Composite peptide nutritional composition for promoting wound healing, preparation process and use

Abstract

The application belongs to the technical field of medical nutritional foods, and particularly relates to a composite peptide nutritional composition for promoting wound healing and a preparation process and application thereof. The composite peptide nutritional composition for promoting wound healing is prepared from raw materials including fish collagen peptide 15%-25%, bovine spleen peptide 0.03%-0.05%, blood protein oligopeptide 0.10%-0.30%, albumin peptide 0.10%-0.30%, ginseng powder 0.01%-0.03%, composite fermentation extract 15%-20%, plant extract 10%-20%, prebiotic 20%-25%, and whey protein matrix supplementing the rest to 100% by weight percentage. The application improves the content of healing-promoting peptide segments and various active ingredients such as anti-inflammatory repair by using self-made fish collagen peptide, composite fermentation extract and other raw materials, and improves the bioavailability of plant extract through embedding technology, thereby significantly improving the wound healing rate and quality.

Description

Technical Field

[0001] This invention belongs to the field of medical nutrition food technology, specifically relating to a compound peptide nutritional composition for promoting wound healing, its preparation process, and its application. Background Technology

[0002] In the field of wound repair and tissue regeneration medicine, accelerating wound healing and improving healing quality through nutritional interventions has always been a key focus of clinical research. Traditional treatments often rely on exogenous growth factors or antibiotics, but these suffer from low bioavailability, the risk of drug resistance, and limitations due to their single mechanism of action. In recent years, compound peptide nutritional compositions combining natural active ingredients and biotechnology have gradually become a research hotspot; however, current technologies still have limitations.

[0003] Collagen peptides, as a key functional component, are easily damaged by conventional enzymatic hydrolysis processes, which disrupt the unique triple helix structure of collagen. This results in insufficient release of short peptides with healing-promoting activity, directly affecting the migration ability of fibroblasts. While some products contain effective ingredients, the composition is often singular, leading to limited improvement in wound healing efficiency, even if it enhances healing. Furthermore, as an oral nutritional product, it is crucial to ensure that sensitive active ingredients are not damaged by gastric acid and other environmental factors, maintaining high bioavailability to be absorbed by the body and exert their effects.

[0004] Therefore, there is an urgent need to develop a compound peptide nutritional composition that can promote wound healing through oral administration. By enriching the content of active short peptides and effective ingredients, it can improve the speed and quality of wound healing and provide an efficient solution for postoperative wounds, trauma, ulcers, dermatitis and other wounds. Summary of the Invention

[0005] The purpose of this invention is to provide a compound peptide nutritional composition, preparation process, and application that promotes wound healing, significantly improving the wound healing rate and quality, and providing an efficient solution for postoperative wounds, trauma, ulcers, dermatitis, and other wounds.

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

[0007] A compound peptide nutritional composition for promoting wound healing, wherein the raw materials for preparation, by weight percentage, include 15%-25% fish collagen peptide, 0.03%-0.05% bovine spleen peptide, 0.10%-0.30% hemoglobin oligopeptide, 0.10%-0.30% albumin peptide, 0.01%-0.03% ginseng powder, 15%-20% compound fermented extract, 10%-20% plant extract, 20%-25% prebiotics, and whey protein matrix to make up the balance to 100%.

[0008] The method for preparing the fish collagen peptide includes the following steps: A1. Take collagen peptide raw material and mix it with sodium citrate solution, stir, centrifuge and wash with water to obtain demineralized fish scales;

[0009] A2. Add water to the demineralized fish scales and adjust the pH. Add alkaline protease and react. Add flavor protease and react to inactivate the enzyme. Use an ultrafiltration membrane for ultrafiltration and then freeze dry.

[0010] Preferably, the preparation method of the fish collagen peptide includes the following steps: A1, take collagen peptide raw material and mix it with 0.1M sodium citrate solution, stir at 0-4℃ for 10-14h, centrifuge and wash with water until neutral to obtain demineralized fish scales;

[0011] A2. Add water to the demineralized fish scales and adjust the pH to 7. Add alkaline protease and react at 25°C for 1-3 hours. Then add flavor protease and react at 25°C for 1-3 hours to inactivate the enzyme. First, use an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to obtain ultrafiltrate. Then, use an ultrafiltration membrane with a molecular weight cutoff of 300 Da to obtain the retentate material. Freeze-dry the obtained retentate material.

[0012] Preferably, the collagen peptide raw material includes fish scales or fish skin.

[0013] Preferably, the fish scales include tilapia scales; the fish skin includes deep-sea cod skin.

[0014] Preferably, the solid-liquid ratio of the collagen peptide raw material to the 0.1M sodium citrate solution is 1g:(8-12)mL.

[0015] Preferably, the mass ratio of the demineralized fish scales to water is 1:(4-6).

[0016] Preferably, the amount of alkaline protease added is 300-400 U / g collagen peptide raw material.

[0017] Preferably, the amount of flavor protease added is 300-400 U / g collagen peptide raw material.

[0018] Preferably, the enzyme inactivation conditions are: a temperature of 85-90℃ and a time of 10-15 min.

[0019] Preferably, the freeze-drying conditions are as follows: after pre-freezing at -40°C for 4 hours, the freeze-drying is carried out at a temperature of -30°C and a pressure of 8-12 Pa for 24 hours.

[0020] By first demineralizing collagen peptide raw materials with sodium citrate, and then preparing fish collagen peptides through a two-stage enzymatic hydrolysis, the product's wound-healing ability can be improved. This is likely because the decalcification of sodium citrate at low temperature protects the triple helix structure. Then, through stepwise enzymatic hydrolysis, alkaline protease is used to directionally cleave hydrophobic peptide bonds in the non-helical regions of collagen, initially releasing large-molecule peptides. Subsequently, flavor protease is added for deep hydrolysis at the same temperature, precisely cleaving specific sites in the helical region to release healing-promoting peptides such as Gly-Pro-Hyp tripeptides. Finally, by controlling the molecular weight cutoff of the ultrafiltration membrane, functional peptides with concentrated molecular weights are obtained, ensuring the bioactivity of collagen peptides while promoting intestinal absorption, thereby increasing the collagen peptides' ability to promote fibroblast migration and accelerating wound healing. Stepwise enzymatic hydrolysis avoids mutual inhibition of enzyme activity due to substrate specificity overlap, ensuring that each enzyme acts independently under optimal conditions, significantly improving the degree of hydrolysis and product yield. Considering cost factors, tilapia scales are generally more economical to use as raw materials. However, deep-sea cod skin is rich in higher-quality collagen, amino acids, and various essential trace elements, and has higher bioactivity, effectively promoting skin cell regeneration and repair. It is often used in high-end products. In actual industrial applications, tilapia scales or deep-sea cod skin can be selected based on specific needs.

[0021] Preferably, the preparation method of the compound fermentation extract includes the following steps: inoculating yeast onto culture medium 1, fermenting to obtain fermentation product 1, adding Cordyceps mycelium to obtain culture medium 2, inoculating Cordyceps militaris and Cordyceps militaris, culturing to obtain fermentation product 2, adding Centella asiatica powder to obtain culture medium 3, inoculating mold, fermenting to obtain fermentation product 3, inoculating Lactobacillus plantarum, fermenting, membrane filtering, concentrating, and spray drying to obtain the final product.

[0022] Preferably, the preparation method of the compound fermentation extract includes the following steps: inoculating yeast onto culture medium 1, fermenting at 28-32℃ and 120-180 rpm for 72 hours to obtain fermentation product 1; adding Cordyceps mycelium to obtain culture medium 2; inoculating Cicadae Periostracum and Cordyceps militaris, and culturing at 23-25℃ for 10 days to obtain fermentation product 2; adding Centella asiatica powder to obtain culture medium 3; inoculating mold, fermenting at 28-30℃ and 180-220 rpm for 96 hours to obtain fermentation product 3; inoculating Lactobacillus plantarum, anaerobic fermenting at 37℃ for 48 hours; filtering with a ceramic membrane with a molecular weight cutoff of 50 kDa; vacuum concentrating at 45℃ until the solid content is ≥30%; and spray drying to obtain the final product.

[0023] Preferably, the yeast includes Candida utilis.

[0024] Preferably, the inoculum size of the yeast is 1×10⁻⁶. 7 CFU / g culture medium 1.

[0025] Preferably, the culture medium 1 comprises, by weight percentage, 5% soybean meal, 3%-4% sucrose, 0.01% sodium selenite, and water to make up the remainder to 100%.

[0026] Preferably, the amount of Cordyceps mycelium added is consistent with the mass of fermentation product 1.

[0027] Preferably, the inoculation amount of both *Cicada Flower* and *Cordyceps militaris* is the same, 2 × 10⁻⁶. 7 -3×10 7 CFU / g medium 2.

[0028] Preferably, the amount of Centella asiatica powder added is 2%-4% of the mass of fermentation product 2.

[0029] Preferably, the mold includes Monascus purpureus and Aspergillus niger.

[0030] Preferably, the inoculum size of Monascus purpureus is 1×10⁻⁶. 7 -2×10 7 CFU / g medium 3; the inoculum size of Aspergillus niger was 2 × 10⁻⁶. 6 -4×10 6 CFU / g medium 3.

[0031] Preferably, the inoculum size of *Lactobacillus plantarum* is 4 × 10⁻⁶. 6 -6×10 6 CFU / g fermentation product 3.

[0032] Preferably, the vacuum degree of the vacuum concentration is 0.08-0.1 MPa.

[0033] Preferably, the specific conditions for spray drying are: inlet air temperature of 150-220℃, outlet air temperature of 80-100℃, and atomizer speed of 20000-30000rpm.

[0034] By preparing a compound fermentation extract, not only can the immune system and anti-inflammatory effects of the prepared compound peptide nutritional composition be improved, but it can also promote tissue repair and regulate intestinal flora, shortening the healing time through a multi-faceted synergistic effect. This may be because, firstly, using soybean meal as a nitrogen source, sucrose as a carbon source, and sodium selenite as a selenium source, the biotransformation of selenium is achieved through fermentation with Candida utilis, generating organic selenium forms such as selenomethionine. Simultaneously, glycolic acid is produced, which can soften the cell walls of Cordyceps mycelium and provide a pretreatment substrate for subsequent fermentation. Then, the stage 1 fermentation broth is mixed with Cordyceps mycelium and inoculated with Cordyceps militaris and Cordyceps militaris. Cordyceps militaris secretes laccase to degrade the chitinous skeleton of the mycelium, and Cordyceps militaris produces cordycepin precursors. The synergistic effect of these two processes increases the cordycepin content in the product. Next, Centella asiatica powder was added, and Monascus purpureus and Aspergillus niger were inoculated. Monascus purpureus secreted red pigment to inhibit other bacteria, while Aspergillus niger produced saccharifying enzymes that activated triterpenoid saponins. The two synergistically improved the conversion rate of triterpenoid compounds. Simultaneously, the asiaticoside produced had a synergistic antioxidant effect with rhodioloside in Rhodiola rosea extract. Finally, Lactobacillus plantarum was inoculated for anaerobic fermentation. Lactic acid bacteria metabolism produced short-chain fatty acids, lowering the pH of the system, promoting the dissolution of active ingredients, and increasing the content of γ-aminobutyric acid (GABA) in the final product, significantly enhancing the anti-inflammatory activity of the composition. The resulting compound fermented extract was rich in cordycepin, selenomethionine, asiaticoside, γ-aminobutyric acid, short-chain fatty acids, and other effective components. Their synergistic effect can shorten the inflammatory phase of wounds, promote fibroblast proliferation and collagen synthesis, accelerate granulation tissue formation, and inhibit excessive scar formation, thereby accelerating wound healing.

[0035] By employing a progressive fermentation method, using the product of the previous fermentation process as the substrate for the next, compared to single fermentation, the generation of active ingredients can be further enhanced, while simultaneously increasing the conversion efficiency of *Lactobacillus plantarum*, thereby increasing the content of effective components in the final product and improving the wound healing effect and efficiency of the compound peptide nutritional composition. This may be because, on the one hand, the fermentation process of this invention has a metabolite cascade amplification effect. The selenoisoflavones in stage 1 can induce cordycepin production in stage 2 (*Cicadae Persicae*), and the cordycepin in stage 2 can inhibit the risk of contamination by *Monascus purpureus* in stage 3, improving the efficiency of the mold reaction. *Lactobacillus plantarum* can degrade byproducts such as phytic acid produced by *Monascus purpureus* fermentation. On the other hand, in the coupled fermentation of *Monascus purpureus* and *Aspergillus niger* in stage 3, the cellulase produced by *Aspergillus niger* can release triterpenoid saponins from the cell walls of *Centella asiatica*, improving the repair and healing effect of the product. In addition, the progressive fermentation broth contains abundant amino acids and short-chain fatty acids, which can provide a carbon source for *Lactobacillus plantarum* in stage 4, promoting its biotransformation efficiency and increasing the content of various effective components in the compound fermentation extract. Moreover, the progressive fermentation method avoids the complex processes of preparing multiple active ingredients and the separation and purification at each stage, thus reducing costs.

[0036] Preferably, the plant extract includes green tea extract and Rhodiola rosea extract.

[0037] Preferably, the mass ratio of the green tea extract to the rhodiola rosea extract is (3-5):1.

[0038] Preferably, the method for preparing the green tea extract includes the following steps:

[0039] B1. After drying green tea, grind it through a 60-mesh sieve to obtain green tea powder. Add an ethanol aqueous solution with a mass fraction of 65%-75%, extract with ultrasonic assistance, filter, and concentrate by rotary evaporation at 45℃ to 1-3 times the mass of green tea powder to obtain a concentrated solution.

[0040] B2. Take the concentrate, wall material liquid and emulsifier, mix them, homogenize twice, freeze dry, and pulverize through a 100-mesh sieve to obtain the final product.

[0041] Preferably, the solid-liquid ratio of the green tea powder and the ethanol aqueous solution with a mass fraction of 65%-75% is 1g:(14-16)mL.

[0042] Preferably, the specific conditions for ultrasound-assisted extraction are: frequency of 38-42kHz, power of 220-270W, time of 25-35min, and temperature of 25-35℃.

[0043] Preferably, the components of the wall material liquid, by weight percentage, include 2% sodium alginate, 0.5% pectin, and water to make up the remainder to 100%.

[0044] Preferably, the emulsifier is lecithin, and the amount added is 0.1% of the mass of the concentrate.

[0045] Preferably, the volume ratio of the concentrate to the wall material liquid is 1:3.

[0046] Preferably, the homogenization pressure is 75-85 MPa and the homogenization time is 3-5 min.

[0047] Preferably, the freeze-drying conditions are as follows: after pre-freezing at -40°C for 2 hours, the freeze-drying is carried out at a temperature of -30°C and a pressure of 8-12 Pa for 24 hours.

[0048] Preferably, the content of rhodioloside in the Rhodiola rosea extract is ≥3%.

[0049] In some preferred embodiments, the Rhodiola rosea extract is sourced from Xi'an Tianyi Biotechnology Co., Ltd.

[0050] By using a sodium alginate-pectin composite wall material to nanoemulsify and encapsulate green tea extract, its oral bioavailability can be improved, resisting gastric acid and increasing the retention rate of active ingredients such as EGCG, thereby enhancing bioavailability. This is likely because, compared to the more stable rhodioloside in Rhodiola rosea extract, the active ingredients such as EGCG in green tea extract are less stable and easily affected by light, heat, and oxidation, and are easily degraded in gastric acid, thus failing to exert the effects of oral administration. Therefore, emulsification and encapsulation are necessary. Sodium alginate and pectin form a double-layer wall material, stabilizing the microcapsule structure through electrostatic composite; lecithin reduces interfacial tension, and high-pressure homogenization achieves nanoscale emulsification; freeze-drying preserves the integrity of the microcapsules, thereby controlling the release rate of active ingredients such as EGCG, prolonging their residence time at the wound site, enhancing antioxidant and anti-inflammatory effects, and also synergistically reducing collagen degradation with rhodioloside.

[0051] Preferably, the prebiotics include resistant dextrin and galactooligosaccharides.

[0052] Preferably, the mass ratio of the resistant dextrin to galactooligosaccharides is 1:(1-2); more preferably, it is 2:3.

[0053] Preferably, the whey protein matrix comprises, by weight percentage, 5%-10% lactose, 1%-2% vitamin C, and whey protein powder to make up the remainder to 100%.

[0054] Preferably, the vitamin C is L-ascorbic acid.

[0055] In some preferred embodiments, the compound peptide nutritional composition of the present invention may also contain an appropriate amount of vitamins, including but not limited to vitamin B1, vitamin B2 and vitamin B6, in an amount of 0.01%-0.02% of the mass of the compound peptide nutritional composition.

[0056] A second aspect of the present invention provides a process for preparing the aforementioned complex peptide nutritional composition for promoting wound healing, comprising the following steps:

[0057] S1. Granulate and crush fish collagen peptides, bovine spleen peptides, hemoglobin oligopeptides, albumin peptides, prebiotics and whey protein matrix to obtain a mixture;

[0058] S2. Add ginseng powder, compound fermented extract and plant extract to the mixture in sequence, mix and obtain the final product.

[0059] Preferably, the preparation process includes:

[0060] S1. Place fish collagen peptides, bovine spleen peptides, hemoglobin oligopeptides, albumin peptides, prebiotics and whey protein matrix in a mixer, dry mix and granulate, and crush to a particle size ≤40 mesh to obtain a mixture;

[0061] S2. Add ginseng powder, compound fermented extract, and plant extract to the mixture in sequence, and mix under nitrogen protection to obtain the final product.

[0062] In step S1, the specific conditions for dry granulation are: rotation speed of 15-25 rpm, mixing time of 5-15 min, roller pressure of 60-70 kN, and roller spacing of 0.8-1.2 mm.

[0063] In step S2, the specific mixing conditions are: a rotation speed of 5-15 rpm and a mixing time of 10-20 min.

[0064] The third aspect of this invention provides the application of the aforementioned complex peptide nutritional composition for promoting wound healing, applied in the field of medical nutritional foods, including postoperative wounds, trauma, ulcers, dermatitis, etc.

[0065] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0066] 1. This invention provides a compound peptide nutritional composition that promotes wound healing. By using self-made fish collagen peptides, compound fermented extracts, and other raw materials, the content of various active ingredients such as healing-promoting peptides and anti-inflammatory repair is increased. Furthermore, the bioavailability of plant extracts is improved through encapsulation technology, thereby significantly enhancing the wound healing rate and quality. It has excellent healing effects, especially on iatrogenic wounds caused by tumor radiotherapy and chemotherapy, as well as pressure sores, diabetic foot ulcers, burns, and other difficult-to-heal wounds. It provides an efficient solution for postoperative wounds, trauma, ulcers, dermatitis, and other wounds.

[0067] 2. This invention improves the product's ability to promote wound healing by first demineralizing fish scales / skin with sodium citrate and then preparing fish collagen peptides through a two-stage enzymatic hydrolysis.

[0068] 3. By preparing a compound fermentation extract, this invention can not only enhance the immune system and anti-inflammatory effects of the prepared compound peptide nutritional composition, but also promote tissue repair and regulate intestinal flora, thereby shortening the healing time through a multi-faceted synergistic effect.

[0069] 4. This invention employs a progressive fermentation method, using the product of the previous fermentation process as the substrate for the next fermentation process. Compared to single fermentation, this method can further enhance the generation of active ingredients and improve the conversion efficiency of Lactobacillus plantarum, thereby increasing the content of effective ingredients in the final product and improving the wound healing effect and efficiency of the compound peptide nutritional composition.

[0070] 5. This invention improves the oral bioavailability of green tea extract by using sodium alginate-pectin composite wall material for nanoemulsification and encapsulation, thereby enhancing its resistance to gastric acid and increasing the retention rate of active ingredients such as EGCG. Detailed Implementation

[0071] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0072] All raw materials used in this invention are commercially available, specifically:

[0073] Bovine spleen peptide, hemoglobin oligopeptide, albumin peptide, and ginseng powder are all from Xi'an Zebang Biotechnology Co., Ltd.

[0074] Alkaline protease, with an enzyme activity of approximately 500,000 U / g, is from Shanghai Huashang Xiangyang Biotechnology Co., Ltd.

[0075] Flavor protease, with an enzyme activity of approximately 30,000 U / g, is from Nanning Dongheng Huadao Biotechnology Co., Ltd.

[0076] Soybean meal, sourced from Shandong Rundong Agricultural Technology Co., Ltd.

[0077] Candida utilis, strain number CCTCC CY 20081274; Monascus purpureus, strain number CCTCC HF2008672; Aspergillus niger, strain number CCTCC AF 2022057; Lactobacillus plantarum, strain number CCTCC AB 2022408; all were obtained from the China Center for Type Culture Collection.

[0078] Cordyceps militaris, strain number cfcc 50943; Cordyceps militaris, strain number cfcc 88944; both were obtained from the China Forestry Microbial Culture Collection Center.

[0079] Cordyceps mycelium, sourced from Fufeng Sinote Biotechnology Co., Ltd.

[0080] Sodium alginate, pectin, and lecithin are all from Shanghai Maclean Biochemical Technology Co., Ltd.

[0081] Rhodiola rosea extract, with a rhodioloside content of ≥3%, is sourced from Xi'an Tianyi Biotechnology Co., Ltd.

[0082] Resistant dextrin, from Roquette, France, NUTRIOSE® FB06.

[0083] Galacto-oligosaccharides, from Shanghai Maclean Biotechnology Co., Ltd.

[0084] Lactose, sourced from Jiangsu Caiwei Biotechnology Co., Ltd.

[0085] Whey protein powder, whey protein concentrate, WPC80, from Shandong Jingyun Food Ingredients Co., Ltd.

[0086] Example 1

[0087] This embodiment provides a compound peptide nutritional composition for promoting wound healing. The raw materials for its preparation, by weight percentage, include 20% fish collagen peptide, 0.04% bovine spleen peptide, 0.20% hemoglobin oligopeptide, 0.20% albumin peptide, 0.02% ginseng powder, 18% compound fermented extract, 15% plant extract, 22% prebiotics, and whey protein matrix to make up the balance to 100%.

[0088] The preparation method of the fish collagen peptide includes the following steps:

[0089] A1. Take collagen peptide raw material and mix it with 0.1M sodium citrate solution, stir at 4℃ for 12h, centrifuge and wash with water until neutral to obtain demineralized fish scales;

[0090] A2. Add water to the demineralized fish scales and adjust the pH to 7. Add alkaline protease and react at 25°C for 2 hours. Then add flavor protease and react at 25°C for 2 hours to inactivate the enzyme. First, use an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to obtain ultrafiltrate. Then, use an ultrafiltration membrane with a molecular weight cutoff of 300 Da to obtain the retained material. Freeze-dry the obtained material.

[0091] The collagen peptide raw material is deep-sea cod skin.

[0092] The solid-liquid ratio of the collagen peptide raw material to the 0.1M sodium citrate solution is 1g:10mL.

[0093] The mass ratio of the demineralized fish scales to water is 1:5.

[0094] The amount of alkaline protease added is 350 U / g collagen peptide raw material.

[0095] The amount of flavor protease added is 350 U / g collagen peptide raw material.

[0096] The enzyme inactivation conditions are: temperature 85℃ and time 15min.

[0097] The freeze-drying conditions are as follows: after pre-freezing at -40℃ for 4 hours, the freeze-drying is carried out at a temperature of -30℃ and a pressure of 10Pa for 24 hours.

[0098] The preparation method of the compound fermentation extract includes the following steps: yeast is inoculated onto culture medium 1, and fermentation is carried out at 30℃ and 150 rpm for 72 hours to obtain fermentation product 1. Cordyceps mycelium is added to obtain culture medium 2. Cordyceps militaris and Cordyceps militaris are inoculated and cultured at 23℃ for 10 days to obtain fermentation product 2. Centella asiatica powder is added to obtain culture medium 3. Mold is inoculated and fermented at 28℃ and 200 rpm for 96 hours to obtain fermentation product 3. Lactobacillus plantarum is inoculated and anaerobic fermentation is carried out at 37℃ for 48 hours. The product is then filtered using a ceramic membrane with a molecular weight cutoff of 50 kDa, vacuum concentrated at 45℃ to a solid content of 30%, and spray-dried to obtain the final product.

[0099] The yeast strain is *Candida utilis*.

[0100] The inoculation amount of the yeast is 1×10⁻⁶. 7 CFU / g culture medium 1.

[0101] The culture medium 1, by weight percentage, comprises 5% soybean meal, 2.5% sucrose, 0.01% sodium selenite, and water to make up to 100%.

[0102] The amount of Cordyceps mycelium added is consistent with the mass of fermentation product 1.

[0103] The inoculation amount of both *Cicada Flower* and *Cordyceps militaris* was the same, 2.5 × 10⁻⁶. 7 CFU / g medium 2.

[0104] The amount of Centella asiatica powder added is 3% of the mass of fermentation product 2.

[0105] The molds include Monascus purpureus and Aspergillus niger.

[0106] The inoculum size of Monascus purpureus was 1.5 × 10⁻⁶. 7 CFU / g medium 3; the inoculum size of Aspergillus niger was 3 × 10⁻⁶. 6 CFU / g medium 3.

[0107] The inoculum size of *Lactobacillus plantarum* was 5 × 10⁻⁶. 6 CFU / g fermentation product 3.

[0108] The vacuum degree of the vacuum concentration is 0.09 MPa.

[0109] The specific conditions for spray drying are: inlet air temperature of 180℃, outlet air temperature of 90℃, and atomizer speed of 25000rpm.

[0110] The plant extracts are green tea extract and Rhodiola rosea extract, in a mass ratio of 4:1.

[0111] The preparation method of the green tea extract includes the following steps:

[0112] B1. After drying green tea, grind it through a 60-mesh sieve to obtain green tea powder. Add a 70% ethanol aqueous solution, extract with ultrasonic assistance, filter, and concentrate by rotary evaporation at 45℃ to twice the mass of green tea powder to obtain a concentrated solution.

[0113] B2. Take the concentrate, wall material liquid and emulsifier, mix them, homogenize twice, freeze dry, and pulverize through a 100-mesh sieve to obtain the final product.

[0114] The solid-liquid ratio of the green tea powder and the 70% ethanol aqueous solution is 1g:15mL.

[0115] The specific conditions for ultrasound-assisted extraction are: frequency of 40kHz, power of 250W, time of 30min, and temperature of 30℃.

[0116] The components of the wall material liquid, by weight percentage, include 2% sodium alginate, 0.5% pectin, and water to make up the remainder to 100%.

[0117] The emulsifier is lecithin, and the amount added is 0.1% of the mass of the concentrate.

[0118] The volume ratio of the concentrate to the wall material liquid is 1:3.

[0119] The homogenization pressure was 85 MPa, and the homogenization time was 4 min.

[0120] The freeze-drying conditions are as follows: after pre-freezing at -40℃ for 2 hours, the freeze-drying is carried out at a temperature of -30℃ and a pressure of 10Pa for 24 hours.

[0121] The prebiotics are resistant dextrin and galactooligosaccharides in a mass ratio of 2:3.

[0122] The whey protein matrix, by weight percentage, comprises 8% lactose, 1.2% vitamin C, and whey protein powder to make up the remainder to 100%.

[0123] The vitamin C mentioned is L-ascorbic acid.

[0124] The preparation process of the compound peptide nutritional composition for promoting wound healing includes the following steps:

[0125] S1. Place fish collagen peptides, bovine spleen peptides, hemoglobin oligopeptides, albumin peptides, prebiotics and whey protein matrix in a mixer, dry mix and granulate, and crush to a particle size ≤40 mesh to obtain a mixture;

[0126] S2. Add ginseng powder, compound fermented extract, and plant extract to the mixture in sequence, and mix under nitrogen protection to obtain the final product.

[0127] In step S1, the specific conditions for dry granulation are: rotation speed of 20 rpm, mixing time of 10 min, roller pressure of 65 kN, and roller spacing of 1.0 mm.

[0128] In step S2, the specific mixing conditions are: a rotation speed of 10 rpm and a mixing time of 15 min.

[0129] Example 2

[0130] The only difference between this embodiment and Embodiment 1 is that the collagen peptide raw material is tilapia scales.

[0131] Comparative Example 1

[0132] The only difference between this comparative example and Example 1 is that: A2, water was added to the demineralized fish scales and the pH was adjusted to 7. Alkaline protease and flavor protease were added, and the mixture was reacted at 25°C for 4 hours. After enzyme inactivation, the mixture was ultrafiltered using an ultrafiltration membrane and then freeze-dried.

[0133] Comparative Example 2

[0134] The only difference between this comparative example and Example 1 is that: A2, water was added to the demineralized fish scales and the pH was adjusted to 7. Alkaline protease was added, and after reacting at 25°C for 2 hours, the enzyme was inactivated. The mixture was then ultrafiltered using an ultrafiltration membrane and freeze-dried.

[0135] Comparative Example 3

[0136] The only difference between this comparative example and Example 1 is the preparation method of the compound fermentation extract, which involves the following steps: yeast is inoculated onto culture medium 1, and fermented at 30°C and 150 rpm for 72 hours to obtain fermentation product 1. Centella asiatica powder is added to obtain culture medium 2, which is then inoculated with mold and fermented at 28°C and 200 rpm for 96 hours to obtain fermentation product 2. Lactobacillus plantarum is inoculated, and anaerobic fermentation is carried out at 37°C for 48 hours. The product is then filtered using a ceramic membrane with a molecular weight cutoff of 50 kDa, vacuum concentrated at 45°C until the solid content is 30%, and then spray-dried to obtain the final product.

[0137] The amount of Centella asiatica powder added is 3% of the mass of fermentation product 1.

[0138] The molds include Monascus purpureus and Aspergillus niger.

[0139] The inoculum size of Monascus purpureus was 1.5 × 10⁻⁶. 7 CFU / g medium 2; the inoculum size of Aspergillus niger was 3 × 10⁻⁶. 6 CFU / g medium 2.

[0140] The inoculum size of *Lactobacillus plantarum* was 5 × 10⁻⁶. 6 CFU / g fermentation product 2.

[0141] Comparative Example 4

[0142] The difference between this comparative example and Example 1 is that the culture medium 1, by weight percentage, includes 5% soybean meal, 2.5% sucrose, and water to make up the remainder to 100%.

[0143] Comparative Example 5

[0144] The only difference between this comparative example and Example 1 is that the preparation method of the compound fermentation extract includes the following steps:

[0145] (1) Yeast was inoculated onto culture medium 1 and fermented at 30°C and 150 rpm for 72 h to obtain fermentation product 1;

[0146] (2) Mix culture medium 2 with cordyceps mycelium to obtain culture medium 2, inoculate cicada flower and cordyceps militaris, and incubate at 23℃ for 10 days to obtain fermentation product 2;

[0147] (3) Mix culture medium 3 and Centella asiatica powder to obtain culture medium 3, inoculate with mold, and ferment at 28℃ and 200rpm for 96h to obtain fermentation product 3;

[0148] (4) After mixing fermentation product 1, fermentation product 2 and fermentation product 3, culture medium 4 is obtained. Lactobacillus plantarum is inoculated and anaerobic fermentation is carried out at 37°C for 48 hours. After filtration with a ceramic membrane with a molecular weight cutoff of 50 kDa, the solid content is concentrated under vacuum at 45°C to 30%, and then spray-dried to obtain the final product.

[0149] Culture media 2 and culture media 3, by weight percentage, both consist of 5% soybean meal, 2.5% sucrose, and water to make up the remainder to 100%.

[0150] The amount of Cordyceps mycelium added was consistent with the mass of culture medium 2.

[0151] The amount of Centella asiatica powder added is 3% of the mass of culture medium 3.

[0152] The inoculum size of *Lactobacillus plantarum* was 5 × 10⁻⁶. 6 CFU / g culture medium 4.

[0153] Comparative Example 6

[0154] The only difference between this comparative example and Example 1 is that the mold used is Monascus purpureus, and the inoculum size is 2×10⁷ CFU / g culture medium 3.

[0155] Comparative Example 7

[0156] The only difference between this comparative example and Example 1 is the preparation method of the green tea extract, which involves: drying green tea, grinding it through a 60-mesh sieve to obtain green tea powder, adding a 70% ethanol aqueous solution, extracting with ultrasonic assistance, filtering, concentrating by rotary evaporation at 45°C to twice the mass of the green tea powder, freeze-drying, pulverizing and passing through a 100-mesh sieve to obtain the final product.

[0157] Experimental Design

[0158] Animal grouping: Male BALB / c mice (6-8 weeks old, 20±2g) were randomly divided into 10 groups, with 20 mice in each group. Experimental groups 1-9: The compound peptide nutritional composition prepared in Examples 1-2 and Comparative Examples 1-7 of this invention was dissolved in water and administered by gavage at a dose of 1g / kg / d; Experimental group 10: Blank group, administered by gavage with an equal volume of physiological saline.

[0159] Modeling and drug administration: After shaving the back, an 8mm punch was used to create a full-thickness skin defect. Immediately after the operation, the drug was administered by gavage once a day for 14 consecutive days.

[0160] Observation indicators: ① Wound healing rate: The wound area was measured daily, and the healing rate was calculated as (initial area - residual area) / initial area × 100%; ② Histological analysis: Wound tissue was taken on day 7, and the proportion of collagen deposition area was calculated by Masson trichrome staining; ③ Detection of inflammatory factors: Blood was collected from the orbital venous plexus on day 7, and the serum was separated by centrifugation after standing at room temperature for 30 minutes. The concentration of TNF-α in the serum was measured by ELISA.

[0161] The evaluation criteria are shown in Table 1.

[0162] Table 1 Evaluation Criteria

[0163]

[0164] The evaluation results are shown in Table 2.

[0165] Table 2 Measurement Results

[0166]

[0167] According to the above data, the composite peptide nutritional composition prepared in Examples 1-2 of the present invention has a fast wound healing speed and significant effect, a high proportion of collagen deposition, and a significant reduction in TNF-α level. Among them, the hydroxyproline content in tilapia scale collagen peptide is lower than that in cod skin, which has a certain impact on the wound healing effect, but it can be ignored. The overall effective rate can still reach 100%. Comparative Example 1: Mixed enzymatic hydrolysis led to substrate competition, disrupting the molecular weight distribution of collagen peptides and reducing healing efficacy. Comparative Example 2: The lack of flavor protease resulted in unhydrolyzed collagen helical regions, reducing the proportion of peptides below 3000 Da. Comparative Example 3: The absence of the Cordyceps militaris stage resulted in the absence of cordycepin production. Comparative Example 4: The absence of selenomethionine in the yeast fermentation product weakened the anti-inflammatory effect, affecting the overall healing efficacy. Comparative Example 5: Inoculation of *Lactobacillus plantarum* after mixing the three-stage fermentation products disrupted the metabolic cascade, leading to inhibition of microbial competition, a decrease in the content of effective products, and affecting the healing efficacy. Comparative Example 6: The absence of *Aspergillus niger* resulted in insufficient conversion of asiaticoside, reduced triterpenoid saponin content, decreased anti-inflammatory effect, and affected the healing efficacy. Comparative Example 7: The lack of encapsulation of green tea extract led to the degradation of its effective components, such as EGCG, by gastric acid, reducing its antioxidant and anti-inflammatory effects and affecting wound healing efficiency. Therefore, the composite peptide nutritional composition prepared using the raw materials and methods described in this application can promote wound healing. The composite peptide nutritional composition heals quickly, achieving significant healing by day 7 and almost complete healing by day 14. At the same time, it has a large collagen deposition area and a rapid decrease in TNF-α concentration. This indicates that by increasing the content of healing-promoting peptides, enhancing the key active ingredients with anti-inflammatory and repair effects, and improving the bioavailability of effective ingredients, it can synergistically promote the inflammatory, proliferative, and remodeling phases of wound healing, thereby improving the speed and quality of wound healing and providing an efficient solution for postoperative wounds, trauma, ulcers, dermatitis, and other wounds.

[0168] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A complex peptide nutritional composition for promoting wound healing, characterized in that, The raw materials used in its preparation, by weight percentage, include 15%-25% fish collagen peptides, 0.03%-0.05% bovine spleen peptides, 0.10%-0.30% hemoglobin oligopeptides, 0.10%-0.30% albumin peptides, 0.01%-0.03% ginseng powder, 15%-20% compound fermented extracts, 10%-20% plant extracts, 20%-25% prebiotics, and whey protein matrix to make up the remainder to 100%. The method for preparing the fish collagen peptide includes the following steps: A1. Mix collagen peptide raw material with sodium citrate solution, stir, centrifuge, and wash with water to obtain demineralized fish scales; the collagen peptide raw material is fish scales. A2. Add water to demineralized fish scales and adjust the pH, add alkaline protease, react, add flavor protease, react, inactivate the enzyme, ultrafilter using an ultrafiltration membrane, and freeze dry; the amount of alkaline protease added is 300-400 U / g collagen peptide raw material; the amount of flavor protease added is 300-400 U / g collagen peptide raw material. The preparation method of the compound fermentation extract includes the following steps: yeast is inoculated onto culture medium 1, and fermented at 28-32℃ and 120-180rpm for 72h to obtain fermentation product 1; Cordyceps mycelium is added to obtain culture medium 2; Cordyceps militaris and Cordyceps militaris are inoculated, and cultured statically at 23-25℃ for 10 days to obtain fermentation product 2; Centella asiatica powder is added to obtain culture medium 3; mold is inoculated, and fermented at 28-30℃ and 180-220rpm for 96h to obtain fermentation product 3; Lactobacillus plantarum is inoculated, and anaerobic fermentation is carried out at 37℃ for 48h; the mixture is filtered using a ceramic membrane with a molecular weight cutoff of 50kDa, and vacuum concentrated at 45℃ until the solid content is ≥30%; and then spray-dried to obtain the final product. The yeast strain is *Candida utilis*, and the inoculum size is 1 × 10⁻⁶. 7 CFU / g culture medium 1; The plant extracts are green tea extract and Rhodiola rosea extract; The molds mentioned are Monascus purpureus and Aspergillus niger; The culture medium 1, by weight percentage, comprises 5% soybean meal, 3%-4% sucrose, 0.01% sodium selenite, and water to make up the remainder to 100%; The prebiotics are resistant dextrin and galactooligosaccharides; The whey protein matrix, by weight percentage, comprises 5%-10% lactose, 1%-2% vitamin C, and whey protein powder to make up the remainder to 100%.

2. A preparation process for the composite peptide nutritional composition for promoting wound healing according to claim 1, characterized in that, Includes the following steps: S1. Granulate and crush fish collagen peptides, bovine spleen peptides, hemoglobin oligopeptides, albumin peptides, prebiotics and whey protein matrix to obtain a mixture; S2. Add ginseng powder, compound fermented extract and plant extract to the mixture in sequence, mix and obtain the final product.

3. The use of the complex peptide nutritional composition for promoting wound healing according to claim 1 in the preparation of special medical foods.

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