Biological additive for promoting poultry feather development and application thereof

Abstract

The application discloses a biological additive for promoting poultry feather development and application thereof, and belongs to the technical field of animal breeding. The biological additive is prepared from scallop skirt oligopeptide and cantharides polypeptide at a mass ratio of 1:1. Animal experiments show that, when meat chickens are fed with 60 mg of the biological additive per 1 kg of basic feed, the growth of the meat chickens can be promoted, and more importantly, the feather development of the meat chickens can be significantly and synergistically promoted, and the length of the main wing feathers is obviously increased. Cytological studies further prove that the biological additive can synergistically promote the proliferation of poultry hair follicle epidermal stem cells, and the effect is better than that of an EGF positive control. Therefore, the biological additive developed by the application is safe, efficient and green, effectively solves the problems of drug residues and drug resistance possibly caused by traditional chemical additives, provides a sustainable solution for poultry breeding, and has important economic and application values.

Description

Technical Field

[0001] This invention belongs to the field of animal husbandry technology, and in particular relates to a biological additive that promotes the development of poultry feathers and its application. Background Technology

[0002] In poultry farming, the condition of feather growth is not only an important indicator of a bird's health but also directly affects the economic benefits of the farming. For poultry, good feathers not only help maintain normal body temperature and support flight ability but also reflect their appearance quality to a certain extent, playing a positive role in the mating selection of breeding birds and the market value of commercial poultry.

[0003] Currently, common methods used in the industry to promote poultry feather development include nutritional regulation, environmental management, and the use of some chemically synthesized additives. However, these traditional methods generally have certain limitations. For example, while some chemically synthesized additives can improve feather quality in the short term, long-term use may lead to a series of problems: on the one hand, they pose a risk of drug residues, affecting the food safety of poultry meat and egg products; on the other hand, they may also cause an imbalance in the gut microbiota of poultry, or even lead to drug resistance, thereby affecting the sustainable development of the overall poultry farming ecosystem.

[0004] Therefore, there is an urgent need to develop a new type of feather growth promoter that is safe, efficient, green, and has comprehensive regulatory functions, so as to effectively promote healthy feather growth while taking into account both nutritional supply and physiological regulation, and avoid adverse effects on the environment and human health. Summary of the Invention

[0005] The purpose of this invention is to provide a biological additive that promotes the development of poultry feathers and its application, thereby safely and effectively promoting the growth of poultry feathers and increasing their economic value.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a biological additive for promoting poultry growth and feather development, the biological additive being composed of scallop skirt oligopeptide and silkworm polypeptide, wherein the mass ratio of the scallop skirt oligopeptide to the silkworm polypeptide is 1:1.

[0008] Preferably, the preparation method of the scallop skirt oligopeptide includes the following steps:

[0009] (1) After cleaning the scallop skirt, blanch it in boiling water, cool it in ice water and drain it.

[0010] (2) After freeze-drying, the powder is pulverized and sieved to obtain scallop skirt powder;

[0011] (3) Add deionized water to the scallop skirt powder at a solid-liquid ratio of 1:10, extract at 4°C for 12 hours to obtain scallop skirt aqueous extract.

[0012] (4) Adjust the scallop skirt water extract to pH 7.5 and 55℃, add a complex protease and hydrolyze for 5 hours. The complex protease is composed of flavor protease, alkaline protease and trypsin in a mass ratio of 5:3:2.

[0013] (5) After inactivation and centrifugation of the enzyme hydrolysate, it is filtered sequentially using 100kDa, 10kDa and 1kDa ultrafiltration membranes, and finally the 1kDa permeate is collected.

[0014] (6) The 1kDa permeate was freeze-dried to obtain scallop skirt oligopeptide.

[0015] Preferably, the preparation method of the silkworm polypeptide includes the following steps:

[0016] (a) Silkworms were pre-frozen in liquid nitrogen, ground into powder, and defatted with petroleum ether;

[0017] (b) Add deionized water at a solid-liquid ratio of 1:10, extract overnight at 4°C to obtain an aqueous extract;

[0018] (c) Adjust the aqueous extract to pH 7.5 and 55°C, add a complex protease and hydrolyze for 5 hours. The complex protease is composed of flavor protease, alkaline protease and trypsin in a mass ratio of 5:3:2.

[0019] (d) The enzyme hydrolysate was inactivated and centrifuged, then purified by Sephadex G-50 gel chromatography column. The fraction corresponding to the absorption peak at 280 nm was collected and freeze-dried to obtain silkworm peptide.

[0020] Preferably, the biological additive is used in feed at a dosage of 60 mg per 1 kg of basal feed.

[0021] The biological additive promotes feather development in poultry by stimulating the proliferation of epidermal stem cells in the hair follicles.

[0022] Preferably, the poultry is chicken.

[0023] Preferably, the type of chicken is AA broiler chicken.

[0024] In a second aspect, the present invention provides the application of a biological additive composed of scallop skirt oligopeptide and silkworm polypeptide in the preparation of feed that synergistically promotes the development of poultry feathers, characterized in that the scallop skirt oligopeptide is prepared by the above-mentioned method for preparing scallop skirt oligopeptide.

[0025] The silkworm peptide was prepared by the above-described method for preparing silkworm peptide.

[0026] In the biological additive, the mass ratio of the scallop skirt oligopeptide to the silkworm polypeptide is 1:1.

[0027] Preferably, the feed consists of a basic feed and biological additives;

[0028] In the feed, the amount of biological additive used per 1 kg of the basic feed is 60 mg.

[0029] Preferably, the poultry is chicken.

[0030] Preferably, the type of chicken is AA broiler chicken.

[0031] In a third aspect, the present invention provides a feed that promotes poultry growth and feather development, the feed comprising a base feed and the aforementioned biological additive;

[0032] In the feed, the amount of biological additive used per 1 kg of the basic feed is 60 mg.

[0033] Preferably, the poultry is chicken.

[0034] Preferably, the type of chicken is AA broiler chicken.

[0035] In a fourth aspect, the present invention provides the application of a biological additive composed of scallop skirt oligopeptide and silkworm polypeptide in the preparation of a cell culture preparation that synergistically promotes the proliferation of poultry hair follicle epidermal stem cells, wherein the scallop skirt oligopeptide is prepared by the above-mentioned method for preparing scallop skirt oligopeptide.

[0036] The silkworm peptide was prepared by the above-described method for preparing silkworm peptide.

[0037] In the biological additive, the mass ratio of the scallop skirt oligopeptide to the silkworm polypeptide is 1:1.

[0038] Preferably, the biological agent consists of biological additives, DMEM basal culture medium, and 10% fetal bovine serum;

[0039] In the biological agent, the concentration of the biological additive is 20 μg / mL.

[0040] Preferably, the poultry is chicken.

[0041] Preferably, the type of chicken is AA broiler chicken.

[0042] The beneficial effects of this invention are as follows:

[0043] This invention provides a biological additive that synergistically promotes poultry growth and feather development. The additive is composed of scallop skirt oligopeptides and silkworm peptides in a 1:1 mass ratio. Compared to traditional chemical additives, this invention has significant advantages:

[0044] Firstly, in promoting poultry growth, the average daily weight gain of broilers with the compound additive significantly increased by 9.43%, demonstrating a good growth-promoting effect. More importantly, in terms of feather development, this invention exhibits a remarkable synergistic effect. Experimental results show that the primary wing feather length of broilers in the compound additive group increased significantly by 24.30%, with a synergistic effect index q value as high as 1.63, far exceeding the effects of single-group addition and high-dose single addition.

[0045] Secondly, this invention explored the mechanism of action in depth and found that the biological additive can synergistically promote the proliferation of chicken hair follicle epidermal stem cells, increasing the proliferation rate by 78.46%, which is superior to the commonly used cell proliferation factor EGF, thereby accelerating feather growth from the source.

[0046] Finally, this invention is based on natural bioactive substances, avoiding the problems of chemical residues and drug resistance, and conforms to the green, safe, and sustainable farming concepts. Therefore, this invention not only significantly improves the economic value and overall health of poultry, but also provides a new, efficient, and environmentally friendly solution for modern animal husbandry. Attached Figure Description

[0047] Figure 1 A comparison chart of the average length of primary wing feathers in different treatment groups;

[0048] Figure 2 A comparison of the proliferation rate of hair follicle epidermal stem cells in different treatment groups;

[0049] exist Figure 1 and Figure 2 In the diagram, * indicates P≤0.05, ** indicates P≤0.01, and *** indicates P≤0.001. Detailed Implementation

[0050] Scallop skirts, a byproduct of seafood processing, are rich in protein, amino acids, and various trace elements. In recent years, they have been widely used in animal feed to enhance nutritional value and promote growth and development in livestock and poultry. Current research mainly focuses on the positive effects of scallop skirts on the growth performance, immune function, and gut health of aquatic animals and livestock. However, research on their application in poultry farming, particularly their potential in feather growth, remains scarce, and no systematic literature reports have been found.

[0051] Currently, research on silkworm pupae mainly focuses on its active medicinal components and their applications in medicine, health care, and some livestock and poultry farming. Silkworm pupae are rich in protein, amino acids, and various bioactive substances, and are widely used in traditional Chinese medicine for sedation, anticonvulsant effects, antibacterial properties, and immune enhancement. In recent years, some studies have also attempted to apply them to livestock production, such as improving animal immunity and promoting growth performance. However, despite the silkworm pupae's excellent nutritional composition and bioactivity, research on its effects on poultry feather growth remains scarce, and no systematic literature reports its impact on feather development.

[0052] Therefore, this invention attempts to prepare scallop skirts and silkworm pupae into oligopeptides and polypeptides respectively, and apply them to poultry farming to promote poultry feather development.

[0053] Example 1

[0054] (1) After thoroughly cleaning the scallop skirt, put it into boiling water and blanch for 2 minutes. After taking it out, immediately put it into ice water to cool it down quickly and soak it for 30 minutes to prevent residual heat from having an adverse effect on the quality of the raw materials.

[0055] (2) After draining, freeze-dry at -40℃, then crush the dried scallop skirt into powder using a pulverizer and pass it through a 100-mesh sieve to obtain scallop skirt powder;

[0056] (3) Add deionized water to the scallop skirt powder at a solid-liquid ratio of 1:10, stir and extract at 4°C for 12 hours to obtain scallop skirt aqueous extract;

[0057] (4) Transfer the scallop skirt water extract to a constant temperature water bath, adjust the temperature to 55°C and pH to 7.5, add 0.03 times the mass of the scallop skirt powder of the complex protease and hydrolyze for 5 hours to obtain scallop skirt hydrolysate. The complex protease is composed of flavor protease, alkaline protease and trypsin in a mass ratio of 5:3:2.

[0058] (5) Heat the enzyme hydrolysate to 95°C and keep it for 15 minutes to completely inactivate the enzyme. After cooling to room temperature, centrifuge at 12,000 rpm for 30 minutes at 4°C and collect the supernatant to obtain scallop skirt peptide solution.

[0059] (6) After removing large particulate impurities from the scallop skirt peptide solution using a 0.45μm filter membrane, it was filtered sequentially using 100kDa, 10kDa and 1kDa ultrafiltration membranes, retaining the permeate from each stage, and finally collecting the 1kDa permeate.

[0060] (7) Place the 1kDa permeate in a vacuum freeze dryer and freeze dry to obtain scallop skirt oligopeptide.

[0061] Example 2

[0062] (1) After cleaning the dried silkworms with deionized water, place the silkworms in liquid nitrogen for pre-freezing and hardening treatment to obtain pre-frozen silkworms.

[0063] (2) Add the pre-frozen silkworms to a mortar pre-cooled with liquid nitrogen, and grind them while continuously adding liquid nitrogen to maintain a low temperature environment until the silkworms are ground into powder.

[0064] (3) Add petroleum ether at a mass-volume ratio of powder to petroleum ether of 1:3, stir at room temperature for 2 hours, centrifuge at 5000 rpm for 20 minutes, and collect the precipitate;

[0065] (4) Add deionized water at a solid-liquid ratio of 1:10, stir overnight at 4°C to obtain silkworm water extract;

[0066] (5) Transfer the silkworm water extract to a constant temperature water bath, adjust the temperature to 55°C and pH to 7.5, add 0.03 times the mass of the silkworm powder of the composite protease and hydrolyze for 5 hours to obtain the silkworm hydrolysate. The composite protease is composed of flavor protease, alkaline protease and trypsin in a mass ratio of 5:3:2.

[0067] (6) Heat the enzyme hydrolysate to 95°C and keep it for 15 minutes to completely inactivate the enzyme. After cooling to room temperature, centrifuge at 12,000 rpm for 30 minutes at 4°C and collect the supernatant to obtain the crude silkworm polypeptide solution.

[0068] (7) After filtration using a 0.45 μm microporous membrane, a Sephadex G-50 gel chromatography column with a height of 40 cm and a diameter of 2 cm was used. Deionized water was used as the mobile phase and eluted at a flow rate of 0.5 mL / min. The eluent was collected in 2 mL fractions per tube. After all elution was completed, all eluent was collected. The eluent fractions corresponding to the 280 nm elution peak were combined to obtain the silkworm peptide solution.

[0069] (8) Place the silkworm polypeptide solution in a vacuum freeze dryer and freeze dry to obtain silkworm polypeptide.

[0070] Example 3

[0071] Animal experiments were conducted to investigate the effects of the scallop skirt oligopeptide and silkworm peptide of the present invention on the growth and feather development of broilers.

[0072] (1) Select 450 AA broiler chickens (roosters) with an initial weight of about 45g and randomly divide them into 5 groups, with 6 replicates in each group and 15 chickens in each replicate;

[0073] The specific grouping arrangements are as follows:

[0074] Control group: fed with basic feed, which was Zhengda Broiler Chicken Compound Feed 510, to provide a standard control for the experiment;

[0075] Low-dose scallop skirt group: fed with basal feed plus scallop skirt oligopeptide (30mg scallop skirt oligopeptide per 1kg basal feed) to study the effects of low-dose scallop skirt oligopeptide alone on broiler growth and feather development.

[0076] Low-dose silkworm peptide group: fed with basal feed + silkworm peptide (30mg silkworm peptide per 1kg basal feed) to study the effects of low-dose silkworm peptide alone on broiler growth and feather development.

[0077] Compound supplement group: fed with basic feed + scallop skirt oligopeptide + silkworm peptide (30mg scallop skirt oligopeptide and 30mg silkworm peptide per 1kg of basic feed) to study whether the two peptides have a synergistic promoting effect when used in combination.

[0078] High-dose scallop skirt group: fed with basal feed plus scallop skirt oligopeptide (60mg of scallop skirt oligopeptide per 1kg of basal feed) to study the effect of high-dose scallop skirt oligopeptide on broiler growth performance.

[0079] High-dose silkworm peptide group: fed with basal feed plus silkworm peptide (60mg silkworm peptide per 1kg basal feed) to study the effect of high-dose silkworm peptide on broiler growth performance.

[0080] Feeding method: Free-feeding is adopted to ensure that broilers have access to sufficient feed at all times to meet their growth needs. Daily trough cleaning is performed to promptly remove any leftover feed, preventing mold or contamination and ensuring the freshness and hygiene of the feed.

[0081] Water Management: Nipple drinkers were used to provide water for the broilers, with fresh water changed daily to ensure cleanliness and hygiene. This reduced the risk of disease caused by water issues and provided excellent drinking conditions for healthy growth. The entire feeding and management process was strictly implemented in accordance with the AA Broiler Management Manual. From controlling environmental conditions such as temperature, humidity, and ventilation to daily hygiene and disease prevention, all procedures were followed to minimize interference with experimental results caused by differences in feeding and management practices.

[0082] Routine vaccine immunization treatment:

[0083] On day 1, administer Marek's disease vaccine subcutaneously; on day 7, administer Newcastle disease + infectious bronchitis bivalent live vaccine via nasal / eye drops; on day 14, administer infectious bursal disease vaccine via drinking water; on day 21, administer Newcastle disease (La Sota strain) vaccine via drinking water; on day 28, administer avian influenza (H5+H7) vaccine subcutaneously in the neck.

[0084] The experimental period was set at 42 days. At the beginning of the experiment (1 day old), the weight of each group of chickens was accurately recorded as initial data. At the end of the experiment (42 days old), the weight of each group of chickens was recorded again. By comparing the weight data at 1 day and 42 days old, growth performance indicators such as average daily weight gain were calculated to examine the effects of the scallop skirt oligopeptide and silkworm peptide of the present invention on the growth of broilers.

[0085] The results are shown in Table 1:

[0086] Table 1. Effects of the scallop skirt oligopeptide and silkworm peptide of the present invention on broiler growth.

[0087]

[0088] As can be seen from the experimental results in Table 1, compared with the control group of broilers fed only basic feed, the addition of scallop skirt oligopeptide, silkworm polypeptide, and the combined addition of the two in the feed all promoted the growth rate of broilers to varying degrees, indicating that the scallop skirt oligopeptide and silkworm polypeptide prepared in this invention have the potential to promote the growth performance of broilers to a certain extent.

[0089] Further comparison of the effects of each experimental group revealed that the group with the combined addition of scallop skirt oligopeptide and silkworm peptide showed a 9.43% increase in growth rate compared to the control group. However, this increase was only slightly higher than the sum of the average daily weight gain increases of the low-dose scallop skirt oligopeptide group and the low-dose silkworm peptide group, and did not show a significant synergistic enhancement effect. Furthermore, the growth-promoting effect of the combined addition group was not statistically significant compared to the experimental groups using high-dose scallop skirt oligopeptide or high-dose silkworm peptide alone. Based on the above analysis, it can be inferred that when scallop skirt oligopeptide and silkworm peptide are used simultaneously as feed additives in broiler farming, their growth-promoting effect is mainly additive.

[0090] Example 4

[0091] (1) After weighing at 42 days of age, five broiler chickens were randomly selected from each replicate and sampled after slaughter;

[0092] (2) Select the right wing of each chicken, locate the 5 longest and fully developed primary wing feathers on the right wing, use hemostatic forceps to clamp 1cm above the feather root, pull them out vertically along the feather growth direction, and put them into the numbered sample bag.

[0093] (3) After cleaning the blood stains at the base with physiological saline, lay the feathers flat on filter paper and place them in a humidity chamber (25℃, RH 50%) for 24 hours;

[0094] (4) Use a vernier caliper to measure the length of the primary wing feathers and calculate the average length of the primary wing feathers for each treatment group.

[0095] The results are shown in Table 2 and Figure 1 As shown.

[0096] Table 2. Average Length Data of Primary Wing Feathers

[0097]

[0098] As shown in Table 2, compared with the control group, the average length of the primary wing feathers of broilers increased in both the low-dose scallop skirt oligopeptide group and the low-dose silkworm polypeptide group, with the increase being more significant in the low-dose silkworm polypeptide group. This result indicates that the scallop skirt oligopeptide and silkworm polypeptide prepared in this invention can both promote broiler feather growth to some extent. However, in terms of overall effect, the promoting effect of either polypeptide on feather growth is still relatively limited when added alone.

[0099] Further comparison revealed that adding scallop skirt oligopeptide and silkworm peptide in low doses resulted in a 24.30% increase in primary wing feather length, significantly higher than the effect of adding either ingredient alone. To verify whether there was a synergistic effect between the two, the synergistic effect was calculated using the formula (q = E(AB) / (E(A) + E(B) - E(A)×E(B))), and the obtained q value was approximately 1.63. Since q≈1.63>1.15, this indicates that scallop skirt oligopeptide and silkworm peptide have a significant synergistic effect in promoting feather growth (primary wing feather growth).

[0100] Furthermore, under the same dosage, the combined additive group showed a 2.08-fold increase in promoting feather growth compared to the high-dose scallop skirt oligopeptide group and a 1.67-fold increase compared to the high-dose silkworm polypeptide group. This result further confirms the non-linear enhancement effect produced by the combined use of the two, which is not only superior to the effects of using either ingredient alone but also to the effects of high-dose single-ingredient additives.

[0101] Example 5

[0102] Obtaining hair follicle epidermal stem cells from AA broiler chickens

[0103] (1) Collect the newly grown feathers (wing area) after plucking, and quickly disinfect the base of the feathers and the surrounding skin with 75% alcohol.

[0104] (2) Gently remove the feather along with the hair follicle with tweezers, and rinse the hair follicle tissue three times in PBS containing 1% penicillin and antibiotics to remove impurities and contaminants;

[0105] (3) After initial cleaning, the hair follicle tissue was transferred into the cell chamber clean bench and rinsed again with PBS containing 2% penicillin and antibiotics.

[0106] (4) Remove the medulla and sheath of the hair follicle, and use sterile scissors to cut the remaining part into small pieces (less than 1 mm³), and then transfer it into a digestion bottle;

[0107] (5) Add 0.2% Collagenase I solution and place in a 37°C water bath for 2 hours to digest. After digestion, add an equal volume of culture medium containing 10% FBS to terminate digestion.

[0108] (6) Pass the digestion solution through a 40 μm cell sieve to remove large pieces of tissue, and collect the cells by centrifugation at 1300 r / min for 5 minutes;

[0109] (7) After washing once with PBS, resuspend the cells in DMEM complete medium, seed the cells in a culture dish, and place them in a 37°C, 5% CO2, saturated humidity incubator;

[0110] (8) After 20 minutes, aspirate the culture medium and inoculate it into the cell culture dish to remove non-stem cells such as fibroblasts with strong adhesion.

[0111] (9) After digesting the cells that have grown to 90% confluence, a portion of the cells were seeded into 96-well plates and identified by CD29 (β1-integrin) and CD49f (α6-integrin) fluorescence staining.

[0112] (10) The remaining cells were passaged and cultured. When CD29 and CD49f were positively expressed and located in the cell membrane, it indicated that the cells were hair follicle epidermal stem cells, which were then used for subsequent experiments.

[0113] Example 6

[0114] From a cellular mechanism perspective, this study examines whether the scallop skirt oligopeptide and silkworm peptide of this invention can synergistically promote the proliferation of hair follicle epidermal stem cells.

[0115] Experimental preparation:

[0116] Scallop skirt oligopeptide and silkworm peptide were prepared into a stock solution of 1 mg / ml using serum-free DMEM / F12 medium;

[0117] Then, the scallop skirt oligopeptide stock solution and silkworm peptide were prepared into a working concentration of 10 μg / mL scallop skirt oligopeptide solution, 10 μg / mL silkworm peptide solution, and a composite solution containing a working concentration of 10 μg / mL scallop skirt oligopeptide and 10 μg / mL silkworm peptide.

[0118] Meanwhile, a 10 ng / mL EGF solution was prepared as a positive control;

[0119] Add 10% FBS before use.

[0120] Experimental steps:

[0121] (1) The hair follicle epidermal stem cells obtained in Example 5 were seeded into collagen-coated 96-well plates, 100 μL per well, with a cell count of 3 × 10⁶ cells / well. 3 indivual;

[0122] (2) After culturing the 96-well plate in a cell culture incubator for 24 hours, the cells were treated according to the following grouping:

[0123]

[0124] (3) After adding the culture medium according to the group, place it in a cell culture incubator and continue to culture for 48 hours;

[0125] (4) At 0h and 48h, add 10μL of CCK-8 solution to each well, incubate at 37℃ in the dark for 2 hours, measure the absorbance at 450nm using a microplate reader, and calculate the proliferation rate: proliferation rate (%) = experimental group 48h OD 450 - 0hOD 450 ×100% / control group 48hOD 450 -0h OD 450 The results are shown in Table 3 and Figure 2 As shown.

[0126] Table 3. Differences in absorbance and proliferation rate among different groups

[0127]

[0128] From Table 3 and Figure 2 The experimental results show that, compared with the control group, the addition of scallop skirt oligopeptide, silkworm polypeptide, the combination of the two, and the EGF positive control can significantly promote the proliferation of chicken hair follicle epidermal stem cells, indicating that these treatment factors all have the effect of promoting the growth of chicken hair follicle epidermal stem cells to a certain extent.

[0129] Further comparison of the effects of each treatment group revealed that the combined addition group (i.e., the combined use of scallop skirt oligopeptide and silkworm polypeptide) had the most significant promoting effect on the proliferation of chicken hair follicle epidermal stem cells. Compared with the control group, its proliferation rate increased by 78.46%, which is not only significantly higher than the single addition group, but also exceeds the effect of the commonly used cell proliferation factor EGF positive control group, demonstrating the superiority of this combined formula in the functional regulation of chicken hair follicle epidermal stem cells.

[0130] Further calculation of the q value shows that q = E(AB) / (E(A) + E(B) - E(A)×E(B))≈1.54. This result indicates that when the scallop skirt oligopeptide and silkworm polypeptide of the present invention are combined for use in chicken hair follicle epidermal stem cells, they can synergistically promote the proliferation of chicken hair follicle epidermal stem cells, thereby helping to promote the growth of chicken feathers.

[0131] Example 7

[0132] A cell culture preparation for promoting the proliferation of poultry hair follicle epidermal stem cells

[0133] This biological agent consists of DMEM / F12 culture medium, 10% fetal bovine serum, 10 μg / mL scallop skirt oligopeptide, and 10 μg / mL silkworm polypeptide.

[0134] This cell culture preparation is used to promote the proliferation of chicken follicular epidermal stem cells.

Claims

1. A biological additive for promoting poultry growth and feather development, characterized in that, The biological additive is composed of scallop skirt oligopeptide and silkworm peptide, wherein the mass ratio of scallop skirt oligopeptide to silkworm peptide is 1:

1. The poultry in question is chicken; The preparation method of the scallop skirt oligopeptide includes the following steps: (1) After cleaning the scallop skirt, blanch it in boiling water, cool it in ice water and drain it. (2) After freeze-drying, the powder is pulverized and sieved to obtain scallop skirt powder; (3) Add deionized water to the scallop skirt powder at a solid-liquid ratio of 1:10, extract at 4°C for 12 hours to obtain scallop skirt aqueous extract. (4) Adjust the scallop skirt water extract to pH 7.5 and 55℃, add a complex protease and hydrolyze for 5 hours. The complex protease is composed of flavor protease, alkaline protease and trypsin in a mass ratio of 5:3:

2. (5) After inactivation and centrifugation of the enzyme hydrolysate, it is filtered sequentially using 100kDa, 10kDa and 1kDa ultrafiltration membranes, and finally the 1kDa permeate is collected. (6) Freeze-dry the 1kDa permeate to obtain scallop skirt oligopeptide; The preparation method of the silkworm peptide includes the following steps: (a) Silkworms were pre-frozen in liquid nitrogen, ground into powder, and defatted with petroleum ether; (b) Add deionized water at a solid-liquid ratio of 1:10, extract overnight at 4°C to obtain an aqueous extract; (c) Adjust the aqueous extract to pH 7.5 and 55°C, add a complex protease and hydrolyze for 5 hours. The complex protease is composed of flavor protease, alkaline protease and trypsin in a mass ratio of 5:3:

2. (d) The enzyme hydrolysate was inactivated and centrifuged, then purified by Sephadex G-50 gel chromatography column. The fraction corresponding to the absorption peak at 280 nm was collected and freeze-dried to obtain silkworm peptide.

2. The biological additive according to claim 1, characterized in that, The biological additive is used in feed at a dosage of 60 mg per 1 kg of basic feed. The biological additive promotes feather development in poultry by stimulating the proliferation of epidermal stem cells in the hair follicles.

3. The application of a biological additive composed of scallop skirt oligopeptides and silkworm polypeptides in the preparation of feed that synergistically promotes poultry feather development, characterized in that, The scallop skirt oligopeptide was prepared by the preparation method described in claim 1; The silkworm peptide was prepared by the preparation method described in claim 1; In the biological additive, the mass ratio of the scallop skirt oligopeptide to the silkworm polypeptide is 1:1; The poultry in question is chicken.

4. The application according to claim 3, characterized in that, The feed consists of a base feed and biological additives; In the feed, the amount of biological additive used per 1 kg of the basic feed is 60 mg.

5. A feed that promotes poultry growth and feather development, characterized in that, The feed consists of a base feed and a biological additive as described in claim 1 or 2; In the feed, the amount of biological additive used per 1 kg of the basic feed is 60 mg; The poultry in question is chicken.

6. The application of a biological additive composed of scallop skirt oligopeptides and silkworm polypeptide in the preparation of a cell culture formulation that synergistically promotes the proliferation of poultry hair follicle epidermal stem cells, characterized in that, The scallop skirt oligopeptide was prepared by the preparation method described in claim 1; The silkworm peptide was prepared by the preparation method described in claim 1; In the biological additive, the mass ratio of the scallop skirt oligopeptide to the silkworm polypeptide is 1:1; The poultry in question is chicken.

7. The application according to claim 6, characterized in that, The biological agent consists of biological additives, DMEM basal culture medium, and 10% fetal bovine serum. In the biological agent, the concentration of the biological additive is 20 μg / mL.

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