Preparation method and application of bovine collagen peptide

Abstract

The application provides a preparation method and application of bovine collagen peptide, and belongs to the technical field of collagen peptide; the preparation method comprises the steps of freeze-thaw-microwave treatment, alkaline hydrolysis, enzyme hydrolysis 1, enzyme hydrolysis 2, enzyme hydrolysis 3, enzyme hydrolysis 4 and antiseptic filtration; in the freeze-thaw-microwave treatment step, bovine skin powder is added into a mixed solution, stirred uniformly, and then soaked for 15-20 min; the mixed solution is placed in a sealed container for freeze-thaw treatment, frozen at-24 to-20 DEG C for 5-6 h, naturally thawed, and then the freeze-thaw treatment is repeated for 1-2 times; the water content is controlled to be 50-52 wt %, and wet material is obtained; the wet material is placed into a microwave device for microwave treatment, and the temperature of the wet material is controlled to be lower than 55 DEG C, so that freeze-thaw-microwave treated bovine skin powder is obtained; the product obtained by the method has high polypeptide content, and when the product is applied to soybeans, potatoes and lettuce, the yield and quality can be effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of collagen peptide technology, specifically relating to a method for preparing bovine skin collagen peptides and their application. Background Technology

[0002] According to existing research, enzymatic hydrolysis of cowhide (mainly collagen from cowhide tanning waste) can be efficiently converted into a mixture rich in amino acids and small molecule peptides. These products are not only rich in nitrogen, providing essential nutrients for crop growth and promoting stem and leaf development, but also have the ability to penetrate the cuticle of plant leaves and be rapidly absorbed and utilized by the plant. In addition, the products have antioxidant properties, which can reduce the damage caused by adverse stress and effectively improve the crop's resistance to stress. They have excellent potential for foliar fertilizer application and also realize the recycling and reuse of waste leather resources.

[0003] Currently, the preparation process of bovine collagen peptides is mostly used in the food and health product industries. The main process includes three steps: raw material degreasing and decolorization pretreatment, enzymatic extraction, and concentration and drying. However, this type of process has obvious shortcomings. The production material-to-liquid ratio is as high as 1:10-20, the water consumption is large, and the concentration of effective components in the produced raw liquid is low. A concentration process must be added to purify the material, which not only increases equipment investment but also increases production energy consumption and processing costs. It cannot meet the actual demand for direct use of high-concentration mother liquor in aqueous solutions.

[0004] Furthermore, tanned bovine collagen forms a stable cross-linked structure, making its molecular structure compact and difficult to degrade. Traditional processes use only a single enzyme for hydrolysis, resulting in limited degradation and poor collagen conversion efficiency. Conventional pretreatment methods have a weak effect on breaking down the dense collagen skeleton, and the hydrolysis reaction is incomplete when feeding the raw materials at once. This results in a high proportion of high molecular weight peptides in the finished product, which are difficult for plants to absorb, ultimately leading to insufficient fertilizer nutrient utilization and limited application effects.

[0005] Therefore, a method for preparing high-concentration, directly usable liquid collagen peptide foliar fertilizer using cowhide is proposed to overcome the shortcomings of existing technologies, such as high material-to-liquid ratio, low product concentration, poor degradation efficiency of tanned collagen, high water and energy consumption, and mismatched product form. This method can be applied to crops to increase chlorophyll content and yield, which is a problem that urgently needs to be solved in existing technologies. Summary of the Invention

[0006] To address the technical problems existing in the prior art, this invention utilizes cowhide enzymatically. By rationally selecting and combining cowhide pretreatment, enzymatic hydrolysis conditions, and the types and ratios of enzyme preparations, an enzymatic hydrolysate with high polypeptide content is obtained for use as a bio-organic fertilizer, effectively improving the yield and quality of crops.

[0007] A method for preparing bovine skin collagen peptides includes freeze-thaw-microwave treatment, alkaline hydrolysis and enzymatic hydrolysis 1, enzymatic hydrolysis 2, enzymatic hydrolysis 3, enzymatic hydrolysis 4, and preservation filtration steps, the specific operations of which are as follows: 1. Freeze-thaw microwave treatment Add the cowhide powder to a mixture of 0.8-1.0 times its weight, stir evenly at 140-160 rpm, let it stand and soak for 15-20 minutes, place it in a sealed container for freeze-thaw treatment at -24 to -20℃ for 5-6 hours, remove it, and let it thaw completely at 24-25℃. After that, freeze and thaw once, stir evenly, and repeat the freeze-thaw process 1-2 times. Then let it stand at room temperature for 25-30 minutes, controlling the moisture content to 50-52 wt%, to obtain wet material. Place 140-160g of wet material into a microwave device, with a material thickness of 2-3cm, microwave power of 260-300W, processing time of 15-20 minutes, and microwave frequency of 2430-2460MHz. During the processing, control the temperature of the wet material below 55℃ to obtain freeze-thaw-microwave treated cowhide powder. The cowhide powder has a particle size of 60-120 mesh, a dry basis moisture content of 5-8 wt%, an ash content of 1-2 wt%, and a collagen content of 90-93%. The mixture is a mixture of deionized water, L-cysteine, disodium ethylenediaminetetraacetate, and sodium chloride, with a mass ratio of 100:0.8-1.0:0.2-0.3:0.6-0.7.

[0008] 2. Alkaline hydrolysis and enzymatic hydrolysis 1 Weigh water, add potassium hydroxide solid, dissolve completely, add freeze-thaw-microwave treated cowhide powder, and perform alkaline hydrolysis at 58-62℃ for 2.0-2.2h to ensure better contact between protein and enzyme in the subsequent process, thus obtaining alkaline hydrolysate. Add citric acid to the alkaline hydrolysate to adjust the pH to 8.0-9.0, add compound enzyme, and perform enzymatic hydrolysis at 58-62℃ for 1.0-1.3h to obtain enzymatic hydrolysate 1. The mass ratio of water, potassium hydroxide solid, and freeze-thaw-microwave treated cowhide powder is 200:24-28:72-78. The amount of the compound enzyme added is 0.4-0.6 wt% of the freeze-thaw-microwave treated bovine hide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 1.8-2.2:1.8-2.2:0.8-1.1. The enzyme activities of the alkaline protease, keratinase, and neutral protease are all 200,000-230,000 U / g.

[0009] 3. Enzymatic hydrolysis 2 Add potassium hydroxide to the enzymatic hydrolysate 1 to adjust the enzyme pH to 8.0-9.0, feed the raw hide powder for freeze-thaw-microwave treatment, add the compound enzyme, and enzymatically hydrolyze at 58-62℃ for 2.0-2.2h to obtain enzymatic hydrolysate 2; The amount of the compound enzyme added is 0.4-0.6 wt% of the freeze-thaw-microwave treated bovine hide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 1.8-2.2:1.8-2.2:0.8-1.1. The enzyme activities of the alkaline protease, keratinase, and neutral protease are all 200,000-230,000 U / g.

[0010] 4. Enzymatic hydrolysis 3 Add potassium hydroxide to the enzymatic hydrolysate 2 to adjust the enzyme pH to 8.0-9.0, feed the raw hide powder for freeze-thaw-microwave treatment, add the compound enzyme, and enzymatically hydrolyze at 60-62℃ for 2.8-3.2h to obtain enzymatic hydrolysate 3; The amount of the compound enzyme added is 0.4-0.6 wt% of the freeze-thaw-microwave treated bovine hide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 1.8-2.2:1.8-2.2:0.8-1.1. The enzyme activities of the alkaline protease, keratinase, and neutral protease are all 200,000-230,000 U / g.

[0011] 5. Enzymatic hydrolysis 4 Add potassium hydroxide to the enzymatic hydrolysate 3 to adjust the enzyme pH to 8.0-9.0. Add the raw hide powder and freeze-thaw-microwave process. Add the compound enzyme and enzymatically hydrolyze at 58-62℃ for 24-25 hours. After the enzymatic hydrolysis is completed, raise the temperature to 84-87℃ and keep it at 20-25 minutes to inactivate the enzyme, and obtain the enzymatic hydrolysate 4. The mass ratio of the freeze-thaw-microwave treated cowhide powder to the compound enzyme is 72-78:2.8-3.2; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 1.8-2.2:1.8-2.2:0.8-1.1. The enzyme activities of the alkaline protease, keratinase, and neutral protease are all 200,000-230,000 U / g.

[0012] 6. Corrosion-resistant filter Add Kathon and propionic acid to the enzymatic hydrolysate 4, mix well, add diatomaceous earth 300# and diatomaceous earth 10#, filter, and obtain bovine skin collagen peptide mother liquor; The volume concentration of Kathon in the enzymatic hydrolysate 4 is 0.4-0.6%; The volume concentration of propionic acid in the enzymatic hydrolysate is 0.4-0.6%; The diatomaceous earth 300# and diatomaceous earth 10# together account for 10-12% of the total mass of cowhide powder; The mass ratio of diatomaceous earth 300# and diatomaceous earth 10# is 1:0.8-1.2.

[0013] The mass ratio of freeze-thaw-microwave treated cowhide powder in the alkaline hydrolysis and enzymatic hydrolysis steps 1, 2, 3, and 4 is 1:1:1:1.

[0014] The bovine collagen peptides obtained by the above preparation method can be applied to soybeans, potatoes, and lettuce.

[0015] This invention utilizes an enzymatic hydrolysis method to prepare bovine collagen peptides from bovine hide powder. Specifically, the bovine hide powder undergoes a freeze-thaw-microwave synergistic treatment. When the bovine hide powder is frozen at low temperatures, the internal impregnation solution crystallizes into ice crystals, causing volume expansion and dispersing the bovine hide collagen fiber bundles from the inside. After natural thawing, numerous micropores and microcracks form within the fibers, breaking the natural dense stacked structure of the bovine hide. Cracks propagate, further dissociating the fiber bundles, thus significantly enhancing the penetration ability of subsequent alkaline and enzymatic solutions. The mixture is composed of cysteine, disodium EDTA, and sodium chloride. Cysteine ​​can gently break the disulfide bonds between bovine hide collagen and keratin molecules, disintegrating the most stubborn cross-linked structures in the hide and promoting... Enzymatic hydrolysis of keratinase; disodium EDTA can chelate metal ions, eliminating the inhibitory effect of metal ions on proteases and protecting the activity of complex enzymes; low concentration of sodium chloride can promote solution penetration and optimize the growth morphology of ice crystals; the combined effect of these three factors keeps the cowhide powder in an activated state; microwave treatment after freeze-thaw cycle can further open up the micropores formed by freeze-thaw, allowing collagen fibers to further expand and disperse, and the temperature is controlled below 55℃ throughout the process, which will not destroy the natural activity of collagen; after freeze-thaw-microwave treatment, subsequent alkaline hydrolysis and multi-stage enzymatic hydrolysis can be carried out more easily, increasing the hydrolysis reaction rate, gradually increasing the peptide content, and ultimately improving the crop's repair performance and growth performance; Bovine hide, as a collagen-like substance, has significant water absorption and swelling properties. During enzymatic hydrolysis, it absorbs a large amount of water from the reaction system, resulting in low concentration and low yield of the enzymatic hydrolysate under traditional single-batch feeding methods. This invention employs a multi-feeding process combined with a specific compound enzyme. By adding bovine hide raw materials in batches, the impact of swelling on the volume of the reaction system is effectively mitigated, significantly increasing the solid content and effective ingredient concentration of the final enzymatic hydrolysate. Ultimately, this promotes crop growth, making it a valuable supplement during critical growth periods and under adverse conditions, thereby improving yield and quality.

[0016] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. The bovine collagen peptides obtained by this invention have a solid content of 27.2-30.8%, a polypeptide content of 50.4-54.6%, a total nitrogen content of 3.7-4.2%, and 78-84% of the polypeptides have a molecular weight <5000 Da, while 47-52% have a molecular weight concentrated between 500-1000 Da. 2. The bovine collagen peptides obtained by this invention can be applied to soybeans. The chlorophyll content of soybeans during flowering is 51.83-53.05 (SPAD value), the yield per pot is 18.13-18.96g, the number of pods per plant is 30.8-31.6, the number of seeds per pod is 2.68-2.75, and the weight of 100 seeds is 21.96-22.51g. 3. The bovine collagen peptides obtained by this invention can be applied to potatoes. The chlorophyll content of potatoes during flowering is 46.2-46.8 (SPAD value), the weight of a single tuber is 0.42-0.48 kg, the number of tubers per plant is 8.5-8.9, and the starch content of the tubers is 17.22-17.58% (fresh basis). 4. The bovine collagen peptides obtained by this invention can be applied to lettuce, which can effectively increase the height of lettuce plants. The fresh weight of the above-ground parts of a single plant is 54.82-56.37 g / plant, the fresh weight of the roots of a single plant is 13.53-16.99 g / plant, and the root-to-shoot ratio is 0.24-0.31. Attached Figure Description

[0017] Figure 1 The image shows soybean plants before the application of the bovine collagen peptide foliar fertilizer prepared in Example 3. The photo was taken on February 27, 2026 at 08:18. Figure 2 The image shows soybean plants after being sprayed with the bovine collagen peptide foliar fertilizer prepared in Example 3. The photo was taken on February 28, 2026 at 16:16. Figure 3 The image shows potato plants before the application of the bovine collagen peptide foliar fertilizer prepared in Example 3. The photo was taken on March 2, 2026 at 08:26. Figure 4 The image shows potato plants after being sprayed with the bovine collagen peptide foliar fertilizer prepared in Example 3. The photo was taken on March 5, 2026 at 12:41. Figure 5 The image shows lettuce plants fertilized with the bovine collagen peptide fertilizer prepared in Example 3. The photo was taken on January 16, 2026 at 08:18. Figure 6 The image shows lettuce plants fertilized with the bovine collagen peptide fertilizer prepared in Comparative Example 2. The photo was taken on January 16, 2026 at 08:18. Detailed Implementation

[0018] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0019] Example 1 1. Freeze-thaw microwave treatment Add the cowhide powder to a 1.0 times its weight of the mixture, stir evenly at 160 rpm, let it stand and soak for 20 minutes, place it in a sealed container for freeze-thaw, freeze at -20℃ for 6 hours, remove it, and let it thaw completely at 25℃. After that, freeze and thaw once, stir evenly, and repeat the freeze-thaw twice more. Then let it stand at room temperature for 30 minutes, control the moisture content to 52 wt%, and obtain wet material. Put 160 g of wet material into a microwave device, with a material thickness of 3 cm, microwave power of 300 W, processing time of 20 minutes, microwave frequency of 2460 MHz, and control the temperature of the wet material below 55℃ during the processing to obtain freeze-thaw-microwave treated cowhide powder. The cowhide powder has a particle size of 120 mesh, a dry basis moisture content of 8 wt%, an ash content of 2 wt%, and a collagen content of 92%. The mixture is a mixture of deionized water, L-cysteine, disodium ethylenediaminetetraacetate, and sodium chloride, with a mass ratio of 100:0.8:0.2:0.7.

[0020] 2. Alkaline hydrolysis and enzymatic hydrolysis 1 Weigh 200g of deionized water, add 28g of potassium hydroxide solid, dissolve completely, add 78g of freeze-thaw-microwave treated cowhide powder, and perform alkaline hydrolysis at 62℃ for 2.2h to ensure better contact between the protein and enzyme in the subsequent process, thus obtaining alkaline hydrolysate. Add citric acid to the alkaline hydrolysate to adjust the pH to 9.0, add the compound enzyme, and perform enzymatic hydrolysis at 62℃ for 1.3h to obtain enzymatic hydrolysate 1. The amount of the compound enzyme added was 0.6 wt% of the freeze-thaw-microwave treated bovine hide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 2.2:1.8:1.1. The enzyme activities of the alkaline protease, keratinase, and neutral protease were all 230,000 U / g.

[0021] 3. Enzymatic hydrolysis 2 Add potassium hydroxide to the enzymatic hydrolysate 1 to adjust the enzyme pH to 9.0, add 78g of freeze-thaw-microwave treated cowhide powder, add compound enzyme, and enzymatically hydrolyze at 62℃ for 2.2h to obtain enzymatic hydrolysate 2; The amount of the compound enzyme added was 0.6 wt% of the freeze-thaw-microwave treated bovine hide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 2.2:1.8:1.1. The enzyme activities of the alkaline protease, keratinase, and neutral protease were all 230,000 U / g.

[0022] 4. Enzymatic hydrolysis 3 Add potassium hydroxide to the enzymatic hydrolysate 2 to adjust the enzyme pH to 9.0, add 78g of freeze-thaw-microwave treated cowhide powder, add the compound enzyme, and enzymatically hydrolyze at 62℃ for 3.2h to obtain enzymatic hydrolysate 3; The amount of the compound enzyme added was 0.6 wt% of the freeze-thaw-microwave treated bovine hide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 2.2:1.8:1.1. The enzyme activities of the alkaline protease, keratinase, and neutral protease were all 230,000 U / g.

[0023] 5. Enzymatic hydrolysis 4 Add potassium hydroxide to the enzymatic hydrolysate 3 to adjust the enzyme pH to 9.0. Add 78g of freeze-thaw-microwave treated cowhide powder and 3.2g of compound enzyme. Enzymatic hydrolysis was carried out at 62℃ for 25h. After the enzymatic hydrolysis was completed, the temperature was raised to 87℃ and kept at 25min to inactivate the enzyme, thus obtaining enzymatic hydrolysate 4. The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 2.2:1.8:1.1. The enzyme activities of the alkaline protease, keratinase, and neutral protease were all 230,000 U / g.

[0024] 6. Corrosion-resistant filter Add Kathon and propionic acid to the enzymatic hydrolysate 4, mix well, add diatomaceous earth 300# and diatomaceous earth 10#, filter, and obtain bovine skin collagen peptide mother liquor; The volume concentration of Kathon in the enzymatic hydrolysate 4 was 0.6%; The volume concentration of propionic acid in the enzymatic hydrolysate is 0.6%; The diatomaceous earth 300# and diatomaceous earth 10# together account for 12% of the total mass of cowhide powder; The mass ratio of diatomaceous earth 300# to diatomaceous earth 10# is 1:1.2.

[0025] The bovine collagen peptide mother liquor is a brown transparent liquid with a solid content of 28.3%, a polypeptide content of 52.5%, a total nitrogen content of 4.0%, and 80% of the polypeptides have a molecular weight <5000Da, while 49% have a molecular weight concentrated between 500-1000Da.

[0026] Example 2 1. Freeze-thaw microwave treatment Add the cowhide powder to a 0.8 times its weight of the mixture, stir evenly at 140 rpm, let it stand and soak for 15 minutes, place it in a sealed container for freeze-thaw, freeze at -24℃ for 5 hours, remove it, and let it thaw completely at 24℃. After that, freeze and thaw once, stir evenly, and repeat the freeze-thaw once more. Then let it stand at room temperature for 25 minutes, control the moisture content to 50 wt%, and obtain wet material. Place 140 g of wet material into a microwave device, with a material thickness of 3 cm, microwave power of 260 W, processing time of 15 minutes, and microwave frequency of 2430 MHz. During the processing, control the temperature of the wet material to be below 55℃ to obtain freeze-thaw-microwave treated cowhide powder. The cowhide powder has a particle size of 60 mesh, a dry basis moisture content of 5 wt%, an ash content of 2 wt%, and a collagen content of 90%. The mixture is a mixture of deionized water, L-cysteine, disodium ethylenediaminetetraacetate, and sodium chloride, with a mass ratio of 100:0.8:0.3:0.6.

[0027] 2. Alkaline hydrolysis and enzymatic hydrolysis 1 Weigh 200g of deionized water, add 24g of potassium hydroxide solid, dissolve completely, add 72g of freeze-thaw-microwave treated cowhide powder, and perform alkaline hydrolysis at 58℃ for 2.0h to ensure better contact between the protein and enzyme in the subsequent process, and obtain alkaline hydrolysate. Add citric acid to the alkaline hydrolysate to adjust the pH to 8.0, add the compound enzyme, and perform enzymatic hydrolysis at 58℃ for 1.0h to obtain enzymatic hydrolysate 1. The amount of the compound enzyme added was 0.4 wt% of the freeze-thaw-microwave treated cowhide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 1.8:2.2:0.8. The enzyme activities of the alkaline protease, keratinase, and neutral protease are all 200,000 U / g.

[0028] 3. Enzymatic hydrolysis 2 Add potassium hydroxide to the enzymatic hydrolysate 1 to adjust the enzyme pH to 8.0, add 72g of freeze-thaw-microwave treated cowhide powder, add compound enzyme, and enzymatically hydrolyze at 58℃ for 2.2h to obtain enzymatic hydrolysate 2; The amount of the compound enzyme added was 0.4 wt% of the freeze-thaw-microwave treated cowhide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 1.8:2.2:0.8. The enzyme activities of the alkaline protease, keratinase, and neutral protease are all 200,000 U / g.

[0029] 4. Enzymatic hydrolysis 3 Add potassium hydroxide to the enzymatic hydrolysate 2 to adjust the enzyme pH to 8.0, add 72g of freeze-thaw-microwave treated cowhide powder, add compound enzyme, and enzymatically hydrolyze at 60℃ for 2.8h to obtain enzymatic hydrolysate 3; The amount of the compound enzyme added was 0.4 wt% of the freeze-thaw-microwave treated cowhide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 1.8:2.2:0.8. The enzyme activities of the alkaline protease, keratinase, and neutral protease are all 200,000 U / g.

[0030] 5. Enzymatic hydrolysis 4 Add potassium hydroxide to the enzymatic hydrolysate 3 to adjust the enzyme pH to 8.0. Add 72g of freeze-thaw-microwave treated cowhide powder and 2.8g of compound enzyme. Enzymatic hydrolysis was carried out at 58℃ for 24h. After the enzymatic hydrolysis was completed, the temperature was raised to 84℃ and kept warm for 20min to inactivate the enzyme, thus obtaining enzymatic hydrolysate 4. The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 1.8:2.2:0.8. The enzyme activities of the alkaline protease, keratinase, and neutral protease are all 200,000 U / g.

[0031] 6. Corrosion-resistant filter Add Kathon and propionic acid to the enzymatic hydrolysate 4, mix well, add diatomaceous earth 300# and diatomaceous earth 10#, filter, and obtain bovine skin collagen peptide mother liquor; The volume concentration of Kathon in the enzymatic hydrolysate 4 was 0.4%; The volume concentration of propionic acid in the enzymatic hydrolysate is 0.4%; The diatomaceous earth 300# and diatomaceous earth 10# together account for 10% of the total mass of cowhide powder; The mass ratio of diatomaceous earth 300# to diatomaceous earth 10# is 1:0.8.

[0032] The bovine collagen peptide mother liquor is a brown transparent liquid with a solid content of 27.2%, a polypeptide content of 50.4%, a total nitrogen content of 3.7%, and 78% of the polypeptides have a molecular weight <5000Da, of which 47% have a molecular weight concentrated between 500-1000Da.

[0033] Example 3 1. Freeze-thaw microwave treatment Add the cowhide powder to a 1.0 times its weight of the mixture, stir evenly at 150 rpm, let it stand and soak for 18 minutes, place it in a sealed container for freeze-thaw, freeze at -22℃ for 6 hours, remove it, and let it thaw completely at 25℃. After that, freeze and thaw once, stir evenly, and repeat the freeze-thaw twice more. Then let it stand at room temperature for 28 minutes, control the moisture content to 50 wt%, and obtain wet material. Put 150 g of wet material into a microwave device, with a material thickness of 2 cm, microwave power of 280 W, processing time of 18 minutes, microwave frequency of 2450 MHz, and control the temperature of the wet material below 55℃ during the processing to obtain freeze-thaw-microwave treated cowhide powder. The cowhide powder has a particle size of 100 mesh, a dry basis moisture content of 6 wt%, an ash content of 1 wt%, and a collagen content of 93%. The mixture is a mixture of deionized water, L-cysteine, disodium ethylenediaminetetraacetate, and sodium chloride, with a mass ratio of 100:1.0:0.2:0.6.

[0034] 2. Alkaline hydrolysis and enzymatic hydrolysis 1 Weigh 200g of deionized water, add 26g of potassium hydroxide solid, dissolve completely, add 75g of freeze-thaw-microwave treated cowhide powder, and perform alkaline hydrolysis at 60℃ for 2.0h to ensure better contact between the protein and enzyme in the subsequent process, thus obtaining alkaline hydrolysate. Add citric acid to the alkaline hydrolysate to adjust the pH to 8.5, add the compound enzyme, and perform enzymatic hydrolysis at 60℃ for 1.2h to obtain enzymatic hydrolysate 1. The amount of the compound enzyme added is 0.5 wt% of the freeze-thaw-microwave treated cowhide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 2.0:2.0:1.0. The enzyme activities of the alkaline protease, keratinase, and neutral protease were all 220,000 U / g.

[0035] 3. Enzymatic hydrolysis 2 Add potassium hydroxide to the enzymatic hydrolysate 1 to adjust the enzyme pH to 8.5, add 75g of freeze-thaw-microwave treated cowhide powder, add compound enzyme, and enzymatically hydrolyze at 60℃ for 2.0h to obtain enzymatic hydrolysate 2; The amount of the compound enzyme added is 0.5 wt% of the freeze-thaw-microwave treated cowhide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 2.0:2.0:1.0. The enzyme activities of the alkaline protease, keratinase, and neutral protease were all 220,000 U / g.

[0036] 4. Enzymatic hydrolysis 3 Add potassium hydroxide to the enzymatic hydrolysate 2 to adjust the enzyme pH to 8.5, add 75g of freeze-thaw-microwave treated cowhide powder, add compound enzyme, and enzymatically hydrolyze at 62℃ for 3.0h to obtain enzymatic hydrolysate 3; The amount of the compound enzyme added is 0.5 wt% of the freeze-thaw-microwave treated cowhide powder; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 2.0:2.0:1.0. The enzyme activities of the alkaline protease, keratinase, and neutral protease were all 220,000 U / g.

[0037] 5. Enzymatic hydrolysis 4 Add potassium hydroxide to the enzymatic hydrolysate 3 to adjust the enzyme pH to 8.5. Add 75g of freeze-thaw-microwave treated cowhide powder and 3.0g of compound enzyme. Enzymatic hydrolyze at 60℃ for 24h. After the enzymatic hydrolysis is completed, raise the temperature to 86℃ and keep it warm for 23min to inactivate the enzyme, and obtain the enzymatic hydrolysate 4. The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 2.0:2.0:1.0. The enzyme activities of the alkaline protease, keratinase, and neutral protease were all 220,000 U / g.

[0038] 6. Corrosion-resistant filter Add Kathon and propionic acid to the enzymatic hydrolysate 4, mix well, add diatomaceous earth 300# and diatomaceous earth 10#, filter, and obtain bovine skin collagen peptide mother liquor; The volume concentration of Kathon in the enzymatic hydrolysate 4 is 0.5%; The volume concentration of propionic acid in the enzymatic hydrolysate is 0.5%; The diatomaceous earth 300# and diatomaceous earth 10# together account for 10% of the total mass of cowhide powder; The mass ratio of diatomaceous earth 300# to diatomaceous earth 10# is 1:1.

[0039] The bovine collagen peptide mother liquor is a brown transparent liquid with a solid content of 30.8%, a polypeptide content of 54.6%, a total nitrogen content of 4.2%, and 84% of the polypeptides have a molecular weight <5000Da, while 52% have a molecular weight concentrated between 500-1000Da.

[0040] Comparative Example 1 Based on Example 3, the following changes were made: The alkaline hydrolysis and enzymatic hydrolysis step 1 is as follows: Weigh 200g of deionized water, add 26g of potassium hydroxide solid, dissolve it completely, add 300g of freeze-thaw-microwave treated cowhide powder, and perform alkaline hydrolysis at 60℃ for 2.0h to obtain alkaline hydrolysate. Add citric acid to the alkaline hydrolysate to adjust the pH to 8.5, add 4.125g of compound enzyme, and perform enzymatic hydrolysis at 60℃ for 30.2h to obtain enzymatic hydrolysate 1. The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease, with a mass ratio of 2.0:2.0:1.0. The enzyme activities of the alkaline protease, keratinase, and neutral protease were all 220,000 U / g; Steps 2, 3, and 4 of enzymatic hydrolysis are omitted. The rest of the operations are exactly the same.

[0041] Comparative Example 1 uses a one-time addition of cowhide powder. The cowhide powder absorbs water and swells, making enzymatic hydrolysis impossible.

[0042] Comparative Example 2 Based on Example 3, the following changes were made: The complex enzymes in the alkaline hydrolysis and enzymatic hydrolysis steps 1, 2, 3, and 4 are all replaced with alkaline protease in equal amounts. The enzyme activity of the alkaline proteases was 220,000 U / g. The rest of the operations are exactly the same.

[0043] The enzymatic hydrolysis effect of Comparative Example 2 was low, with more residue and slower filtration speed; the bovine collagen peptide mother liquor obtained from Comparative Example 2 had a solid content of 21.3%, a polypeptide content of 45.2%, and a total nitrogen content of 2.5%.

[0044] Comparative Example 3 Based on Example 3, the following changes were made: The freeze-thaw-microwave treatment step is omitted, and the untreated cowhide powder is directly subjected to alkaline hydrolysis and enzymatic hydrolysis step 1. The cowhide powder has a particle size of 100 mesh, a dry basis moisture content of 6 wt%, an ash content of 1 wt%, and a collagen content of 93%. Replace the freeze-thaw-microwave treated cowhide powder in enzymatic hydrolysis 2, enzymatic hydrolysis 3, and enzymatic hydrolysis 4 with untreated cowhide powder in equal amounts; The rest of the operations are exactly the same.

[0045] The bovine collagen peptide mother liquor obtained in Comparative Example 3 had a solid content of 23.7%, a polypeptide content of 47.5%, and a total nitrogen content of 3.1%.

[0046] Application performance 1. Application in soybeans A pot experiment was conducted using the Qihuang 34 soybean variety. All plants were planted in uniform cultivation pots with 4 plants per pot. The experiment was divided into 5 groups, and each group had 40 pots as replicates. The bovine collagen peptide stock solution prepared in Examples 1-3 and Comparative Examples 2-3 was diluted 800 times to obtain foliar fertilizer, and then sprayed on the leaves. The amount sprayed per plant was controlled at 25 ml, and the leaves were evenly moistened without dripping. The plant was sprayed twice during the growing season, with an interval of 5 days between the two sprays. The interval between spraying should not be too short. The chlorophyll content of soybean plants during the flowering period was tested. After harvest, the yield per pot, number of pods per plant, number of seeds per pod, and weight of 100 seeds were recorded. The results are as follows:

[0047] The experimental results showed that, 2 to 3 days after spraying, the yellowed leaves in the example could be visibly turned green again, and the color of the leaf veins and leaf tissues was uniformly restored to normal. The example group treated with bovine collagen peptides showed that all indicators were significantly better than the comparative example. The chlorophyll content during the flowering period of the example was 51.83-53.05%, which was 22.91%-25.80% higher than that of comparative example 2 and 16.42%-19.16% higher than that of comparative example 3, effectively improving the photosynthetic capacity of soybeans. In terms of yield and constituent traits, the yield per pot in Example 1 was 18.13–19.56 g, which was 65.72%–78.79% higher than that in Comparative Example 2 and 43.66%–54.99% higher than that in Comparative Example 3. The number of pods per plant, the number of seeds per pod, and the weight of 100 seeds per plant were also significantly higher than those in the two control groups, with increases of 15.36%–24.90%, 10.29%–19.05%, and 12.90%–20.25%, respectively. Example 3 showed the best overall yield performance.

[0048] Comparing the three sets of examples, it can be seen that each treatment has stable effects and its own advantages: Example 3 has the highest chlorophyll content during flowering, number of pods per plant, number of seeds per pod, 100-seed weight and yield per pot, and the best overall growth promotion and yield increase effect; Example 1 has all indicators at a medium level and its overall performance is stable; Example 2 has overall indicators slightly lower than the other two groups, but is still significantly better than the two control treatments.

[0049] The results of this study show that the bovine collagen peptide mother liquor of this invention has a significant effect on promoting soybean growth and yield. The active peptides and amino acid components contained in the bovine collagen peptide can improve the photosynthetic performance of soybean leaves, regulate the absorption and translocation of nutrients in plants, optimize yield composition indicators, and achieve a significant increase in yield by increasing the number of pods and improving grain quality. This substance is suitable for the growth and development needs of soybeans and has good application value for high-yield soybean cultivation.

[0050] 2. Application in potatoes The bovine collagen peptide stock solutions prepared in Examples 1-3 and Comparative Examples 2-3 were diluted 800 times with water to obtain foliar fertilizers. The foliar fertilizers were then sprayed evenly on the surface of potato plant leaves. Each plant was sprayed with 25 ml of diluted solution, ensuring that both sides of the leaves were evenly moistened without dripping. The local main potato variety, Lu Yin No. 1, was selected for the pot experiment. All plants were planted in uniform pots, with one plant per pot. Each experimental treatment had 40 pots as replicates. The plants were sprayed twice during the growing season, with a 5-day interval between the two sprays. They were not allowed to be sprayed on the same day. The chlorophyll content of potatoes during the flowering period was tested. After harvest, the tuber weight per plant, the number of tubers per plant, and the starch content of the tubers were recorded. The results are as follows:

[0051] The experimental results showed that 2 to 3 days after spraying the pesticide, the yellowed leaves of the plants visibly and rapidly turned green, and the color of the leaf veins and leaf tissues evenly returned to normal. The potatoes in the example group treated with bovine collagen peptide stock solution showed significantly better performance in all indicators compared to the control group. The chlorophyll content in the example group was 46.2-46.8%, an increase of 28.69%-30.36% compared to control group 2, and an increase of 17.26%-18.78% compared to control group 3, indicating a significant enhancement in photosynthetic capacity. Regarding yield-related indicators, the example group showed a higher single-plant tuber weight (single-pot yield) of 0.42-0.48 kg and a higher single-plant tuber count of 8. Compared to Comparative Example 2, the tuber weight per plant increased by 35.48%-54.84%, and the number of tubers increased by 34.92%-41.27%; compared to Comparative Example 3, the tuber weight per plant increased by 20.00%-37.14%, and the number of tubers increased by 25.00%-30.88%, with Example 2 showing the best yield. In terms of processing quality, the starch content of the tubers in Example 2 was 17.22%-17.58%, which was 14.12%-16.50% higher than that in Comparative Example 2 and 8.58%-10.84% ​​higher than that in Comparative Example 3, indicating improved processing quality.

[0052] The results of this study show that there are significant differences in the promoting effect of bovine collagen peptide mother liquor on potato growth after application. The active peptides and amino acid components contained in bovine collagen peptide can regulate plant physiological metabolism, promote nutrient absorption and photosynthetic product distribution, and coordinate potato vegetative growth and tuber development. Due to the lack of corresponding active substances for regulation, the photosynthetic capacity, tuber formation capacity and tuber quality of the control treatment were all at a low level.

[0053] In summary, bovine collagen peptide treatment can achieve the dual effects of increasing potato yield and improving quality, and has good application value in potato cultivation.

[0054] 3. Application in lettuce 3.1 Materials and Methods 3.1.1 Test Site and Time The experiment was conducted in the greenhouse of Shandong Seres Agricultural Technology Co., Ltd., using potted plants, and took place from January 5, 2026 to January 31, 2026.

[0055] 3.1.2 Test Methods The experiment consisted of 5 experimental groups: Examples 1-3 and Comparative Examples 2-3. Each experimental group had 40 replicates, with one lettuce planted in each pot. The bovine collagen peptide mother liquors prepared in Examples 1-3 and Comparative Examples 2-3 were diluted 500 times to obtain fertigation fertilizers, which were then applied to lettuce. 200 ml of fertilizer solution was applied to each pot, and fertilization was carried out every 5 days. The first fertilization was carried out on January 5, 2026, and the above-ground plant height (distance from the base of the stem to the highest point of the plant) was measured at the time of the first fertilization. Plant height was measured every 7 days. At harvest, the fresh weight of the lettuce roots and the fresh weight of the above-ground parts were measured.

[0056] 3.1.3 Data Analysis Calculate the average plant height, plant height growth rate ((current plant height - initial plant height) / initial plant height × 100%), fresh weight, and root-to-shoot ratio for each group and each measurement. 3.2 Results and Analysis During the lettuce growth period, the lettuce plant height treated with bovine collagen peptide mother liquor in Example 3 was consistently higher than that in the control group. By January 31, the lettuce plant height treated with bovine collagen peptide in Example 3 increased by 12.15% compared to the control group 2. This indicates that the 500-fold treatment with bovine collagen peptide in Example 3 was more effective in promoting lettuce plant height growth. The specific results are shown in the table below:

[0057] The yield results show that the bovine collagen peptide of the present invention performs better in terms of fresh weight of the aboveground parts of a single plant and has a higher root-to-shoot ratio, which indicates that the bovine collagen peptide treatment is more inclined to promote the growth of the aboveground parts. The specific results are shown in the table below:

[0058] The results of this study indicate that the application of the bovine collagen peptide mother liquor of this invention has significant differences in its effect on promoting lettuce growth. Regarding plant height, the bovine collagen peptide exhibits a superior effect on promoting the elongation of the aboveground parts of lettuce, mainly due to its unique amino acids and active peptide components, which effectively regulate plant cell elongation and division, promoting rapid and robust stem and leaf growth. In terms of biomass accumulation, the bovine collagen peptide of this invention significantly increases the fresh weight accumulation of the aboveground parts of lettuce, complementing its growth-promoting effect and better meeting the growth requirements of high-quality lettuce cultivation. Comparative Example 2 showed weaker overall growth. The difference between the two is mainly due to the differences in the amino acids and molecular weight obtained from enzymatic hydrolysis, affecting the regulation of nutrient absorption and transport, and the distribution of photosynthetic products. Overall, the nutrient composition of the bovine collagen peptide is more aligned with the growth and development patterns of lettuce, and can better coordinate the overall growth of the plant.

[0059] Unless otherwise stated, all percentages used in this invention are mass percentages.

[0060] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing bovine skin collagen peptides, characterized in that, The preparation method is as follows: Weigh water, add potassium hydroxide solid, add freeze-thaw-microwave treated cowhide powder for the first time, and obtain alkaline hydrolysate after alkaline hydrolysis. Adjust the pH value to 8.0-9.0, add a compound enzyme, and obtain enzymatic hydrolysate 1 after enzymatic hydrolysis. Adjust the pH value of enzymatic hydrolysate 1 to 8.0-9.0, add freeze-thaw-microwave treated cowhide powder for the second time, add a compound enzyme, and obtain enzymatic hydrolysate 2 after enzymatic hydrolysate. Adjust the pH value of enzymatic hydrolysate 2 to 8.0-9.0, add freeze-thaw-microwave treated cowhide powder for the third time, add a compound enzyme, and obtain enzymatic hydrolysate 3 after enzymatic hydrolysate. Adjust the pH value of enzymatic hydrolysate 3 to 8.0-9.0, add freeze-thaw-microwave treated cowhide powder for the fourth time, add a compound enzyme, and obtain enzymatic hydrolysate 4 after enzymatic hydrolysis and enzyme inactivation. After preservation and filtration, obtain cowhide collagen peptide mother liquor. The mass ratio of the first addition of freeze-thaw-microwave treated cowhide powder, the second addition of freeze-thaw-microwave treated cowhide powder, the third addition of freeze-thaw-microwave treated cowhide powder, and the fourth addition of freeze-thaw-microwave treated cowhide powder is 1:1:1:1; The complex enzyme is a mixture of alkaline protease, keratinase, and neutral protease; The method for preparing freeze-thaw-microwave treated cowhide powder is as follows: cowhide powder is added to a mixture, stirred evenly, and allowed to stand and soak for 15-20 minutes. Then, it is placed in a sealed container for freeze-thaw treatment at -24 to -20°C for 5-6 hours. After natural thawing, the freeze-thaw process is repeated 1-2 times to control the moisture content to 50-52 wt%, resulting in wet material. The wet material is then placed in a microwave device for microwave treatment, with the temperature of the wet material controlled below 55°C, to obtain freeze-thaw-microwave treated cowhide powder. The mixture is a mixture of deionized water, L-cysteine, disodium ethylenediaminetetraacetate, and sodium chloride.

2. The method for preparing bovine collagen peptides according to claim 1, characterized in that, In the method for preparing freeze-thaw-microwave treated cowhide powder, the cowhide powder has a particle size of 60-120 mesh, a dry basis moisture content of 5-8 wt%, an ash content of 1-2 wt%, and a collagen content of 90-93%. In the mixture, the mass ratio of deionized water, L-cysteine, disodium ethylenediaminetetraacetate, and sodium chloride is 100:0.8-1.0:0.2-0.3:0.6-0.

7.

3. The method for preparing bovine collagen peptides according to claim 1, characterized in that, In the preparation of the enzymatic hydrolysate 1, the alkaline hydrolysis temperature is 58-62℃ and the alkaline hydrolysis time is 2.0-2.2h; the enzymatic hydrolysis temperature is 58-62℃ and the enzymatic hydrolysis time is 1.0-1.3h. The mass ratio of water, potassium hydroxide solid, and the first addition of freeze-thaw-microwave treated cowhide powder is 200:24-28:72-78. The amount of the compound enzyme added is 0.4-0.6 wt% of the amount of the first addition of the freeze-thaw-microwave treated cowhide powder.

4. The method for preparing bovine collagen peptides according to claim 1, characterized in that, In the preparation of the enzymatic hydrolysate 2, the enzymatic hydrolysis temperature is 58-62℃ and the enzymatic hydrolysis time is 2.0-2.2h; The amount of the compound enzyme added is 0.4-0.6 wt% of the amount of the freeze-thaw-microwave treated cowhide powder added in the second addition.

5. The method for preparing bovine collagen peptides according to claim 1, characterized in that, In the preparation of the enzymatic hydrolysate 3, the enzymatic hydrolysis temperature is 60-62℃ and the enzymatic hydrolysis time is 2.8-3.2h; The amount of the compound enzyme added is 0.4-0.6 wt% of the bovine hide powder subjected to the third freeze-thaw-microwave treatment.

6. The method for preparing bovine collagen peptides according to claim 1, characterized in that, In the preparation of the enzymatic hydrolysate 4, the enzymatic hydrolysis temperature is 58-62℃, the enzymatic hydrolysis time is 24-25h, the enzyme inactivation temperature is 84-87℃, and the enzyme inactivation time is 20-25min; The mass ratio of the freeze-thaw-microwave treated cowhide powder to the compound enzyme added in the fourth addition is 72-78:2.8-3.

2.

7. The method for preparing bovine collagen peptides according to claim 1, characterized in that, In the complex enzyme, the mass ratio of alkaline protease, keratinase, and neutral protease is 1.8-2.2:1.8-2.2:0.8-1.1; The enzyme activities of the alkaline protease, keratinase, and neutral protease are all 200,000-230,000 U / g.

8. The method for preparing bovine collagen peptides according to claim 1, characterized in that, The anti-corrosion filtration step is as follows: add Kathon and propionic acid to the enzymatic hydrolysate 4, mix them evenly, add diatomaceous earth 300# and diatomaceous earth 10#, and filter to obtain bovine skin collagen peptide mother liquor. The volume concentration of Kathon in the enzymatic hydrolysate 4 is 0.4-0.6%; The volume concentration of propionic acid in the enzymatic hydrolysate is 0.4-0.6%; The total amount of diatomaceous earth 300# and diatomaceous earth 10# accounts for 10-12% of the total mass of the freeze-thaw-microwave treated cowhide powder; The mass ratio of diatomaceous earth 300# and diatomaceous earth 10# is 1:0.8-1.

2.

9. An application of a bovine skin collagen peptide, characterized in that, Application of bovine collagen peptides prepared by the preparation method according to any one of claims 1-7 in increasing the yield of soybeans, potatoes and lettuce.

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