use; Step 4: Filter hydrolysate I using 3 kDa, 5 kDa and 10 kDa ultrafiltration membranes to obtain hydrolysate fractions with molecular weights of 3-5 kDa and >10 kDa. Combine the two fractions, add 15 g / L hydrolytic enzyme II, and enzymatically hydrolyze at 40-50℃ for 1-1.5 h. Heat to 80℃ to inactivate the enzyme, centrifuge, and obtain hydrolysate II for later use. Step 5: Filter hydrolysate II using a 5 kDa ultrafiltration membrane to obtain hydrolysate with a molecular weight <5 kDa. Concentrate and dry to obtain the initial product. Dissolve the initial product in water and load it onto a Sephadex G25 gel column. Use water as the elution solvent for elution. Collect the eluent and analyze it. Combine the eluents containing short peptides with a molecular weight of 500-5000 Da, concentrate and dry to obtain the target short peptide.

[0028] Comparative Example 4 Step 1, Preparation of hydrolase I: Bacillus licheniformis AB 2016311 (purchased from the China Center for Type Culture Collection) was activated according to the instructions and inoculated 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. The medium was then cultured in a shaker (30℃, 150 rpm) for 24 h to obtain seed solution I. Seed solution I was then inoculated at 10% (v / v) 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. The medium was cultured at 35℃ and 180 rpm for 4 h with shaking. 20 g / L Chlorella powder was added, and fermentation was stopped after 36 h. The mixture was filtered, and the filtrate was concentrated and dried to obtain hydrolase I, with a protease activity of 1503 U / g. Step 2, Preparation of hydrolase II: The *Aspergillus oryzae* strain CCTCC AF 2016006 purchased from the China Center for Type Culture Collection was activated according to the instructions and inoculated into seed culture medium II containing 200g potato, 20g glucose, 20g agar, and 1L water. The medium was incubated statically at 30℃ for 48 hours. The spores were scraped off, shaken to disperse, filtered to remove mycelium, and diluted with sterile physiological saline to a spore count of 1×10⁻⁶. 6 The spores were collected at a concentration of 100 spores / mL to obtain a spore suspension. The spore suspension was inoculated at a concentration of 5% (v / v) into fermentation medium II containing 30% wheat bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride, and 50% water. After culturing at 30°C for 72 h, fermentation was stopped, and the mixture was filtered and dried to obtain hydrolytic enzyme II, in which the protease activity was 2128 U / g. Step 3: Dry and pulverize the sunflower head to obtain sunflower head powder for later use; take 1 kg of sunflower head powder, add 5 L of water and mix well, add 10 g of Bacillus subtilis with an effective viable count of 20 billion CFU / g, ferment at 35℃ for 6 h, then heat to boiling for 20 min, cool to room temperature, add 0.3 kg of gluten powder and mix well, add 10 g / L hydrolytic enzyme I, enzymatically hydrolyze at 50℃ for 2 h, centrifuge to obtain hydrolysate I for later use; Step 4: Filter hydrolysate I using 3 kDa, 5 kDa, and 10 kDa ultrafiltration membranes to obtain hydrolysate fractions with molecular weights <3 kDa and 5-10 kDa. Combine the two fractions, add 15 g / L hydrolase II and 8 g / L aminopeptidase (enzyme activity 5000 U / g), and enzymatically hydrolyze at 40℃ for 1.5 h. Heat to 80℃ to inactivate the enzymes, centrifuge, and obtain hydrolysate II for later use. Step 5: Filter hydrolysate II using a 5 kDa ultrafiltration membrane to obtain a fraction with a molecular weight <5 kDa. Concentrate and dry the fraction to obtain the initial product. Dissolve the initial product in water and load it onto a Sephadex G25 gel column. Use water as the elution solvent for elution. Collect the eluent and analyze it. Combine the eluents containing short peptides with a molecular weight of 500-5000 Da, concentrate and dry the eluent to obtain the target short peptide.

[0029] Determination of the types and contents of amino acids in short peptides The amino acid types and contents of the target short peptide prepared in Example 1 were determined using an amino acid analyzer, and the results are shown in Table 1.

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

[0031] Performance testing: The effect of short peptides on improving the immune function of laying hens 1. Materials and Methods The experiment was conducted at our company's farm. 30-week-old laying hens with similar body weight and condition were randomly divided into 8 groups of 30 birds each. One group served as a control group, fed a basal diet with the chicken house temperature maintained at approximately 20°C. Another group served as a control group, fed a basal diet with the chicken house temperature maintained at approximately 30°C. The remaining groups were fed a basal diet supplemented with different short peptides (1% addition), with the chicken house temperature maintained at approximately 30°C. The pre-trial period was one week, and the formal trial period was four weeks.

[0032] Basic feed formula: corn 57.9%, wheat bran 4%, soybean meal 21.5%, fish meal 2%, rapeseed meal 4%, dicalcium phosphate 1.3%, limestone powder 8%, 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 veins after fasting. Serum was then collected. The levels 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 measured according to the kit instructions.

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

[0035]

[0036] The results are expressed as mean ± standard deviation. Data were analyzed using SPSS 22.0 one-way ANOVA. A p < 0.05 was considered statistically significant between groups.

[0037] 3. Results and Analysis Under high-temperature conditions, the immune function of laying hens declines, and the mortality rate increases. As shown in Table 1, the levels of IgA, IgG, and IgM in the control group of normally fed laying hens decreased significantly under high-temperature conditions, and the spleen index also decreased. This result verifies that the immune performance of laying hens is reduced under high-temperature conditions. The results in Table 1 also show that adding the short peptides prepared in Examples 1-3 of this invention to the basic feed can significantly increase the levels of IgA, IgG, and IgM in the serum of laying hens and improve their spleen index. This indicates that the short peptides prepared in this invention have the effect of enhancing the immune function of laying hens, and their effect is significantly better than that of the short peptides prepared in Comparative Examples 1-4.

[0038] Table 2 Comparison of Immune Performance in Laying Hens

[0039] Note: * indicates that P < 0.05 compared to the control group. Under high-temperature heat stress conditions, laying hens exhibit abnormal metabolism, with enhanced oxidative metabolism and increased peroxides, leading to oxidative damage or oxidative stress. Table 2 shows that the control group of laying hens exhibited decreased T-AOC, significantly reduced SOD, GSH-Px, and CAT activities, and a significantly increased MDA content. This indicates that high-temperature environments cause oxidative stress in laying hens, consistent with existing technologies. Adding the short peptides prepared in Examples 1-3 of this invention during feeding significantly reduced oxidative stress, increased T-AOC, increased SOD, GSH-Px, and CAT activities, and decreased MDA content, demonstrating a significantly better effect than the short peptides prepared in Comparative Examples 1-4.

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

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

Claims

1. A method for preparing a short peptide to improve the immune performance of laying hens, characterized in that, Includes the following steps: Step 1, Preparation of hydrolase I: After activating Bacillus licheniformis, it was inoculated 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. The medium was then placed in a shaker incubator and cultured for 24 h to obtain seed solution I. Seed solution I was then inoculated into fermentation culture 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 medium was cultured at 35 °C and 180 r / min for 4 h with shaking. Chlorella powder was added, and fermentation was stopped after 36 h. The medium was then filtered, and the filtrate was concentrated and dried to obtain hydrolase I. Step 2, preparation of hydrolase II: After activating Aspergillus oryzae, it was inoculated into seed culture medium II containing 200g potato, 20g glucose, 15-20g agar and 1L water. It was incubated at 30℃ for 48h. The spores were scraped off, shaken to disperse, filtered to remove mycelium, and diluted with sterile physiological saline to obtain a spore suspension. The spore suspension was inoculated into fermentation culture medium II containing 30% wheat bran, 20% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% calcium chloride and 50% water. Fermentation was stopped after incubation at 30℃ for 72h. The mixture was then filtered and dried to obtain hydrolase II. Step 3: Dry and pulverize the sunflower head to obtain sunflower head powder for later use; mix the sunflower head powder with water evenly, add Bacillus subtilis, ferment at 35℃ for 6 hours, then heat to boiling for 20 minutes, cool to room temperature, add gluten powder and mix well, add hydrolytic enzyme I, and enzymatically hydrolyze at 40-50℃ for 2-3 hours, centrifuge to obtain hydrolysate I for later use. Step 4: Filter hydrolysate I using 3 kDa, 5 kDa and 10 kDa ultrafiltration membranes to obtain hydrolysate fractions with molecular weights of 3-5 kDa and >10 kDa. After combining the fractions, add hydrolase II and aminopeptidase, and enzymatically hydrolyze at 40-50℃ for 1-1.5 h. Heat to 80℃ to inactivate the enzymes, centrifuge, and obtain hydrolysate II for later use. Step 5: Filter hydrolysate II using a 5 kDa ultrafiltration membrane to obtain hydrolysate with a molecular weight <5 kDa. Concentrate and dry to obtain the initial product. Dissolve the initial product in water and load it onto a Sephadex G25 gel column. Use water as the elution solvent for elution. Collect the eluent and analyze it. Combine the eluents containing short peptides with a molecular weight of 500-5000 Da, concentrate and dry to obtain the target short peptide.

2. The preparation method according to claim 1, characterized in that, In step 1, the inoculation amount of seed liquid I is 10%; the amount of Chlorella powder added is 20g / L.

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

4. The preparation method according to claim 1, characterized in that, In step 2, the spore quantity in the spore suspension is 1×10⁻⁶. 6 spores / mL; the inoculation amount of spore suspension in step 2 is 5%.

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

6. The preparation method according to claim 1, characterized in that, In step 3, the amount of Bacillus subtilis added is 1% of the weight of sunflower head powder; the amount of wheat gluten added in step 3 is 30% of the weight of sunflower head powder; and the amount of hydrolytic enzyme I added in step 3 is 10 g / L.

7. The preparation method according to claim 1, characterized in that, 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 function of laying hens, characterized in that, The short peptide is prepared by the preparation method according to any one of claims 1-7.

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