Preparation method of collagen peptide, nutrition powder composition for promoting wound healing and preparation method, product and application of nutrition powder composition

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method of collagen peptide, a nutrition powder composition for promoting wound healing and a preparation method, a product and application of the nutrition powder composition. The preparation method of the collagen peptide comprises the following steps: firstly, adding water into bovine bone particles, heating, soaking and filtering to obtain bovine bone protein liquid; laccase, subtilisin and flavourzyme are sequentially added into the bovine bone protein liquid for enzymolysis, centrifugation is conducted, and supernate is collected; and finally, adding chitosan into the supernate, and carrying out ultrafiltration and drying, so as to obtain a finished product. The prepared collagen peptide is high in yield, good in taste and beneficial to human body absorption, the invention further provides a nutrition powder composition which can accelerate wound healing by promoting wound angiogenesis, collagenous fiber deposition and epidermis regeneration, and therefore the collagen peptide has good application prospects in skin tissue wound repairing.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing collagen peptides, a nutritional powder composition for promoting wound healing, its preparation method, products, and applications. Background Technology

[0002] Skin wounds refer to damage to the integrity of skin tissue, which may even be accompanied by disruption of the structure and function of underlying fat, muscle, and bone tissue. This can be caused by a variety of factors, including trauma, burns, and surgery. Wound healing involves complex interactions between epidermal and dermal cells, the extracellular matrix, and plasma-derived proteins, all of which are regulated by a range of signaling molecules, such as cytokines and growth factors. Wound healing is generally considered a four-step process: hemostasis, inflammation, cell proliferation / granulation, and remodeling / maturation. The successful completion of this process depends not only on clinical antimicrobial wound-healing medications but also on nutritional fortification therapy. This is especially important and crucial for patients with large-area deep burns, destructive trauma, and chronic, difficult-to-heal wounds, where protein and multinutrient supplementation is essential for treatment.

[0003] Early postoperative enteral nutrition has been shown to increase hydroxyproline levels and improve surgical wound healing. Nutritional deficiencies or malnutrition can delay wound healing by prolonging the inflammatory phase, reducing fibroblast proliferation, and altering collagen synthesis. Malnourished patients may develop pressure sores, infections, and experience delayed wound healing, leading to chronic, difficult-to-heal wounds. The "Guidelines for the Clinical Application of Micronutrients in Chinese Adult Patients (2024 Edition)" states that micronutrient deficiencies can lead to poor wound healing and weakened immune responses. For adult burn patients, in addition to ensuring adequate energy and protein intake, a variety of vitamins and trace elements should be consumed. For example, vitamin C participates in the entire process of wound healing (inflammation, proliferation, and remodeling). Vitamin C supplementation helps complete the inflammatory phase, and when sufficient vitamin C circulates in the body, it stimulates fibroblast migration, matrix deposition, and angiogenesis, promoting wound healing. Zinc is a component of human enzymes and a cofactor for many metalloenzymes required for cell membrane repair, cell proliferation, tissue growth, and maintaining the function of the immune system. Zinc deficiency can lead to growth retardation, impaired immune function, skin lesions, and impaired wound healing. Copper is an important cofactor for cytochrome oxidase and the cytoplasmic antioxidant superoxide dismutase. It is also essential for the interaction of lysyl oxidase, which is crucial for the cross-linking and strengthening of the collagen framework.

[0004] For example, Chinese invention patent publication number CN119014550A discloses a medical nutritional composition for promoting wound repair and its preparation method. This medical nutritional composition includes the following components: 10-15 parts whey protein powder, 5-15 parts soybean protein peptide powder, 5-10 parts bovine spleen peptide powder, 10-15 parts collagen tripeptide powder, 5-8 parts collagen peptide powder, 10-12 parts wheat oligopeptide powder, 5-10 parts hemoglobin oligopeptide powder, 40-55 parts low-GI complex carbohydrates, 3-7 parts fat, 5-15 parts complex plant extracts, 0.2 parts vitamin C, 0.001 parts vitamin A, 0.015 parts vitamin E, 0.0018 parts vitamin B1, 0.0018 parts vitamin B2, 0.0015 parts vitamin B6, and 0.0015 parts vitamin B6. 12 This medical nutrition composition contains 0.000002 parts, 2-4 parts hyaluronic acid, 10-18 parts traditional Chinese medicine components, and 1 part inorganic salt combination packet. It has a relatively scientific and comprehensive effect of strengthening nutrition, reducing the risk of infection, improving the patient's immunity, and promoting the wound healing process.

[0005] Another Chinese invention patent publication, CN117297109A, discloses a nutritional composition, nutritional powder, functional food, and its applications. The nutritional composition comprises the following components in parts by weight: 5-10 parts collagen tripeptide and 3-5 parts low-methoxyl pectin. The collagen tripeptide promotes wound healing and improves skin health, while the low-methoxyl pectin has significant effects in anti-oxidation, anti-inflammation, and enhancing immunity, thus helping to improve the efficiency and quality of the wound repair process. This invention achieves a synergistic and complementary effect by compounding collagen tripeptide and low-methoxyl pectin in a specific ratio, comprehensively enhancing the effects of wound repair and immune enhancement.

[0006] However, the nutritional compositions prepared in the aforementioned patent documents are still not ideal in increasing wound healing rate and accelerating wound closure, and their complex composition and high cost make them unsuitable for industrial production.

[0007] Collagen peptides are products of collagen hydrolysis. They are substances between amino acids and large protein molecules. Two or more amino acids dehydrate and condense to form several peptide bonds, thus forming a peptide. Multiple peptides undergo multilevel folding to form a protein molecule. Collagen peptide extraction methods can be divided into indirect and direct extraction methods. Indirect extraction involves first extracting collagen from the raw material using methods such as hot water extraction, acid extraction, alkali extraction, or salt extraction, and then further hydrolyzing the collagen to obtain collagen peptides. Collagen hydrolysis methods include chemical degradation and enzymatic degradation. Direct extraction methods, depending on the target organism, can be divided into natural organism extraction, chemical synthesis, recombinant DNA synthesis, and in vitro protein hydrolysis. Compared to traditional acid and alkali hydrolysis, enzymatic hydrolysis is gentler, safer, and more specific, with shorter degradation time, less nutrient loss, and no environmental pollution.

[0008] For example, Chinese invention patent publication number CN118909095A discloses a fresh bovine bone collagen peptide that supplements joint nutrition and increases bone density, and its preparation method. The preparation method includes enzymatic hydrolysis, in which a complex protease carboxypeptidase, aminopeptidase, neutral protease, phytase, and trypsin are used. The mass ratio of carboxypeptidase, aminopeptidase, neutral protease, phytase, and trypsin is 0.5-1:0.5-1:2-4:1-3:1-3. The molecular weight of the prepared collagen peptide is less than 5000 Da. This collagen peptide can effectively improve bone density, bone mineral content, bone biomechanical properties, and bone metabolism indicators, and has good health care and therapeutic effects on elderly patients with osteoporosis and fractures. However, this invention uses five enzymes in the enzymatic hydrolysis process, which is complex, costly, and results in a low collagen peptide yield.

[0009] Therefore, there is an urgent need in the art to provide a method for preparing collagen peptides with high yield and good taste, as well as a nutritional composition that effectively promotes wound healing and has simple components. Summary of the Invention

[0010] This invention addresses the problems existing in the prior art by providing a method for preparing collagen peptides, a nutritional powder composition for promoting wound healing, the preparation method thereon, the product, and its application.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing collagen peptides includes the following steps: S101: First, add water to the bovine bone granules, heat and soak, then filter to obtain bovine bone protein solution; S102: Add laccase, subtilisin and flavor protease to the bovine bone protein solution in sequence for enzymatic hydrolysis, centrifuge and collect the supernatant. S103: Finally, add chitosan to the supernatant, ultrafilter, and dry to obtain the final product.

[0012] Preferably, in step S101, the particle size of the bovine bone particles is 8-15 mm; the mass ratio of the bovine bone particles to water is 1:1-3; the heating and soaking temperature is 100-125℃, and the heating and soaking time is 90-120 min.

[0013] Preferably, in step S102, the enzymatic hydrolysis process includes: first, adding 1-2 wt% laccase according to the mass of the bovine bone protein solution, and hydrolyzing for 1-2 h at pH=5-6 and 45-55℃; after inactivating the enzyme, adding 2-3 wt% Bacillus subtilis protease according to the mass of the bovine bone protein solution, and hydrolyzing for 3-4 h at pH=7-7.5 and 55-65℃; finally, adding 1-1.5 wt% flavor protease according to the mass of the bovine bone protein solution, and hydrolyzing for 1-2 h at 50-60℃ to inactivate the enzyme, thus obtaining the final product.

[0014] Preferably, the enzyme inactivation conditions are: incubation at 95-100℃ for 20-40 min.

[0015] Preferably, the centrifugation speed is 4500-5500 rpm and the centrifugation time is 20-30 min.

[0016] Preferably, the amount of chitosan added in step S103 is 1-2% of the mass of the supernatant.

[0017] Preferably, the ultrafiltration in step S103 uses an ultrafiltration membrane with a density of 2000-3000 Da, and the filtrate is collected.

[0018] Preferably, the drying in step S103 is vacuum drying or freeze drying.

[0019] The present invention also provides a nutritional powder composition for promoting wound healing, comprising the following components: collagen peptides, whey protein, fructooligosaccharides, isomaltooligosaccharides, calcium β-hydroxy-β-methylbutyrate, compound vitamins, and compound minerals prepared by the above preparation method.

[0020] Preferably, the compound vitamins are selected from vitamin A, vitamin D, vitamin E, vitamin B1, vitamin B2, vitamin B6, and vitamin B6. 12 It contains at least two of the following: vitamin C, folic acid, niacin, biotin, and pantothenic acid.

[0021] Preferably, the compound minerals are selected from at least two of iron, calcium, magnesium, zinc, copper, selenium, sodium, potassium, manganese, phosphorus, iodine and chromium.

[0022] More preferably, the compound minerals are selected from at least two of iron, calcium, magnesium, zinc, copper and selenium.

[0023] More preferably, the compound minerals are zinc, copper, and selenium.

[0024] Preferably, the nutritional powder composition further includes excipients selected from one or more of white sugar, edible flavorings, fruit and vegetable powders, and maltodextrin.

[0025] More preferably, the edible flavoring is selected from at least one of self-blending flavoring, reactive flavoring, fermented flavoring, enzymatically hydrolyzed flavoring, and lipid oxidation flavoring.

[0026] More preferably, the fruit and vegetable powder is selected from at least one of coconut milk powder, mango powder, orange powder, strawberry powder, banana powder, pineapple powder, lemon powder, passion fruit powder, and pumpkin powder.

[0027] Preferably, the nutritional powder composition comprises, by weight, the following components: 2.5-10 parts collagen peptide, 20-65 parts whey protein, 0.5-10 parts fructooligosaccharide, 0.5-10 parts isomaltooligosaccharide, 1-6 parts calcium β-hydroxy-β-methylbutyrate, 0.1-1 parts compound vitamins, 0.1-5 parts compound minerals, and 2-15 parts excipients.

[0028] More preferably, the nutritional powder composition comprises, by weight, the following components: 5-10 parts collagen peptide, 45-65 parts whey protein, 5-9 parts fructooligosaccharide, 5-9 parts isomaltooligosaccharide, 3-6 parts calcium β-hydroxy-β-methylbutyrate, 0.1-0.5 parts compound vitamins, 0.1-1 parts compound minerals, and 2-8 parts excipients.

[0029] Preferably, the calcium β-hydroxy-β-methylbutyrate is in the form of microcapsule powder. The preparation method of calcium β-hydroxy-β-methylbutyrate in microcapsule powder form includes: dissolving hydroxypropyl-β-cyclodextrin and calcium β-hydroxy-β-methylbutyrate in water, stirring until homogeneous, and obtaining a mixture; then adding maltitol, mixing until homogeneous, and spray drying to obtain the final product.

[0030] Preferably, the molar ratio of hydroxypropyl-β-cyclodextrin to calcium β-hydroxy-β-methylbutyrate is 2-3:1; the temperature of the water is 50-60℃; the stirring conditions are stirring at 1500-2000 rpm for 2-4 h; the mass ratio of maltitol to hydroxypropyl-β-cyclodextrin is 2-3:1; and the inlet temperature of the spray dryer is 155-175℃, and the outlet temperature is 80-90℃.

[0031] Calcium β-hydroxy-β-methylbutyrate, abbreviated as HMB-Ca, has the chemical formula C. 10 H 18CaO6 has functions such as promoting muscle growth, regulating cholesterol metabolism, and promoting wound healing. However, calcium β-hydroxy-β-methylbutyrate itself has an unpleasant bitter taste and sulfurous odor, affecting the palatability, and is prone to dust generation, which is not conducive to production and use. Therefore, this invention utilizes hydroxypropyl-β-cyclodextrin as the main wall material to embed the hydrophobic "β-hydroxy-β-methylbutyric acid" portion of the HMB-Ca molecule into it, forming an inclusion complex. This can directly mask the bitterness and off-odor at the molecular level and improve the stability of HMB-Ca. Maltitol is used as a filler. During the drying process, maltitol and hydroxypropyl-β-cyclodextrin together form the capsule wall, preventing microcapsule rupture and partially offsetting the residual bitterness of HMB-Ca, thus improving the palatability of HMB-Ca. Finally, calcium β-hydroxy-β-methylbutyrate is formed in the form of microcapsule powder.

[0032] The present invention also provides a method for preparing the above-mentioned nutritional powder composition, comprising the following steps: A nutritional powder composition is obtained by mixing collagen peptides, whey protein, fructooligosaccharides, isomaltooligosaccharides, calcium β-hydroxy-β-methylbutyrate in microcapsule form, compound vitamins, compound minerals, and excipients.

[0033] The present invention also provides a product for promoting wound healing, comprising the above-described nutritional powder composition or the nutritional powder composition prepared by the above-described preparation method.

[0034] Preferably, the dosage form of the product is any one of tablets, capsules, granules, jelly, gel, powder, and liquid preparations.

[0035] The present invention also provides the application of the above-described nutrient powder composition or the nutrient powder composition prepared by the above-described preparation method in the preparation of products that promote wound healing.

[0036] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention utilizes the synergistic effect of laccase, subtilisin and flavor protease to prepare collagen peptides with high yield and good taste. Furthermore, by adding chitosan, the bitter taste of collagen peptides is removed, which is beneficial to human absorption.

[0037] (2) The present invention uses hydroxypropyl-β-cyclodextrin as the main wall material, which can directly mask the bitterness and off-flavor of calcium β-hydroxy-β-methylbutyrate at the molecular level and improve its stability; it also uses maltitol as a filler. During the drying process, maltitol and hydroxypropyl-β-cyclodextrin together form the capsule wall to prevent microcapsule rupture and can also partially offset the residual bitterness of HMB-Ca, improve the taste of HMB-Ca, and finally form calcium β-hydroxy-β-methylbutyrate in the form of microcapsule powder.

[0038] (3) The present invention also provides a nutritional powder composition that can promote angiogenesis, epidermal regeneration and collagen formation in the wound area, increase collagen deposition, significantly improve wound healing rate, accelerate tissue remodeling and re-epithelialization process, and has good application prospects for wound repair. Attached Figure Description

[0039] Figure 1 The images show the wound appearance of the model group, control group 1, control group 2, and example group 1.

[0040] Figure 2 The results of H&E staining are for the normal group, model group, control group 1, control group 2, and example group 1.

[0041] Figure 3 Masson staining results for the normal group, model group, control group 1, control group 2, and example group 1.

[0042] Figure 4 The results of immunohistochemical CD31 analysis are for the normal group, model group, control group 1, control group 2, and example group 1. Detailed Implementation

[0043] It is worth noting that all raw materials used in this invention are commercially available products. Specifically, whey protein (model 392) was purchased from Fonterra; fructooligosaccharides (model QHT-FOS-P95S) were purchased from Guangdong Quantum High-Tech Biotechnology Co., Ltd.; isomaltooligosaccharides (model IMO-90) were purchased from Baolingbao Biotechnology Co., Ltd.; the compound vitamins (vitamin A 1364 μg, vitamin D 35.36 μg, vitamin E 75.76 mg, vitamin B1 9.19 mg, vitamin B2 7.51 mg, vitamin B6 7.51 mg, vitamin C 389 mg, folic acid 404 μg, niacin 69.45 mg, biotin 177 mg, and pantothenic acid 24.62 mg) were purchased from Zhejiang Jinkangpu Food Technology Co., Ltd.; the compound minerals (zinc sulfate 81.32 mg, copper sulfate 7071 μg, sodium selenite 152 μg) were purchased from Zhejiang Jinkangpu Food Technology Co., Ltd.; and Fortimel (a brand of vitamin C supplement) was also purchased. Repair, purchased from Nutricia; Junbeiqing Special Medical Purpose Protein Component Formula Food, provided by Guangdong Junyue Nutritional Medicine Co., Ltd.; Laccase, product number S10189, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Bacillus subtilis protease, product number S10051, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Flavor protease, CAS: 9001-92-7, purchased from Beijing Solarbio Technology Co., Ltd.

[0044] Example 1 A method for preparing collagen peptides, comprising the following steps: (1) First, crush the beef bones into beef bone particles with a particle size of 8-15 mm, add 2 times the mass of water to the beef bone particles, heat and soak at 120℃ for 120 min, filter to remove fat and bone residue, and obtain beef bone protein liquid. (2) Add 2% laccase (by weight of bovine bone protein solution) to the bovine bone protein solution and hydrolyze for 2 h at pH 6 and 50℃. After inactivating the enzyme at 100℃ for 30 min, add 2.5% Bacillus subtilis protease (by weight of bovine bone protein solution) and hydrolyze for 3.5 h at pH 7.5 and 60℃. Finally, add 1.5 wt% flavor protease (by weight of bovine bone protein solution) and hydrolyze for 1.5 h at 60℃. Inactivate the enzyme at 100℃ for 30 min. Centrifuge the hydrolysate at 5000 rpm for 25 min and collect the supernatant. (3) Finally, add chitosan to the supernatant (the amount of chitosan added is 1.5% of the mass of the supernatant), mix well, pass through a 3000 Da ultrafiltration membrane, collect the filtrate, freeze dry, and obtain collagen peptides with a yield of 71.4%.

[0045] The yield of collagen peptides is calculated using the following formula: Collagen peptide yield (%) = Collagen peptide mass ÷ Total protein mass in bovine bone protein solution × 100%.

[0046] A nutritional powder composition for promoting wound healing, by weight, comprises the following components: 7.5 parts collagen peptide, 65 parts whey protein, 8 parts fructooligosaccharide, 8 parts isomaltooligosaccharide, 3.5 parts calcium β-hydroxy-β-methylbutyrate in microcapsule form, 0.3 parts compound vitamins, 0.15 parts compound minerals, and 8 parts pineapple powder.

[0047] The preparation method of the above-mentioned nutritional powder composition is as follows: (1) Hydroxypropyl-β-cyclodextrin and calcium β-hydroxy-β-methylbutyrate with a molar ratio of 2.5:1 were dissolved in 25 times their mass of water at 50°C and stirred at 2000 rpm for 3 h to obtain a mixture; then maltitol (mass ratio of maltitol to hydroxypropyl-β-cyclodextrin was 2.5:1) was added, mixed well, and spray-dried (inlet temperature was 165°C, outlet temperature was 80°C) to obtain calcium β-hydroxy-β-methylbutyrate in microcapsule form; (2) Finally, the collagen peptides, whey protein, fructooligosaccharides, isomaltooligosaccharides, calcium β-hydroxy-β-methylbutyrate in microcapsule form, compound vitamins, compound minerals and pineapple powder are mixed to obtain the nutritional powder composition.

[0048] Example 2 A method for preparing collagen peptides, comprising the following steps: (1) First, crush the beef bones into beef bone particles with a particle size of 8-15 mm, add 1 times the mass of water to the beef bone particles, heat and soak at 100℃ for 120 min, filter, remove fat and bone residue, and obtain beef bone protein liquid. (2) Add 1% laccase (by weight of bovine bone protein solution) to the bovine bone protein solution and hydrolyze for 2 h at pH 6 and 45℃. After inactivating the enzyme at 100℃ for 20 min, add 2% Bacillus subtilis protease (by weight of bovine bone protein solution) and hydrolyze for 3 h at pH 7 and 55℃. Finally, add 1 wt% flavor protease (by weight of bovine bone protein solution) and hydrolyze for 1 h at 60℃. Inactivate the enzyme at 100℃ for 30 min. Centrifuge the hydrolysate at 4500 rpm for 30 min and collect the supernatant. (3) Finally, add chitosan to the supernatant (the amount of chitosan added is 1% of the mass of the supernatant), mix well, pass through a 3000 Da ultrafiltration membrane, collect the filtrate, freeze dry, and obtain collagen peptides with a yield of 70.1%.

[0049] A nutritional powder composition for promoting wound healing, by weight, comprises the following components: 10 parts collagen peptides, 45 parts whey protein, 5 parts fructooligosaccharides, 5 parts isomaltooligosaccharides, 3 parts calcium β-hydroxy-β-methylbutyrate in microcapsule form, 1 part compound vitamins, 1 part compound minerals, and 8 parts maltodextrin.

[0050] The preparation method of the above-mentioned nutritional powder composition is as follows: (1) Hydroxypropyl-β-cyclodextrin and calcium β-hydroxy-β-methylbutyrate in a molar ratio of 2:1 were dissolved in 20 times their mass of water at 50°C and stirred at 1500 rpm for 2 h to obtain a mixture; then maltitol (mass ratio of maltitol to hydroxypropyl-β-cyclodextrin was 2:1) was added, mixed well, and spray-dried (inlet temperature was 155°C, outlet temperature was 90°C) to obtain calcium β-hydroxy-β-methylbutyrate in microcapsule form; (2) Finally, the collagen peptides, whey protein, fructooligosaccharides, isomaltooligosaccharides, calcium β-hydroxy-β-methylbutyrate in microcapsule form, compound vitamins, compound minerals and maltodextrin are mixed to obtain the nutritional powder composition.

[0051] Example 3 A method for preparing collagen peptides, comprising the following steps: (1) First, crush the beef bones into beef bone particles with a particle size of 8-15 mm, add 1 times the mass of water to the beef bone particles, heat and soak at 125℃ for 100 min, filter, remove fat and bone residue, and obtain beef bone protein liquid. (2) Add 2% laccase (by weight of bovine bone protein solution) to the bovine bone protein solution and hydrolyze for 1 h at pH 5 and 55℃. After inactivating the enzyme at 95℃ for 40 min, add 3% Bacillus subtilis protease (by weight of bovine bone protein solution) and hydrolyze for 4 h at pH 7.5 and 65℃. Finally, add 1.5 wt% flavor protease (by weight of bovine bone protein solution) and hydrolyze for 2 h at 50℃. Inactivate the enzyme at 95℃ for 40 min. Centrifuge the hydrolysate at 5500 rpm for 20 min and collect the supernatant. (3) Finally, add chitosan to the supernatant (the amount of chitosan added is 2% of the mass of the supernatant), mix well, pass through a 2000 Da ultrafiltration membrane, collect the filtrate, freeze dry, and obtain collagen peptides with a yield of 67.8%.

[0052] A nutritional powder composition for promoting wound healing, by weight, comprises the following components: 2.5 parts collagen peptide, 65 parts whey protein, 0.5 parts fructooligosaccharide, 0.5 parts isomaltooligosaccharide, 6 parts calcium β-hydroxy-β-methylbutyrate in microcapsule form, 0.1 parts compound vitamins, 0.1 parts compound minerals, and 2 parts maltodextrin.

[0053] The preparation method of the above-mentioned nutritional powder composition is as follows: (1) Hydroxypropyl-β-cyclodextrin and calcium β-hydroxy-β-methylbutyrate in a molar ratio of 3:1 were dissolved in 30 times their mass of water at 60°C and stirred at 2000 rpm for 2 h to obtain a mixture; then maltitol (mass ratio of maltitol to hydroxypropyl-β-cyclodextrin was 3:1) was added, mixed well, and spray-dried (inlet temperature was 175°C, outlet temperature was 80°C) to obtain calcium β-hydroxy-β-methylbutyrate in microcapsule form; (2) Finally, the collagen peptides, whey protein, fructooligosaccharides, isomaltooligosaccharides, calcium β-hydroxy-β-methylbutyrate in microcapsule form, compound vitamins, compound minerals and maltodextrin are mixed to obtain the nutritional powder composition.

[0054] Comparative Example 1 Compared with Example 1, the only difference is that only Bacillus subtilis protease and flavor protease are added during the preparation of collagen peptides for enzymatic hydrolysis, ultimately yielding a nutritional powder composition that promotes wound healing.

[0055] A method for preparing collagen peptides, comprising the following steps: (1) First, crush the beef bones into beef bone particles with a particle size of 8-15 mm, add 2 times the mass of water to the beef bone particles, heat and soak at 120℃ for 120 min, filter to remove fat and bone residue, and obtain beef bone protein liquid. (2) Add 3.8% of the bovine bone protein solution by weight of Bacillus subtilis protease to the bovine bone protein solution and hydrolyze it at pH=7.5 and 60℃ for 4.9 h; finally add 2.2 wt% of the bovine bone protein solution by weight of flavor protease and hydrolyze it at 60℃ for 2.1 h, inactivate the enzyme at 100℃ for 30 min, centrifuge the hydrolysate at 5000 rpm for 25 min, and collect the supernatant. (3) Finally, add chitosan to the supernatant (the amount of chitosan added is 1.5% of the mass of the supernatant), mix well, pass through a 3000 Da ultrafiltration membrane, collect the filtrate, freeze dry, and obtain collagen peptides with a yield of 48.7%.

[0056] Comparative Example 2 Compared with Example 1, the only difference is that only laccase is added for enzymatic hydrolysis during the preparation of collagen peptides, ultimately yielding a nutritional powder composition that promotes wound healing.

[0057] A method for preparing collagen peptides, comprising the following steps: (1) First, crush the beef bones into beef bone particles with a particle size of 8-15 mm, add 2 times the mass of water to the beef bone particles, heat and soak at 120℃ for 120 min, filter to remove fat and bone residue, and obtain beef bone protein liquid. (2) Add 6% of bovine bone protein solution by weight of laccase to the bovine bone protein solution and enzymatically hydrolyze for 7 h at pH=6 and 50℃. After inactivating the enzyme at 100℃ for 30 min, centrifuge the hydrolysate at 5000 rpm for 25 min and collect the supernatant. (3) Finally, add chitosan to the supernatant (the amount of chitosan added is 1.5% of the mass of the supernatant), mix well, pass through a 3000 Da ultrafiltration membrane, collect the filtrate, freeze dry, and obtain collagen peptides with a yield of 36.1%.

[0058] Comparative Example 3 Compared with Example 1, the only difference is that only laccase and subtilisin are added during the preparation of collagen peptides, resulting in a nutritional powder composition that promotes wound healing.

[0059] A method for preparing collagen peptides, comprising the following steps: (1) First, crush the beef bones into beef bone particles with a particle size of 8-15 mm, add 2 times the mass of water to the beef bone particles, heat and soak at 120℃ for 120 min, filter to remove fat and bone residue, and obtain beef bone protein liquid. (2) Add 2.7% of bovine bone protein solution by weight of laccase to the bovine bone protein solution and hydrolyze for 2.5 h at pH=6 and 50℃. After inactivating the enzyme at 100℃ for 30 min, add 3.3% of bovine bone protein solution by weight of Bacillus subtilis protease and hydrolyze for 4.5 h at pH=7.5 and 60℃. Centrifuge the hydrolysate at 5000 rpm for 25 min and collect the supernatant. (3) Finally, add chitosan to the supernatant (the amount of chitosan added is 1.5% of the mass of the supernatant), mix well, pass through a 3000 Da ultrafiltration membrane, collect the filtrate, freeze dry, and obtain collagen peptides with a yield of 42.5%.

[0060] Comparative Example 4 Compared with Example 1, the only difference is that only laccase and flavor protease are added during the preparation of collagen peptides, resulting in a nutritional powder composition that promotes wound healing.

[0061] A method for preparing collagen peptides, comprising the following steps: (1) First, crush the beef bones into beef bone particles with a particle size of 8-15 mm, add 2 times the mass of water to the beef bone particles, heat and soak at 120℃ for 120 min, filter to remove fat and bone residue, and obtain beef bone protein liquid. (2) Add 3.4% of bovine bone protein solution by weight of laccase to the bovine bone protein solution and hydrolyze for 4 h at pH=6 and 50℃. After inactivating the enzyme at 100℃ for 30 min, add 2.6 wt% of bovine bone protein solution by weight of flavor protease and hydrolyze for 3 h at 60℃. Inactivate the enzyme at 100℃ for 30 min. Centrifuge the hydrolysate at 5000 rpm for 25 min and collect the supernatant. (3) Finally, add chitosan to the supernatant (the amount of chitosan added is 1.5% of the mass of the supernatant), mix well, pass through a 3000 Da ultrafiltration membrane, collect the filtrate, freeze dry, and obtain collagen peptides with a yield of 46.2%.

[0062] Comparative Example 5 Compared to Example 1, the only difference is that maltitol is replaced with chitosan in the preparation of the nutritional powder composition, ultimately resulting in a nutritional powder composition that promotes wound healing. Specifically, the preparation method of this nutritional powder composition is as follows: (1) Hydroxypropyl-β-cyclodextrin and calcium β-hydroxy-β-methylbutyrate with a molar ratio of 2.5:1 were dissolved in 25 times their mass of water at 50°C and stirred at 2000 rpm for 3 h to obtain a mixture; then chitosan (the mass ratio of chitosan to hydroxypropyl-β-cyclodextrin was 2.5:1) was added, mixed well, and spray-dried (inlet temperature was 165°C, outlet temperature was 80°C) to obtain calcium β-hydroxy-β-methylbutyrate in microcapsule form; (2)Finally, mix collagen peptide, whey protein, fructo-oligosaccharide, isomaltooligosaccharide, calcium β-hydroxy-β-methylbutyrate in the form of microcapsule powder, compound vitamins, compound minerals and pineapple powder to obtain the nutritional powder composition.

[0063] Comparative Example 6 Compared with Example 1, the difference is only that the formula of the nutritional powder composition is different, specifically: A nutritional powder composition for promoting wound healing, which is composed of the following components by weight: 20 parts of collagen peptide, 45 parts of whey protein, 8 parts of fructo-oligosaccharide, 10 parts of isomaltooligosaccharide, 8 parts of calcium β-hydroxy-β-methylbutyrate in the form of microcapsule powder, 0.5 part of compound vitamins, 0.3 part of compound minerals and 8.2 parts of pineapple powder.

[0064] Test Example 1 1. Experimental purpose To investigate the effect of the nutritional powder composition of the present invention on the wound healing rate.

[0065] 2. Test samples The nutritional powder compositions prepared in Examples 1-3 and Comparative Examples 1-6.

[0066] 3. Experimental animals Male Wistar rats, 7-8 weeks old, weighing 300-310 g, provided by Spf (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2024-0001. The rats were raised in a barrier environment animal laboratory. The laboratory temperature was 20-26 °C, and the relative humidity was 30-70%. A 12 h:12 h day-night intermittent lighting was used.

[0067] 4. Animal modeling 24 h before the experiment, the back hair of the rats was removed, and the hair removal area was 3 cm × 3 cm. The rats were fasted overnight before the operation. During the operation, the body weight of the rats was weighed, and general anesthesia and local anesthetic were injected according to the body weight. After the rats were anesthetized, the back skin was depilated and disinfected, and a circular shape with a diameter of 1.5 cm was cut on the back of the rats to prepare a skin wound model.

[0068] 5. Experimental method The rats were randomly divided into a model group, Examples 1-3 groups and Comparative Examples 1-6 groups according to body weight, with 6 rats in each group. The rats in the model group, Examples 1-3 groups and Comparative Examples 1-6 groups were all subjected to skin wound modeling. Starting from the first day after modeling, each rat in Examples 1-3 groups and Comparative Examples 1-6 groups was intragastrically administered 6.8 g / kg BW of the test sample, and the model group was given an equal amount of normal saline, and the drug was administered continuously for 14 days.

[0069] 6. Data processing The rat wounds were photographed and recorded on days 0, 7, 10, and 14, with the shooting position and lighting fixed, and the wound area was calculated. The wounds were cleaned, photographed, and re-bandaged each time.

[0070] The wound healing rate is calculated using the following formula: Wound area (cm²) 2 = Maximum longitudinal diameter × Maximum transverse diameter × π / 4 Wound healing rate (%) = (original wound area) Wound area at various time points after injury ÷ original wound area × 100% Note: π is 3.14.

[0071] Experimental data are expressed as mean ± standard deviation (X ± S). Repeated measures ANOVA or factorial design ANOVA was used for overall comparisons across multiple groups, and Dunnett's method was used for pairwise comparisons within groups. t Test. With P <0.05 indicates that the difference is statistically significant.

[0072] 7. Experimental Results The wound healing rate results are shown in Table 1. According to the results in the table, there were significant differences between the Example 1-3 groups and the model group compared to the model group. P <0.05 or P <0.01), there was no significant difference between comparative examples 1-5 and the model group; compared with example 1 group, there was no statistically significant difference between example 2-3 groups and example 1 group, while there was a statistically significant difference between comparative examples 1-5 and example 1 group. P <0.01). This indicates that the collagen peptides prepared by the present invention utilize the synergistic effect of laccase, subtilisin, and flavor protease to achieve high yield and good taste. By adding chitosan, the bitter and fishy taste of the collagen peptides is removed, which facilitates absorption. Furthermore, the synergistic combination of the raw materials in the nutritional powder composition of the present invention can significantly promote wound healing.

[0073] Table 1. Wound healing rate results of the nutritional powder composition of the present invention (n=6)

[0074] Note: @ indicates comparison with model groups. P <0.05, @@ indicates P <0.01; compared with Example 1, # indicates P <0.05.

[0075] Experimental Example 2 1. Experimental Objective The wound healing effects of the nutritional powder composition of Example 1 and commercially available products were examined.

[0076] 2. Test samples The nutritional powder composition prepared in Example 1, Control 1 (Fortimel Repair), and Control 2 (Junbeiqing Special Medical Purpose Protein Component Formula Food).

[0077] 3. Experimental animals Male Wistar rats, 7 - 8 weeks old, weighing 300 - 310 g, provided by Spf (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2024 - 0001. The rats were raised in a barrier environment animal laboratory. The laboratory temperature was 20 - 26°C, the relative humidity was 30 - 70%, and a 12 h:12 h day - night intermittent lighting was used.

[0078] 4. Animal modeling Same as Test Example 1.

[0079] 5. Experimental method The rats were randomly divided into a normal group, a model group, a Control 1 group (i.e., Control 1), a Control 2 group (i.e., Control 2), and an Example 1 group, with 18 rats in each group. Rats in the model group, Control 1 group, Control 2 group, and Example 1 group were all subjected to skin wound modeling. Starting from the 1st day after modeling, each rat in the Example 1 group was intragastrically administered with 6.82 g / kg·BW of the Example 1 sample, the Control 1 group was given a sample with the same energy as the Example 1 group (i.e., 19.16 mL / kg BW), the Control 2 group was given a sample with the same protein content as the Example 1 group (i.e., 4.59 g / kg BW), and the normal group and the model group were given an equal amount of normal saline. Each group was continuously administered for 14 days.

[0080] 6. Data processing 6.1 At 0 d (day 0), 7 d (day 7), 10 d (day 10), and 14 d (day 14), the wounds of the rats were photographed. The shooting position and light were fixed, and the wound area was calculated. Each time, the wound needed to be cleaned, photographed, and redressed and bandaged.

[0081] The wound healing rate was calculated by the following formula: Wound area (cm 2 ) = maximum longitudinal diameter × maximum transverse diameter × π / 4 Wound healing rate (%) = (original wound area - wound area at each time point after injury) ÷ original wound area × 100% Note: π is taken as 3.14.

[0082] At 7, 10 and 14 days, 6 rats in each group were anesthetized and sacrificed. Skin from the back (3 cm × 3 cm around the wound) was taken and fixed with 4% paraformaldehyde fixative for subsequent skin tissue morphology examination.

[0083] 6.2 Skin tissue morphological examination: ① Three rats from each group were subjected to H&E staining at each time point to observe the morphology of wound tissue and the infiltration of inflammatory cells; ② Masson staining was performed to observe the number and arrangement of collagen fibers in the rat wound; ③ Immunohistochemical CD31 examination was performed to observe the angiogenesis of rat skin wounds.

[0084] Experimental data are expressed as mean ± standard deviation (X ± S). Repeated measures ANOVA or factorial design ANOVA was used for overall comparisons across multiple groups, and Dunnett's method was used for pairwise comparisons within groups. t Test. With P <0.05 indicates that the difference is statistically significant.

[0085] 7. Experimental Results 7.1 Wound healing rate See the image of the wound. Figure 1 The wound healing rate is shown in Table 2.

[0086] Table 2. Wound healing rate in rats (%, n=6)

[0087] Note: # indicates comparison with model groups. P <0.05; compared with control group 2, '&' indicates P <0.05.

[0088] As shown in Table 2, compared with the model group, control group 1, control group 2, and example group 1 all showed varying degrees of significant promotion of wound healing at 7d, 10d, and 14d. P <0.05), indicating that appropriate nutritional intervention helps wound healing; compared with control group 1, there was no significant difference in wound healing rate at 7d, 10d, and 14d in example group 1; compared with control group 2, the wound healing rate of example group 1 was significantly improved at 7d, 10d, and 14d ( P <0.05), the above shows that Example 1 can not only effectively promote wound healing, but also the promoting effect is reflected in the entire healing process, and the effect is better than a commercially available control product and comparable to another product.

[0089] 7.2 H&E staining H&E stained pathological sections are shown Figure 2H&E staining results showed that the epidermis, dermis, dermal appendages, subcutaneous fat layer and muscle layer of the skin tissue of the normal group rats at all time points were normal.

[0090] At 7 days, the wounds of rats in the model group were covered with thick necrotic tissue, with a severe inflammatory cell infiltration zone between the necrotic tissue and the wound base. Some necrotic tissue sloughed off, and new blood vessels were distributed at the base. Small-scale epithelialization began at the wound edges. The wounds of control groups 1 and 2 were also covered with necrotic tissue and inflammatory infiltration zones, but the severity was less than that of the model group. The extent of new blood vessels at the base and epithelialization at the wound edges was greater than that of the model group. It is worth noting that the wounds of group 1 had the least necrotic tissue and the least inflammatory infiltration, and the structure was the clearest. The extent of new blood vessels at the base and epithelialization at the wound edges was the greatest.

[0091] At 10 days, necrotic tissue sloughed off from the wounds of rats in the model group, the number of newly formed microvessels at the base of the wound increased, and the overall degree of inflammation was more severe. The extent of epithelialization at the wound edges and the number of newly formed microvessels in control groups 1 and 2 were significantly higher than those in the model group, and the degree of inflammatory infiltration was also less than that in the model group. The degree of inflammatory infiltration, the number of newly formed microvessels at the base of the wound, and the extent of epithelialization at the wound edges were the best among the four groups in Example 1.

[0092] At 14 days, the area of ​​wound edge epithelialization in the model group rats further expanded, and the remaining unhealed wound surface was still covered with necrotic tissue, with a more severe inflammatory response and more abundant neovascularization. The area of ​​wound edge epithelialization and the number of newly formed microvessels in control group 1 and control group 2 were still significantly higher than those in the model group, and the degree of inflammatory infiltration was less than that in the model group. The wound edge epithelialization rate of rats in Example 1 group was the fastest among the four groups.

[0093] 7.3 Masson staining Masson-stained pathological sections are shown Figure 3 Masson staining results showed that the skin of the normal group rats at each time point had a normal structure and collagen fibers were arranged in a neat and orderly manner.

[0094] At 7 days, neovascularization was observed at the base of the wound in the model group rats, and new collagen fibers gradually deposited and grew from the periphery and base of the wound, but the center of the wound still showed a "low-lying state". The wound base of control group 1 and control group 2 had abundant new microvascularization, and the degree of collagen deposition and growth was higher than that of the model group. The collagen arrangement was also relatively regular. It is worth noting that among the four groups, the degree of new microvascularization, collagen deposition and regular arrangement, and the extent of epithelialization at the wound edge were the highest in the wound base of the example group 1.

[0095] At 10 days, the model group rats had abundant new microvessels at the base of the wound and collagen deposition and growth, but the residual wound area was still large. The new microvessels and collagen deposition and growth at the base of the wound in control group 1 and control group 2 were significantly higher than those in the model group. The new microvessels, collagen deposition and growth at the base of the wound, and the extent of epithelialization at the wound edge in Example 1 group were the best among the four groups.

[0096] At 14 days, collagen deposition and growth in the model group rats had increased to the same level as the epidermis, and the area of ​​the residual wound had also decreased further compared to 10 days. The collagen deposition and growth at the base of the wound and the wound healing area in control group 1 and control group 2 were significantly higher than those in the model group. The collagen deposition and growth at the base of the wound, the maturity of neovascularization, and the extent of epithelialization at the wound edge were the best among the four groups in Example 1.

[0097] 7.4 Immunohistochemical marker CD31 CD31 immunohistochemical staining pathology results Figure 4 CD31 staining results showed that the skin of the normal group rats at each time point had a normal structure, with scattered small blood vessels distributed in the dermis and subcutaneous tissue.

[0098] At 7 days, a large number of new microvessels were formed at the base of the wound in the model group rats. Similarly, the number of new microvessels in Example 1 group, Control 1 group, and Control 2 group was also significantly increased. When comparing the four groups, the number of new blood vessels in Control 2 group was significantly higher than that in Control 1 group, and the number of new blood vessels in Control 1 group was significantly higher than that in the model group. Among them, Example 1 group had the highest number of new blood vessels.

[0099] At 10 days, the number of new blood vessels in the wounds of the four groups of rats was further increased. When comparing the four groups, the number of new blood vessels in control group 2 was significantly higher than that in control group 1, and control group 1 was significantly more than that in the model group. Among them, group 1 of Example 1 had the highest number of new blood vessels.

[0100] At 14 days, the number of new blood vessels in the wounds of rats in the model group increased further compared to 10 days. However, the blood vessels in the wounds of control group 1, control group 2 and example group 1 had begun to mature and allow blood flow, but their number remained relatively stable and did not increase further.

[0101] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing collagen peptides, characterized in that, Includes the following steps: S101: First, add water to the bovine bone granules, heat and soak, then filter to obtain bovine bone protein solution; S102: Add laccase, subtilisin and flavor protease to the bovine bone protein solution in sequence for enzymatic hydrolysis, centrifuge and collect the supernatant. S103: Finally, add chitosan to the supernatant, ultrafilter, and dry to obtain the final product.

2. The preparation method according to claim 1, characterized in that, In step S101, the particle size of the bovine bone particles is 8-15 mm; the mass ratio of the bovine bone particles to water is 1:1-3; the heating and soaking temperature is 100-125℃, and the heating and soaking time is 90-120 min.

3. The preparation method according to claim 1, characterized in that, In step S102, the enzymatic hydrolysis process includes: first, adding 1-2 wt% laccase according to the mass of the bovine bone protein solution, and hydrolyzing for 1-2 h at pH 5-6 and 45-55℃; after enzyme inactivation, adding 2-3 wt% Bacillus subtilis protease according to the mass of the bovine bone protein solution, and hydrolyzing for 3-4 h at pH 7-7.5 and 55-65℃; finally, adding 1-1.5 wt% flavor protease according to the mass of the bovine bone protein solution, and hydrolyzing for 1-2 h at 50-60℃ to inactivate the enzyme, thus obtaining the product. The enzyme inactivation conditions are as follows: incubation at 95-100℃ for 20-40 min; centrifugation speed of 4500-5500 rpm for 20-30 min; in step S103, the amount of chitosan added is 1-2% of the supernatant mass; the ultrafiltration is performed using 2000-3000... The ultrafiltration membrane of Da is used to collect the filtrate; the drying is vacuum drying or freeze drying.

4. A nutritional powder composition for promoting wound healing, characterized in that, It comprises the following components: collagen peptides, whey protein, fructooligosaccharides, isomaltooligosaccharides, calcium β-hydroxy-β-methylbutyrate, compound vitamins, and compound minerals prepared by the preparation method according to any one of claims 1-3.

5. The nutritional powder composition according to claim 4, characterized in that, The compound vitamins are selected from vitamin A, vitamin D, vitamin E, vitamin B1, vitamin B2, vitamin B6, and vitamin B1. 12 The compound contains at least two of the following: vitamin C, folic acid, niacin, biotin, and pantothenic acid; the compound minerals are selected from at least two of the following: iron, calcium, magnesium, zinc, copper, selenium, sodium, potassium, manganese, phosphorus, iodine, and chromium; the nutritional powder composition also includes excipients selected from one or more of white sugar, edible flavorings, fruit and vegetable powders, and maltodextrin, wherein the fruit and vegetable powders are selected from at least one of coconut milk powder, mango powder, orange powder, strawberry powder, banana powder, pineapple powder, lemon powder, passion fruit powder, and pumpkin powder.

6. The nutritional powder composition according to claim 5, characterized in that, By weight, the nutritional powder composition comprises the following components: 2.5-10 parts collagen peptide, 20-65 parts whey protein, 0.5-10 parts fructooligosaccharides, 0.5-10 parts isomaltooligosaccharides, 1-6 parts calcium β-hydroxy-β-methylbutyrate, 0.1-1 part compound vitamins, 0.1-5 parts compound minerals, and 2-15 parts excipients.

7. The nutritional powder composition according to any one of claims 4-6, characterized in that, The calcium β-hydroxy-β-methylbutyrate is in microencapsulated powder form. The preparation method of the microencapsulated calcium β-hydroxy-β-methylbutyrate includes: dissolving hydroxypropyl-β-cyclodextrin and calcium β-hydroxy-β-methylbutyrate in water, stirring until homogeneous, and obtaining a mixture; then adding maltitol, mixing, and spray drying to obtain the final product; the molar ratio of hydroxypropyl-β-cyclodextrin to calcium β-hydroxy-β-methylbutyrate is 2-3:1; the temperature of the water is 50-60℃; the stirring conditions are stirring at 1500-2000 rpm for 2-4 h; the mass ratio of maltitol to hydroxypropyl-β-cyclodextrin is 2-3:1; the inlet temperature of the spray dryer is 155-175℃, and the outlet temperature is 80-90℃.

8. A method for preparing the nutritional powder composition as described in claim 7, characterized in that, The process includes the following steps: mixing collagen peptides, whey protein, fructooligosaccharides, isomaltooligosaccharides, calcium β-hydroxy-β-methylbutyrate in microcapsule form, compound vitamins, compound minerals and excipients to obtain a nutritional powder composition.

9. A product for promoting wound healing, characterized in that, This includes the nutritional powder composition according to any one of claims 4-7 or the nutritional powder composition prepared by the preparation method according to claim 8.

10. The use of a nutritional powder composition as described in any one of claims 4-7 or a nutritional powder composition prepared by the preparation method of claim 8 in the preparation of a product that promotes wound healing.

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