Codfish skin collagen peptide as well as preparation method and application thereof

Abstract

The invention discloses cod skin collagen peptide as well as a preparation method and application thereof. The preparation method of the cod skin collagen peptide comprises the following steps: mixing an alkaline solution and cod skin, and performing pretreatment under an ultrasonic-assisted condition; mixing the pretreated cod skin with water, carrying out heat treatment on the obtained mixture by using microwave-assisted heating, and drying to prepare cod skin collagen; the method comprises the following steps: extracting cod skin collagen by adopting a supercritical CO2 extraction method to prepare degreased cod skin collagen; carrying out enzymolysis on the defatted cod skin collagen by using protease to prepare an enzymolysis product; carrying out enzyme deactivation on the enzymolysis product, decolorizing by adopting an activated carbon fiber membrane, mixing the decolorized product with mesoporous silica, and carrying out purification separation and nanofiltration to prepare the cod skin collagen peptide. The prepared cod skin collagen peptide is relatively high in quality.

Description

Cod skin collagen peptides, their preparation methods and applications Technical Field

[0001] This application belongs to the field of collagen peptide preparation technology, and relates to cod skin collagen peptides, their preparation methods and applications. Background Technology

[0002] Cod, also known as bighead cod or largemouth cod, refers specifically to fish of the genus Gadus, including Atlantic cod, Greenland cod, and Pacific cod. With the rapid development of the cod processing industry, the large amount of fish skin generated during the meat processing can be fully utilized for extraction, thus saving resources and increasing the added value of aquatic product processing. Cod skin is thick and wide, rich in collagen, a high-quality anti-wrinkle and beauty substance. However, the complex structure of large-molecule proteins and their limitation by cell membrane permeability result in low bioavailability and unstable oral absorption. Therefore, developing low-molecular-weight, high-bioavailability, highly bioactive, and high-quality cod skin collagen peptides has become a new research hotspot. Cod skin collagen peptides can be effectively absorbed by the human body. Through a unique biological signaling mechanism, they stimulate the proliferation of dermal fibroblasts, resist skin oxidation, repair skin damage, increase collagen synthesis, restore skin elasticity and radiance, reduce wrinkles, and achieve significant anti-aging effects. At the same time, it can promote the metabolism of skin cells, accelerate the shedding of aging cells, keep the skin youthful, and further enhance the vitality and regenerative ability of skin cells.

[0003] Cod skin is rich in protein, but due to its unique growing environment, it also contains a significant amount of fat, especially in haddock skin. Furthermore, deep-sea cod has a strong fishy odor, all of which severely impact the quality of cod skin collagen peptide products. Currently, the quality of cod skin collagen peptide products is poor and cannot meet market demand. Summary of the Invention

[0004] Therefore, it is necessary to provide a high-quality cod skin collagen peptide, its preparation method, and its application.

[0005] In some embodiments, a method for preparing cod skin collagen peptides is provided, comprising the following steps:

[0006] An alkaline solution is mixed with cod skin and pretreated under ultrasonic-assisted conditions; the alkaline solution comprises sodium hydroxide, calcium chloride, and water, wherein the mass fraction of sodium hydroxide is 0.5%~1% and the mass fraction of calcium chloride is 1%~2%.

[0007] Pretreated cod skin and water were mixed, and the resulting mixture was heat-treated using microwave-assisted heating and then dried to prepare cod skin collagen.

[0008] The cod skin collagen was extracted using supercritical CO2 extraction to prepare defatted cod skin collagen.

[0009] The defatted cod skin collagen was enzymatically hydrolyzed using a protease to prepare the enzymatic hydrolysate;

[0010] After inactivating the enzyme in the enzymatic hydrolysis product, it is decolorized using an activated carbon fiber membrane. The resulting decolorized product is mixed with mesoporous silica for purification, separation, and nanofiltration to prepare cod skin collagen peptides.

[0011] In some embodiments, the provided method for preparing cod skin collagen peptides satisfies one or more of the following conditions:

[0012] (1) In the step of heat treatment of the obtained mixture using microwave-assisted heating, the temperature of the mixture is controlled at 120℃~140℃ and the heat treatment time is 20min~40min;

[0013] (2) The power of microwave heating is 300W~600W; and

[0014] (3) The extraction conditions include: temperature of 35℃~60℃, pressure of 20MPa~40MPa, supercritical CO2 flow rate of 0.5L / min~2.5L / min, and extraction time of 1h~4h.

[0015] In some embodiments, the method for preparing cod skin collagen peptides includes a step of preparing enzymatic hydrolysate in which the defatted cod skin collagen is mixed with water, alkaline protease and protease A are added for a first enzymatic hydrolysis, and then flavor protease is added for a second enzymatic hydrolysis.

[0016] The protease A includes one of neutral protease, papain, fig protease, and trypsin.

[0017] In some embodiments, the provided method for preparing cod skin collagen peptides satisfies one or more of the following conditions:

[0018] (1) The mass ratio of the defatted cod skin collagen to the water is 1:(4~6);

[0019] (2) The mass of the alkaline protease is 0.5% to 1.5% of the mass of the defatted cod skin collagen protein;

[0020] (3) The mass of the protein protein protein A is 0.05% to 0.3% of the mass of the defatted cod skin collagen protein;

[0021] (4) The mass of the flavor protease is 0.05% to 0.1% of the mass of the defatted cod skin collagen protein;

[0022] (5) The temperature of the first enzymatic hydrolysis is 55℃~60℃, the time of the first enzymatic hydrolysis is 4h~6h, and the pH value of the first enzymatic hydrolysis is 8.0~9.5; and

[0023] (6) The temperature of the second enzymatic hydrolysis is 55℃~60℃, the time of the second enzymatic hydrolysis is 2h~4h, and the pH value of the second enzymatic hydrolysis is 6.8~7.2.

[0024] In some embodiments, the provided method for preparing cod skin collagen peptides satisfies one or more of the following conditions:

[0025] (1) During the pretreatment process, ultrasonic treatment is performed every 6h to 8h. The frequency of each ultrasonic treatment is 40kHz to 60kHz, the duration of each ultrasonic treatment is 6min to 12min, and the ultrasonic treatment is performed for 5s to 6s with a pause of 4s to 5s.

[0026] (2) During the pretreatment process, the alkaline solution should be replaced every 6 to 8 hours;

[0027] (3) The mass ratio of the alkaline solution to the cod skin is (8~12):1;

[0028] (4) Pretreatment is carried out at 0℃~4℃; and

[0029] (5) The pretreatment time is 20h~30h.

[0030] In some embodiments, the method for preparing cod skin collagen peptides includes a purification and separation step in which the decolorized product is mixed with the mesoporous silica, stirred for 15 to 20 minutes, allowed to stand, and then filtered.

[0031] The mesoporous silica has a particle size of 5μm to 10μm, a pore size of 10nm to 20nm, and a mass of 1% to 3% of the defatted cod skin collagen protein.

[0032] In some embodiments, the method for preparing cod skin collagen peptides provides a method where the activated carbon fiber membrane has 6 to 8 layers.

[0033] In some embodiments, the method for preparing cod skin collagen peptides uses a nanofiltration membrane with a molecular weight cutoff of 150 Da to 200 Da in the nanofiltration step.

[0034] In some embodiments, a cod skin collagen peptide is provided, which is prepared by the preparation method described above.

[0035] In some embodiments, the cod skin collagen peptides are provided for use in the preparation of products that satisfy one or more of the following conditions:

[0036] (1) The product has antioxidant function;

[0037] (2) The product has the function of promoting cell proliferation;

[0038] (3) The product has the function of promoting cell repair; and

[0039] (4) The product has the function of promoting collagen synthesis.

[0040] The aforementioned method for preparing cod skin collagen peptides utilizes an alkaline solution comprising sodium hydroxide, calcium chloride, and water, combined with ultrasonic-assisted treatment. This effectively removes impurities such as proteins and pigments, achieving efficient deodorization. Microwave-assisted heating is used to fully extract collagen, and the pretreatment of cod skin with microwave-assisted heating induces cavitation, promoting more thorough separation of collagen components from impurities such as fat. Supercritical CO2 extraction can effectively remove fat components, facilitating subsequent enzymatic hydrolysis of the collagen substrate. Activated carbon fiber membrane treatment of the cod skin enzymatic hydrolysate provides good decolorization and further deodorization. The provided preparation method thoroughly removes fat, efficiently and fully enzymatically degrades collagen, effectively removes residual macromolecular substances, and is cost-effective, making it suitable for industrial production. The prepared cod skin collagen peptides have a more concentrated molecular weight distribution, higher quality, and can significantly promote skin cell proliferation, repair, anti-oxidation, and collagen synthesis. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0042] Figure 1 shows the effect of cod skin collagen peptides on the proliferation of CCK8 cells;

[0043] Figure 2 shows the effect of cod skin collagen peptides on ROS and MDA content in cells;

[0044] Figure 3 shows the effect of cod skin collagen peptides on cell repair;

[0045] Figure 4 shows the effect of cod skin collagen peptides on the expression levels of Collagen I and MMP1 proteins. Detailed Implementation

[0046] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0047] It should be noted that when a component is said to be "fixed to" another component, it can be directly attached to the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0049] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0050] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more 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 using 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 solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B."

[0051] 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.

[0052] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0053] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., 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.

[0054] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0055] 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.

[0056] 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.

[0057] In this invention, the terms "first aspect," "second aspect," "third aspect," and "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," and "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0058] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0059] 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.

[0060] 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℃.

[0061] 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.

[0062] In this application, "room temperature" generally refers to 5℃~30℃, and more preferably 25±5℃.

[0063] Cod skin collagen peptides: A mixture of small molecule peptides prepared from cod skin through enzymatic hydrolysis and refining.

[0064] Cod skin collagen peptide yield (%) = cod skin collagen peptide mass / cod skin mass after pretreatment × 100.

[0065] Hydroxyproline content (Hyp, %): Mass of hydroxyproline in cod skin collagen peptide sample / Mass of cod skin collagen peptide sample × 100.

[0066] OD 380 Value: The absorbance of the decolorized solution at 380 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0067] Cod skin is rich in protein, but also contains a significant amount of fat and fishy-smelling components. Therefore, effective pretreatment of cod skin (including decolorization and deodorization) is crucial for preparing high-quality collagen peptide products. Currently, the process of preparing cod skin collagen peptides involves washing and soaking the cod skin with NaOH solution and n-butanol, but this has very limited effectiveness in removing impurities and deodorizing, resulting in poor-quality collagen peptides. Furthermore, the enzymatic hydrolysate is filtered sequentially through 10000D, 3000D, and 1000D membranes, making the process complex, resulting in low product yield and high production costs. While ultrasonic pretreatment and high-pressure homogenization are used to dissociate collagen fibers, the extracted collagen components are not adequately separated from other components such as fat, hindering the overall enzymatic degradation process and ultimately leading to poor-quality cod collagen peptide products. Microwave treatment is also used during the enzymatic hydrolysis of the cod skin and cod bone mixture. However, microwaves reduce the activity of the protease preparation, resulting in low enzymatic hydrolysis efficiency. The prepared cod collagen peptide extract was subjected to multiple membrane treatments with different pore sizes, and the retentate with a pore size of 1000 Da to 5000 Da was finally collected as the target substance. The prepared cod collagen peptides had a relatively large molecular weight, limiting their bioavailability and bioactivity. The entire separation process was cumbersome, complex, and costly, making it unsuitable for industrial production, and the yield of the target product was very low. Therefore, this application is submitted.

[0068] In some embodiments, a method for preparing cod skin collagen peptides is provided, comprising the following steps:

[0069] The cod skin was mixed with an alkaline solution and pretreated under ultrasonic-assisted conditions. The alkaline solution consisted of sodium hydroxide, calcium chloride, and water, with sodium hydroxide having a mass fraction of 0.5%–1% and calcium chloride having a mass fraction of 1%–2%.

[0070] Pretreated cod skin and water were mixed, and the resulting mixture was heat-treated using microwave-assisted heating and then dried to prepare cod skin collagen.

[0071] Supercritical CO2 extraction was used to extract collagen from cod skin to prepare defatted cod skin collagen.

[0072] The degreased cod skin collagen was enzymatically hydrolyzed using protease to prepare the hydrolysate;

[0073] After inactivating the enzyme in the enzymatic hydrolysis product, it was decolorized using an activated carbon fiber membrane. The resulting decolorized product was then mixed with mesoporous silica for purification, separation, and nanofiltration to prepare cod skin collagen peptides.

[0074] Understandably, in an alkaline solution, the mass fraction of sodium hydroxide can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc., or any range of any two of the aforementioned values; in an alkaline solution, the mass fraction of calcium chloride can be 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, etc., or any range of any two of the aforementioned values.

[0075] In some embodiments, in the method for preparing cod skin collagen peptides, in the step of heat treatment of the obtained mixture using microwave-assisted heating, the temperature of the mixture is controlled at 120°C to 140°C, and the heat treatment time is 20 min to 40 min. For example, the temperature of the mixture can be 120°C, 125°C, 130°C, 135°C, 140°C, etc., or it can be any range of two of the aforementioned values. For example, the heat treatment time can be 20 min, 25 min, 30 min, 35 min, 40 min, etc., or it can be any range of two of the aforementioned values.

[0076] In some embodiments, the microwave heating power in the provided method for preparing cod skin collagen peptides is 300W to 600W. For example, the microwave heating power can be 300W, 400W, 500W, 600W, etc., or it can be any combination of the two values ​​mentioned above.

[0077] In some embodiments, the water content in cod skin collagen is less than 10%.

[0078] In some embodiments, the extraction conditions in the provided method for preparing cod skin collagen peptides include: a temperature of 35℃~60℃, a pressure of 20MPa~40MPa, a supercritical CO2 flow rate of 0.5L / min~2.5L / min, and an extraction time of 1h~4h. For example, the extraction temperature can be 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, etc., or any combination of the aforementioned values; the extraction pressure can be 20MPa, 30MPa, 40MPa, etc., or any combination of the aforementioned values; the supercritical CO2 flow rate can be 0.5L / min, 1L / min, 1.5L / min, 2.5L / min, etc., or any combination of the aforementioned values; and the extraction time can be 1h, 2h, 3h, 4h, etc., or any combination of the aforementioned values.

[0079] In some embodiments, the method for preparing cod skin collagen peptides includes a step of preparing enzymatic hydrolysate in which defatted cod skin collagen is mixed with water, alkaline protease and protease A are added for a first enzymatic hydrolysis, and then flavor protease is added for a second enzymatic hydrolysis.

[0080] Proteinase A includes one of the following: neutral protease, papain, fig protease, and trypsin.

[0081] In some embodiments, in the method for preparing cod skin collagen peptides, the mass ratio of defatted cod skin collagen to water is 1:(4~6). For example, the mass ratio of defatted cod skin collagen to water can be 1:4, 1:5, 1:6, etc., or it can be any range of the two aforementioned ratios.

[0082] In some embodiments, in the method for preparing cod skin collagen peptides, the mass of alkaline protease is 0.5% to 1.5% of the mass of defatted cod skin collagen protein. For example, the mass of alkaline protease can be 0.5%, 1%, 1.5% of the mass of defatted cod skin collagen protein, or it can be a range of any two of the aforementioned values.

[0083] In some embodiments, in the method for preparing cod skin collagen peptides, the mass of proteinase A is 0.05% to 0.3% of the defatted cod skin collagen protein. For example, the mass of proteinase A can be 0.05%, 0.1%, 0.2%, 0.3% of the defatted cod skin collagen protein, or it can be a range of any two of the aforementioned values.

[0084] In some embodiments, in the method for preparing cod skin collagen peptides, the mass of the flavor protease is 0.05% to 0.1% of the defatted cod skin collagen protein. For example, the mass of the flavor protease can be 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% of the defatted cod skin collagen protein, or it can be a range of any two of the aforementioned values.

[0085] In some embodiments, in the provided method for preparing cod skin collagen peptides, the temperature of the first enzymatic hydrolysis is 55℃~60℃, the time of the first enzymatic hydrolysis is 4h~6h, and the pH value of the first enzymatic hydrolysis is 8.0~9.5. For example, the temperature of the first enzymatic hydrolysis can be 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, etc., or it can be any range of two of the aforementioned values; the time of the first enzymatic hydrolysis can be 4h, 5h, 6h, etc., or it can be any range of two of the aforementioned values; the pH value of the first enzymatic hydrolysis can be 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, etc., or it can be any range of two of the aforementioned values.

[0086] In some embodiments, in the provided method for preparing cod skin collagen peptides, the temperature of the second enzymatic hydrolysis is 55℃~60℃, the time of the second enzymatic hydrolysis is 2h~4h, and the pH value of the second enzymatic hydrolysis is 6.8~7.2. For example, the temperature of the second enzymatic hydrolysis can be 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, etc., or any range of any two of the aforementioned values; the time of the second enzymatic hydrolysis can be 2h, 3h, 4h, etc., or any range of any two of the aforementioned values; the pH value of the second enzymatic hydrolysis can be 6.8, 6.9, 7.0, 7.1, 7.2, etc., or any range of any two of the aforementioned values.

[0087] In some embodiments, in the method for preparing cod skin collagen peptides, during the pretreatment process, ultrasonic treatment is performed every 6 to 8 hours, with a frequency of 40 kHz to 60 kHz and a duration of 6 to 12 minutes for each ultrasonic treatment, and each ultrasonic treatment is performed for 5 to 6 seconds of ultrasonic operation followed by a 4 to 5 second pause.

[0088] In some embodiments, in the method for preparing cod skin collagen peptides, the alkaline solution is replaced every 6 to 8 hours during the pretreatment process.

[0089] In some embodiments, in the method for preparing cod skin collagen peptides, the mass ratio of alkaline solution to cod skin is (8~12):1. For example, the mass ratio of alkaline solution to cod skin can be 8:1, 9:1, 10:1, 11:1, 12:1, etc., or it can be any range of the two aforementioned ratios.

[0090] In some embodiments, the cod skin collagen peptide preparation method provides a pretreatment process performed at 0°C to 4°C.

[0091] In some embodiments, the pretreatment time in the provided method for preparing cod skin collagen peptides is 20h~30h.

[0092] In some embodiments, the method for preparing cod skin collagen peptides includes a purification and separation step in which the decolorized product is mixed with mesoporous silica, stirred for 15 to 20 minutes, allowed to stand, and then filtered.

[0093] The mesoporous silica has a particle size of 5μm to 10μm, a pore size of 10nm to 20nm, and a mass of 1% to 3% of the collagen protein content of defatted cod skin. For example, the particle size of the mesoporous silica can be 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, etc., or any combination of two of the aforementioned values; the pore size of the mesoporous silica can be 10nm, 11nm, 12nm, 13nm, 14nm, 15nm, 16nm, 17nm, 18nm, 19nm, 20nm, etc., or any combination of two of the aforementioned values; the mass of the mesoporous silica can be 1%, 2%, 3% of the collagen protein content of defatted cod skin, etc., or any combination of two of the aforementioned values.

[0094] Understandably, using recyclable and reusable mesoporous silica to treat cod skin enzymatic hydrolysate removes residual collagen peptides with relatively large molecular weights, thus producing cod skin collagen peptide products with a more concentrated molecular weight distribution and higher quality.

[0095] In some embodiments, the enzyme inactivation conditions include incubation at 85-95°C for 10-20 minutes.

[0096] In some embodiments, after enzyme inactivation, centrifugation is performed to remove any remaining large insoluble particles. In some embodiments, the centrifugation speed is 4000 r / m to 5000 r / m and the centrifugation time is 15 min to 20 min.

[0097] In some embodiments, the method for preparing cod skin collagen peptides provides that the activated carbon fiber membrane has 6 to 8 layers, for example, 6, 7, or 8 layers, or any combination of the aforementioned two values.

[0098] In some embodiments, the method for preparing cod skin collagen peptides uses a nanofiltration membrane with a molecular weight cutoff of 150 Da to 200 Da in the nanofiltration step. Understandably, using a nanofiltration membrane with a molecular weight cutoff of 150 Da to 200 Da can remove free amino acids and sodium chloride, achieving sample purification. For example, the molecular weight cutoff of the nanofiltration membrane can be 150 Da, 160 Da, 170 Da, 180 Da, 190 Da, 200 Da, etc., or it can be a range consisting of any two of the aforementioned values.

[0099] In some embodiments, a cod skin collagen peptide is provided, which is prepared by the aforementioned preparation method.

[0100] In some embodiments, the use of cod skin collagen peptides in the preparation of products with antioxidant properties is provided.

[0101] In some embodiments, the use of cod skin collagen peptides in the preparation of products that promote cell proliferation is provided.

[0102] In some embodiments, the use of cod skin collagen peptides in the preparation of products that promote cell repair is provided.

[0103] In some embodiments, the use of cod skin collagen peptides in the preparation of products that promote collagen synthesis is provided.

[0104] The following are specific embodiments. They are intended to provide a more detailed description of this application to help those skilled in the art and researchers better understand it. The technical conditions described do not constitute any limitation on this application. Any modifications made within the scope of the claims of this application are protected by the claims.

[0105] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods not specifying particular conditions in the examples were performed under conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0106] Example 1

[0107] This embodiment provides a cod skin collagen peptide and its preparation method, as follows:

[0108] 1. Pretreatment to remove fishy smell and impurities: Cut the cleaned cod skin into 0.5cm × 0.5cm slices, add 8 times its weight of alkaline solution, and stir the mixture continuously at 4℃ for 24 hours. Change the solution every 8 hours, and perform ultrasonic treatment every 8 hours at a frequency of 40kHz for 12 minutes (6 seconds of operation followed by a 4-second pause constitutes one work cycle). Finally, rinse with deionized water until neutral. The alkaline solution is a mixture of sodium hydroxide and calcium chloride, with a sodium hydroxide concentration of 0.5% and a calcium chloride concentration of 1%.

[0109] 2. Microwave Thermal Extraction of Collagen: Cod skin was subjected to high-pressure heat treatment using microwave heating. The pretreated cod skin was transferred to a pressure-resistant bottle, and four times its weight of purified water was added before sealing. Microwave treatment was conducted intermittently, with the temperature inside the pressure-resistant bottle set to 120℃ and kept constant. The microwave was activated (300W) when the monitored temperature dropped 3℃ below the set value, and stopped when the temperature rose 3℃ above the set value. This cycle was repeated. The solution in the pressure-resistant bottle underwent high-pressure heat treatment for a total of 40 minutes. The extracted collagen solution was then freeze-dried under vacuum (-60℃, 10Pa) for 48 hours, and the resulting cod skin collagen powder (moisture content less than 10%) was collected.

[0110] 3. Supercritical CO2 Extraction for Fat Removal: The obtained cod skin collagen powder is evenly placed in an extraction vessel. Supercritical CO2 is used to dynamically extract the fat components from the cod skin collagen, thereby achieving efficient degreasing. The temperature in the extraction vessel is set to 35℃, the pressure to 20MPa, the CO2 flow rate to 1.0L (liquid CO2) / min, and the extraction time to 1h. After extraction, the system pressure is slowly reduced (the depressurization rate needs to be controlled at 1MPa / min) until atmospheric pressure is reached. The extraction vessel is then opened, and the degreased cod skin collagen raw material is removed.

[0111] 4. Stepwise enzymatic hydrolysis: Dissolve the degreased cod skin collagen completely in 4 times its weight of purified water. Heat the collagen solution to 60°C and adjust the pH to 9.0 with alkali. Add 1.0% (by weight) of alkaline protease and 0.1% (by weight) of neutral protease from the degreased cod skin collagen powder, and hydrolyze for 4 hours. Then adjust the pH to 7.0, add 0.05% (by weight) of flavor protease from the degreased cod skin collagen powder, and continue hydrolysis for 3 hours.

[0112] 5. Enzyme inactivation: After the enzymatic hydrolysis is completed, heat the hydrolysate to 90℃ and keep it warm for 15 minutes.

[0113] 6. Decolorization and deodorization: Centrifuge the enzymatic hydrolysate (5000 r / m, 15 min) to remove residual large insoluble particles, and then pass the supernatant through 6 layers of activated carbon fiber membrane.

[0114] 7. Purification and separation: 1% by weight of defatted cod skin collagen powder and 10μm average particle size mesoporous silica (SBA-15, pore size 20nm) were added to the obtained enzymatic hydrolysate. After stirring at room temperature for 20 minutes, the mixture was allowed to stand and filtered to obtain the purified cod skin collagen peptide solution, in order to further remove the cod skin collagen peptides with relatively large molecular weight that were not fully hydrolyzed in the enzymatic hydrolysate.

[0115] 8. 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%.

[0116] 9. Sterilization: The prepared liquid is sterilized using a 0.22μm sterilization membrane.

[0117] 10. Drying: The sterilized liquid is subjected to vacuum freeze drying (-60℃, 20Pa) for 48 hours to obtain high-quality cod skin collagen peptide powder.

[0118] Example 2

[0119] This embodiment provides a cod skin collagen peptide, prepared by the following method:

[0120] 1. Pretreatment to remove fishy smell and impurities: Cut the cleaned cod skin into 0.5cm × 0.5cm slices, add 8 times its weight of alkaline solution, and stir the mixture continuously at 4℃ for 24 hours. Change the solution every 8 hours, and perform ultrasonic treatment every 8 hours at a frequency of 60kHz for 6 minutes (6 seconds of operation followed by a 4-second pause constitutes one work cycle). Finally, rinse with deionized water until neutral. The alkaline solution is a mixture of sodium hydroxide and calcium chloride, with a sodium hydroxide concentration of 1% and a calcium chloride concentration of 1.5%.

[0121] 2. Microwave Thermal Extraction of Collagen: Cod skin was subjected to high-pressure heat treatment using microwave heating. The pretreated cod skin was transferred to a pressure-resistant bottle, and four times its weight of purified water was added before sealing. Microwave treatment was conducted intermittently, with the temperature inside the pressure-resistant bottle set to 130℃ and kept constant. The microwave was activated (600W) when the monitored temperature dropped 3℃ below the set value, and paused when the temperature rose 3℃ above the set value. This cycle was repeated. The solution in the pressure-resistant bottle underwent high-pressure heat treatment for a total of 20 minutes. The extracted collagen solution was then freeze-dried under vacuum (-60℃, 10Pa) for 48 hours, yielding cod skin collagen powder (moisture content less than 10%).

[0122] 3. Supercritical CO2 Extraction for Fat Removal: The obtained cod skin collagen powder is evenly placed in an extraction vessel. Supercritical CO2 is used to dynamically extract the fat components from the cod skin collagen, thereby achieving efficient defatting. The temperature in the extraction vessel is set to 60℃, the pressure to 30MPa, the CO2 flow rate to 2.5L (liquid CO2) / min, and the extraction time to 2h. After extraction, the system pressure is slowly reduced (the depressurization rate needs to be controlled at 2MPa / min) until atmospheric pressure is reached. The extraction vessel is then opened, and the defat-free cod skin collagen raw material is removed.

[0123] 4. Stepwise enzymatic hydrolysis: Dissolve the degreased cod collagen completely in 4 times its weight of purified water. Heat the collagen solution to 60°C and adjust the pH to 9.0 with alkali. Add 1.0% (by weight) of alkaline protease and 0.1% (by weight) of papain of degreased cod skin collagen powder, respectively, and hydrolyze for 4 hours. Then adjust the pH to 7.0, add 0.05% (by weight) of flavor protease of degreased cod skin collagen powder, and continue enzymatic hydrolysis for 3 hours.

[0124] 5. Enzyme inactivation: After the enzymatic hydrolysis is completed, heat the hydrolysate to 95℃ and keep it warm for 10 minutes.

[0125] 6. Decolorization and deodorization: The enzymatic hydrolysate is first centrifuged (5000r / m, 15min) to remove residual large insoluble particles. The prepared supernatant is then passed through 8 layers of activated carbon fiber membrane.

[0126] 7. Purification and separation: 1% by weight of defatted cod skin collagen powder and 10μm average particle size mesoporous silica (SBA-15, pore size 20nm) were added to the obtained enzymatic hydrolysate. After stirring at room temperature for 20 minutes, the mixture was allowed to stand and filtered to obtain the purified cod skin collagen peptide solution, in order to further remove the cod skin collagen peptides with relatively large molecular weight that were not fully hydrolyzed in the enzymatic hydrolysate.

[0127] 8. 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%.

[0128] 9. Sterilization: The prepared liquid is sterilized using a 0.22μm sterilization membrane.

[0129] 10. Drying: The sterilized liquid is subjected to vacuum freeze drying (-60℃, 20Pa) for 48 hours to obtain high-quality cod skin collagen peptide powder.

[0130] Example 3

[0131] This embodiment provides a cod skin collagen peptide, prepared by the following method:

[0132] 1. Pretreatment to remove fishy smell and impurities: Cut the cleaned cod skin into 1cm×1cm pieces, add 10 times its weight of alkaline solution, and stir the mixture continuously at 4℃ for 24 hours. Change the solution every 8 hours, and perform ultrasonic treatment every 8 hours at a frequency of 50kHz for 10 minutes (6 seconds of operation followed by a 4-second pause constitutes one work cycle). Finally, rinse with deionized water until neutral. The alkaline solution is a mixture of sodium hydroxide and calcium chloride, with a sodium hydroxide solution concentration of 0.5% and a calcium chloride solution concentration of 1.5%.

[0133] 2. Microwave Thermal Extraction of Collagen: Cod skin was subjected to high-pressure heat treatment using microwave heating. The pretreated cod skin was transferred to a pressure-resistant bottle, and four times its weight of purified water was added before sealing. Microwave treatment was conducted intermittently, with the temperature inside the pressure-resistant bottle set to 120℃ and kept constant. The microwave was activated (400W) when the monitored temperature dropped 3℃ below the set value, and paused when the temperature rose 3℃ above the set value. This cycle was repeated. The solution in the pressure-resistant bottle underwent high-pressure heat treatment for a total of 20 minutes. The extracted collagen solution was then freeze-dried under vacuum (-60℃, 10Pa) for 48 hours, yielding cod skin collagen powder (moisture content less than 10%).

[0134] 3. Supercritical CO2 Extraction for Fat Removal: The obtained cod skin collagen powder is evenly placed in an extraction vessel. Supercritical CO2 is used to dynamically extract the fat components from the cod skin collagen, thereby achieving efficient defatting. The temperature in the extraction vessel is set to 45℃, the pressure to 40MPa, the CO2 flow rate to 2.5L (liquid CO2) / min, and the extraction time to 2h. After extraction, the system pressure is slowly reduced (the depressurization rate needs to be controlled at 1MPa / min) until atmospheric pressure is reached. The extraction vessel is then opened, and the defat-free cod skin collagen raw material is removed.

[0135] 4. Stepwise enzymatic hydrolysis: Dissolve the degreased cod collagen completely in 4 times its weight of purified water. Heat the collagen solution to 60℃ and adjust the pH to 9.0-9.5 with alkali. Add 1.2% (by weight) of alkaline protease from the defatted cod collagen powder and 0.05% (by weight) of fig protease from the defatted cod skin collagen powder, and hydrolyze for 6 hours. Then adjust the pH to 7.0, add 0.1% (by weight) of flavor protease from the defatted cod skin collagen powder, and continue enzymatic hydrolysis for 2 hours.

[0136] 5. Enzyme inactivation: After the enzymatic hydrolysis is completed, heat the hydrolysate to 95℃ and keep it warm for 10 minutes.

[0137] 6. Decolorization and deodorization: The enzymatic hydrolysate is first centrifuged (5000r / m, 15min) to remove residual large insoluble particles. The prepared supernatant is then passed through 8 layers of activated carbon fiber membrane.

[0138] 7. Purification and separation: 1% by weight of defatted cod skin collagen powder and 10μm average particle size mesoporous silica (SBA-15, pore size 20nm) were added to the obtained enzymatic hydrolysate. After stirring at room temperature for 20 minutes, the mixture was allowed to stand and filtered to obtain the purified cod skin collagen peptide solution, in order to further remove the cod skin collagen peptides with relatively large molecular weight that were not fully hydrolyzed in the enzymatic hydrolysate.

[0139] 8. 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%.

[0140] 9. Sterilization: The prepared liquid is sterilized using a 0.22μm sterilization membrane.

[0141] 10. Drying: The sterilized liquid is subjected to vacuum freeze drying (-60℃, 20Pa) for 48 hours to obtain high-quality cod skin collagen peptide powder.

[0142] Comparative Example 1

[0143] This comparative example is similar to Example 1, except that in this comparative example, in step 1 of the pretreatment to remove fishy smells and impurities, the alkaline solution used is a sodium hydroxide solution with a mass concentration of 0.5%.

[0144] Comparative Example 2

[0145] This comparative example is similar to Example 1, except that in this comparative example, in step 1 of the pretreatment for deodorization and impurity removal, the alkaline solution is a mixed solution of sodium hydroxide and calcium chloride, wherein the mass concentration of sodium hydroxide solution is 0.1% and the mass concentration of calcium chloride solution is 1%.

[0146] Comparative Example 3

[0147] This comparative example is similar to Example 1, except that in this comparative example, in step 1 of the pretreatment for deodorization and impurity removal, the alkaline solution is a mixed solution of sodium hydroxide and calcium chloride, wherein the mass concentration of the sodium hydroxide solution is 0.5% and the mass concentration of the calcium chloride solution is 1%.

[0148] Comparative Example 4

[0149] This comparative example is similar to Example 1, except that step 2 is replaced with heat extraction of collagen: cod skin is subjected to high-temperature heat treatment. The pretreated cod skin is transferred to a flask, 4 times its weight of purified water is added, and the flask is sealed and kept at 100°C for 40 min. The extracted collagen solution is then freeze-dried under vacuum (-60°C, 10 Pa) for 48 h, and the resulting cod skin collagen powder (moisture content less than 10%) is collected.

[0150] Comparative Example 5

[0151] This comparative example is similar to Example 1, except that in this comparative example, step 3 uses lipase to remove fat: Six times the weight of purified water is added to the obtained cod skin collagen powder to adjust the pH to 8.5. Liquid lipase at 1% of the weight of the cod skin collagen powder is added, and the mixture is incubated at 60°C for 3 hours. After inactivation, the enzyme is inactivated at 85°C for 15 minutes, followed by vacuum freeze-drying (-60°C, 10 Pa) for 48 hours. The fat-removed cod skin collagen powder is then collected.

[0152] Comparative Example 6

[0153] This comparative example is similar to Example 1, except that in this comparative example, step 3 uses n-butanol to remove fat: 8 times the weight of 10% n-butanol solution is added to the obtained cod skin collagen powder, the mixture is continuously stirred at 4°C for 24 hours, the solution is changed every 8 hours to remove fat components, and finally it is rinsed with deionized water until neutral, and then vacuum freeze-dried (-60°C, 10Pa) for 48 hours, and the cod skin collagen powder after fat removal is collected.

[0154] Comparative Example 7

[0155] This comparative example is similar to Example 1, except that in this comparative example, step 6 uses activated carbon particles for decolorization: the enzymatic hydrolysate is first centrifuged (5000 r / m, 15 min) to remove residual large insoluble particles. Activated carbon powder at 6% of the weight of defatted cod skin collagen powder is added to the supernatant, and the mixture is incubated at 60°C for 35 min. Solid-liquid separation is then performed by vacuum filtration to obtain a clear and transparent cod skin enzymatic hydrolysate.

[0156] Comparative Example 8

[0157] This comparative example is similar to Example 1, except that in this comparative example, step 7 is purified and separated using the following method: Anhydrous ethanol is added to the decolorized enzymatic hydrolysate to a final ethanol concentration of 85% v / v. The mixture is allowed to stand at 4°C for 18 hours to allow sufficient precipitation. The precipitate is removed by centrifugation at 4000 rpm for 15 minutes, and the supernatant is then filtered. Ethanol is removed from the hydrolysate by rotary evaporation until the enzymatic hydrolysate has no alcohol odor.

[0158] The yield, fishy smell value, hydroxyproline content, fat content, weight-average molecular weight, and OD of cod skin collagen peptide samples prepared in each example and comparative example were measured. 380 The values ​​are shown in Table 1.

[0159] The enzyme preparations involved in this application are all commercially available proteases.

[0160] A panel of 10 trained members with sensory evaluation experience scored the cod skin collagen peptide samples (reconstituted at a mass concentration of 4%) at room temperature according to the sensory scoring criteria in Table 1. The average score was taken, and the results were rounded to two decimal places as appropriate.

[0161] Table 1 Sensory Evaluation Criteria

[0162]

[0163] This application uses a hydroxyproline (Hyp) assay kit to determine the Hyp content in cod skin collagen peptides.

[0164] The weight-average molecular weight of cod skin collagen peptides was determined by high performance liquid chromatography (HPLC) in accordance with GB31645-2018.

[0165] The acid hydrolysis method, as specified in GB 5009.6-2025, is applicable to the determination of fat in food.

[0166] The results of the examples and comparative examples are shown in Table 2.

[0167] Table 2. Physicochemical properties of samples prepared in each embodiment and comparative example.

[0168]

[0169] As shown in Table 2, compared with the cod skin collagen peptide sample prepared in the examples, the fishy smell value of the sample in Comparative Example 1 was significantly increased. This is because calcium chloride can effectively remove the fishy smell through crystal permeation and salting out. Meanwhile, the fishy smell value and OD of the sample in Comparative Example 2 were significantly lower. 380 The values ​​all increased, indicating that the low-concentration sodium hydroxide alkaline solution has limited ability to remove fishy odor and pigments from cod skin. In Comparative Example 3, the fishy odor value of the sample increased significantly, while the Hyp content decreased, indicating that ultrasonic treatment can promote the dissolution of fishy substances. Ultrasonic treatment generates bubbles, and the continuous formation, growth, and collapse of these bubbles increase the dissolution rate of mass transfer, mixing, large-particle-to-small-particle breakage, surface area, extraction kinetics, and cavitation forces in the reaction system. As can be seen from Comparative Examples 1-3, the pretreatment method for cod skin in the examples is a complete and systematic combination that can achieve efficient removal of impurities and fishy odor.

[0170] The fat content and sample yield of the cod skin collagen peptide sample prepared in Comparative Example 4 were significantly increased, while the Hyp content was reduced. This indicates that the cavitation effect caused by microwave heating promoted the separation of large collagen molecules and fat components in cod skin, thereby achieving efficient and thorough removal of fat and reducing the steric hindrance between collagen substrate and enzyme.

[0171] Compared with the cod skin collagen peptide samples prepared in the examples, the fat content of the samples prepared in Comparative Examples 5 and 6 was significantly increased. This indicates that the traditional lipase degradation method and the physical extraction method using organic solvents for "like dissolves like" have limited ability to remove fat components from cod skin. In contrast, the supercritical CO2 extraction method, as the latest and most mature process, can achieve more thorough and efficient removal of fat components, resulting in a high-quality final product.

[0172] As can be seen from the data of the examples and Comparative Example 7, the fishy smell value and OD of the sample in Comparative Example 7 are... 380 The values ​​all increased significantly, indicating that activated carbon fiber membranes have a better decolorization effect than activated carbon fine powder, and can further remove fishy odors. Moreover, activated carbon fiber membranes can be recycled multiple times after regeneration treatment, with low overall cost, making them suitable for industrial production.

[0173] As can be seen from the data of the examples and Comparative Example 8, the weight-average molecular weight of the sample in Comparative Example 8 increased significantly. This indicates that compared with the traditional high-concentration ethanol precipitation method, the novel material with strong adsorption function—mesoporous silica—can better separate and remove collagen peptide components with relatively large molecular weights in the mixed liquid, thereby preparing cod skin collagen peptide products with more concentrated molecular weight distribution and higher quality. Furthermore, mesoporous silica can be regenerated through high-temperature hot water treatment, allowing for multiple recycling and reuse. It is highly safe, has a lower overall cost, and is suitable for industrial production.

[0174] Effect verification

[0175] The cod skin collagen peptides prepared in Example 1 were used to conduct the following experiments.

[0176] 1.1 Effects of cod skin collagen peptides on cell proliferation

[0177] Methods: The effect of cod skin collagen peptides on cell proliferation was detected using the CCK8 assay. Cells were seeded in 96-well plates, with different concentrations (0, 1, 2, 3, 4, 5, 6 mg / mL) of cod skin collagen peptides in treatment groups and a blank control group. After culturing at 37°C and 5% CO2 for 12 h, CCK8 reagent was added and incubated at 37°C for another 20 min. The absorbance at 450 nm was then measured using a microplate reader. Cell proliferation activity was calculated based on the absorbance values. 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 drug was added.

[0178]

[0179] The results are shown in Figure 1. CCK-8 assay results indicated that different concentrations of cod skin collagen peptides could promote cell proliferation to some extent, showing an overall trend of initial increase followed by a slight decrease. Compared with the control group, the proliferation rate of each treatment group was increased, and no significant cytotoxicity was observed. The 5 mg / mL group had the highest proliferation rate, significantly higher than other groups (P < 0.05), indicating that this concentration had the most significant promoting effect on cell proliferation. There was no significant difference among the 2-4 mg / mL groups, but all were significantly higher than the low concentration group (1 mg / mL) and the 6 mg / mL group. Although 6 mg / mL still had a certain promoting effect, the effect was lower than that of the medium concentration, possibly due to increased cellular metabolic burden or saturation at higher concentrations. Overall, cod skin collagen peptides have a good proliferative effect in the range of 2-5 mg / mL, with 5 mg / mL being the optimal concentration.

[0180] 1.2 Effects of cod skin collagen peptides on intracellular ROS and MDA

[0181] Methods: HSF cells were cultured at a concentration of 9 × 10⁻⁶. 4 Cells were seeded at a density of 1 / 2 well in 6-well plates, and an appropriate amount of complete culture medium was added. The plates were then incubated at 37°C with 5% CO2 for approximately 12 hours to allow for full cell adhesion and entry into the logarithmic growth phase. Cells were then randomly divided into a blank control group, a UVB model group, and a cod skin collagen peptide (5 mg / mL) treatment group. Intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA fluorescent probe method. After treatment, the culture medium was discarded, and DCFH-DA was diluted with serum-free medium to a suitable working concentration (usually 10 μmol / L). This diluted DCFH-DA was added to the cells and incubated at 37°C in the dark for 20–30 minutes to allow the probe to fully penetrate the cells and be hydrolyzed by esterases to form DCF that can be oxidized by ROS. After incubation, the cells were gently washed three times with PBS to remove any probes that had not penetrated the cells, and then observed and photographed under a fluorescence microscope.

[0182] Malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) method. After treatment, cells from each group were collected, lysis buffer was added for thorough lysis, and the mixture was mixed with TBA working solution according to the kit instructions. The mixture was reacted in a 95°C water bath for approximately 15–30 min to allow the MDA and TBA to form a colored complex. After the reaction, the cells were rapidly cooled and centrifuged, and the supernatant was collected. The absorbance was measured at 532 nm, and the MDA content was calculated based on a standard curve to assess the level of cellular lipid peroxidation.

[0183] The results are shown in Figure 2. Figure 2A shows that the green fluorescence in the cells of the blank control group was weak, indicating a low basal ROS level. After UVB irradiation, the cell fluorescence significantly increased, indicating that UVB can significantly induce the generation of large amounts of reactive oxygen species in cells. After treatment with cod skin collagen peptides, the fluorescence intensity was significantly weaker than that of the UVB group, but still slightly higher than that of the control group, indicating that it can alleviate UVB-induced oxidative stress damage to some extent. The quantitative results of relative fluorescence intensity shown in Figure 2B are consistent with those in Figure 2A. The UVB group was significantly higher than the control group (P < 0.05), while the ROS level significantly decreased after cod skin collagen peptide intervention (P < 0.05), indicating that it has a good antioxidant effect. Figure 2C shows that the MDA content significantly increased after UVB irradiation, indicating an aggravated degree of cellular lipid peroxidation. After treatment with cod skin collagen peptides, the MDA level significantly decreased, but was still higher than that of the blank control group (P < 0.05). In summary, UVB can induce oxidative damage in HSF cells, while cod skin collagen peptides can effectively reduce ROS generation and lipid peroxidation levels, thus providing some protection to cells.

[0184] 1.3 Effects of cod skin collagen peptides on cell repair

[0185] 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 sterile pipette tip was used to scratch the wells perpendicular to the drawn lines. Floating cells were washed away with PBS. A blank control group, a UVB model group, and a cod skin collagen peptide (5 mg / mL) treatment group were set up. Cells in each group were cultured under appropriate conditions, and the scratch healing process was observed and photographed under a microscope at 0 h, 24 h, and 48 h post-scratching. The effect of cod skin collagen peptide on cell repair capacity was evaluated by measuring the change in scratch width at different time points.

[0186] As shown in Figure 3, the scratches in the blank control group were basically healed within 48 hours, demonstrating the strong self-repair ability of the cells. The scratch healing rate in the UVB model group was significantly slower, with a large unhealed area remaining after 48 hours, indicating that UVB irradiation significantly inhibited cell repair. The scratch healing in the cod skin collagen peptide treatment group fell between the two groups; the scratch width was significantly smaller than that in the UVB model group at 24 hours, and the degree of healing was also better than that in the UVB model group at 48 hours, indicating that cod skin collagen peptides can promote cell repair after UVB damage to a certain extent.

[0187] 1.4 Effects of cod skin collagen peptides on cellular collagen synthesis

[0188] Methods: The expression levels of type I collagen (Collagen I) and matrix metalloproteinase 1 (MMP1) in cells of each group were detected by Western blot.

[0189] A blank control group, a UVB treatment group, and a cod skin collagen peptide (5 mg / mL) treatment group were set up. Cells were seeded in culture plates, and when cell confluence reached 70%-80%, the blank control group received no special treatment. The UVB treatment group received 90 minutes of UVB irradiation, while the cod skin collagen peptide treatment group received UVB treatment followed by the addition of a 5 mg / mL cod skin collagen peptide solution for further culture. After 12 hours of culture, cell samples were collected, and total protein was extracted by cell lysis. After quantification, proteins were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The PVDF membrane was incubated with a specific primary antibody to identify collagen I and MMP1, followed by incubation with a secondary antibody. Finally, chemiluminescence staining was performed, and images were acquired using an imaging system. The gray values ​​of each band were analyzed using relevant software to reflect the expression levels of Collagen I and MMP1.

[0190] Western blot was used to detect the effects of cod skin collagen peptides on the expression of Collagen I and MMP1 in UVB-induced cells. As shown in Figure 4, the results indicated that compared with the blank control group, Collagen I protein expression was significantly decreased and MMP1 expression was significantly increased after UVB irradiation, suggesting that UVB can inhibit collagen synthesis and promote its degradation. Compared with the UVB group, the expression level of Collagen I protein was significantly upregulated and MMP1 expression decreased after cod skin collagen peptide intervention, indicating that cod skin collagen peptides can, to some extent, reverse the collagen metabolism imbalance caused by UVB. In conclusion, cod skin collagen peptides can protect against UVB-induced cell damage by promoting collagen synthesis and inhibiting its degradation.

[0191] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0192] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing cod skin collagen peptides, characterized in that, The process includes the following steps: mixing an alkaline solution with cod skin and pretreating it under ultrasonic-assisted conditions; the alkaline solution comprises sodium hydroxide, calcium chloride, and water, wherein the mass fraction of sodium hydroxide is 0.5%~1% and the mass fraction of calcium chloride is 1%~2%; mixing the pretreated cod skin with water, heat-treating the resulting mixture using microwave-assisted heating, and drying it to prepare cod skin collagen; extracting the cod skin collagen using supercritical CO2 extraction to prepare defatted cod skin collagen; enzymatically hydrolyzing the defatted cod skin collagen with a protease to prepare an enzymatic hydrolysate; after inactivating the enzyme in the enzymatic hydrolysate, decolorizing it using an activated carbon fiber membrane, mixing the resulting decolorized product with mesoporous silica, purifying and separating it, and nanofiltration to prepare cod skin collagen peptides.

2. The method for preparing cod skin collagen peptides according to claim 1, characterized in that, One or more of the following conditions must be met: (1) In the heat treatment step of the obtained mixture using microwave-assisted heating, the temperature of the mixture is controlled at 120℃~140℃ and the heat treatment time is 20min~40min; (2) The power of microwave heating is 300W~600W. And (3) The extraction conditions include: temperature of 35℃~60℃, pressure of 20MPa~40MPa, supercritical CO2 flow rate of 0.5L / min~2.5L / min, and extraction time of 1h~4h.

3. The method for preparing cod skin collagen peptides according to claim 1, characterized in that, In the step of preparing the enzymatic hydrolysate, the defatted cod skin collagen is mixed with water, and alkaline protease and protease A are added for the first enzymatic hydrolysis, followed by the addition of flavor protease for the second enzymatic hydrolysis; the protease A includes one of neutral protease, papain, fig protease and trypsin.

4. The method for preparing cod skin collagen peptides according to claim 3, characterized in that, The following conditions must be met: (1) the mass ratio of the defatted cod skin collagen to the water is 1:(4~6); (2) the mass of the alkaline protease is 0.5%~1.5% of the defatted cod skin collagen protein; (3) the mass of the protease A is 0.05%~0.3% of the defatted cod skin collagen protein; (4) the mass of the flavor protease is 0.05%~0.1% of the defatted cod skin collagen protein; (5) the temperature of the first enzymatic hydrolysis is 55℃~60℃, the time of the first enzymatic hydrolysis is 4h~6h, and the pH value of the first enzymatic hydrolysis is 8.0~9.5; and (6) the temperature of the second enzymatic hydrolysis is 55℃~60℃, the time of the second enzymatic hydrolysis is 2h~4h, and the pH value of the second enzymatic hydrolysis is 6.8~7.

2.

5. The method for preparing cod skin collagen peptides according to any one of claims 1 to 4, characterized in that, The following conditions shall be met: (1) During the pretreatment process, ultrasonic treatment shall be performed every 6h to 8h, the frequency of each ultrasonic treatment shall be 40kHz to 60kHz, the duration of each ultrasonic treatment shall be 6min to 12min, and the ultrasonic treatment shall be performed for 5s to 6s and paused for 4s to 5s; (2) During the pretreatment process, the alkaline solution shall be replaced every 6h to 8h; (3) The mass ratio of the alkaline solution to the cod skin shall be (8 to 12): 1; (4) The pretreatment shall be performed at 0℃ to 4℃; and (5) The pretreatment time shall be 20h to 30h.

6. The method for preparing cod skin collagen peptides according to any one of claims 1 to 4, characterized in that, In the purification and separation step, the decolorized product is mixed with the mesoporous silica, stirred for 15 min to 20 min, allowed to stand, and then filtered; wherein the particle size of the mesoporous silica is 5 μm to 10 μm, the pore size of the mesoporous silica is 10 nm to 20 nm, and the mass of the mesoporous silica is 1% to 3% of the mass of the defatted cod skin collagen protein.

7. The method for preparing cod skin collagen peptides according to any one of claims 1 to 4, characterized in that, The activated carbon fiber membrane has 6 to 8 layers.

8. The method for preparing cod skin collagen peptides according to any one of claims 1 to 4, characterized in that, In the nanofiltration step, a nanofiltration membrane with a molecular weight cutoff of 150 Da to 200 Da is used.

9. A cod skin collagen peptide, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 8.

10. The application of the cod skin collagen peptide according to claim 9 in the preparation of the product, characterized in that, The product satisfies one or more of the following conditions: (1) the product has antioxidant function; (2) the product has cell proliferation promotion function; (3) the product has cell repair promotion function; and (4) the product has collagen synthesis promotion function.

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