Andrias davidianus skin collagen peptide, and preparation method and application thereof

Abstract

This application belongs to the field of biotechnology, specifically relating to giant salamander skin collagen peptides, their preparation methods, and applications. This application provides a method for preparing giant salamander skin collagen peptides. The method first pre-treats the fish skin, then performs ultrasonic-assisted extraction under acidic conditions to effectively promote collagen dissolution. The extract undergoes flocculation to remove impurities, significantly reducing interference from other proteins. The enzymatic hydrolysis stage employs a stepwise enzymatic hydrolysis strategy: first, a combination of alkaline protease and another specific protease is used for hydrolysis, sequentially cleaving the complex structure of giant salamander skin collagen to improve initial hydrolysis efficiency and generate specific intermediate peptides; subsequently, a flavor protease is added for further deep hydrolysis to achieve complete hydrolysis. This method has high enzymatic hydrolysis efficiency, controllable hydrolysis degree, concentrated molecular weight distribution of the obtained collagen peptides, high yield of the target product, and stable process.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to giant salamander skin collagen peptides, their preparation methods, and applications. Background Technology

[0002] Collagen peptides, as important bioactive substances, have wide applications in the food, cosmetics, and pharmaceutical fields. Giant salamander skin is rich in high-quality collagen, making it an ideal raw material for preparing collagen peptides. However, existing methods for preparing giant salamander skin collagen peptides suffer from problems such as incomplete removal of impurities, low enzymatic hydrolysis efficiency, and low product purity, which seriously affect the product's quality and application value.

[0003] Currently, the preparation of collagen peptides from giant salamander skin faces several technical bottlenecks, including insufficient pretreatment, low collagen extraction efficiency, limited enzymatic hydrolysis processes, and complex separation and purification. Traditional methods often employ a single enzyme preparation for hydrolysis and lack effective means of removing impurities, resulting in high impurity content, uneven molecular weight distribution, and limited bioactivity in the product, making it difficult to meet the requirements for high-quality products.

[0004] The problems of incomplete removal of impurities and low enzymatic hydrolysis efficiency in the preparation of giant salamander skin collagen peptides urgently need to be solved. The presence of impurities not only affects the purity and bioactivity of the product, but also reduces the enzymatic hydrolysis efficiency, resulting in an uneven molecular weight distribution of the final product, making it difficult to obtain high-quality collagen peptide products with low molecular weight and high bioavailability. Summary of the Invention

[0005] Based on this, one embodiment of this application provides giant salamander skin collagen peptides, their preparation method, and their applications.

[0006] This application provides a method for preparing giant salamander skin collagen peptides, comprising:

[0007] Giant salamander skin is provided, and the giant salamander skin is pretreated;

[0008] The pretreated giant salamander skin was placed in an acidic extract and extracted under ultrasonic conditions; the extract was then collected.

[0009] After salting out and dialysis, the extract was subjected to a first collagen solution.

[0010] The first collagen solution is subjected to flocculation and impurity removal treatment to prepare a second collagen solution;

[0011] The second collagen solution was subjected to stepwise enzymatic hydrolysis to prepare enzymatic hydrolysate; and,

[0012] The enzymatic hydrolysate was purified to prepare giant salamander skin collagen peptides;

[0013] The stepwise enzymatic hydrolysis process includes: mixing and hydrolyzing with a first protease and a second protease, and then adding a third protease to continue the enzymatic hydrolysis;

[0014] The first protease includes an alkaline protease;

[0015] The second protease includes one of neutral protease, papain, and trypsin;

[0016] The third protease includes flavor protease.

[0017] In some embodiments, the weight of the first protease added is 0.5%-1% of the weight of the pretreated giant salamander skin.

[0018] In some embodiments, the weight of the second protease added is 0.05%-0.1% of the weight of the pretreated giant salamander skin.

[0019] In some embodiments, the added weight of the third protease is 0.005%-0.05% of the weight of the pretreated giant salamander skin.

[0020] In some embodiments, the stepwise enzymatic hydrolysis process includes:

[0021] The first and second proteases are added for the first enzymatic hydrolysis step; and the third protease is added for the second enzymatic hydrolysis step.

[0022] The conditions for the first step of enzymatic hydrolysis include: temperature of 54℃-56℃, pH of 8.4-8.6, and time of 3.5 h-5.5 h.

[0023] The conditions for the second step of enzymatic hydrolysis include: a temperature of 54℃-56℃, a pH of 7.4-7.6, and a time of 2.5 h-3.5 h.

[0024] In some embodiments, the pretreatment includes: slicing the giant salamander skin, removing fat with a n-butanol solution, and then removing pigment and non-collagen components with a strongly alkaline solution.

[0025] In some embodiments, the pretreatment conditions include: the concentration of n-butanol in the n-butanol solution is 8 w / v%-12 w / v.

[0026] In some embodiments, the pretreatment conditions include: the strongly alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution, and the concentration of sodium hydroxide in the strongly alkaline solution is 0.05 mol / L - 0.2 mol / L.

[0027] In some embodiments, the acidic extract includes hydrochloric acid or acetic acid.

[0028] In some embodiments, the ultrasound conditions include a frequency of 20 kHz to 40 kHz, a duration of 30 min to 60 min, and an interval of 10 h to 12 h.

[0029] In some embodiments, the flocculation and impurity removal process uses phosphoric acid and calcium hydroxide for flocculation reaction.

[0030] In some embodiments, the purification process includes removing macromolecules using macroporous adsorption resins, followed by removal of free amino acids and sodium chloride via nanofiltration membranes.

[0031] In some embodiments, the concentration of the acidic extract is 0.3 mol / L to 0.8 mol / L.

[0032] In some embodiments, the macroporous adsorption resin is SD300 type macroporous adsorption resin, and the influent liquid flow rate is 4 BV / h - 6 BV / h.

[0033] In some embodiments, the nanofiltration membrane has a molecular weight cutoff of ≤200 Da. In some embodiments, the conditions for the flocculation reaction using phosphoric acid and calcium hydroxide include a mass ratio of phosphoric acid to calcium hydroxide of 1:(1.5-2).

[0034] In some embodiments, the conditions for using phosphoric acid and calcium hydroxide for flocculation include: the weight of added phosphoric acid is 1%-2% of the weight of the pretreated giant salamander skin.

[0035] In some embodiments, the conditions for flocculation reaction using phosphoric acid and calcium hydroxide include: the added weight of calcium hydroxide is 1.5%-2% of the weight of the pretreated giant salamander skin.

[0036] Another aspect of this application provides a method for preparing the giant salamander skin collagen peptides.

[0037] This application also provides the use of the aforementioned giant salamander skin collagen peptides in the preparation of products that promote skin cell proliferation and / or collagen synthesis.

[0038] This application provides a method for preparing collagen peptides from giant salamander skin. The method first pre-treats the fish skin, followed by ultrasonic-assisted extraction under acidic conditions to effectively promote collagen dissolution. The extract is then subjected to flocculation to remove impurities, significantly reducing interference from other proteins. The enzymatic hydrolysis stage employs a stepwise enzymatic hydrolysis strategy: first, a combination of an alkaline protease and another specific protease (one of a neutral protease, papain, or trypsin) is used for hydrolysis, sequentially cleaving the complex structure of giant salamander skin collagen to improve initial hydrolysis efficiency and generate specific intermediate peptides; subsequently, a flavor protease is added for further deep hydrolysis to achieve complete hydrolysis. This method offers high enzymatic hydrolysis efficiency, controllable hydrolysis degree, concentrated molecular weight distribution of the obtained collagen peptides, high yield of the target product, and stable process, making it suitable for large-scale industrial production. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 The proliferation rate of HSF fibroblasts was detected after 12 hours of treatment with giant salamander skin collagen peptides provided in an embodiment of this application.

[0041] Figure 2 The scratch test results show the effect of giant salamander skin collagen peptides on cell repair provided in an embodiment of this application.

[0042] Figure 3 The results of the detection of the effect of giant salamander skin collagen peptides on intracellular ROS levels provided in an embodiment of this application;

[0043] Figure 4 This is a verification result of the effect of the giant salamander skin collagen peptide provided in one embodiment of this application on cellular collagen synthesis. Detailed Implementation

[0044] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0045] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0046] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0047] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0048] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0049] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0050] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0051] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0052] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0053] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0054] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0055] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0056] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0057] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0058] The term "giant salamander skin collagen peptide" refers to a mineral-rich peptide prepared from giant salamander skin through enzymatic hydrolysis and refining. 2+ A mixture of small molecule peptides. In this field, collagen peptides refer to peptides that have been degraded from large collagen molecules into smaller molecular weight fragments through enzymatic hydrolysis to improve their bioavailability and bioactivity. Giant salamander skin collagen peptides possess biological functions such as promoting skin cell proliferation and repair, anti-oxidation, and promoting collagen synthesis, and have broad application prospects in cosmetics, health products, and pharmaceuticals.

[0059] The term "phosphate-calcium hydroxide flocculation system" refers to a technique that utilizes the reaction of phosphoric acid and calcium hydroxide in a specific ratio to generate calcium phosphate precipitate, thereby removing impurities from a solution. In this field, flocculation refers to the process of adding a flocculant to aggregate colloidal particles or suspended matter into larger flocs, leading to precipitation and separation. The phosphate-calcium hydroxide flocculation system not only effectively removes impurities but also introduces calcium ions into the system. Calcium ions act as enzyme activity enhancers, significantly improving the efficiency of subsequent enzymatic hydrolysis.

[0060] The term "macroporous adsorption resin SD300" refers to a macroporous adsorption resin without active functional groups, primarily used in the field for the separation and purification of biomolecules. Macroporous adsorption resins are high-molecular-weight polymers with large pore sizes, capable of removing large molecular weight impurities from solution through physical adsorption. SD300 resin possesses suitable pore size distribution and surface properties, effectively adsorbing large-molecule collagen peptides while allowing smaller target peptides to pass through, thereby achieving separation and purification. Specific sub-concepts include influent flow rates of 4 BV / h, 5 BV / h, and 6 BV / h.

[0061] The term "nanofiltration membrane" refers to a separation membrane with a specific molecular weight cutoff, primarily used in this field for removing small molecule impurities and concentrating ions. Nanofiltration membranes are pressure-driven membranes with a molecular weight cutoff between 200 and 1000 Da, capable of separating different components through size exclusion and the Donnan effect. A 200 Da nanofiltration membrane can effectively remove free amino acids and monovalent sodium ions, while also exhibiting good retention of divalent calcium ions, thus achieving a low-sodium, high-calcium composition in the product.

[0062] This application provides a method for preparing giant salamander skin collagen peptides, comprising:

[0063] Giant salamander skin is provided, and the giant salamander skin is pretreated;

[0064] The pretreated giant salamander skin was placed in an acidic extract and extracted under ultrasonic conditions. The extract was collected. The extract was then subjected to salting out and dialysis to prepare a first collagen solution.

[0065] The first collagen solution is subjected to flocculation and impurity removal treatment to prepare a second collagen solution;

[0066] The second collagen solution was subjected to stepwise enzymatic hydrolysis to prepare enzymatic hydrolysate;

[0067] The enzymatic hydrolysate was purified to prepare giant salamander skin collagen peptides;

[0068] The stepwise enzymatic hydrolysis process includes: mixing and hydrolyzing with a first protease and a second protease, and then adding a third protease to continue the enzymatic hydrolysis;

[0069] The first protease includes an alkaline protease;

[0070] The second protease includes one of neutral protease, papain, and trypsin;

[0071] The third protease includes flavor protease.

[0072] The stepwise enzymatic hydrolysis process employs a mixture of alkaline protease and a second protease for hydrolysis, followed by the addition of flavor protease for further hydrolysis, achieving highly efficient degradation of collagen to obtain small peptides with uniform molecular weight distribution. The purification process utilizes a combination of macroporous adsorption resin and nanofiltration membrane to ensure product purity and molecular weight control. This technical solution solves the problems of incomplete removal of impurities and low enzymatic hydrolysis efficiency in traditional methods, producing giant salamander skin collagen peptides with high purity, low molecular weight, and excellent bioactivity.

[0073] In some embodiments, the weight of the first protease added is 0.5%-1% of the weight of the pretreated giant salamander skin. For example, the weight of the first protease added is 0.5%, 0.6%, 0.7%, or 0.8% of the weight of the pretreated giant salamander skin.

[0074] 0.9% or 1.0% and any value in between.

[0075] In some embodiments, the second protease is added at a weight of 0.05%-0.1% of the pretreated giant salamander skin. For example, the amount of the second protease added is 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, or 0.1% of the pretreated giant salamander skin, or any value in between.

[0076] In some embodiments, the added weight of the third protease is 0.005% of the weight of the pretreated giant salamander skin—for example, the added weight of the third protease is 0.005%, 0.015%, 0.025%, 0.035%, or 0.045% of the weight of the pretreated giant salamander skin, and any value in between.

[0077] In some embodiments, the stepwise enzymatic hydrolysis process includes:

[0078] The first and second proteases were added for the first enzymatic hydrolysis; and the third protease was added for the second enzymatic hydrolysis.

[0079] The conditions for the first enzymatic hydrolysis treatment included: a temperature of 54℃-56℃, a pH of 8.4-8.6, and a time of 3.5 h-5.5 h.

[0080] The conditions for the second enzymatic hydrolysis treatment included: a temperature of 54℃-56℃, a pH of 7.4-7.6, and a time of 2.5 h-3.5 h.

[0081] Specifically, the second collagen solution is first heated to 54℃-56℃, the pH is adjusted to 8.4-8.6, the first and second proteases are added, and enzymatic hydrolysis is carried out for 3.5 h-5.5 h; then the pH is adjusted to 7.4-7.6, the third protease is added, and enzymatic hydrolysis is continued for 2.5 h-3.5 h.

[0082] For example, the first enzymatic hydrolysis treatment can be heated to 54℃, 55℃, or 56℃, and the pH can be adjusted to 8.4, 8.5, or 8.6; the hydrolysis time can be 3.5 h, 3.6 h, 3.7 h, 3.8 h, 3.9 h, 4.0 h, 4.1 h, 4.2 h, 4.3 h, 4.4 h, 4.5 h, 4.6 h, 4.7 h, 4.8 h, 4.9 h, 5.0 h, 5.1 h, 5.2 h, 5.3 h, 5.4 h, or 5.5 h, or any value in between.

[0083] The second enzymatic hydrolysis treatment was performed at a temperature of 54℃, 55℃, or 56℃, a pH of 8.4, 8.5, or 8.6, and a hydrolysis time of 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3.0h, 3.1h, 3.2h, 3.3h, 3.4h, or 3.5h, or any value in between.

[0084] In some embodiments, the pretreatment includes: slicing the giant salamander skin, removing fat with a n-butanol solution, and then removing pigments and non-collagenous components with a strongly alkaline solution. This pretreatment step effectively removes fat, pigments, and non-collagenous components from the giant salamander skin, providing a pure raw material basis for subsequent extraction and purification, and reducing the interference of impurities on subsequent processes.

[0085] In some embodiments, the concentration of the n-butanol solution is 8 w / v%-12 w / v%. For example, concentrations of 8 w / v%, 9 w / v%, 10 w / v%, 11 w / v%, or 12 w / v%, and any value in between.

[0086] In some embodiments, the strongly alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution, and the concentration of the strongly alkaline solution is 0.05 mol / L - 0.2 mol / L. For example, the concentration is 0.05 mol / L, 0.10 mol / L, 0.15 mol / L, or 0.20 mol / L, or any value in between.

[0087] In some embodiments, the acid solution includes hydrochloric acid or acetic acid.

[0088] In some embodiments, the concentration of the acid solution is 0.3 mol / L to 0.8 mol / L. For example, the concentration of the acid solution is 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, or 0.8 mol / L, or any value in between.

[0089] In some embodiments, the ultrasonic treatment frequency is 20 kHz - 40 kHz, the duration is 30 min - 60 min, and the interval is 10 h - 12 h. For example, the frequency is 20 kHz, 22 kHz, 24 kHz, 26 kHz, 28 kHz, 30 kHz, 32 kHz, 34 kHz, 36 kHz, 38 kHz, or 40 kHz, and any value in between; the duration is 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, 42 min, 44 min, 46 min, 48 min, 50 min, 52 min, 54 min, 56 min, 58 min, or 60 min, and any value in between.

[0090] When extracting collagen using acidic solutions, this application employs ultrasonic-assisted treatment. Ultrasonic waves generate bubbles through alternating high and low pressures. The continuous formation, growth, and collapse of these bubbles increase mass transfer, mixing, particle breakage from large to small, surface area, extraction kinetics, and cavitation force dissolution rate in the reaction system, thus more effectively promoting the dissolution of collagen under acidic conditions.

[0091] In some embodiments, the flocculation and impurity removal process includes a flocculation reaction using phosphoric acid and calcium hydroxide. This application effectively removes impurities and proteins using a phosphoric acid-calcium hydroxide flocculation system, while simultaneously introducing calcium ions as an enzyme activity enhancer, significantly improving the efficiency of subsequent enzymatic hydrolysis.

[0092] In some embodiments, the mass ratio of phosphoric acid to calcium hydroxide is 1:(1.5-2). For example, ratios of 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.0, and any values ​​in between. This application uses a complex enzyme preparation to perform stepwise enzymatic hydrolysis of giant salamander skin collagen, and adds Ca, which has the function of enhancing and stabilizing enzyme activity. 2+ The overall enzymatic hydrolysis efficiency is higher, and the degree of enzymatic hydrolysis is more complete.

[0093] In some embodiments, the added phosphoric acid is 1%-2% of the weight of the pretreated giant salamander skin. For example, the added phosphoric acid is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% of the weight of the pretreated giant salamander skin, or any value in between.

[0094] In some embodiments, the added calcium hydroxide is 1.5%-2% of the weight of the pretreated giant salamander skin. For example, the added calcium hydroxide is 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% of the weight of the pretreated giant salamander skin, or any value in between.

[0095] In some embodiments, the purification process includes: removing macromolecular substances using macroporous adsorption resin, followed by removing free amino acids and sodium chloride via nanofiltration membrane.

[0096] In some embodiments, the macroporous adsorption resin is SD300 type macroporous adsorption resin, and the influent liquid flow rate is 4 BV / h - 6 BV / h.

[0097] In some embodiments, the nanofiltration membrane has a molecular weight cutoff of ≤200 Da.

[0098] This application utilizes column chromatography separation and purification technology to effectively remove residual, small amounts of high molecular weight substances from the enzymatic hydrolysis products. Combined with nanofiltration and other refining processes, this effectively reduces the sodium content of the product, removes free amino acids, and improves the quality of the final giant salamander skin collagen peptide product. Furthermore, the prepared giant salamander skin collagen peptide product is rich in calcium ions, which are beneficial to human health and can be used as a nutritional fortifier.

[0099] Another aspect of this application provides a method for preparing the giant salamander skin collagen peptides.

[0100] The weight-average molecular weight of this giant salamander skin collagen peptide is 800 Da-1000 Da, with a hydroxyproline content of ≥9.7%, a calcium ion content of ≥2000 mg / 100g, and a sodium ion content of ≤400 mg / 100g.

[0101] This giant salamander skin collagen peptide can promote fibroblast proliferation, achieving a cell proliferation rate of 1.26% at a concentration of 2 mg / mL. The giant salamander skin collagen peptide product prepared in this application is rich in calcium ions, which are beneficial to human health and can be used as a nutritional fortifier. Furthermore, calcium ions and the giant salamander skin collagen peptide can work synergistically to promote skin cell proliferation, repair, and collagen synthesis, resulting in strong cosmetic effects.

[0102] This application also provides the use of the aforementioned giant salamander skin collagen peptides in the preparation of products that promote skin cell proliferation and / or collagen synthesis.

[0103] The giant salamander skin collagen peptides described herein can reduce intracellular ROS levels and promote cellular collagen synthesis. This technical effect demonstrates that the product of this application possesses significant antioxidant activity and the ability to promote collagen synthesis, resisting skin oxidative damage, increasing collagen synthesis, restoring skin elasticity and radiance, reducing wrinkles, and achieving a significant anti-aging effect.

[0104] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0105] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0106] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0107] Example 1

[0108] This embodiment provides a method for preparing collagen peptides from giant salamander skin, including the following steps:

[0109] 1. Remove fat: Cut the cleaned giant salamander skin (the skin of artificially raised giant salamanders) into 0.5cm×0.5cm pieces, add 10 times its weight of 10% n-butanol solution, stir the mixture continuously at 4℃ for 24 hours, change the solution every 8 hours to remove fat components, and finally rinse with deionized water until neutral.

[0110] 2. Removal of pigments and non-collagenous components: Soak the fish in 15 times its weight of alkaline solution at room temperature for 24 hours, changing the alkaline solution every 8 hours to thoroughly remove pigments, black membranes, and non-collagenous components naturally present in the giant salamander skin. After soaking, rinse with deionized water until the conductivity of the effluent is below 1000 μS / cm. The alkaline solution is a sodium hydroxide solution with a mass concentration of 0.1 mol / L.

[0111] 3. Extraction of acid-soluble collagen: Ten times the weight of acid solution was added to the pretreated giant salamander skin and swollen at 4℃ for 48 hours until the tissue was fragmented. During this period, ultrasonic treatment was performed every 12 hours at a frequency of 20 kHz for 40 minutes. After ultrasonic treatment and acid extraction, the mixture was filtered to obtain a filtrate, which was then precipitated with 2.6 mol / L sodium chloride at 4℃ for 48 hours. The precipitate was centrifuged at 18000×g for 30 minutes (4℃), and the precipitate was dissolved in 0.1 mol / L acetic acid. Dialysis was performed with 0.1 mol / L acetic acid and deionized water (molecular weight cutoff 25 kDa). The resulting dialysate was freeze-dried to obtain giant salamander skin collagen. The acid solution was acetic acid solution with a mass concentration of 0.75 mol / L.

[0112] 4. Flocculation to remove impurities: Add the giant salamander skin collagen to deionized water and dissolve it completely at a mass ratio of 1:20. At room temperature, add 1% phosphoric acid and 1.5% calcium hydroxide by weight of the pretreated giant salamander skin, with a mass ratio of 1:1.5. Stir and flocculate for 30 minutes, then let it stand for 1 hour. Filter the solution through a 0.45μm membrane to obtain the collagen solution, and then adjust the pH to neutral with an appropriate amount of hydrochloric acid.

[0113] 5. Stepwise enzymatic hydrolysis: The collagen solution was heated to 55℃, and the pH was adjusted to 8.5 with alkali. 0.5% (by weight) of alkaline protease and 0.1% (by weight) of neutral protease preparations from the pretreated giant salamander skin were added separately, and enzymatic hydrolysis was carried out for 4 hours. Then, the pH was adjusted to 7.5, and 0.05% (by weight) of flavor protease from the pretreated giant salamander skin was added, and enzymatic hydrolysis continued for 3 hours.

[0114] 6. Enzyme inactivation: After the enzymatic hydrolysis is completed, heat the hydrolysate to 85℃ and keep it warm for 20 min.

[0115] 7. Decolorization and deodorization: Centrifuge the enzymatic hydrolysate (5000 r / m for 15 min) to remove residual large insoluble particles. Add 6% of the weight of the pretreated giant salamander skin to the prepared supernatant and keep it at 65℃ for 40 min.

[0116] 8. Solid-liquid separation: After the heat preservation is completed, diatomaceous earth is added to the liquid as a filter aid to separate the enzymatic hydrolysate from the activated carbon. Then, a ceramic membrane (50nm) is used for further solid-liquid separation to achieve a clear and transparent enzymatic hydrolysate.

[0117] 9. Purification and separation: The obtained enzymatic hydrolysate is passed through macroporous adsorption resin SD300 (without active functional groups) and the influent flow rate is controlled at 4 BV / h to further remove the collagen peptides that have not been fully hydrolyzed and have a relatively large molecular weight.

[0118] 10. Nanofiltration: The obtained feed solution is passed through a 200 Da nanofiltration membrane to remove free amino acids and sodium chloride, thereby purifying the sample. At the same time, the feed solution is pre-concentrated, and the solid content reaches 15-20%.

[0119] 11. Sterilization: The prepared liquid is sterilized using a 0.22μm sterilization membrane.

[0120] 12. Drying: The sterilized liquid material is subjected to vacuum freeze drying (-60℃, 20Pa) for 48 hours to obtain giant salamander skin collagen peptide powder.

[0121] Example 2

[0122] This embodiment provides a method for preparing collagen peptides from giant salamander skin, including the following steps:

[0123] 1. Remove fat: Cut the cleaned giant salamander skin into 1cm×1cm pieces, add 10 times its weight of 10% n-butanol solution, stir the mixture continuously at 4℃ for 24 hours, change the solution every 8 hours to remove fat components, and finally rinse with deionized water until neutral.

[0124] 2. Removal of pigments and non-collagenous components: Soak the fish in 15 times its weight of alkaline solution at room temperature for 24 hours, changing the alkaline solution every 8 hours to thoroughly remove pigments, black membranes, and non-collagenous components naturally present in the giant salamander skin. After soaking, rinse with deionized water until the conductivity of the effluent is below 1000 μS / cm. The alkaline solution is potassium hydroxide solution with a mass concentration of 0.1 mol / L.

[0125] 3. Extraction of acid-soluble collagen: Pretreated giant salamander skin was added to a 10-fold volume of acid solution and swollen at 4°C for 48 hours until the tissue was fragmented. During this period, it was treated with ultrasound every 12 hours at a frequency of 40 kHz for 30 minutes. After ultrasound treatment and acid extraction, the mixture was filtered to obtain a filtrate, which was then precipitated with 2.6 mol / L sodium chloride at 4°C for 48 hours. The precipitate was centrifuged at 18000×g for 30 minutes (4°C), and the precipitate was dissolved in 0.1 mol / L acetic acid. Dialysis was performed with 0.1 mol / L acetic acid and deionized water (molecular weight cutoff 25 kDa). The dialysate was freeze-dried to obtain giant salamander skin collagen. The acid solution was hydrochloric acid with a mass concentration of 0.3 mol / L.

[0126] 4. Flocculation to remove impurities: Add the giant salamander skin collagen to deionized water and dissolve it completely at a mass ratio of 1:20. At room temperature, add 1% phosphoric acid and 1.5% calcium hydroxide by weight of the pretreated giant salamander skin, with a mass ratio of 1:1.5. Stir and flocculate for 30 minutes, then let it stand for 1 hour. Filter the solution through a 0.45μm membrane to obtain the collagen solution, and then adjust the pH to neutral with an appropriate amount of hydrochloric acid.

[0127] 5. Stepwise enzymatic hydrolysis: Heat the collagen solution to 55℃, adjust the pH to 8.5 with alkali, add 0.5% alkaline protease and 0.1% papain enzyme preparation (based on the weight of pretreated giant salamander skin), and hydrolyze for 4 hours. Then adjust the pH to 7.5, add 0.05% flavor protease (based on the weight of pretreated giant salamander skin), and continue enzymatic hydrolysis for 3 hours.

[0128] 6. Enzyme inactivation: After the enzymatic hydrolysis is completed, heat the hydrolysate to 85℃ and keep it warm for 20 min.

[0129] 7. Decolorization and deodorization: Centrifuge the enzymatic hydrolysate (5000 r / m for 15 min) to remove any remaining large insoluble particles. Add 6% of the weight of the pretreated giant salamander skin to the prepared supernatant and keep it at 65℃ for 40 min.

[0130] 8. Solid-liquid separation: After the heat preservation is completed, diatomaceous earth is added to the liquid as a filter aid to separate the enzymatic hydrolysate from the activated carbon. Then, a ceramic membrane (50nm) is used for further solid-liquid separation to achieve a clear and transparent enzymatic hydrolysate.

[0131] 9. Purification and separation: The obtained enzymatic hydrolysate is passed through macroporous adsorption resin SD300 (without active functional groups) with the influent flow rate controlled at 6 BV / h to further remove unhydrolyzed and residual collagen peptides with relatively large molecular weights.

[0132] 10. Nanofiltration: The obtained feed solution is passed through a 200 Da nanofiltration membrane to remove free amino acids and sodium chloride, thereby purifying the sample. At the same time, the feed solution is pre-concentrated, and the solid content reaches 15-20%.

[0133] 11. Sterilization: The prepared liquid is sterilized using a 0.22μm sterilization membrane.

[0134] 12. Drying: The sterilized liquid material is subjected to vacuum freeze drying (-60℃, 20Pa) for 48 hours to obtain giant salamander skin collagen peptide powder.

[0135] Example 3

[0136] This embodiment provides a method for preparing collagen peptides from giant salamander skin, including the following steps:

[0137] 1. Remove fat: Cut the cleaned giant salamander skin into 0.5cm×0.5cm pieces, add 10 times its weight of 10% n-butanol solution, stir the mixture continuously at 4℃ for 24 hours, change the solution every 8 hours to remove fat components, and finally rinse with deionized water until neutral.

[0138] 2. Removal of pigments and non-collagenous components: Soak the fish in 15 times its weight of alkaline solution at room temperature for 24 hours, changing the alkaline solution every 8 hours to thoroughly remove pigments, black membranes, and non-collagenous components naturally present in the giant salamander skin. After soaking, rinse with deionized water until the conductivity of the effluent is below 1000 μS / cm. The alkaline solution is a sodium hydroxide solution with a mass concentration of 0.1 mol / L.

[0139] 3. Extraction of acid-soluble collagen: Ten times the weight of acid solution was added to the pretreated giant salamander skin and swollen at 4℃ for 48 hours until the tissue was fragmented. During this period, ultrasonic treatment was performed every 12 hours at a frequency of 20 kHz for 40 minutes. After ultrasonic treatment and acid extraction, the mixture was filtered to obtain a filtrate, which was then precipitated with 2.6 mol / L sodium chloride at 4℃ for 48 hours. The precipitate was centrifuged at 18000×g for 30 minutes (4℃), and the precipitate was dissolved in 0.1 mol / L acetic acid. Dialysis was performed with 0.1 mol / L acetic acid and deionized water (molecular weight cutoff 25 kDa). The resulting dialysate was freeze-dried to obtain giant salamander skin collagen. The acid solution was acetic acid solution with a mass concentration of 0.75 mol / L.

[0140] 4. Flocculation to remove impurities: Add the giant salamander skin collagen to deionized water and dissolve it completely at a mass ratio of 1:20. At room temperature, add 1% phosphoric acid and 1.8% calcium hydroxide by weight of the pretreated giant salamander skin, with a mass ratio of 1:1.8. Stir and flocculate for 30 minutes, then let it stand for 1 hour. Filter the solution through a 0.45μm membrane to obtain the collagen solution, and then adjust the pH to neutral with an appropriate amount of hydrochloric acid.

[0141] 5. Stepwise enzymatic hydrolysis: Heat the collagen solution to 55℃, adjust the pH to 8.5 with alkali, and add 0.5% (by weight) of alkaline protease and 0.1% (by weight) of trypsin preparation from the pretreated giant salamander skin, respectively, and hydrolyze for 3 hours. Then adjust the pH to 7.5, add 0.05% (by weight) of flavor protease from the pretreated giant salamander skin, and continue enzymatic hydrolysis for 2 hours.

[0142] 6. Enzyme inactivation: After the enzymatic hydrolysis is completed, heat the hydrolysate to 85℃ and keep it warm for 20 min.

[0143] 7. Decolorization and deodorization: Centrifuge the enzymatic hydrolysate (8000r / m for 10min) to remove residual large insoluble particles. Add 6% of the weight of pretreated giant salamander skin to the prepared supernatant and keep it at 65℃ for 40min.

[0144] 8. Solid-liquid separation: After the heat preservation is completed, diatomaceous earth is added to the liquid as a filter aid to separate the enzymatic hydrolysate from the activated carbon. Then, a ceramic membrane (50nm) is used for further solid-liquid separation to achieve a clear and transparent enzymatic hydrolysate.

[0145] 9. Purification and separation: The obtained enzymatic hydrolysate is passed through macroporous adsorption resin SD300 (without active functional groups) and the influent flow rate is controlled at 4 BV / h to further remove the collagen peptides that have not been fully hydrolyzed and have a relatively large molecular weight.

[0146] 10. Nanofiltration: The obtained feed solution is passed through a 200 Da nanofiltration membrane to remove free amino acids and sodium chloride, thereby purifying the sample. At the same time, the feed solution is pre-concentrated, and the solid content reaches 15-20%.

[0147] 11. Sterilization: The prepared liquid is sterilized using a 0.22μm sterilization membrane.

[0148] 12. Drying: The sterilized liquid material is subjected to vacuum freeze drying (-60℃, 20Pa) for 48 hours to obtain giant salamander skin collagen peptide powder.

[0149] Example 4

[0150] This embodiment is basically the same as Embodiment 1, except that:

[0151] Step 2: Removal of pigments and non-collagenous components: Soak the fish in 15 times its weight of alkaline solution at room temperature for 24 hours, changing the alkaline solution every 8 hours to thoroughly remove pigments, black membranes, and non-collagenous components naturally present in the giant salamander skin. After soaking, rinse with deionized water until the conductivity of the effluent is below 1000 μS / cm. The alkaline solution is a sodium carbonate solution with a mass concentration of 0.2 mol / L.

[0152] Example 5

[0153] This embodiment is basically the same as Embodiment 1, except that:

[0154] Step 3 was modified to extract acid-soluble collagen: Ten times the weight of acid solution was added to the pretreated giant salamander skin, and the mixture swelled at 4°C for 48 hours until the tissue was fragmented. During this period, ultrasonic treatment was performed every 12 hours at a frequency of 20 kHz for 40 minutes. After ultrasonic treatment and acid extraction, the mixture was filtered to obtain a filtrate, which was then precipitated with 2.6 mol / L sodium chloride at 4°C for 48 hours. The precipitate was centrifuged at 18000×g for 30 minutes (4°C), and the precipitate was dissolved in 0.1 mol / L acetic acid. Dialysis was then performed with 0.1 mol / L acetic acid and deionized water (molecular weight cutoff 25 kDa). The resulting dialysate was freeze-dried to obtain giant salamander skin collagen. The acid solution was citric acid solution with a mass concentration of 0.75 mol / L.

[0155] Example 6

[0156] This embodiment is basically the same as Embodiment 1, except that:

[0157] In Example 1, step 4 was changed to prepare a giant salamander skin collagen solution: the giant salamander skin collagen was added to deionized water and fully dissolved at a mass ratio of 1:20, and calcium chloride was added at 0.55% of the weight of the pretreated giant salamander skin.

[0158] Example 7

[0159] This embodiment is basically the same as Embodiment 1, except that:

[0160] Step 4 is changed to flocculation to remove impurities: Giant salamander skin collagen is added to deionized water and fully dissolved at a mass ratio of 1:20. At room temperature, 1% phosphoric acid and 1.5% calcium hydroxide by weight of the pretreated giant salamander skin are added respectively, with a mass ratio of 1:1.5. After stirring and flocculating for 30 minutes, the mixture is allowed to stand for 1 hour. The collagen solution is obtained by filtration through a 0.45μm membrane. 0.60% sodium carbonate by weight of the pretreated giant salamander skin is added to the solution. After the calcium ions in the solution are fully precipitated, the solution is filtered through a 0.45μm membrane again. The final collagen solution is adjusted to neutral pH with an appropriate amount of hydrochloric acid.

[0161] Example 8

[0162] This embodiment is basically the same as Embodiment 1, except that:

[0163] Step 4 is changed to flocculation to remove impurities: Giant salamander skin collagen is added to deionized water and fully dissolved at a mass ratio of 1:20. At room temperature, 0.1% polyacrylamide (PAM) by weight of pretreated giant salamander skin is added. After stirring and flocculating for 30 minutes, it is allowed to stand for 1 hour. After filtration through a 0.45μm membrane, a collagen solution is obtained. 2% sodium chloride by weight of pretreated giant salamander skin is added.

[0164] Comparative Example 1

[0165] This comparative example is basically the same as Example 1, except that:

[0166] Step 3 in Example 1 was modified to extract acid-soluble collagen: Ten times the weight of an acid solution was added to the pretreated giant salamander skin, and the mixture swelled at 4°C for 48 hours until the tissue was fragmented. After acid extraction, the mixture was filtered to obtain a filtrate, which was then precipitated with 2.6 mol / L sodium chloride at 4°C for 48 hours. The precipitate was centrifuged at 18000×g for 30 minutes (4°C), and the precipitate was dissolved in 0.1 mol / L acetic acid. The precipitate was then dialyzed with 0.1 mol / L acetic acid and deionized water (molecular weight cutoff 25 kDa). The dialysate was freeze-dried to obtain giant salamander skin collagen. The acid solution was acetic acid solution with a mass concentration of 0.75 mol / L.

[0167] Comparative Example 2

[0168] This comparative example is basically the same as Example 1, except that:

[0169] Step 5 was changed to stepwise enzymatic hydrolysis: The collagen solution was heated to 55℃, the pH was adjusted to 7.5 with alkali, and 0.05% (by weight) of flavor protease from the pretreated giant salamander skin was added. Enzymatic hydrolysis was carried out for 3 hours. Then the pH was adjusted to 8.5, and 0.5% (by weight) of alkaline protease and 0.1% (by weight) of neutral protease from the pretreated giant salamander skin were added respectively. Enzymatic hydrolysis was continued for 4 hours.

[0170] Comparative Example 3

[0171] This comparative example is basically the same as Example 1, except that:

[0172] Step 5 is changed to enzymatic hydrolysis: the collagen solution is heated to 55℃, the pH is adjusted to 8.5 with alkali, and 0.5% alkaline protease and 0.1% neutral protease preparation of pretreated giant salamander skin are added respectively, and enzymatic hydrolysis is carried out for 4 hours.

[0173] Comparative Example 4

[0174] This comparative example is basically the same as Example 1, except that:

[0175] Delete step 9.

[0176] Comparative Example 5

[0177] This comparative example is basically the same as Example 1, except that:

[0178] Delete step 10.

[0179] Comparative Example 6

[0180] This comparative example is basically the same as Example 1, except that:

[0181] The first protease is a neutral protease, and the second protease is trypsin.

[0182] Result verification:

[0183] The yield, hydroxyproline content, and Ca content of the collagen peptide samples prepared in each example and comparative example were compared. 2+ Content, Na in collagen peptide samples + The content, weight-average molecular weight, and OD380 value are shown in Table 1.

[0184] The enzyme preparations involved in this application are all commercially available proteases.

[0185] This application uses a hydroxyproline (Hyp) assay kit to determine the Hyp content in collagen peptides from giant salamander skin.

[0186] The weight-average molecular weight of collagen peptides from giant salamander skin was determined by high performance liquid chromatography (HPLC) in accordance with GB31645-2018.

[0187] Determination of Na in collagen peptides from giant salamander skin by flame atomic absorption spectrometry, according to GB 5009.91-2017, Method I. + content.

[0188] Determination of Ca in collagen peptides from giant salamander skin by inductively coupled plasma atomic emission spectrometry (ICP-AES) according to GB 5009.92-2016, Method III. 2+ content.

[0189] Table 1 Physicochemical properties of samples prepared in each embodiment and comparative example

[0190]

[0191] As shown in Table 1, the methods for preparing giant salamander skin collagen peptides provided in Examples 1-8 ensure efficient and high-quality acquisition of the target product from complex fish skin raw materials, with overall performance superior to Comparative Examples 1-6. The significantly reduced yields (approximately 4-5 percentage points) in Comparative Example 1 (removal of ultrasound assistance) and Comparative Examples 2 and 3 (change in enzymatic hydrolysis sequence or combination) demonstrate that the absence or misordering of key steps in this scheme can compromise the overall system efficiency, highlighting the importance of the integrity of the process design.

[0192] The data from Comparative Example 1 show that the yield of collagen peptides prepared in Comparative Example 1 was significantly reduced, indicating that ultrasonic treatment effectively promoted collagen extraction under acidic conditions. Ultrasonic waves generate bubbles through alternating high and low pressures. The continuous formation, growth, and collapse of these bubbles increase mass transfer, mixing, particle breakage from large to small, surface area, extraction kinetics, and the dissolution rate of cavitation forces in the reaction system. The results of Comparative Example 1 demonstrate that ultrasonic-assisted treatment is an indispensable part of the extraction of acid-soluble collagen, and the examples show that this method has systematic integrity.

[0193] As can be seen from the pretreatment stage, the phosphoric acid-calcium hydroxide flocculation system (Examples 1-3) has proven to be the preferred method for removing non-collagenous impurities. In Example 4, the Hyp content of the sample decreased when the alkaline solution was changed to a 0.2 mol / L sodium carbonate solution, while the OD360 value of the liquid after solid-liquid separation increased significantly. This indicates that the higher mass concentration (2 mol / L) of sodium carbonate solution is weakly alkaline and has limited ability to remove organic pigments and impurities from giant salamander skin. As a result, the prepared sample contains a small amount of non-collagenous peptides, and the sample preparation yield data (16.90%) also verifies this.

[0194] The yield of collagen peptide samples in Example 5 was also reduced, indicating that citric acid was less effective than hydrochloric acid and acetic acid in separating and dissolving collagen during the extraction process.

[0195] As can be seen from the data in Example 6, the Hyp content of the collagen peptide sample in Example 6 was reduced, indicating that the flocculation reaction in the example could further remove non-collagen components from the system, and the preparation yield data of the sample in Example 6 (17.05%) also verified this. As can be seen from Examples 3, 6, and 7, Ca... 2+ It can significantly promote the enzymatic degradation process of collagen in giant salamander skin.

[0196] It can be seen that Ca 2+ Calcium ions can directly activate protease activity, and they can bind to specific sites on the enzyme molecule, acting as a "molecular glue" to stabilize its three-dimensional structure and prevent autolysis or denaturation inactivation, thus stabilizing the protease structure. The effect of calcium ion activity on protease activity is concentration-dependent; for example, in Example 6, Ca... 2+ At higher concentrations, the enzymatic hydrolysis time can be effectively shortened to achieve the same hydrolysis effect, while in Example 7, Ca... 2+ The concentration was low, the enzymatic hydrolysis of giant salamander skin collagen was not effective, and the weight-average molecular weight (Mw 1010) of the prepared sample was relatively large.

[0197] It can be seen that calcium ions (Ca) 2+The concentration management of calcium ions has a dual nature. On the one hand, an appropriate amount of calcium ions in the flocculation step (as in Examples 1-3) helps remove impurities and proteins; on the other hand, as shown in Example 7, the calcium content of the final product (1150 mg / 100g) can be significantly reduced by subsequently adding sodium carbonate to precipitate excess calcium ions, which provides a way to adjust the process according to the product positioning (such as high calcium or low calcium).

[0198] The data from Example 8 show that even lower concentrations of PAM can achieve good flocculation effects, and the appropriate amount of Na in the system... + The content has a relatively small direct impact on protease. + Mainly by maintaining optimal ionic strength (system osmotic pressure) to provide a suitable physicochemical environment for the protease to function. High concentrations of Na... + It will compete with enzymes for water molecules, destroy the hydration layer on the surface of proteins, and lead to reduced protease activity or even denaturation and inactivation. The significant increase in the weight-average molecular weight of the sample prepared in Example 8 is a good verification of this.

[0199] The enzymatic hydrolysis stage reveals that the core technology lies in the sequential enzymatic hydrolysis strategy of "alkaline / neutral proteases hydrolyzing first, followed by flavor proteases modifying later." Comparative Example 2 (reversed order) and Comparative Example 3 (lacking flavor proteases) both showed significantly reduced yields to approximately 12%, confirming that this hydrolysis sequence and combination are indispensable for efficient and thorough collagen hydrolysis and potentially improved product flavor. Specifically, alkaline / neutral proteases, acting as endopeptides, are responsible for the rapid degradation of large molecules, while flavor proteases, acting as exopeptides, play a crucial role in further cleavage and flavor modulation. Alkaline proteases play a major role in collagen hydrolysis. Neutral and alkaline proteases, being endopeptides, first randomly cleave large protein molecules internally, generating numerous peptides of varying lengths, thus achieving rapid degradation of giant salamander skin collagen. Flavor proteases, being exopeptides, excel at cleaving from the ends of peptide chains, eliminating undesirable flavors. Therefore, using only flavor proteases is insufficient for effective collagen degradation.

[0200] In the refining and purification stage: the combined use of macroporous adsorption resin (SD300) and nanofiltration (200 Da) is key to controlling the molecular weight distribution and inorganic salt content of the product. Removing the macroporous resin step (Comparative Example 4) nearly doubled the weight-average molecular weight (1795 Da), demonstrating its effective adsorption and removal of residual large peptides; removing the nanofiltration step (Comparative Example 5) caused the sodium ion content to surge to 890 mg / 100g, highlighting the irreplaceable role of nanofiltration in deep desalination, removal of free amino acids, and pre-concentration. Simultaneously, the calcium ion retention effect of nanofiltration allows the product to retain the natural calcium components derived from the flocculation process.

[0201] Furthermore, data from Comparative Example 5 show that the weight-average molecular weight of the sample prepared in Comparative Example 5 is reduced, while Na... + The significant increase in content indicates that nanofiltration membranes can effectively remove free amino acids and monovalent sodium ions from the preparation solution. Furthermore, due to size exclusion and the Donnan effect of nanofiltration membranes, the content of Ca is significantly increased. 2+ It has a good retention effect, so the calcium ion content of the samples in Example 1 and Comparative Example 5 remained basically unchanged. Data from Example 8 and Comparative Example 5 show that excessive exogenous Na... + This results in a high sodium ion content in the enzymatic hydrolysis solution, which puts significant stress on the nanofiltration process, accelerates membrane wear, shortens its lifespan, and substantially increases manufacturing costs—a situation undesirable in actual industrial production. Furthermore, a high sodium content in the prepared collagen peptide product is detrimental to health, limiting its application in the end-market.

[0202] Effect verification

[0203] 1.1 Effects of giant salamander skin collagen peptides on fibroblast proliferation

[0204] Methods: HSF fibroblasts in the logarithmic growth phase were harvested, digested with trypsin, and resuspended to adjust the cell density to 5 × 10⁶ cells / year. 4 Cells / mL. Cell suspension was seeded into 96-well plates, 200 μL per well, and incubated for 12 h. Subsequently, cells were treated with different concentrations (0, 0.1, 0.5, 1, 1.5, 2, 2.5 mg / mL) of giant salamander skin collagen peptides for 12 h. 20 μL of CCK8 solution was added to each well, and after incubation at 37 ℃ for 30 min, the absorbance at 450 nm was measured for each well. Cell proliferation rate was calculated as follows: the blank group contained only culture medium and no cells; the control group contained only cells and no drugs were added.

[0205]

[0206] Results analysis: Figure 1This study demonstrates the proliferation rate of HSF fibroblasts after 12 hours of treatment with giant salamander skin collagen peptides. When the concentrations of giant salamander skin collagen peptides were 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 2.5 mg / mL, the proliferation rates were 1.01%, 1.02%, 1.04%, 1.10%, 1.26%, and 1.17%, respectively, with the highest proliferation rate observed at a concentration of 2 mg / mL. The results show that giant salamander skin collagen peptides significantly promote the proliferation of HSF fibroblasts. Furthermore, when the concentration of giant salamander skin collagen peptides reached 1.5 mg / mL, the proliferation rate of fibroblasts generally showed an increasing trend. Based on this, further experimental studies will be conducted using giant salamander skin collagen peptides at concentrations of 1.5 mg / mL, 2 mg / mL, and 2.5 mg / mL.

[0207] 1.2 Effects of giant salamander skin collagen peptides on cell repair

[0208] Methods: A straight line was drawn on the back of a 6-well plate using a ruler, with 5 horizontal lines passing through each well. HSF cells were seeded into the 6-well plates. After the cells reached confluence, a 200 μL pipette tip was used to scratch the lines perpendicularly. Floating cells were washed away with PBS. Then, the cells were treated with different concentrations (1.5 mg / mL, 2 mg / mL, 2.5 mg / mL) of giant salamander skin collagen peptides and incubated at 37 ℃. Samples were taken and photographed at 0 h, 12 h, and 24 h.

[0209] Results Analysis: The scratch test results are as follows: Figure 2 As shown, at 0 h, the scratch widths of all concentration groups were basically the same, and the cell distribution was relatively uniform. With the progression of culture time, at 12 h, cells treated with *Andrias davidianus* collagen peptides at concentrations of 1.5 mg / mL, 2 mg / mL, and 2.5 mg / mL all began to migrate towards the scratched area. The 2 mg / mL concentration group showed relatively faster cell migration, and the scratch width narrowed significantly. By 24 h, the scratched areas of the 2 mg / mL and 2.5 mg / mL concentration groups were basically filled with cells, and the cells were densely packed. While the scratched area of ​​the 1.5 mg / mL concentration group still had some unfilled cells, it showed significant improvement compared to 12 h. These results indicate that *Andrias davidianus* collagen peptides can promote the migration of HSF cells and have a positive effect on cell repair. Furthermore, within a certain concentration range, the 2 mg / mL concentration of *Andrias davidianus* collagen peptides showed a relatively better effect in promoting cell repair.

[0210] 1.3 Effects of giant salamander skin collagen peptides on intracellular ROS levels

[0211] Methods: HSF cells were seeded at a density of 8 × 10⁴ cells / well in 6-well plates and cultured for 12 h. Subsequently, cells were treated with different concentrations (1.5, 2, 2.5 mg / mL) of giant salamander skin collagen peptides for 12 h. DCFH-DA was diluted 1:1000 with serum-free culture medium to a final concentration of 10 μmol / L. The cell culture medium was removed, and an appropriate volume of diluted DCFH-DA was added. The cells were incubated at 37°C for 20 min. The cells were washed three times with serum-free cell culture medium to thoroughly remove any undiluted DCFH-DA. Finally, the 6-well plates were photographed under a fluorescence microscope.

[0212] Results Analysis: The ROS experimental results are as follows: Figure 3 As shown, compared with the control group, the UV-treated group had a significantly increased intracellular ROS level, indicating that UV treatment successfully induced intracellular oxidative stress. After treatment with different concentrations of giant salamander skin collagen peptides, the intracellular ROS levels showed varying degrees of decrease. Among them, the 2 mg / mL and 2.5 mg / mL concentrations of giant salamander skin collagen peptides resulted in the most significant decrease in intracellular ROS levels, indicating that at these concentrations, the giant salamander skin collagen peptides had a strong inhibitory effect on intracellular oxidative stress, helping to alleviate cell damage caused by oxidative stress.

[0213] 1.4 Effects of giant salamander skin collagen peptides on cellular collagen synthesis

[0214] Methods: HSF cells were cultured at 8 × 10⁻⁶ cells / day. 5 HSF cells were seeded at a density of 1 / 2 well in 6 mm dishes and incubated for 12 h. Subsequently, HSF cells were treated for 12 h under different conditions (UV irradiation, UV irradiation + 2 mg / mL giant salamander skin collagen peptide). Total protein concentration was extracted from each group using RIPA protein lysis buffer. 20 mg of total protein from each group was loaded for SDS-PAGE gel electrophoresis, transferred to a PVDF membrane after 2 h, blocked with 5% BSA for 1 h, and incubated with Type I Collagen primary antibody overnight at 4°C. After primary antibody recovery, the cells were washed three times with TBST and incubated with rabbit anti-IgG secondary antibody at room temperature for 1 h. After incubation, the cells were washed three times with TBST and detected using fluorescence imaging. The relative expression levels of each target protein were analyzed using ImageJ software.

[0215] Results Analysis: The results are as follows Figure 4As shown, compared with the blank control group, the relative expression level of Type I Collagen in the UV irradiation group was significantly reduced, indicating that UV irradiation had a significant inhibitory effect on cellular collagen synthesis. However, in the UV irradiation + 2 mg / mL giant salamander skin collagen peptide treatment group, the relative expression level of Type I Collagen was significantly increased compared with the UV irradiation group, and close to that of the blank control group, indicating that giant salamander skin collagen peptide can, to some extent, reverse the inhibitory effect of UV irradiation on cellular collagen synthesis and promote cellular collagen synthesis.

[0216] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing collagen peptides from giant salamander skin, characterized in that, include: Giant salamander skin is provided, and the giant salamander skin is pretreated; The pretreated giant salamander skin was placed in an acidic extract and extracted under ultrasonic conditions; the extract was then collected. After salting out and dialysis, the extract was subjected to a first collagen solution. The first collagen solution is subjected to flocculation and impurity removal treatment to prepare a second collagen solution; The second collagen solution was subjected to stepwise enzymatic hydrolysis to prepare enzymatic hydrolysate; as well as, The enzymatic hydrolysate was purified to prepare giant salamander skin collagen peptides; The stepwise enzymatic hydrolysis process includes: mixing and hydrolyzing with a first protease and a second protease, and then adding a third protease to continue the hydrolysis. The first protease includes an alkaline protease; The second protease includes one of neutral protease, papain, and trypsin; The third protease includes flavor protease.

2. The method for preparing giant salamander skin collagen peptides according to claim 1, characterized in that, The weight of the first protease added is 0.5%-1% of the weight of the pretreated giant salamander skin; and / or The second protease was added at a weight of 0.05%-0.1% of the pretreated giant salamander skin weight; and / or The added weight of the third protease is 0.005%-0.05% of the weight of the pretreated giant salamander skin.

3. The method for preparing giant salamander skin collagen peptides according to claim 1, characterized in that, The steps of the stepwise enzymatic hydrolysis process include: First and second proteases are added for the first enzymatic hydrolysis step; and third protease is added for the second enzymatic hydrolysis step. The conditions for the first step of enzymatic hydrolysis include: temperature of 54℃-56℃, pH of 8.4-8.6, and time of 3.5 h-5.5 h; The conditions for the second step of enzymatic hydrolysis include: a temperature of 54℃-56℃, a pH of 7.4-7.6, and a time of 2.5 h-3.5 h.

4. The method for preparing giant salamander skin collagen peptides according to claim 1, characterized in that, Preprocessing includes: The giant salamander skin was sliced, and the fat was removed using a n-butanol solution. Then, the pigment and non-collagen components were removed using a strong alkaline solution. The preprocessing satisfies one or more of the following conditions: (1) The concentration of n-butanol in the n-butanol solution is 8 w / v%-12 w / v%; (2) The strongly alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution, and the concentration of sodium hydroxide in the strongly alkaline solution is 0.05 mol / L - 0.2 mol / L.

5. The method for preparing giant salamander skin collagen peptides according to any one of claims 1 to 4, characterized in that, The preparation method satisfies one or more of the following conditions: (1) The acidic extract includes hydrochloric acid or acetic acid; (2) The conditions for ultrasound include: frequency of 20 kHz-40 kHz, time of 30 min-60 min, and interval of 10 h-12 h; (3) The flocculation and impurity removal treatment uses phosphoric acid and calcium hydroxide for flocculation reaction; (4) The purification process includes using macroporous adsorption resin to remove macromolecular substances, and then using nanofiltration membrane to remove free amino acids and sodium chloride.

6. The method for preparing giant salamander skin collagen peptides according to claim 5; characterized in that, The concentration of the acidic extract is 0.3 mol / L-0.8 mol / L.

7. The method for preparing giant salamander skin collagen peptides according to claim 5; characterized in that, The macroporous adsorption resin is SD300 type macroporous adsorption resin, and the influent liquid flow rate is 4 BV / h - 6 BV / h; and / or The molecular weight cutoff of the nanofiltration membrane is ≤200 Da.

8. The method for preparing giant salamander skin collagen peptides according to claim 5, characterized in that, The flocculation reaction using phosphoric acid and calcium hydroxide meets one or more of the following conditions: (1) The mass ratio of the phosphoric acid to the calcium hydroxide is 1:(1.5-2); (2) The added phosphoric acid is 1%-2% of the weight of the pretreated giant salamander skin; (3) The amount of calcium hydroxide added is 1.5%-2% of the weight of the pretreated giant salamander skin.

9. The giant salamander skin collagen peptide prepared by the method described in any one of claims 1 to 8.

10. The use of the giant salamander skin collagen peptide of claim 9 in the preparation of products that promote skin cell proliferation and / or promote collagen synthesis.

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