Preparation method of high-activity fresh collagen peptide by ultrasonic synergistic enzymolysis and application for promoting collagen production

Abstract

This invention provides a method for preparing highly active fresh collagen peptides through ultrasound-assisted enzymatic hydrolysis and its application in promoting collagen production. It belongs to the field of bioactive peptide preparation technology. The method includes: pulverizing collagen raw materials and mixing them with purified water to form a homogenate; adding a flavored protease and stirring at a constant temperature to obtain a deodorized and modified homogenate; then placing the homogenate in an ultrasonic reactor and treating it with dual-frequency alternating pulse ultrasound combined with gradient temperature programming to obtain a loose modified collagen raw material solution; subsequently adding a complex protease and stirring at a constant temperature for gentle and directional enzymatic hydrolysis to obtain a crude collagen peptide hydrolysate; then subjecting it to high-temperature enzyme inactivation treatment, followed by sequential purification through sieve filtration, microfiltration, and ultrafiltration to obtain a refined peptide solution; finally, vacuum concentration and drying to obtain highly active fresh collagen peptide powder. This invention solves the technical problems of traditional processes, such as the easy destruction of the natural structure of collagen during pretreatment, severe loss of active peptides during processing, and difficulty in ensuring product quality under high-production conditions.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide preparation technology, and in particular to a method for preparing highly active fresh collagen peptides by ultrasound-assisted enzymatic hydrolysis and its application in promoting collagen production. Background Technology

[0002] Collagen peptides possess excellent biocompatibility and physiological activity, and are widely used in health foods, cosmetics, and biomedical materials. Currently, industrial production primarily employs enzymatic hydrolysis to prepare collagen peptides, a method characterized by its mild reaction and high safety. To break down dense collagen fibers and improve processing efficiency, the industry often incorporates ultrasonic pretreatment; ultrasound-assisted processing is currently the mainstream physical processing method.

[0003] With the technological iteration of the industry, collagen peptide processing is increasingly pursuing high extraction rates, small molecule size, and flavor purity. However, existing processing techniques generally suffer from common technical problems. Physical pretreatment methods are often crude, relying heavily on high-intensity external forces or high temperatures to treat raw materials, which can easily damage the natural spatial structure of collagen. Simultaneously, the deodorization and purification processes are often delayed, mostly employing non-selective adsorption methods, which easily lead to the loss of active peptides. Furthermore, most processes unilaterally pursue processing output indicators, focusing solely on increasing raw material extraction rates and achieving small peptide molecule size, while neglecting the regulation of bioactivity levels in the processing flow. This ignores the main function of the finished product in promoting collagen production, resulting in products with generally weak bioactivity and low levels of effective components.

[0004] To optimize existing processes, existing patent documents have made corresponding improvements, but significant technical shortcomings still exist. Patent document CN113789360B uses constant power continuous ultrasonic treatment of raw materials, which improves extraction efficiency. However, continuous ultrasonic treatment at a single frequency damages the triple helix active structure of collagen, resulting in poor bioavailability of the finished product, failing to meet the processing requirements of high raw material extraction rate and high bioactivity of the finished product. Patent document CN111118094A uses a high-temperature pretreatment combined with post-activated carbon purification. High temperatures cause protein denaturation and inactivation, and activated carbon adsorption depletes active peptides, causing double damage to product activity.

[0005] In summary, existing improved processes still suffer from structural damage and loss of activity, thus necessitating a method for preparing highly active fresh collagen peptides through ultrasound-assisted enzymatic hydrolysis. Summary of the Invention

[0006] In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing highly active fresh collagen peptides by ultrasound-assisted enzymatic hydrolysis and its application in promoting collagen production. This method solves the technical problems of traditional processes, such as the easy destruction of the natural structure of collagen during pretreatment, the serious loss of active peptides during processing, and the difficulty in ensuring product quality under high-yield conditions.

[0007] To achieve the above objectives, the present invention provides the following solution: On one hand, the present invention provides a method for preparing highly active fresh collagen peptides by ultrasound-assisted enzymatic hydrolysis, comprising the following steps: Animal skin, bone or fish scale collagen raw materials are crushed and mixed with purified water to make a homogenate. Then, flavor protease is added and stirred at a constant temperature to obtain a deodorized and modified homogenate. The obtained deodorized modified homogenate was placed in an ultrasonic reactor and treated with dual-frequency alternating pulse ultrasonication combined with gradient temperature program to obtain a loose modified gum raw material solution. A complex protease composed of endopeptide and exopeptide was added to the loose modified collagen raw material solution after the temperature was stabilized. The solution was then stirred at a constant temperature to carry out gentle and directional enzymatic hydrolysis, resulting in crude collagen peptide hydrolysate. The crude enzymatic hydrolysate of collagen peptides was subjected to high-temperature enzyme inactivation treatment, and then purified by sieve filtration, microfiltration and ultrafiltration to obtain a refined peptide solution. The refined peptide solution was then concentrated under vacuum and dried to finally obtain highly active fresh collagen peptide powder.

[0008] Preferably, the process of pulverizing animal skin, bone, or fish scale collagen raw materials and mixing them with purified water to form a homogenate, followed by adding flavor protease and stirring at a constant temperature to obtain a deodorizing modified homogenate is as follows: The clean collagen raw materials are pulverized to a particle size of 1-5 mm, and the pulverized raw materials are mixed with purified water at a mass ratio of 1:8-1:15 to form a homogenate; 0.1%-0.5% of the total mass of flavor protease is added to the homogenate, and the mixture is stirred at a constant temperature of 20-30℃, pH 6.0-7.0, and stirring speed of 50-100 rpm for 30-90 minutes to pre-hydrolyze and remove the fishy odor precursor proteins and lipid oxidation products from the surface and gaps of the raw materials, thereby obtaining the deodorizing modified homogenate.

[0009] Preferably, the process of placing the obtained deodorized modified homogenate in an ultrasonic reactor and using dual-frequency alternating pulse ultrasonic treatment combined with gradient programmed heating to obtain a loose modified rubber raw material solution is as follows: setting the ultrasonic low frequency to 20kHz and the high frequency to 40kHz, controlling the ultrasonic power density to 150~250W / L, the total ultrasonic treatment time to 15~25min, and maintaining the material solution temperature not higher than 35℃ throughout the process; after the ultrasonic treatment, the material solution is uniformly heated to 48~52℃ at a rate of 0.8~1.2℃ / min to obtain the loose modified rubber raw material solution.

[0010] Preferably, the dual-frequency alternating pulse ultrasound is a low-frequency and high-frequency alternating working mode. Each frequency operates in a pulse cycle mode of 2 seconds working and 4 seconds intermittently. After completing a single-frequency pulse cycle, the other frequency is switched to perform pulse operation.

[0011] Preferably, the process of adding a complex protease composed of endopeptides and exopeptides to the loose modified gum raw material solution after temperature stabilization, and performing gentle and directional enzymatic hydrolysis at a constant temperature to obtain crude collagen peptide hydrolysate is as follows: The complex protease, prepared by mixing endopeptides and exopeptides at a mass ratio of 3:1 to 5:1, is added to the loose modified gum raw material solution at a constant temperature of 48-52°C; the hydrolysis is continued at a constant temperature for 3-5 hours under gentle conditions of pH 6.5-7.5 and stirring speed of 80-120 rpm. The endopeptides break down the collagen backbone, and the exopeptides modify the peptide chain ends, simultaneously removing bitter substances to obtain crude collagen peptide hydrolysate.

[0012] Preferably, the endopeptide is selected from at least one of neutral protease or alkaline protease, and the exopeptide is a flavor protease; the total amount of the complex protease added is 0.8% to 1.5% of the dry weight of the collagen raw material.

[0013] Preferably, the process of subjecting the obtained crude collagen peptide hydrolysate to high-temperature enzyme inactivation treatment, followed by sequential filtration through a sieve, microfiltration, and ultrafiltration to obtain a refined peptide solution, and then vacuum concentrating and drying the refined peptide solution to finally obtain highly active fresh collagen peptide powder is as follows: After enzymatic hydrolysis, the temperature is raised to 85~90℃ and held for 10~15 minutes to complete enzyme inactivation; insoluble residues are removed by filtration through a 100~200 mesh sieve; the filtrate is then sequentially treated through a 0.1~0.22μm microfiltration membrane and an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and the ultrafiltration permeate is collected as the refined peptide solution; the refined peptide solution is vacuum concentrated at ≤50℃ and a vacuum degree ≥-0.08MPa to a solid content of 25%~35%, and then spray dried to obtain collagen peptide powder.

[0014] Preferably, the spray drying process parameters are: inlet air temperature 160~175℃, outlet air temperature 75~85℃.

[0015] On the other hand, the present invention also provides a highly active fresh collagen peptide prepared by the above preparation method.

[0016] Specifically, based on the application of the aforementioned highly active fresh collagen peptides in promoting collagen production, the highly active fresh collagen peptides are adapted to any application form, such as powder or liquid, to induce and enhance the production and expression of collagen in the body.

[0017] Compared with the prior art, the present invention discloses at least the following technical effects: (1) This invention employs a pre-treatment process combining enzymatic deodorization and dual-frequency alternating pulsed ultrasound. The pre-treatment method removes fishy odor impurities from the source, avoiding the loss of active peptides caused by traditional post-adsorption processes. Based on the ultrasonic cavitation effect, the collagen fiber structure is loosened, enhancing the mass transfer capacity of the material. At the same time, the dual-frequency pulsed ultrasound combined with the gradient heating mode can reduce the damage caused by ultrasonic thermal effects and mechanical forces, effectively preserving the natural triple helix structure of collagen, and solving the problem that traditional pretreatment methods easily damage protein structure and cause serious loss of activity.

[0018] (2) The present invention uses a combination system of endopeptidase and exopeptidase to carry out multi-stage purification treatment. The combination protease can achieve mild and targeted enzymatic hydrolysis, which can not only reduce the amount of enzyme preparation and shorten the enzymatic hydrolysis time, but also regulate the peptide structure, remove bitter substances, and improve the purity of peptide solution. Combined with graded filtration, membrane separation and low temperature concentration and drying treatment, it can enrich highly active small molecule peptides, avoid the activity destruction caused by high temperature processing, and the resulting collagen peptide powder has good solubility and improved penetration absorption rate, which significantly improves the physical and chemical quality of the product.

[0019] (3) The fresh collagen peptides prepared by this invention have complete structure and high bioactivity, which can effectively induce collagen synthesis in the body, improve skin elasticity and luster, and have good skin care application value. At the same time, the preparation method provided is simple and controllable, with high raw material utilization rate, and is suitable for large-scale industrial production. It effectively solves the technical problem of high yield but poor quality in traditional processes, and achieves a joint improvement in production efficiency, product quality and biological efficacy. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a flowchart of a method for preparing highly active fresh collagen peptides by ultrasound-assisted enzymatic hydrolysis according to the present invention. Figure 2 This is a comparison curve of type I collagen content provided in different embodiments and comparative examples of the present invention; Figure 3 This is a bar chart comparing the increase in hydroxyproline content between Example 1 and Comparative Example 2 of the present invention. Figure 4 A bar chart comparing the improvement in skin elasticity in the experimental group, control group, and placebo group of this invention. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0023] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] like Figure 1 As shown, this invention provides a method for preparing highly active fresh collagen peptides through ultrasound-assisted enzymatic hydrolysis, comprising the following steps: Step 100: After crushing the collagen raw materials of animal skin, bone or fish scales, mix them with purified water to make a homogenate, then add flavor protease and stir at a constant temperature to obtain a deodorized and modified homogenate.

[0025] Specifically, this step involves pulverizing clean collagen raw materials to a particle size of 1-5 mm, mixing the pulverized raw materials with purified water at a mass ratio of 1:8 to 1:15 to form a uniform slurry, and controlling the addition of flavor protease at a controlled amount of 0.1% to 0.5%. The mixture is stirred at a constant temperature for 30-90 minutes under a mild environment of 20-30℃, pH 6.0-7.0, and stirring speed of 50-100 rpm. Utilizing the specific hydrolytic action of flavor protease, the fishy odor precursor proteins and lipid oxidation products on the surface and between the raw materials are decomposed and removed in advance, thus removing fishy odor impurities from the source. This solves the problem of active peptide loss in traditional post-deodorization processes and further ensures the purity of the raw materials.

[0026] Step 200: Place the obtained deodorized modified homogenate in an ultrasonic reactor, and treat it with dual-frequency alternating pulse ultrasonication and gradient temperature program to obtain loose modified gum raw material liquid.

[0027] Specifically, this step uses a dual-frequency ultrasonic mode of 20kHz low frequency combined with 40kHz high frequency, controlling the ultrasonic power density at 150~250W / L, maintaining a total processing time of 15~25min, and controlling the temperature throughout to not exceed 35℃ to avoid high temperature damage to the protein structure. It adopts a pulse cycle of 2s single-frequency operation followed by 4s intermittent operation, using the ultrasonic cavitation effect to physically loosen the dense collagen fibers and enhance the mass transfer efficiency of the material. After ultrasonic treatment, the temperature is uniformly raised to 48~52℃ at a rate of 0.8~1.2℃ / min to smoothly transition the temperature of the solution, creating a suitable and mild environment for the subsequent enzymatic hydrolysis reaction and ensuring the natural triple helix structure of the whole collagen.

[0028] Step 300: Add a complex protease composed of endopeptide and exopeptide to the loose modified collagen raw material solution after the temperature has stabilized, and carry out gentle and directional enzymatic hydrolysis by stirring at a constant temperature to obtain crude collagen peptide hydrolysate.

[0029] Specifically, in this step, under constant temperature conditions of 48~52℃, a complex protease composed of endopeptidase and exopeptidase in a mass ratio of 3:1~5:1 is added. The total amount of the complex protease is controlled to be 0.8%~1.5% of the dry weight of the collagen raw material. Neutral or alkaline protease is selected as the endopeptidase and flavor protease as the exopeptidase. Under constant temperature conditions of pH 6.5~7.5 and stirring at 80~120 rpm for 3~5 hours, the endopeptidase breaks the collagen macromolecular backbone, and the exopeptidase modifies the peptide chain ends, simultaneously degrading bitter impurities, to achieve mild and targeted enzymatic hydrolysis, regulate the peptide structure and improve the purity of the peptide solution.

[0030] Step 400: The obtained crude collagen peptide hydrolysate is subjected to high-temperature enzyme inactivation treatment, and then purified by sieve filtration, microfiltration and ultrafiltration to obtain refined peptide solution. The refined peptide solution is concentrated under vacuum and then dried to finally obtain highly active fresh collagen peptide powder.

[0031] Specifically, after enzymatic hydrolysis, the temperature is raised to 85-90℃ and held for 10-15 minutes to achieve rapid enzyme inactivation. Insoluble residues are initially removed using a 100-200 mesh sieve. Then, the product is purified by fractional purification using a 0.1-0.22μm microfiltration membrane combined with a 3000Da molecular weight cutoff ultrafiltration membrane to precisely remove large molecular impurities and extraneous proteins, while enriching highly active small molecule peptides. The product is then concentrated at low temperature to a solid content of 25%-35% under a vacuum environment of ≤50℃ and ≥-0.08MPa. Finally, it is powdered using a spray drying process with an inlet air temperature of 160-175℃ and an outlet air temperature of 75-85℃. The entire process is carried out at low temperature to avoid heat damage, maximize the preservation of collagen peptide bioactivity, and ensure the solubility and uniformity of the powder.

[0032] The above content will be further described below through specific implementation methods.

[0033] Example 1 This embodiment aims to provide a method for preparing highly active fresh collagen peptides by ultrasound-assisted enzymatic hydrolysis, which includes the following specific steps: Raw material pretreatment for deodorization: Fresh fish scales are selected as collagen raw material, cleaned and crushed to a particle size of 1-5 mm. The crushed raw material is mixed with purified water at a mass ratio of 1:10 to form a homogenate. 0.3% of flavor protease by total mass is added to the homogenate. The temperature is controlled at 25℃, pH value at 6.5, and stirring speed at 80 rpm. The mixture is stirred at a constant temperature for 60 min to obtain a deodorized modified homogenate. Dual-frequency pulse ultrasonic pretreatment: The deodorized modified homogenate was placed in an ultrasonic reactor, with a low frequency of 20kHz, a high frequency of 40kHz, and an ultrasonic power density of 200W / L. The mixture was treated for 20 minutes using an alternating pulse mode of 2 seconds working and 4 seconds intermittent. The temperature of the mixture was controlled to be ≤35℃ throughout the process. After ultrasonic treatment, the temperature was gradually increased to 50℃ at a rate of 1.0℃ / min to obtain a loose modified gum raw material solution. Targeted enzymatic hydrolysis with compound enzyme: Add compound protease to the raw material solution at a constant temperature of 50℃. The compound protease is composed of neutral protease and flavor protease in a mass ratio of 4:1, and the total amount added is 1.2% of the dry weight of collagen raw material. Adjust the pH value to 7.0, stir at 100 rpm, and enzymatically hydrolyze at a constant temperature for 4 hours to obtain crude enzymatic hydrolysate of collagen peptides. Purification and drying: The crude enzymatic hydrolysate was heated to 88℃ and kept at that temperature for 12 min to inactivate the enzyme; the residue was removed by filtration through a 150-mesh sieve, and the filtrate was purified by passing it through a 0.22μm microfiltration membrane and a 3000Da ultrafiltration membrane. The ultrafiltration permeate was collected to obtain a purified peptide solution; the solution was concentrated to a solid content of 30% at a temperature of 45℃ and a vacuum of -0.09MPa, and then spray-dried to prepare powder with an inlet air temperature of 168℃ and an outlet air temperature of 80℃, finally yielding highly active fresh collagen peptides.

[0034] Example 2 In this embodiment, bovine bone was used as the raw material, the mass ratio of crushed raw material to purified water was 1:8, the amount of flavor protease added was 0.1%, and the mixture was stirred at a constant temperature for 30 min; the ultrasonic power density was 150 W / L, the treatment time was 15 min, and the temperature was gradually increased to 48℃; the compound protease ratio was 3:1, the amount added was 0.8%, and the enzymatic hydrolysis time was 3 h; the remaining process parameters were the same as in Example 1.

[0035] Example 3 In this embodiment, pig skin was used as the raw material. The mass ratio of crushed raw material to purified water was 1:15. The amount of flavor protease added was 0.5%. The mixture was stirred at a constant temperature for 90 minutes. The ultrasonic power density was 250W / L, the treatment time was 25 minutes, and the temperature was gradually increased to 52℃. The ratio of compound protease was 5:1, the amount added was 1.5%, and the enzymatic hydrolysis time was 5 hours. The remaining process parameters were the same as in Example 1.

[0036] Comparative Example 1 The ultrasonic treatment was performed continuously for 20 minutes at a constant power of 20 kHz, without pulse intervals, high and low frequency alternation, or gradient heating. The remaining steps were the same as in Example 1.

[0037] Comparative Example 2 The raw materials were pretreated at 55℃ for 30 minutes. The pre-processed flavor protease was omitted to remove the fishy smell. After enzymatic hydrolysis, activated carbon was added for adsorption and deodorization. The remaining steps were the same as in Example 1.

[0038] Comparative Example 3 Only neutral protease was added for enzymatic hydrolysis, and the combination system of endopeptidase and exopeptidase was cancelled. The total amount of protease added remained unchanged, and the remaining steps were the same as in Example 1.

[0039] Comparative Example 4 Ultrasonic pretreatment was eliminated, and the raw materials were directly crushed by high-temperature stirring. All purification processes were carried out after enzymatic hydrolysis, and non-selective adsorption purification was used. The remaining steps were the same as in Example 1.

[0040] Based on Examples 1-3 and Comparative Examples 1-4 provided above, the products prepared therefrom were subjected to performance testing and experimental verification, specifically including: (1) In vitro cell experiments (human skin fibroblasts HSF cells) To investigate the bioactivity of collagen peptides prepared by different processes, this experiment used human skin fibroblasts for in vitro culture to verify the effects of various samples on fibroblast proliferation and endogenous collagen synthesis. During the experiment, the cultured human skin fibroblasts were divided into several experimental groups: a blank control group, three sample groups from the embodiments of this invention, and four comparative sample groups. All groups maintained a consistent cell culture environment and were supplemented with the same mass concentration of collagen peptides, and cultured continuously at a constant temperature for 48 hours. After culture, the content of type I collagen in the cells was quantitatively detected using an enzyme-linked immunosorbent assay (ELISA) kit. Simultaneously, cell proliferation activity was measured using the CCK-8 assay, and the absorption and utilization rate of collagen peptide fragments by the cells was statistically calculated. Furthermore, the relative expression level of the key collagen synthesis gene COL1A1 was detected using real-time quantitative polymerase chain reaction (qPCR), and the total antioxidant capacity in the cells was measured using the ferric reduction / antioxidant capacity (FRAP) method, thus characterizing the in vitro bioactivity of collagen peptides from multiple dimensions.

[0041] The ferric reducing / antioxidant capacity (FRAP) method was used to determine the FRAP, including: collecting HSF cells cultured for 48 h from each group, adding lysis buffer (containing PMSF protease inhibitor), lysing on ice for 30 min, centrifuging at 12000 rpm for 10 min at 4 °C, and collecting the supernatant. The FRAP working solution was prepared by mixing 300 mmol / L acetate buffer (pH 3.6), 10 mmol / L TPTZ solution, and 20 mmol / L FFeCl3 solution in a 10:1:1 ratio. 10 μL of the test sample was mixed with 300 μL of the FRAP working solution, reacted in a 37 °C water bath for 10 min, and the absorbance was measured at 593 nm. Quantification was performed using the FeSO4·7H2O standard curve, and the results are expressed as mmol / g prot. Each group had three replicates, and the experiment was repeated three times. The baseline value of the blank control group was 0.820 mmol / g prot, which was obtained through parallel measurements in this experiment. Increase (%) = (Experimental group value - Blank control group value) / Blank control group value × 100%.

[0042] To clearly and intuitively demonstrate the differences in in vitro detection among the samples, the key indicators of cell experiments are summarized in Table 1 below.

[0043] Table 1. Results of in vitro cell experiments

[0044] Table 1, based on the experimental results, shows that the blank control group cells maintained only a basic normal proliferation state, with a cell proliferation rate of 100.00%, a basic intracellular type I collagen content of 82.36 ng / mL, and a relative expression level of the COL1A1 gene of 1.00. Combined with... Figure 2 The comparison curves of type I collagen content and the experimental results shown demonstrate that the samples prepared in this invention effectively promoted fibroblast growth and proliferation, induced collagen synthesis, and significantly enhanced the total antioxidant capacity of cells. Example 1 exhibited the best overall effect, with a cell proliferation rate of 124.62%, an intracellular type I collagen content of 131.57 ng / mL, a cell absorption and utilization rate of active peptides reaching 85.21%, an upregulation of the relative expression level of the COL1A1 gene to 1.48, and an increase in total antioxidant capacity to 1.045 mmol / g prot, representing an increase of 27.44%.

[0045] The sample detection data of Example 2 and Example 3 were similar, with cell proliferation rates of 118.35% and 121.74%, respectively, peptide uptake rates maintained within the range of 82% to 84%, and total cellular antioxidant capacity of 1.028 mmol / g prot and 1.038 mmol / g prot, respectively, with increases of 25.37% and 26.59%, respectively, and stable activity.

[0046] In contrast, the cell proliferation rates of comparative samples 1 to 4 were all between 107% and 113%, the type I collagen content was all below 110 ng / mL, the peptide absorption and utilization rate was generally below 78%, the upregulation of the COL1A1 gene was less than 1.20, and the total antioxidant capacity of comparative samples 1 to 4 ranged from 0.898 to 0.942 mmol / g prot, with an increase of only 9.51% to 14.88%. The antioxidant improvement effect was significantly weaker than that of the embodiments of the present invention, indicating that the collagen peptides prepared by the present invention can more effectively retain the antioxidant active groups of collagen peptides, and its cell protection effect is significant.

[0047] Comprehensive analysis of in vitro cell experiments confirms that this invention can preserve the bioactivity of collagen peptides to the greatest extent and ensure the cellular permeability and absorption capacity of peptide segments. In contrast, traditional processing methods such as continuous ultrasound at a single frequency, high-temperature pretreatment, and single-protease hydrolysis all damage the spatial structure and active sites of collagen peptides to varying degrees, reducing the sample's ability to promote cell proliferation and induce collagen synthesis.

[0048] (2) Mouse animal experiments To further verify the effects of the collagen peptides of this invention on organisms, a skin aging animal model was constructed using healthy SPF-grade mice. The effects of the samples on improving the body's skin collagen synthesis capacity, moisturizing capacity, and antioxidant capacity were comprehensively evaluated.

[0049] The experimental mice were randomly divided into a blank control group, an aging model group, a sample administration group of Example 1, and a sample administration group of Comparative Example 2. Ten experimental mice were set in each group. Except for the mice in the blank control group, which were fed normally without treatment, the mice in the other groups were subjected to continuous irradiation with medium-wave ultraviolet light to construct a skin photoaging model.

[0050] After modeling, mice in the drug-treated group were administered collagen peptide samples (200 mg / kg body weight) by gavage daily at regular intervals. The blank control group and the model group were fed the same dose of physiological saline. This intervention was continued for 45 days. At the end of the experimental period, skin tissue from the backs of the mice was dissected, and the following indicators were measured: hydroxyproline content (reflecting collagen levels) was detected using the alkaline hydrolysis method; total collagen content was detected using the BCA protein quantification method; superoxide dismutase (SOD) activity was detected using the WST-1 method; skin moisture content was determined using the tissue drying method; and epidermal thickness was observed by paraffin sectioning and H&E staining. All tests were performed in triplicate, and data are expressed as mean ± standard deviation.

[0051] The original test values ​​(mean values) of various indicators of skin tissue in each group of mice are shown in Table 2 below. Among them, the unit of hydroxyproline content is μg / mg tissue wet weight, the unit of total collagen content is mg / g tissue wet weight, the unit of SOD activity is U / mgprot, and the unit of skin moisture content is %. Improvement rate (%) = (value of drug treatment group - value of aging model group) / value of aging model group × 100%.

[0052] To visually compare the differences in skin test results among the different groups of mice, the core detection indicators of the animal experiments are summarized in Table 2 below.

[0053] Table 2 Results of Skin Tissue Detection in Animal Experiments

[0054] Table 2, based on the results of the animal experiments, shows that the mice in the model group treated with ultraviolet light exhibited obvious signs of aging. Compared with the blank control group, their hydroxyproline content decreased by 46.1%, total collagen content decreased by 38.1%, SOD activity decreased by 40.0%, skin moisture content decreased by 38.0%, and the epidermis became significantly thinner and looser in structure.

[0055] Compared to the aging model group, the skin physiological state of mice in the treatment group of Example 1 was significantly improved: such as Figure 3 As shown, hydroxyproline content increased by 22.33% (from 2.06 μg / mg to 2.52 μg / mg); total collagen content increased by 19.71% (from 42.37 mg / g to 50.72 mg / g); SOD activity increased by 16.43% (from 51.24 U / mg prot to 59.66 U / mg prot); skin moisture content increased by 17.53% (from 38.56% to 45.32%); and the epidermis thickened uniformly, significantly improving the integrity of the skin barrier.

[0056] The comparative example 2 sample, prepared using a traditional high-temperature pretreatment combined with a post-deodorization process, showed a significantly weaker improvement in various detection indicators compared to Example 1. Figure 3 As shown, hydroxyproline increased by 10.19%; total collagen increased by 8.95%; SOD activity increased by 7.63%; skin moisture content increased by 8.27%; and the epidermis only thickened slightly, indicating limited improvement.

[0057] Furthermore, animal experiments have confirmed that the fresh collagen peptides prepared in this invention can effectively delay skin aging damage caused by ultraviolet radiation, inhibit the loss of collagen within the skin, and enhance the skin's own antioxidant capacity. The gentle pre-treatment process and low-temperature physical pretreatment employed in this invention avoid damage to active peptides caused by high-temperature processing, ensuring the efficacy of collagen peptides in vivo. Compared to collagen peptides prepared through traditional high-temperature processing, this invention exhibits superior in vivo nourishing effects.

[0058] (3) Human oral clinical trials To evaluate the actual skin care effects of the collagen peptides of this invention after human consumption, this study selected 90 healthy subjects aged 25-45 years with skin problems such as dull skin, weak skin elasticity, and slight fine lines to conduct a human oral test.

[0059] All subjects were randomly and equally divided into a placebo group, an experimental group, and a control group, with 30 subjects in each group. Subjects in the experimental group took a fixed amount of highly active fresh collagen peptide powder prepared according to Example 1 of this invention orally every day. Subjects in the control group took conventional collagen peptides available on the market. Subjects in the placebo group took an equal amount of placebo (maltodextrin, which has the same appearance and taste as collagen peptide powder) orally every day. The daily dosage of the three groups was the same (5g / day). The trial period was set to be eight weeks. During the trial, subjects maintained a regular schedule and diet and did not take any other skin care or health care products.

[0060] Before the start of the trial (week 0) and after the end of the trial (week 8), non-invasive skin analysis was performed on the subjects' facial skin using a professional skin analysis instrument. Key indicators included: Skin tone radiance: The visual simulation scoring method is used (0~10 points, the higher the score, the better the radiance). Stratum corneum moisture content (%): determined by a skin moisture meter; Skin firmness (F4 value, in mm): The smaller the value, the firmer the skin. Skin elasticity (R2 value): The closer to 1, the better the elasticity; Fine line improvement: Visual simulation scoring method (0~10 points, the higher the score, the fewer fine lines); Pore ​​fineness: Visual simulation scoring method (0~10 points, the higher the score, the finer the pores). Skin brightness L* value: measured with a colorimeter.

[0061] All tests were performed by the same operator under the same environmental conditions. The average value of each set of data was taken, and the results are as follows: Skin radiance (score): Before the trial, the baseline values ​​of the three groups were similar, with an average of 4.50. After eight weeks, the placebo group scored 4.63 (an increase of 0.13 points, or 2.9%), the control group scored 4.98 (an increase of 0.48 points, or 10.7%), and the experimental group scored 6.08 (an increase of 1.58 points, or 35.1%).

[0062] Stratum corneum moisture content (%): The baseline average was 30.00%. After eight weeks, the placebo group had 30.90% (an increase of 3.0%), the control group had 33.60% (an increase of 12.0%), and the experimental group had 38.40% (an increase of 28.0%).

[0063] Skin firmness (F4 value, mm): A lower F4 value indicates firmer skin. The baseline average value was 3.00 mm. After eight weeks, the placebo group had a firmness of 2.94 mm (2.0% improvement); the control group had a firmness of 2.67 mm (11.0% improvement); and the experimental group had a firmness of 2.28 mm (24.0% improvement). Improvement margin = (baseline value - post-experiment value) / baseline value × 100%.

[0064] Skin elasticity (R2 value): The baseline mean value was 0.650. After eight weeks, the placebo group had a value of 0.660 (an improvement of 1.5%), the control group had a value of 0.715 (an improvement of 10.0%), and the experimental group had a value of 0.793 (an improvement of 22.0%).

[0065] Fine line improvement score (points): The baseline average score was 4.00. After eight weeks, the placebo group scored 4.10 (an increase of 0.10 points, or 2.5%), the control group scored 4.48 (an increase of 0.48 points, or 12.0%), and the experimental group scored 5.20 (an increase of 1.20 points, or 30.0%).

[0066] Pore ​​refinement score (points): The baseline average score was 4.00. After eight weeks, the placebo group scored 4.12 (an increase of 0.12 points, or 3.0%), the control group scored 4.48 (an increase of 0.48 points, or 12.0%), and the experimental group scored 5.00 (an increase of 1.00 point, or 25.0%).

[0067] Skin brightness L* value: The baseline value was 55.00. After eight weeks, the placebo group had a value of 55.50 (an improvement of 0.9%), the control group had a value of 58.30 (an improvement of 6.0%), and the experimental group had a value of 63.80 (an improvement of 16.0%).

[0068] Combination Figure 4As shown in the bar chart illustrating the improvement in skin elasticity and the original data above, the placebo group showed improvement of less than 3% in all indicators, indicating that natural skin improvement without intervention was extremely limited. In contrast, the control group, after taking commercially available ordinary collagen peptides, showed improvement of 6%–12% in all indicators, demonstrating a certain degree of improvement. After the experimental group took the highly active fresh collagen peptides prepared in Example 1 of this invention, all skin indicators showed significant improvement: skin radiance increased by 35.1% (from 4.50 to 6.08), stratum corneum moisture content increased by 28.0% (from 30.00% to 38.40%), skin firmness improved by 24.0% (F4 value decreased from 3.00mm to 2.28mm), skin elasticity increased by 22.0% (from 0.650 to 0.793), fine line improvement score increased by 30.0% (from 4.00 to 5.20), pore fineness score increased by 25.0% (from 4.00 to 5.00), and skin brightness L* value increased by 16.0% (from 55.00 to 63.80). All improvements were significantly better than those in the control group (p<0.01).

[0069] To visually quantify the differences in skin improvement among human patients, the key indicators of human clinical trials are summarized in Table 3 below.

[0070] In addition, no adverse reactions such as gastrointestinal discomfort or skin allergies occurred in any of the subjects during the trial period, and the experimental group samples had no fishy or odor and were palatable.

[0071] Table 3. Results of skin quality testing in human clinical trials.

[0072] Based on the results of human clinical trials, it has been confirmed that the fresh collagen peptides prepared in this invention have good absorption and utilization rates in the human body. Long-term oral supplementation can effectively increase the collagen content in the dermis, improve skin problems such as sagging, fine lines, and enlarged pores, and optimize the skin's translucency and radiance. Simultaneously, the pre-treatment bio-enzymatic deodorization process effectively removes the fishy smell of the raw materials, improving the product's palatability and making it suitable for long-term continuous use. Compared with commercially available ordinary collagen peptides, the product of this invention shows significant advantages in various skincare indicators, and demonstrates significant efficacy compared to the placebo group.

[0073] Based on the results of the above in vitro cell experiments, in vivo animal experiments, and human oral clinical trials, it is clear that the preparation method of this invention, which involves dual-frequency alternating pulse ultrasound pretreatment, directional enzymatic hydrolysis with endonuclease and exonuclease complex enzymes, pre-treatment with biological enzymes to remove odor, and multi-stage membrane separation and purification, can perfectly preserve the natural triple helix structure of collagen during processing and reduce the loss of active peptides and damage to active sites.

[0074] Furthermore, the fresh collagen peptides prepared by this invention exhibit stable physicochemical properties and good permeability and absorption capacity. They demonstrate stable and excellent collagen-promoting ability and skin-care effects in cell, animal, and human trials. Simultaneously, the preparation method is simple and controllable, with mild processing conditions, high raw material utilization, and moderate production costs, making it suitable for continuous large-scale industrial production. This solves the technical problems commonly found in traditional collagen peptide preparation processes, such as high yields but difficulty in maintaining quality, low product bioactivity, and poor flavor and taste. Therefore, it has promising application prospects and promotional value in the fields of skincare, health care, and functional foods.

[0075] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0076] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing highly active fresh collagen peptides by ultrasound-assisted enzymatic hydrolysis, characterized in that, Includes the following steps: Animal skin, bone or fish scale collagen raw materials are crushed and mixed with purified water to make a homogenate. Then, flavor protease is added and stirred at a constant temperature to obtain a deodorized and modified homogenate. The obtained deodorized modified homogenate was placed in an ultrasonic reactor and treated with dual-frequency alternating pulse ultrasonication combined with gradient temperature program to obtain a loose modified gum raw material solution. A complex protease composed of endopeptide and exopeptide was added to the loose modified collagen raw material solution after the temperature was stabilized. The solution was then stirred at a constant temperature to carry out gentle and directional enzymatic hydrolysis, resulting in crude collagen peptide hydrolysate. The crude enzymatic hydrolysate of collagen peptides was subjected to high-temperature enzyme inactivation treatment, and then purified by sieve filtration, microfiltration and ultrafiltration to obtain a refined peptide solution. The refined peptide solution was then concentrated under vacuum and dried to finally obtain highly active fresh collagen peptide powder.

2. The preparation method according to claim 1, characterized in that, The process of pulverizing animal skin, bone, or fish scale collagen raw materials and mixing them with purified water to form a homogenate, followed by adding flavor protease and stirring at a constant temperature to obtain a deodorizing modified homogenate, is as follows: Clean collagen raw materials are pulverized to a particle size of 1-5 mm, and the pulverized raw materials are mixed with purified water at a mass ratio of 1:8-1:15 to form a homogenate; 0.1%-0.5% of the total mass of flavor protease is added to the homogenate, and the mixture is stirred at a constant temperature of 20-30℃, pH 6.0-7.0, and stirring speed of 50-100 rpm for 30-90 minutes to pre-hydrolyze and remove the fishy odor precursor proteins and lipid oxidation products from the surface and gaps of the raw materials, thus obtaining the deodorizing modified homogenate.

3. The preparation method according to claim 1, characterized in that, The process of placing the obtained deodorized and modified homogenized slurry in an ultrasonic reactor and treating it with dual-frequency alternating pulse ultrasonication combined with gradient programmed heating to obtain a loose modified rubber raw material solution is as follows: the ultrasonic low frequency is set to 20kHz and the high frequency to 40kHz, the ultrasonic power density is controlled to be 150~250W / L, the total ultrasonic treatment time is 15~25min, and the temperature of the material solution is maintained not higher than 35℃ throughout the process; after the ultrasonic treatment, the material solution is heated to 48~52℃ at a rate of 0.8~1.2℃ / min to obtain a loose modified rubber raw material solution.

4. The preparation method according to claim 3, characterized in that, The dual-frequency alternating pulse ultrasound operates in a low-frequency and high-frequency alternating mode. Each frequency operates in a pulse cycle mode of 2 seconds of operation followed by a 4-second interval. After completing a single-frequency pulse cycle, it switches to another frequency for pulse operation.

5. The preparation method according to claim 1, characterized in that, The process of adding a complex protease composed of endopeptides and exopeptides to the loose modified gum raw material solution after temperature stabilization, and performing gentle and directional enzymatic hydrolysis at a constant temperature to obtain crude collagen peptide hydrolysate is as follows: The complex protease, which is prepared by mixing endopeptides and exopeptides at a mass ratio of 3:1 to 5:1, is added to the loose modified gum raw material solution at a constant temperature of 48~52℃; under gentle conditions of pH 6.5~7.5 and stirring speed of 80~120rpm, the enzymatic hydrolysis is carried out at a constant temperature for 3~5h. The endopeptides break the collagen backbone and the exopeptides modify the peptide chain ends, while simultaneously removing bitter substances, to obtain crude collagen peptide hydrolysate.

6. The preparation method according to claim 5, characterized in that, The endopeptide is selected from at least one of neutral protease or alkaline protease, and the exopeptide is a flavor protease; the total amount of the complex protease added is 0.8% to 1.5% of the dry weight of the collagen raw material.

7. The preparation method according to claim 1, characterized in that, The process of subjecting the crude enzymatic hydrolysate of collagen peptides to high-temperature enzyme inactivation treatment, followed by sequential filtration through a sieve, microfiltration, and ultrafiltration to obtain a refined peptide solution, and then vacuum concentrating and drying the refined peptide solution to finally obtain highly active fresh collagen peptide powder is as follows: After enzymatic hydrolysis, the temperature is raised to 85~90℃ and held for 10~15 minutes to complete enzyme inactivation; insoluble residues are removed by filtration through a 100~200 mesh sieve; the filtrate is then sequentially treated through a 0.1~0.22μm microfiltration membrane and an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and the ultrafiltration permeate is collected as the refined peptide solution; the refined peptide solution is vacuum concentrated at ≤50℃ and a vacuum degree ≥-0.08MPa to a solid content of 25%~35%, and then spray dried to obtain collagen peptide powder.

8. The preparation method according to claim 7, characterized in that, The spray drying process parameters are: inlet air temperature 160~175℃, outlet air temperature 75~85℃.

9. A highly active fresh collagen peptide prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the highly active fresh collagen peptide according to claim 9 in promoting collagen production, characterized in that, The highly active fresh collagen peptides are adaptable to any application form, either in powder or liquid form, to induce and enhance the expression of collagen in the body.

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