Preparation and application of a bioactive peptide complex feed additive

Abstract

The application discloses a kind of preparation and application of bioactive peptide complex new feed additive.The bioactive peptide complex new feed additive is specifically to bioactive peptide as main material and is composed of auxiliary material, wherein the main material bioactive peptide is chemically synthesized peptide, auxiliary material 1 is the peptide mixture with molecular weight below 2000 Da obtained by marine microalgae enzymolysis, and auxiliary material 2 is stone powder screened by 20-80 mesh screen.The main material and auxiliary material are composed in a certain ratio.The complex new feed additive composed is the nutrient supplement of animal, is used to protect the intestinal tract and prevent intestinal inflammation.The bioactive peptide complex is non-toxic and has no side effect, does not remain in the body, can effectively avoid the threat of veterinary drug residues to human health and economic loss.The application will promote the rapid development of new feed additive product based on bioactive peptide in breeding industry and feed adding aspect, make great contribution to the common problem of healthy breeding in China and even the whole world.

Description

Technical Field

[0001] This invention pertains to the field of animal nutrition, specifically to the preparation of a novel bioactive peptide complex feed additive and its application in preventing gastrointestinal inflammation. Background Technology

[0002] Numerous studies have shown that small molecule peptides possess characteristics such as rapid absorption, low carrier saturation, low energy consumption, and high efficiency. With the assistance of intestinal transport carriers, they can be directly and completely absorbed. Supplying amino acids in small peptide form avoids the inhibitory effect caused by free amino acids competing for binding sites, thus promoting the absorption and conversion of protein in feed. Therefore, the nutritional functions and mechanisms of action of small molecule peptides have attracted widespread attention in the fields of animal nutrition and medicine. Bioactive peptides possess diverse physicochemical properties and can play a positive role in various metabolic pathways and physiological processes. Due to their multiple functions, bioactive peptides have become a research hotspot worldwide.

[0003] Bioactive peptides have a wide range of sources. One type is plant-derived bioactive peptides; studies have reported the extraction of such peptides from plant fruits and seeds, which exhibit antibacterial activity against various pathogens, although the mechanism of action remains unclear. Another type is animal-derived bioactive peptides; studies have reported the extraction of an immunosuppressive peptide from scorpion venom, which can inhibit the Kv13 channel in peripheral blood lymphocytes, thus treating some autoimmune diseases. A third type is microbial-derived bioactive peptides, which have been reported to also regulate immune function. Milk-derived bioactive peptides possess various physiological functions, including promoting intestinal absorption and utilization of minerals, regulating immunity, antithrombotic effects, antihypertensive effects, and exhibiting morphine-like and antibacterial activities. Studies have also reported that bioactive peptides primarily promote the absorption of nutrients by intestinal epithelial cells, improve oxidative stress in piglets caused by weaning and inflammatory bowel disease (IBD), inhibit various harmful pathogens to improve the intestinal microecological environment, and promote the proliferation and function of macrophages and lymphocytes to strengthen the intestinal immune barrier. In marine organisms such as fish, bioactive peptides mainly promote the growth of aquatic organisms, improve their survival rate, and promote the absorption and utilization of mineral elements. In feed, bioactive peptides improve palatability and enhance animal production performance; for example, adding bioactive peptides to pig feed can significantly increase lean meat percentage, reduce ketone body fat content, and improve pork quality. They can also reduce diarrhea in piglets, promote bone growth, improve eggshell quality, and enhance the metabolic capacity of ruminants. The above studies mainly focus on the antibacterial and gut microbiota-modifying effects of bioactive peptides, while detailed reports on the effects of seaweed-derived bioactive peptides on intestinal inflammation and growth promotion in animals have not yet been found. Summary of the Invention

[0004] This invention discloses the preparation and application of a novel bioactive peptide complex feed additive. Specifically, the novel bioactive peptide complex feed additive is composed of bioactive peptides as the main ingredient and compound excipients. The main ingredient, bioactive peptides, are chemically synthesized peptides; excipient 1 is a mixture of peptides with a molecular weight below 2000 Da obtained by enzymatic hydrolysis of marine microalgae; and dressing 2 is limestone powder that has passed through a 20-80 mesh sieve. The main ingredient and excipients are combined in a specific ratio. The resulting compound feed additive serves as a nutritional supplement for animals, protecting the intestines and preventing intestinal inflammation. This bioactive peptide complex is non-toxic, has no side effects, and does not leave residues in the body, effectively avoiding the threat to human health and economic losses caused by veterinary drug residues. This invention will promote the rapid development of novel bioactive peptide-based feed additives in the aquaculture industry and feed additives, making a significant contribution to addressing common health issues in aquaculture in my country and worldwide.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] Preparation and application of a novel bioactive peptide complex feed additive. The novel feed additive consists of a main ingredient and excipients. The main ingredient is an active peptide prepared using a solid-phase synthesis method. The excipient, microalgae proteolytic peptides, is a mixture of peptides with a molecular weight of 1000 Da obtained by trypsin hydrolysis of Schizochytrium meal. The ratio of the main ingredient to the excipients is 1:10~100. The resulting novel feed additive is a nutritional supplement for animals, used to protect the intestines and prevent intestinal inflammation.

[0007] The preparation of the above-mentioned novel bioactive peptide complex feed additive, wherein the preparation method of the main bioactive peptide includes, but is not limited to, the bioactive peptide prepared by chemical synthesis, with a purity of over 95%, i.e., a peptide with a molecular formula of 6 amino acids obtained by dehydration condensation of amino acids and having the molecular formula C 24~53 H 25~61 N 7~17 O 12 The sequence is BOXZJM, where B is one of the amino acids Arg and Lys, O is one of the amino acids Phe, Tyr, and Thr, X is one of the amino acids Thr, Phe, and Tyr, Z is one of the amino acids Leu, Val, Ala, and Ile, J is one of the amino acids Val, Leu, Ile, and Pro, and M is one of the amino acids Ala and Gla.

[0008] The preparation of the above-mentioned novel bioactive peptide complex feed additive, wherein the excipient 1, microalgae proteolytic peptides, is a mixture of peptides with a molecular weight of less than 1000 Da obtained by trypsin hydrolysis of Schizochytrium algal meal as raw material; the specific preparation steps are as follows:

[0009] (1) Extracting oils from the liquid of marine Schizochytrium or Wukenella;

[0010] (2) The algal meal after oil separation is subjected to protease degradation. The proteases used include pepsin, trypsin and papain. The ratio of protease to substrate is 1% to 5%. The enzymatic hydrolysis temperature is 37℃ and the enzymatic hydrolysis time is 2 to 4 hours.

[0011] (3) The hydrolysate after enzymatic hydrolysis is filtered through a 2000 Da membrane;

[0012] (4) Concentrate using a concentration instrument, and then freeze-dry into powder;

[0013] (5) The resulting powdery substance is excipient 1 - microalgae protease hydrolysate peptide.

[0014] In the preparation of the above-mentioned novel bioactive peptide complex feed additive, the auxiliary material 2 is composed of stone powder that has passed through a 20-80 mesh sieve.

[0015] The preparation of the above-mentioned novel bioactive peptide complex feed additive involves a ratio of main ingredients to auxiliary ingredients determined according to different feed applications, including but not limited to the following:

[0016] (1) The ratio of main ingredient to auxiliary ingredient 1 and auxiliary ingredient 2 is 1:10:10, which is suitable for nutritional supplements for laying hens, broilers and other poultry.

[0017] (2) The ratio of main ingredient to auxiliary ingredient 1 and auxiliary ingredient 2 is 1:10:5, which is suitable for nutritional supplements for livestock such as pigs;

[0018] (3) The ratio of main ingredient to auxiliary ingredient is 1:10, which is suitable for nutritional supplements for fish and other aquatic products;

[0019] (4) The ratio of main feed to supplementary feed is 1:10, which is suitable for any waterline feeding as a nutritional supplement.

[0020] The application of the above-mentioned novel bioactive peptide complex feed additive, which protects the intestines from inflammation, mainly involves the active peptides inhibiting the expression of pro-inflammatory factors in the intestines, such as interleukins IL-1β, IL-6, and IL-17, thereby inhibiting inflammation and thus protecting the intestines.

[0021] The significant advantages of this invention are:

[0022] The peptide molecule of 6 amino acids obtained by the dehydration condensation of amino acids in this invention has C 24~53 H 25~61 N 7~ 17 O 12The sequence is BOXZJM, where B is one of the amino acids Arg and Lys, O is one of the amino acids Phe, Tyr, and Thr, X is one of the amino acids Thr, Phe, and Tyr, Z is one of the amino acids Leu, Val, Ala, and Ile, J is one of the amino acids Val, Leu, Ile, and Pro, and M is one of the amino acids Ala and Gla.

[0023] The nutritional supplement prepared by adding adjuvant 1 and dressing 2 is non-toxic, harmless, and has no side effects. It protects the intestines, prevents intestinal inflammation in livestock and poultry, is superior to existing antibiotics, and has significant efficacy. It is a green feed additive with significant economic and social value. Attached Figure Description

[0024] Figure 1 : Rate of change in the weight of laying hens.

[0025] Figure 2 Disease activity index score for laying hens.

[0026] Figure 3 Occult blood in the feces of laying hens.

[0027] Figure 4 Egg quality indicators.

[0028] Figure 5 Changes in intestinal inflammatory factors. Detailed Implementation

[0029] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Example

[0031] Preparation of bioactive peptide LHT – Solid-phase synthesis:

[0032] 1) Resin activation: Accurately weigh 0.2 mmol of Wang resin into a solid-phase synthesis tube, then pour 5 mL of solvent into the solid-phase synthesis tube, place it in a shaker and shake for 30 min to allow the Wang resin to fully swell. After swelling is complete, use a vacuum filter to dry the solution and set it aside for later use.

[0033] 2) Resin-amino acid linkage: Accurately weigh 0.6 mmol Fmoc-amino acid, 0.6 mmol condensing agent, and 0.6 mmol HOBt, and add them to a solid-phase synthesis tube for later use. Then, measure 5 mL of solvent and 1.2 mmol DIEA into a beaker and mix them evenly. Pour the mixture into the solid-phase synthesis tube and shake it manually beforehand to disperse the solid and solvent evenly. Then, place it in a shaker and shake for 3 hours. Filter dry, wash three times, and filter the filtrate for later use.

[0034] 3) Resin end sealing: After the first amino acid is condensed with the resin, 10 mL of acetic anhydride / pyridine (1:1) is measured as the resin end sealing solution and poured into a solid phase synthesis tube containing the resin. The mixture is shaken on a shaker at room temperature for 2 hours. Then, the waste liquid is removed and filtered out. The mixture is washed three times alternately with an appropriate amount of MeOH and then filtered dry for later use.

[0035] 4) Removal of amino acid protecting groups on resin: Measure 5 mL of 20% PIP / DMF solution and add it to a solid-phase synthesis tube containing resin. Place the tube in a shaker and shake at room temperature. After the reaction is complete, filter out the waste liquid, wash 3 times, and filter dry for later use.

[0036] 5) Condensation of amino acids: Accurately weigh 0.6 mmol of Fmoc-amino acid, 0.6 mmol of HATU, and 0.6 mmol of HOAt into a beaker. Add 5 mL of solvent and 1.2 mmol of DIEA, mix thoroughly, pour into a solid-phase synthesis tube, and shake well to mix with the resin particles. Shake at room temperature for 3 hours. After condensation is complete, filter out the waste liquid and wash. Repeat the above operation to condense the remaining amino acids.

[0037] 6) Resin cleavage: After all amino acids have condensed, the resin is completely filtered dry. Then, 5 mL of cleavage solution is added to a solid-phase synthesis tube, and the mixture is shaken for 2.5 h. The filtrate is then filtered and retained. The filtrate is poured into a rotary evaporator flask and filtered under reduced pressure at 45 °C until the liquid level no longer decreases. The remaining liquid is poured into a 50 mL centrifuge tube, and 6 times the volume of methyl tert-butyl ether is added. A white solid precipitate is observed to form. The precipitate is allowed to stand at 0 °C for 30 min. The precipitate is then centrifuged at 4000 rpm / min and 4 °C for 10 min. The supernatant is discarded, and the precipitate is washed three times with methyl tert-butyl ether to obtain a white precipitate. This precipitate is dissolved in sterile water and lyophilized to obtain a powdered crude peptide. Further purification yields a high-purity peptide.

[0038] Excipient 1 – Preparation of microalgae enzymatically hydrolyzed peptides:

[0039] 1) Extracting oils from the liquid of marine Schizochytrium or Wukenella;

[0040] 2) The algal meal after oil separation is subjected to protease degradation. The proteases used include pepsin, trypsin and papain. The ratio of protease to substrate is 1% to 5%. The enzymatic hydrolysis temperature is 37℃ and the enzymatic hydrolysis time is 2 to 4 hours.

[0041] 3) The hydrolysate after enzymatic hydrolysis is filtered through a 2000 Da membrane;

[0042] 4) Concentrate using a concentration instrument, then freeze-dry into powder;

[0043] 5) The resulting powdery substance is excipient 1 – microalgae protease hydrolysate peptide.

[0044] Auxiliary material 2 – stone powder:

[0045] Purchase stone powder, requiring it to pass through a sieve of 20-80 mesh.

[0046] Preparation of novel feed additives containing bioactive peptide complexes:

[0047] 1) The ratio of main ingredient to auxiliary ingredient 1 and auxiliary ingredient 2 is 1:10:10, which is suitable for nutritional supplements for laying hens, broilers and other poultry.

[0048] 2) The ratio of main ingredient to auxiliary ingredient 1 and auxiliary ingredient 2 is 1:10:5, which is suitable for nutritional supplements for livestock such as pigs;

[0049] 3) The ratio of main ingredient to auxiliary ingredient is 1:10, which is suitable for nutritional supplements for fish and other aquatic products;

[0050] 4) The ratio of main feed to supplementary feed is 1:10, which is suitable for any waterline feeding as a nutritional supplement.

[0051] The bioactive peptides obtained above are used in specific implementations:

[0052] Application Example 1

[0053] The efficacy of the prepared nutritional supplement (LHT) in regulating enteritis was tested.

[0054] 1) Establishment of the DSS enteritis model in laying hens: Thirty-two healthy 50-day-old Jingfen No. 6 laying hens were randomly divided into four treatment groups: blank group, negative group, positive group, LHT group (peptide B-Tyr-X-Val-J-Gla composed of the above molecular sequence), and O7 peptide group (peptide Lys-O-Phe-Z-Val-M composed of the above molecular sequence), with eight replicates in each group, and an experimental period of 30 days. During the experiment, the blank group laying hens were fed a basal diet and drinking water, while the LHT group was fed a basal diet and active peptide solution. The active peptide solution was replaced with drinking water, and the administration was carried out at the same time every day for 30 days until the end of the experiment. Starting from day 7 of the experiment, 2.5% DSS solution was provided to the negative, positive, and LHT groups to replace drinking water for free drinking for 7 days. During this period, the LHT group laying hens were also given active peptide, and the positive group laying hens were given ciprofloxacin. The feed intake and body weight of the laying hens in different groups were recorded and counted daily during the experiment. The results are as follows Figure 1 .

[0055] Depend on Figure 1 It can be seen that the rate of change in body weight of laying hens with enteritis can be roughly divided into three stages: the rising stage (0-10 days), the falling stage (11-13 days), the balancing stage (14-20 days), and the recovery stage (21-30 days).

[0056] 2) Disease Activity Index (DAI) Assessment: The Disease Activity Index (DAI) was developed for diagnosis. Three indicators were used to assess enteritis status: animal weight change rate, stool characteristics, and fecal occult blood. First, a score was assigned to each indicator, and then the average of these three indicators was calculated as the DAI score for each animal. Throughout the experimental period, each group of animals was weighed at the same time each day, and their activity level, defecation and fecal blood characteristics, coat color, food intake, and water consumption were observed. A DAI scoring table (Table 1) was used for comprehensive scoring to assess the disease status of each animal.

[0057] Table 1 Disease Activity Index Score*

[0058]

[0059] The Disease Activity Index (DAI) is a comprehensive score combining three factors: body mass index (BMI) and stool consistency. The BAI score is calculated by combining the patient's (sick animal's) weight loss rate (0 for no change in weight, 1-5 for 1 point, 5-10 for 2 points, 10-15 for 3 points, and greater than 15 for 4 points), stool consistency (0 for normal, 2 for loose stool, and 4 for diarrhea), and stool bleeding (0 for normal, 2 for occult blood, and 4 for overt bleeding). The total score of these three factors is divided by 3 to obtain the DAI value. That is, DAI = (BMI + Stool consistency + Bleeding) / 3.

[0060] The results are as follows Figure 2 .

[0061] 3) Effect of Active Peptide Veterinary Drug Alternatives on Fecal Occult Blood in Enteritis-Induced Hens: The semi-quantitative detection method using the Pilamydole test was employed to test for occult blood in fecal samples from laying hens. This method utilizes Pilamydole as an indicator; after the addition of acid and hydrogen peroxide, the iron in hemoglobin catalyzes the Pilamydole to turn blue-purple. The darker the color and the faster the color development, the higher the occult blood content in the sample. According to the instructions for using the fecal occult blood test strip, a suitable amount of fecal sample was picked up with a wooden stick and smeared onto the squares on the back of the test card. The cap was closed, and then a drop of colorimetric reagent A was dropped into the square on the front of the test card. After complete penetration, a drop of colorimetric reagent B was dropped. The test strip was scored based on the speed of the blue-purple color development, and the results were interpreted within 2 minutes. From day 1 to day 9 of DSS administration, fecal bleeding in laying hens was measured using the fecal occult blood test strip at a fixed time each day, and the experimental results were recorded as follows: Figure 3 As shown.

[0062] like Figure 3As shown, the symptoms of fecal occult blood in laying hens generally showed a pattern of gradually worsening, then slowly decreasing until recovery, with the most severe symptoms occurring on day 7 after administration of DSS, persisting until day 10. Day 7 was the last day of DSS administration, and drinking water was switched back on day 8. Over the first 10 days, the degree of fecal occult blood in each group gradually worsened. The negative group showed the most severe symptoms, with the longest duration of severity, from the appearance of occult blood on day 3 to recovery on day 12. The active peptide group showed relatively milder symptoms, with a shorter duration of severity. The LHT group showed occult blood from day 5 but recovered by day 9.

[0063] Application Example 2

[0064] Growth-promoting efficacy testing of the prepared nutritional supplement (LHT):

[0065] Laying hen production performance test: Jingfen No. 6 laying hens aged 64 weeks (late laying period) were selected. The hens were divided into a control group and an experimental group. House No. 9 was designated as the experimental group fed with bioactive peptide LHT, and House No. 11 as the control group fed a normal basal diet. The initial number of laying hens in each house was 48,000. The experimental period was 60 days. The experimental group was fed a certain dose of bioactive peptide LHT daily. During the experiment, the growth status and egg production of the hens in each house were recorded and statistically analyzed at the same time each day. Indicators such as egg production, laying rate, average egg weight, mortality rate, and feed conversion ratio were statistically analyzed. These indicators are important factors in evaluating laying hen production performance. During the laying hen experiment, to further explore the effects of LHT on laying hens, 10 eggs were randomly selected from each house daily for egg quality index testing over a period of 21 days. The following indicators of the eggs were measured and analyzed: egg shape index, yolk color, eggshell thickness, albumen height, and Haugh units. The results are shown below. Figure 4 As shown.

[0066] like Figure 4As shown, the egg shape index values ​​of the control group and the experimental group fluctuated between 1.28 and 1.36. Since an egg shape index between 1.24 and 1.37 is considered normal, the egg shape index values ​​of the control group and the experimental group were within the normal range, and therefore there was no significant difference in the egg shape index values ​​between the two groups. Observation of the yolk color of the control group and the experimental group showed that the data trends of both groups were relatively stable, while the yolk color of the experimental group was generally higher than that of the control group, fluctuating between 14 and 15. Although the depth of yolk color does not directly reflect the quality of the egg, a deeper yolk color may indicate a better diet for the chickens, thus having a positive impact on egg quality. Therefore, administering the hen active peptide LHT can effectively deepen the yolk color and has a positive impact on improving egg quality. Observation of the eggshell thickness index showed that the eggshell thickness of the control group and the experimental group showed a trend of first decreasing and then fluctuating gradually. This may be due to the influence of feeding conditions, causing the eggshells of the laying hens to become thinner. There was no significant difference in eggshell thickness between the control group and the experimental group. Observation of egg albumen height and Haugh unit index revealed that both the control and experimental groups showed a stable fluctuation trend in albumen height, while the experimental group's height was slightly higher than the control group's. This indicates that the administration of the active peptide LHT can increase the albumen height and protein content of eggs. Regarding the Haugh unit index, the experimental group's value was generally higher than the control group's, with fluctuations remaining stable between 70-80, which is within the normal Haugh unit range. Since a higher Haugh unit value indicates better egg protein quality, this reflects that the active peptide LHT can increase the Haugh unit value of eggs, thereby improving the quality of egg protein.

[0067] Application Example 3

[0068] 1) Thirty C57 mice were used. They were divided into a control group, an experimental group, and a positive control group, and were administered PBS, the nutritional supplement of this project (active peptide + excipient 1), and norfloxacin by gavage for 7 consecutive days.

[0069] 2) Establishment of intestinal damage caused by administering 2.5% DSS (sodium dodecyl sulfate) via drinking water. While administering DSS, continue the above three groups of experiments by gavage. On the 6th day after administering DSS, when the weight of the control group decreased to the lowest point (not less than 70% of the original weight), the mice were sacrificed and dissected, and the intestinal mucosa was scraped.

[0070] 3) mRNA was extracted and RT-PCR was performed to examine changes in intestinal inflammatory factors, particularly IL-1α, IL-1β, IL-17, and TNF-α. The results are as follows: Figure 5 As shown.

[0071] Depend on Figure 5The results shown indicate that the nutritional supplements in the experimental group of this invention significantly inhibited the secretion of intestinal inflammatory factors IL-1α, IL-1β, IL-17 and TNF-α, thereby inhibiting the occurrence of inflammation and protecting the intestines.

Claims

1. A method for preparing a bioactive peptide complex feed additive, characterized in that: The bioactive peptide complex feed additive is composed of main ingredient and auxiliary ingredient 1, or main ingredient, auxiliary ingredient 1 and auxiliary ingredient 2; the ratio of main ingredient to auxiliary ingredient is 1:10 to 1:

100. The main ingredient is a hexapeptide prepared by solid-phase synthesis, and the amino acid sequence of the hexapeptide is Arg-Tyr-Phe-Val-Val-Gla. The specific preparation steps for auxiliary material 1 are as follows: (1) Extracting oil from marine Schizochytrium; (2) The algal meal after oil separation is subjected to protease degradation. The proteases used include pepsin, trypsin and papain. The ratio of protease to substrate is 1% to 5%. The enzymatic hydrolysis temperature is 37°C and the enzymatic hydrolysis time is 2 to 4 hours. (3) The hydrolysate after enzymatic hydrolysis is filtered through a 2000 Da membrane; (4) Concentrate using a concentration instrument, and then freeze-dry into powder; (5) The powdery substance is excipient 1 - microalgae protease hydrolysate peptide.

2. The method for preparing the bioactive peptide complex feed additive according to claim 1, characterized in that: The auxiliary material 2 is stone powder that has passed through a 20-80 mesh sieve.

3. The method for preparing the bioactive peptide complex feed additive according to claim 2, characterized in that: The ratio of main ingredients to auxiliary ingredients is determined according to different feed uses, including but not limited to the following: (1) The ratio of main ingredient to auxiliary ingredient 1 and auxiliary ingredient 2 is 1:10:10, which is suitable for nutritional supplements for laying hens and broilers; (2) The ratio of main feed to supplementary feed is 1:10, which is suitable for any waterline feeding as a nutritional supplement.

4. The application of the bioactive peptide complex feed additive prepared by the method of claim 1 in the preparation of drugs for inhibiting intestinal inflammation, characterized in that: The inhibition of intestinal inflammation is achieved by suppressing the expression of intestinal pro-inflammatory factors IL-1β, IL-6, and IL-17.

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