A wool protein peptide, and a preparation method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN SUNHY BIOLOGICAL
- Filing Date
- 2026-05-20
- Publication Date
- 2026-08-07
AI Technical Summary
[0004]本发明的目的是克服现有技术中羊毛鳞片层结构致密导致其蛋白利用率低的问题
[0020]本发明提供的这种羊毛蛋白肽制备方法通过角质酶、角蛋白酶、氨肽酶的分步酶解，并结合马克思克鲁维酵母的菌酶协同作用，突破鳞片层脂质屏障和角蛋白致密结构的降解难题，将羊毛鳞片层中的角蛋白转化为饲用蛋白肽。在水产饲料中添加采用该方法制备的羊毛蛋白肽可使草鱼增重率提高，显著提升饲料应用价值，为羊毛副产物的资源化利用提供了新路径。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological feed technology, specifically relating to a wool protein peptide, its preparation method, and its application. Background Technology
[0002] Wool is an important natural protein fiber containing a large number of scale layers. The wool scale layer has a dense structure, with an outer layer encased by a lipid barrier and an inner layer rich in keratin. However, due to the unique structure of the scale layer, keratin is difficult to degrade using conventional technologies and cannot be converted into a usable protein resource.
[0003] In the feed industry, the demand for high-quality protein peptides and functional additives is increasing. Traditional protein peptide preparation methods suffer from limited raw material sources and high costs; while functional additives are mostly chemically synthesized products, posing certain safety risks. Meanwhile, the large accumulation of wool byproducts not only wastes resources but also puts pressure on the environment. Therefore, developing a pretreatment technology that can efficiently degrade wool cuticle keratin has significant economic and environmental value. Summary of the Invention
[0004] The purpose of this invention is to overcome the problem of low protein utilization caused by the dense scale layer structure of wool in the prior art.
[0005] Therefore, the present invention provides a method for preparing wool protein peptides, comprising the following steps:
[0006] Pretreated wool is obtained by enzymatic hydrolysis of wool raw materials using keratinase.
[0007] Pretreated wool was subjected to synergistic fermentation of keratinase and Max Kluyveromyces simultaneously. After fermentation, the first liquid phase product was obtained by separation.
[0008] The first liquid phase product was treated with a nuclease to separate the second liquid phase product.
[0009] The first liquid phase product was enzymatically treated with aminopeptidase to obtain wool protein peptides.
[0010] Specifically, the preparation process of the above-mentioned pretreated wool is as follows: after the wool raw material is crushed, a buffer solution with a pH of 7.0-8.0 is added, keratinase is added, and enzymatic hydrolysis is carried out at 40℃-50℃ for 2-4 hours to obtain pretreated wool.
[0011] Specifically, based on the weight of the wool raw material, the amount of keratinase added is 0.6%-1.0%; the enzyme activity of the keratinase is ≥500U / g.
[0012] Specifically, based on the weight of the wool raw material, the amount of keratinase added is 1.3%-1.7%, and the amount of Kluyveromyces martensii added is 1%-5%; the keratinase activity is ≥10000 U / g; and the Kluyveromyces martensii activity is ≥1×10⁻⁶. 8 CFU / g.
[0013] Specifically, the temperature for the synergistic fermentation of the above-mentioned bacteria and enzymes is 35℃-40℃, and the fermentation time is 12-24h.
[0014] Specifically, based on the weight of the wool raw material, the amount of nuclease added is 0.1%-0.2%; the enzyme activity of the nuclease is ≥2000.
[0015] Specifically, based on the weight of the wool raw material, the amount of aminopeptidase added is 0.3%-0.7%; the enzyme activity of the aminopeptidase is ≥1000 U / g.
[0016] Specifically, the enzymatic hydrolysis temperature of the above-mentioned nuclease is 45℃-65℃, and the hydrolysis time is 0.5h-2h; the enzymatic hydrolysis temperature of the aminopeptidase is 37℃-42℃, and the hydrolysis time is 3-6h.
[0017] The present invention also provides a wool protein peptide, which is prepared by any of the preparation methods described above.
[0018] The present invention also provides the application of the above-mentioned wool protein peptides in aquatic feed.
[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0020] The wool protein peptide preparation method provided by this invention overcomes the degradation challenges of the lipid barrier in the wool scale layer and the dense structure of keratin by stepwise enzymatic hydrolysis using keratinase, keratinase, and aminopeptidase, combined with the synergistic effect of enzymes from *Kluyveromyces martensii*. This process converts keratin in the wool scale layer into feed-grade protein peptides. Adding wool protein peptides prepared using this method to aquatic feed can increase the weight gain rate of grass carp, significantly enhancing the feed's application value and providing a new pathway for the resource utilization of wool by-products. Detailed Implementation
[0021] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.
[0022] This invention provides a method for preparing wool protein peptides, comprising the following steps:
[0023] S1, Preprocessing
[0024] Wool raw materials are washed, dried, and pulverized to a fineness of 60 mesh or higher. A buffer solution with a pH of 7.0-8.0 is added at a material-to-liquid ratio of 1:10-1:20. Based on the weight of the wool raw materials, a certain percentage of keratinase (enzyme activity ≥500 U / g) is added. Enzymatic hydrolysis is carried out at 40℃-50℃ for 2-4 hours. After the reaction, the mixture is filtered and washed with water until neutral to obtain pretreated wool powder. Keratinase can specifically degrade the lipid components on the surface of the wool cuticle layer, disrupting its dense lipid barrier and opening channels for subsequent enzymatic hydrolysis reactions.
[0025] S2, co-fermentation of bacteria and enzymes
[0026] Add the pretreated wool powder to a buffer solution with a pH of 8.0-9.0 at a material-to-liquid ratio of 1:15-1:25. Add keratinase (enzyme activity ≥10000U / g) by weight of the wool raw material, and simultaneously inoculate with 1%-5% of Kluyveromyces martensii culture (viable count ≥1×10⁻⁶). 8 The product (CFU / g) was subjected to bacterial-enzyme co-fermentation degradation at 35℃-40℃ for 12-24 hours. After fermentation, solid-liquid separation was performed to obtain the first liquid phase product containing peptides.
[0027] Keratinase can specifically hydrolyze the peptide bonds of keratin, breaking down large keratin molecules into polypeptide fragments; Kluyveromyces martensii can secrete a variety of coenzymes and utilize the small molecules produced during degradation for growth and metabolism, further promoting keratin degradation and improving degradation efficiency.
[0028] S3, Nucleic Acid Removal Processing
[0029] Adjust the pH of the first liquid phase product system to the desired level. Add 0.1%-0.2% nuclease (preferably a DNase / RNase complex with an enzyme activity ≥2000, based on the weight of the wool raw material). Treat at 45℃-65℃ for 0.5-2 hours to obtain the second liquid phase product. This step can completely degrade DNA / RNA released from cell lysis, preventing it from forming complexes with peptides that affect absorption, while also reducing the potential risk of genetic material residues in animal feed.
[0030] S4, Small Peptide Targeted Purification
[0031] Adjust the pH of the second liquid phase product to 7.5-8.5, and add 0.3%-0.7% aminopeptidase (enzyme activity ≥1000 U / g) based on the weight of the wool raw material. Perform enzymatic hydrolysis at 37℃-42℃ for 3-6 hours. Aminopeptidase can stepwise hydrolyze peptide bonds from the amino terminus of polypeptide chains, breaking down large peptides into small peptides with a molecular weight below 1000 Da, i.e., feed-grade protein peptides.
[0032] S5. Product purification and drying
[0033] After enzymatic hydrolysis with aminopeptidase, macromolecular impurities are removed by ultrafiltration (preferably with a molecular weight cutoff of 1000 Da). The filtrate is then spray-dried to obtain a white to pale yellow powder, which is high-purity wool protein peptide powder. The finished product contains ≥65% small peptides and <5ppm of residual nucleic acid, meeting the safety standards for green feed.
[0034] The effects of the wool protein peptide of the present invention, its preparation method, and its application are studied through specific embodiments below.
[0035] Example 1:
[0036] This embodiment provides a wool protein peptide, which is prepared using the following steps.
[0037] S1, Preprocessing
[0038] 1000g of wool raw material was washed to remove impurities, dried at 60℃ to constant weight, and then pulverized to a fineness of 60 mesh or higher. Phosphate buffer with a pH of 7.5 was added at a material-to-liquid ratio of 1:15, and 8g of keratinase (enzyme activity 500U / g) was added. The mixture was enzymatically hydrolyzed at 45℃ for 3 hours. After the reaction was completed, the mixture was filtered and washed with water until neutral to obtain pretreated wool powder.
[0039] S2, co-fermentation of bacteria and enzymes
[0040] Add pretreated wool powder to a borate buffer solution with a pH of 8.5 at a material-to-liquid ratio of 1:20, add 15g of keratinase (enzyme activity 1000U / g), and simultaneously inoculate with 50g of Kluyveromyces macrocephalae culture (crystal concentration 1×10⁻⁶). 8 CFU / was subjected to bacterial-enzyme co-fermentation degradation at 37°C for 18 hours. After fermentation, solid-liquid separation was performed to obtain the first liquid phase product containing peptides.
[0041] S3, Nucleic Acid Removal Processing
[0042] The pH of the first liquid phase product system was adjusted to 8.0, 2g of nuclease (DNase / RNase complex, enzyme activity 2000) was added, and the system was treated at 55℃ for 1 hour to separate the second liquid phase product.
[0043] S4, Small Peptide Targeted Purification
[0044] The pH of the second liquid phase product was adjusted to 8.0, and 5g of aminopeptidase (enzyme activity of 1000 U / g) was added. The mixture was then enzymatically hydrolyzed at 40℃ for 4 hours.
[0045] S5. Product purification and drying
[0046] After enzymatic hydrolysis with aminopeptidase, the product was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the filtrate was spray-dried to obtain wool protein peptide powder. The content of small peptides and residual nucleic acids in the finished product were tested, and the results are shown in Table 1, which meet the safety standards for green feed.
[0047] Example 2:
[0048] This embodiment provides a wool protein peptide, which is prepared using the following steps.
[0049] S1, Preprocessing
[0050] 1000g of wool raw material was washed to remove impurities, dried at 60℃ to constant weight, and then pulverized to a fineness of 60 mesh or higher. Phosphate buffer with a pH of 7.5 was added at a material-to-liquid ratio of 1:15, and 8g of keratinase (enzyme activity 500U / g) was added. The mixture was enzymatically hydrolyzed at 45℃ for 3 hours. After the reaction was completed, the mixture was filtered and washed with water until neutral to obtain pretreated wool powder.
[0051] S2, co-fermentation of bacteria and enzymes
[0052] Add pretreated wool powder to borate buffer solution with a pH of 8.5 at a material-to-liquid ratio of 1:20, add 15g of keratinase (enzyme activity 10000U / g), and simultaneously inoculate with 10g of Kluyveromyces macrocephalae culture (crystal concentration 1×10⁻⁶). 8 CFU / was subjected to bacterial-enzyme co-fermentation degradation at 37°C for 18 hours. After fermentation, solid-liquid separation was performed to obtain the first liquid phase product containing peptides.
[0053] S3, Nucleic Acid Removal Processing
[0054] The pH of the first liquid phase product system was adjusted to 8.0, 1g of nuclease (DNase / RNase complex, enzyme activity 2000) was added, and the system was treated at 55℃ for 1 hour to separate the second liquid phase product.
[0055] S4, Small Peptide Targeted Purification
[0056] The pH of the second liquid phase product was adjusted to 8.0, and 5g of aminopeptidase (enzyme activity of 1000 U / g) was added. The mixture was then enzymatically hydrolyzed at 40℃ for 4 hours.
[0057] S5. Product purification and drying
[0058] After enzymatic hydrolysis with aminopeptidase, the product was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the filtrate was spray-dried to obtain wool protein peptide powder. The content of small peptides and residual nucleic acids in the finished product were tested, and the results are shown in Table 1, which meet the safety standards for green feed.
[0059] Comparative Example 1:
[0060] This comparative example provides a wool protein peptide prepared using the following steps.
[0061] S1, Preprocessing
[0062] 1000g of wool raw material was washed to remove impurities, dried at 60℃ to constant weight, and then pulverized to a fineness of 60 mesh or higher. Phosphate buffer with a pH of 7.5 was added at a material-to-liquid ratio of 1:15, and 8g of keratinase (enzyme activity 500U / g) was added. The mixture was enzymatically hydrolyzed at 45℃ for 3 hours. After the reaction was completed, the mixture was filtered and washed with water until neutral to obtain pretreated wool powder.
[0063] S2, keratinase hydrolysis
[0064] Pretreated wool powder was added to borate buffer solution with a pH of 8.5 at a material-to-liquid ratio of 1:20, and 15g of keratinase (enzyme activity 10000U / g) was added. The mixture was degraded at 37℃ for 18 hours. After the enzymatic hydrolysis was completed, solid-liquid separation was performed to obtain the first liquid phase product containing peptides.
[0065] S3, Nucleic Acid Removal Processing
[0066] The pH of the first liquid phase product system was adjusted to 8.0, 2g of nuclease (DNase / RNase complex, enzyme activity 2000) was added, and the system was treated at 55℃ for 1 hour to separate the second liquid phase product.
[0067] S4, Small Peptide Targeted Purification
[0068] The pH of the second liquid phase product was adjusted to 8.0, and 5g of aminopeptidase (enzyme activity of 1000 U / g) was added. The mixture was then enzymatically hydrolyzed at 40℃ for 4 hours.
[0069] S5. Product purification and drying
[0070] After enzymatic hydrolysis with aminopeptidase, the product was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da. The filtrate was then spray-dried to obtain wool protein peptide powder. The content of small peptides and residual nucleic acids in the finished product were tested, and the results are shown in Table 1.
[0071] Comparative Example 2:
[0072] This embodiment provides a wool protein peptide, which is prepared using the following steps.
[0073] S1, Preprocessing
[0074] 1000g of wool raw material was washed to remove impurities, dried at 60℃ to constant weight, and then pulverized to a fineness of 60 mesh or higher. Phosphate buffer with a pH of 7.5 was added at a material-to-liquid ratio of 1:15, and 8g of keratinase (enzyme activity 500U / g) was added. The mixture was enzymatically hydrolyzed at 45℃ for 3 hours. After the reaction was completed, the mixture was filtered and washed with water until neutral to obtain pretreated wool powder.
[0075] S2, co-fermentation of bacteria and enzymes
[0076] Add the pretreated wool powder to a borate buffer solution with a pH of 8.5 at a material-to-liquid ratio of 1:20, add 15g of keratinase (enzyme activity 10000U / g), and simultaneously inoculate with 50g of Kluyveromyces macrocephalae culture (crystal concentration 1×10⁻⁶). 8 CFU / was subjected to bacterial-enzyme co-fermentation degradation at 37°C for 18 hours. After fermentation, solid-liquid separation was performed to obtain the first liquid phase product containing peptides.
[0077] S3, Small Peptide Targeted Purification
[0078] The pH of the first liquid phase product was adjusted to 8.0, and 5g of aminopeptidase (enzyme activity of 1000 U / g) was added. The mixture was then enzymatically hydrolyzed at 40℃ for 4 hours.
[0079] S5. Product purification and drying
[0080] After enzymatic hydrolysis with aminopeptidase, the product was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da. The filtrate was then spray-dried to obtain wool protein peptide powder. The content of small peptides and residual nucleic acids in the finished product were tested, and the results are shown in Table 1.
[0081] Table 1. Wool protein peptide content and nucleic acid residue
[0082]
[0083] Example 3:
[0084] In this embodiment, a monogastric animal biomimetic digestive system was used to evaluate the protein digestibility of Examples 1-2, Comparative Examples 1-2, and commercially available enzymatically hydrolyzed feather meal. The results are shown in Table 2. As can be seen from Table 2, the wool protein peptide powder prepared in this invention has a higher protein digestibility compared to commercially available feather meal.
[0085] Table 2 Protein digestibility
[0086]
[0087] Example 4:
[0088] This embodiment studies the application of wool protein peptides in aquatic feed.
[0089] Seventy-five hundred healthy grass carp at the same growth stage were randomly divided into five groups, with three replicates per group and fifty fish per replicate. The control group was fed a basal diet, while the experimental groups were fed the wool protein peptides prepared in Examples 1-2 and Comparative Examples 1-2 at a dosage of 5%. The experiment lasted for 60 days, and the results are shown in Table 3. As can be seen from Table 3, the addition of the wool protein peptide powder prepared in this invention can significantly improve the weight gain rate of grass carp. A 5% addition can increase the weight gain rate of grass carp by more than 16%, significantly improving the feed application value.
[0090] Table 3. Grass carp weight gain rate
[0091]
[0092] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.
Claims
1. A method for preparing wool protein peptides, characterized in that, Includes the following steps: Pretreated wool is obtained by enzymatic hydrolysis of wool raw materials using keratinase. Pretreated wool was subjected to synergistic fermentation of keratinase and Max Kluyveromyces simultaneously. After fermentation, the first liquid phase product was obtained by separation. The first liquid phase product was treated with a nuclease to separate the second liquid phase product. The first liquid phase product was enzymatically treated with aminopeptidase to obtain wool protein peptides.
2. The method for preparing wool protein peptides as described in claim 1, characterized in that, The specific preparation process of the pretreated wool is as follows: after the wool raw material is crushed, a buffer solution with a pH of 7.0-8.0 is added, keratinase is added, and enzymatic hydrolysis is carried out at 40℃-50℃ for 2-4 hours to obtain pretreated wool.
3. The method for preparing wool protein peptides as described in claim 2, characterized in that: The amount of keratinase added is 0.6%-1.0% based on the weight of the wool raw material; the enzyme activity of the keratinase is ≥500U / g.
4. The method for preparing wool protein peptides as described in claim 1, characterized in that: Based on the weight of the wool raw material, the amount of keratinase added is 1.3%-1.7%, and the amount of Kluyveromyces martensii added is 1%-5%; the keratinase activity is ≥10000 U / g; and the Kluyveromyces martensii activity is ≥1×10⁻⁶. 8 CFU / g.
5. The method for preparing wool protein peptides as described in claim 1, characterized in that: The co-fermentation temperature of the bacteria and enzymes is 35℃-40℃, and the fermentation time is 12-24h.
6. The method for preparing wool protein peptides as described in claim 1, characterized in that: The amount of nuclease added is 0.1%-0.2% based on the weight of the wool raw material; the enzyme activity of the nuclease is ≥2000.
7. The method for preparing wool protein peptides as described in claim 1, characterized in that: The amount of aminopeptidase added is 0.3%-0.7% based on the weight of the wool raw material; the enzyme activity of the aminopeptidase is ≥1000 U / g.
8. The method for preparing wool protein peptides as described in claim 1, characterized in that: The enzymatic hydrolysis temperature of the nuclease is 45℃-65℃, and the hydrolysis time is 0.5h-2h; the enzymatic hydrolysis temperature of the aminopeptidase is 37℃-42℃, and the hydrolysis time is 3-6h.
9. A wool protein peptide, characterized in that: The wool protein peptide is prepared using the preparation method described in any one of claims 1-8.
10. The application of the wool protein peptide as described in claim 9 in aquatic feed.