LU501617B1

LU501617B1 - COLLAGEN PEPTIDE CAPABLE OF ACTIVATING AQP3 mRNA IN DERMAL FIBROBLASTS AND PREPARATION METHOD THEREOF

Info

Publication number: LU501617B1
Application number: LU501617A
Authority: LU
Inventors: Po Li; Dan Cheng; Yingxia Zhang
Original assignee: Lithy One Health Group
Priority date: 2022-03-08
Filing date: 2022-03-08
Publication date: 2022-10-10

Abstract

The collagen peptide includes at least 99% by mass of a collagen peptide A with a molecular weight < 10,000; the collagen peptide A includes at least 60% by mass of a collagen peptide a with a molecular weight < 1,000; the collagen peptide a includes at least 40% by mass of a small-molecule collagen peptide with a molecular weight < 500; the small-molecule collagen peptide includes a dipeptide, a tripeptide, and a tetrapeptide; the collagen peptide has a transmittance of 90 or higher at a wavelength of 450 nm; and the collagen peptide has a transmittance of 97 or higher at a wavelength of 620 nm. The collagen peptide of the present disclosure includes a variety of functional small-molecule peptides. According to in vitro tests, the collagen peptide has the effects of inhibiting the expression of matrix metalloproteinase (MMP) and promoting the expression of genes AQP3, FBN2, and LOX.

Description

COLLAGEN PEPTIDE CAPABLE OF ACTIVATING AQP3 mRNA IN DERMAL LU501617

FIBROBLASTS AND PREPARATION METHOD THEREOF

TECHNICAL FIELD The present disclosure relates to the field of biotechnology, and in particular to a collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts and a preparation method thereof.

BACKGROUND In recent years, oral beauty products (such as collagen and hyaluronic acid (HA)) have become more and more popular in the market, because oral collagen is easily absorbed by the body through the intestinal tract and thus reaches the dermis compared with collagen for external use. The dermis is mainly an extracellular matrix (ECM) composed of collagen, proteoglycan, HA, and the like, and due to aging of the dermis itself and external factors (such as light aging), matrix metalloproteinases (MMPs) in the dermis will be activated to degrade collagen, causing skin aging, wrinkles, and other problems. Supplemented collagen peptides can be absorbed through the intestinal tract and reach the dermis through the human circulatory system, thereby activating fibroblasts to synthesize components of the dermis ECM such as collagen.

However, although collagen peptides can activate fibroblasts to secrete ECM components, collagen peptides cannot inhibit MMP, and thus cannot block collagen breakdown caused by self-aging and external factors. In addition, although collagen peptides can activate fibroblasts to produce HA and thus can theoretically increase the moisture-holding capacity of the skin, the transfer of moisture among skin cells requires the aquaporin AQP3. In the aged skin, the expression of AQP3 decreases. That is, even though collagen can theoretically increase the moisturizing ability of the skin, there is still an obstacle for the transfer of moisture among skin cells due to the inability to repair AQP3.

SUMMARY The present disclosure provides a collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts.

The present disclosure specifically adopts the following technical solutions: Specifically, an embodiment of the present disclosure provides a collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts, including: at least 99% by mass of a collagen peptide A with a molecular weight < 10,000;

where the collagen peptide A includes at least 60% by mass of a collagen peptide a with a LUS01617 molecular weight < 1,000; the collagen peptide a includes at least 40% by mass of a small-molecule collagen peptide with a molecular weight < 500; the small-molecule collagen peptide includes a dipeptide, a tripeptide, and a tetrapeptide; the collagen peptide has a transmittance of 90 or higher at a wavelength of 450 nm; and the collagen peptide has a transmittance of 97 or higher at a wavelength of 620 nm.

The present disclosure provides a preparation method of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts described above, including the following steps: pretreatment of a raw material: mixing fish skin and water in a container B according to a mass ratio of less than 1:5, and heating at 60°C to 90°C for 1 hour to 6 hours; cooking and enzymolysis: adding an alkaline protease, a neutral protease, and a collagenase to the container B to extract the collagen peptide; enzymatic inactivation: after the enzymolysis is completed, heating in a boiling water bath to inactivate the enzymes; and separating the collagen peptide.

In some embodiments, in the pretreatment of a raw material, the mass ratio of the fish skin to the water may be 1:4.

In some embodiments, in the pretreatment of a raw material, the heating may be conducted at 70°C for 2 hour.

In some embodiments, the alkaline protease and the neutral protease may be derived from Bacillus subtilis (B. subtilis).

In some embodiments, in the step of cooking and enzymolysis, a total addition amount of the enzymes may account for 0.3% to 0.9% of a total mass of the fish skin; and the enzymolysis may be conducted for 1 hour to 6 hours at a temperature of 35°C to 65°C and a pH of 7 to 7.5.

In some embodiments, the cooking and enzymolysis may include the following steps: first cooking: placing the pretreated mixed raw material in the container B, heating to 35°C to 45°C, adding the alkaline protease, the neutral protease, and the collagenase, and cooking for 1 hour to 2 hours to obtain a first collagen; second cooking: further heating some of the first collagen to 45°C to 55°C, and cooking for 1 hour to 2 hours to obtain a second collagen; and third cooking: further heating some of the second collagen to 55°C to 65°C, and cooking for 1 hour to 2 hours to obtain a third collagen.

In some embodiments, addition amounts of the alkaline protease, the neutral protease, and the collagenase may respectively account for 0.1% to 0.3%, 0.1% to 0.3%, and 0.1% to 0.3%

of the total mass of the fish skin. LUS01617 In some embodiments, the first collagen, the second collagen, and the third collagen obtained in the step of cooking and enzymolysis may have a ratio of (X1-Y1):(X2-Y2):(X3-Y3).

In some embodiments, separating the collagen peptide may include the following steps: primary filtration; ultrafiltration; activated carbon filtration; secondary filtration; vacuum concentration; and spray drying.

Based on the above content, compared with the prior art, the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts provided by the present disclosure can activate the expression of the gene AQP3 mRNA in dermal fibroblasts, thereby activating the skin aquaporin and increasing the transfer of moisture among skin cells to increase a moisture content in the skin (especially in the stratum corneum).

Other features and beneficial effects of the present disclosure will be illustrated in the following description, and some of these will become apparent from the description or be understood by implementing the present disclosure. The objectives and other beneficial effects of the present disclosure can be implemented or obtained by structures specifically indicated in the description, claims, and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS To describe the technical solutions in examples of the present disclosure or in the prior art more clearly, the accompanying drawings required for describing the examples or the prior art will be briefly described below. Apparently, the accompanying drawings in the following description show some examples of the present disclosure, and a person of ordinary skill in the art may still derive other drawings from these accompanying drawings without creative efforts. Unless otherwise specified, the positional relationships described in the accompanying drawings in the following description are based on the drawing directions of components in the accompanying drawings.

FIG. 1 is a histogram illustrating a relative gene expression level at each collagen peptide concentration; FIG. 2 shows the improvement of fine lines on days 0, 14, and 28 after the application of the collagen peptide provided by the present disclosure; and FIG. 3 shows the improvement of erythema on days 0, 14, and 28 after the application of the collagen peptide provided by the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In order to make the objectives, technical solutions, and advantages of the embodiments LUS01617 of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure are clearly and completely described below with reference to the accompanying drawings in the embodiments of the present disclosure. Apparently, the described embodiments are merely some rather than all of the embodiments of the present disclosure. The technical features designed in different embodiments of the present disclosure described below can be combined with each other as long as they do not conflict with each other. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present disclosure without creative work fall within the protection scope of the present disclosure.

In the description of the present disclosure, it should be noted that all terms (including technical terms and scientific terms) used in the present disclosure have the same meanings as commonly understood by those of ordinary skill in the art to which the present disclosure belongs, and should not be construed as limiting the present disclosure. It should be further understood that the terms used in the present disclosure should be understood to have meanings consistent with meanings of these terms in the context of this specification and in the relevant art, and should not be taken in an idealized or overly formal sense, unless explicitly defined in the present disclosure.

As used herein, the article "a/an" refers to one or more, and does not necessarily restrict a noun modified thereby as singular.

As used herein, unless otherwise stated, the room temperature refers to 25°C. The standard temperature and pressure respectively refer to 25°C and 1 atm. In general, unless otherwise stated, the term "about" is intended to include + 10% variance or range and an experimental or instrumental error associated with the acquisition of a related value, and preferably include the greater one among these.

An embodiment of the present disclosure provides a collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts, including: at least 99% by mass of a collagen peptide A with a molecular weight < 10,000; where the collagen peptide A includes at least 60% by mass of a collagen peptide a with a molecular weight < 1,000; the collagen peptide a includes at least 40% by mass of a small-molecule collagen peptide with a molecular weight < 500; the small-molecule collagen peptide includes a dipeptide, a tripeptide, and a tetrapeptide; the collagen peptide has a transmittance of 90 or higher at a wavelength of 450 nm; and the collagen peptide has a transmittance of 97 or higher at a wavelength of 620 nm.

An embodiment of the present disclosure provides a preparation method of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts, including the following LUS01617 steps: Step 1: Acquisition of a raw material: Tilapia skin is used as the raw material, which can be directly purchased from a fish slaughtering factory. After coming out from the fish slaughtering factory, the skin is cleaned, quick-frozen, transported by a refrigerated truck, and directly fed.

Traditionally, dried fish scales and fish meal are used as a raw material, which not only requires a high temperature and a large enzyme amount in the subsequent enzymolysis treatment, but also makes a collagen peptide product have a strong fishy odor.

In addition, unless otherwise specified, the raw material used can also be other fish skin products, such as codfish skin and salmon skin, or can be a fish skin product prepared by a conventional method in the art.

Step 2: The skin is cut.

Step 3: Pretreatment of a raw material: the fish skin is mixed with water in a container B according to a mass ratio of less than 1:5, and then heated at 60°C to 90°C for 1 hour to 6 hours.

The pretreatment is conducted mainly to denature the protein, thereby facilitating the subsequent enzymolysis. The present disclosure adopts a small water amount and a low pretreatment temperature, which can cooperate with the combination of an alkaline protease and a neutral protease derived from B. subtilis and a collagenase to produce excellent hydrolysis and enzymolysis effects.

Generally, the pretreatment requires a large amount of water (at least 5 times or more), a high temperature (80°C to 100°C), and a long time, which causes waste of resources and energy. Moreover, a collagen peptide produced by enzymolysis of fish skin pretreated at a high temperature (such as 90°C or higher) has a distinct fishy odor, and the hydrolysis and enzymolysis efficiency is low, which is not conducive to market promotion.

Step 4: Cooking and enzymolysis: an alkaline protease and a neutral protease derived from B. subtilis are added; and the addition amounts of enzymes (0.3% to 0.9%), the temperature (35°C to 65°C), the pH (7 to 7.5), and the time (1 hour to 6 hours) are controlled to conduct enzymolysis to obtain a collagen peptide. This process requires a small amount of protease and a low enzymolysis temperature, which is of great significance for cost saving. Moreover, the collagen peptide prepared can activate the expression of the gene AQP3 mRNA in dermal fibroblasts, thereby activating the skin aquaporin and increasing the transfer of moisture among skin cells to increase a moisture content in the skin (especially in the stratum corneum). In addition, the collagen peptide can inhibit the expression of MMP to reduce the degradation of skin collagen caused by natural aging and external factors, which reduces the LUS01617 degradation of collagen while stimulating dermal fibroblasts to synthesize collagen, such that the efficacy of oral collagen to improve skin wrinkles is amplified.

Step 5: Enzymatic inactivation: after the enzymolysis is completed, heating is conducted in a boiling water bath to inactivate the enzymes.

Step 6: The collagen peptide is separated, which specifically includes the following steps: primary filtration: a 400-mesh duplex filter is used; ultrafiltration: an ultrafiltration membrane with a molecular weight cut-off (MWCO) of 5,000 Da is used for ultrafiltration; activated carbon filtration: activated carbon is added at an amount 2% to 5% of an ultrafiltrate for decolorization and deodorization; secondary filtration: a 400-mesh duplex filter is used; and vacuum concentration: a filtrate is subjected to vacuum concentration until a brix is 35% to 45% to obtain a concentrate of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts.

Step 7: Drying for powdering and packaging: the concentrate is spray dried at 85°C to 95°C for powdering, and then packaged to obtain a finished product of the deep-sea fish collagen peptide.

The following examples are provided to illustrate the present disclosure in more detail without limiting the present disclosure in any way.

Example 1 A preparation method of a collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts was provided, including the following steps: Step 1: Acquisition of a raw material: Tilapia skin was used as the raw material.

Step 2: The fish skin was chopped.

Step 3: Pretreatment of the raw material: the fish skin was mixed with water in a container B according to a mass ratio of 1:4, and then heated at 70°C for 2 hours.

Step 4: Cooking and enzymolysis: an alkaline protease and a neutral protease derived from B. subtilis and a collagenase were added; and enzymolysis was conducted for 4.5 hours at a temperature of 35°C to 65°C and a pH of 7 to 7.5 to obtain the collagen peptide, where a total addition amount of the enzymes was 0.6% of a total mass of the fish skin.

Specifically, the pretreated mixed raw material was placed in the container B and heated to 40°C, then the alkaline protease (at an amount 0.2% of the total mass of the fish skin), the neutral protease (at an amount 0.2% of the total mass of the fish skin), and the collagenase (at an amount 0.2% of the total mass of the fish skin) were added, and a resulting mixture was cooked for 1.5 hours to obtain a first collagen; LUS01617 some of the first collagen was further heated to 50°C and cooked for 1.5 hours to obtain a second collagen; and some of the second collagen was further heated to 60°C and cooked for 1.5 hours to obtain a third collagen.

The first collagen, the second collagen, and the third collagen had a ratio of 64:22:14.

Step 5: Enzymatic inactivation: after the enzymolysis was completed, heating was conducted in a boiling water bath to inactivate the enzymes.

Step 6: The collagen peptide was separated, which specifically included the following steps: primary filtration: a 400-mesh duplex filter was used; ultrafiltration: an ultrafiltration membrane with an MWCO of 5,000 Da was used for ultrafiltration; activated carbon filtration: activated carbon was added at an amount 2% to 5% of an ultrafiltrate for decolorization and deodorization; secondary filtration: a 400-mesh duplex filter was used; and vacuum concentration: a filtrate was subjected to vacuum concentration until a brix was 35% to 45% to obtain a concentrate of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts.

Step 7: Drying for powdering and packaging: the concentrate was spray dried at 85°C to 95°C for powdering, and then packaged to obtain a finished product of the deep-sea fish collagen peptide.

Example 2 A preparation method of a collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts was provided, including the following steps: Step 1: Acquisition of a raw material: Tilapia skin was used as the raw material.

Step 2: The fish skin was chopped.

Step 3: Pretreatment of the raw material: the fish skin was mixed with water in a container B according to a mass ratio of 1:3, and then heated at 60°C for 5 hours.

Step 4: Cooking and enzymolysis: an alkaline protease and a neutral protease derived from B. subtilis and a collagenase were added; and enzymolysis was conducted for 6 hours at a temperature of 35°C to 65°C and a pH of 7 to 7.5 to obtain the collagen peptide, where a total addition amount of the enzymes was 0.9% of a total mass of the fish skin.

Specifically, the pretreated mixed raw material was placed in the container B and heated to 45°C, then the alkaline protease (at an amount 0.3% of the total mass of the fish skin), the neutral protease (at an amount 0.3% of the total mass of the fish skin), and the collagenase (at LUS01617 an amount 0.3% of the total mass of the fish skin) were added, and a resulting mixture was cooked for 2 hours to obtain a first collagen; some of the first collagen was further heated to 55°C and cooked for 2 hours to obtain a second collagen; and some of the second collagen was further heated to 65°C and cooked for 2 hours to obtain a third collagen.

The first collagen, the second collagen, and the third collagen had a ratio of 70:20:10.

Example 3 A preparation method of a collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts was provided, including the following steps: Step 1: Acquisition of a raw material: Tilapia skin was used as the raw material.

Step 2: The fish skin was chopped.

Step 3: Pretreatment of the raw material: the fish skin was mixed with water in a container B according to a mass ratio of 1:4, and then heated at 90°C for 1 hour.

Step 4: Cooking and enzymolysis: an alkaline protease and a neutral protease derived from B. subtilis and a collagenase were added; and enzymolysis was conducted for 3.6 hours at a temperature of 35°C to 65°C and a pH of 7 to 7.5 to obtain the collagen peptide, where a total addition amount of the enzymes was 0.3% of a total mass of the fish skin.

Specifically, the pretreated mixed raw material was placed in the container B and heated LUS01617 to 36°C, then the alkaline protease (at an amount 0.1% of the total mass of the fish skin), the neutral protease (at an amount 0.2% of the total mass of the fish skin), and the collagenase (at an amount 0.3% of the total mass of the fish skin) were added, and a resulting mixture was cooked for 1.2 hours to obtain a first collagen; some of the first collagen was further heated to 47°C and cooked for 1.2 hours to obtain a second collagen; and some of the second collagen was further heated to 55°C and cooked for 1.2 hours to obtain a third collagen.

The first collagen, the second collagen, and the third collagen had a ratio of 60:23:17.

It should be noted that specific parameters or some common reagents in the above examples are specific examples or preferred examples under the concept of the present disclosure, and do not limit the present disclosure. Those skilled in the art can make adaptive adjustments within the concept and protection scope of the present disclosure.

The collagen peptides in the above examples and comparative examples were tested as follows: (1) Detection of enzymolysis effects: The collagen peptides obtained in the examples and comparative examples were tested by a method in GB 31645-2018, and results are shown in Table 1.

Table 1 LU501617 Item Molecular weight distribution (MWD) of collagen peptides (%) Molecular weight < Molecular weight < Molecular weight < 500 1,000 10,000 (2) Detection of light transmittance of finished collagen peptide products: The finished collagen peptide products obtained in the examples were tested and evaluated for transmittance (clarity) according to a method in QB2732-2005, and results are shown in Table 2 below.

Table 2 Item Light transmittance (%) at 450 Light transmittance (%) at nm 620 nm (3) The impact of the collagen peptide (different mass percentages) on the expression of AQP3 mRNA in fibroblasts was tested, and the product obtained in Example 1 was used for the test. Results are shown in Table 3 and FIG. 1.

Table 3 Relative gene expression levels

100.00% 100.00% 100.00% 100.00%

175.50% 36.97% 103.82% 382.28%

581.28% 25.72% 44.74% 482.69%

86.26% 38.45% 225.09% 360.31%

0.05% 215.48% 73.22% 231.90% 519.56%

0.01% 197.69% 151.56% 223.44% 490.19% LU501617 Cell experiments show that the collagen peptide (different concentrations) can promote the expression of AQP3 mRNA in fibroblasts and inhibit MMP, which exhibits similar effects to epidermal growth factor (EGF). The collagen peptide can promote the expression of AQP3, FBN2, and LOX, and also can inhibit MMP1.

It should be noted that bars in FIG. 1 represent blank, positive, 1%, 0.5%, 0.05%, and

0.01% sequentially from left to right.

(4) Clinical experiments of the collagen peptide: The before-after study was conducted on 30 people, where 5 g of the collagen peptide was taken daily for 14 days and 28 days, and the skin melanin, skin moisture, skin gloss, skin elasticity, and other indexes were investigated. Results are shown in Table 4 and 5 below.

Table 4 Moisturizing effect of the collagen peptide

57.4143 125% 17.2851 63% Table 5 Effects of the collagen peptide to increase skin elasticity and reduce roughness and erythema Skin elasticity Skin roughness Skin erythema 0, 0.53+0.02 18.00+2.22 35.00+3.25

0.62+0.03** 14.00+1.89** 30.00+3.15**

0.69+0.02** 13.20+1.67** 27.00+2.63** The collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts provided by the present disclosure includes a variety of functional small-molecule peptides. According to in vitro tests, the collagen peptide has the effects of inhibiting the expression of MMP and promoting the expression of genes AQP3, FBN2, and LOX. According to human clinical trials, as shown in FIG. 2 and FIG. 3, the collagen peptide can improve skin elasticity and retain moisture.

In summary, compared with the prior art, the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts provided by the present disclosure can activate the expression of the gene AQP3 mRNA in dermal fibroblasts, thereby activating the skin aquaporin and increasing the transfer of moisture among skin cells to increase a moisture content in the skin LUS01617 (especially in the stratum corneum).

In addition, the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts provided by the present disclosure can inhibit the expression of MMP to reduce the degradation of skin collagen caused by natural aging and external factors, which reduces the degradation of collagen while stimulating dermal fibroblasts to synthesize collagen, such that the efficacy of oral collagen to improve skin wrinkles is amplified.

In addition, those skilled in the art should understand that, although there are many problems in the prior art, each embodiment or technical solution of the present disclosure can achieve the improvement in only one or some aspects, and is not necessary to simultaneously solve all technical problems listed in the prior art or the background art. Those skilled in the art should understand that any content not mentioned in the claims should not be construed as a limitation to the claims.

Although terms such as collagen peptide, fish skin, cooking, enzymolysis, and enzymatic inactivation are frequently used in the present disclosure, it is possible to use other terms. These terms are used only to describe and explain the essence of the present disclosure more conveniently, and it is contrary to the spirit of the present disclosure to interpret the terms as any additional limitation. The terms "first", "second", and the like (if present) in the description and claims of the embodiments of the present disclosure and the above accompanying drawings are used to distinguish similar objects, but do not necessarily indicate a specific order or sequence.

Finally, it should be noted that the above examples are merely intended to describe the technical solutions of the present disclosure, rather than to limit the present disclosure. Although the present disclosure is described in detail with reference to the above examples, persons of ordinary skill in the art should understand that modifications may be made to the technical solutions described in the above examples or equivalent replacements may be made to some or all technical features thereof, which do not make the essence of corresponding technical solutions depart from the scope of the technical solutions in the examples of the present disclosure.

Claims

CLAIMS LU501617

1. A collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts, comprising: at least 99% by mass of a collagen peptide A with a molecular weight < 10,000; wherein the collagen peptide A comprises at least 60% by mass of a collagen peptide a with a molecular weight < 1,000; the collagen peptide a comprises at least 40% by mass of a small-molecule collagen peptide with a molecular weight < 500; the small-molecule collagen peptide comprises a dipeptide, a tripeptide, and a tetrapeptide; the collagen peptide has a transmittance of 90 or higher at a wavelength of 450 nm; and the collagen peptide has a transmittance of 97 or higher at a wavelength of 620 nm.

2. A preparation method of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts according to claim 1, comprising the following steps: pretreatment of a raw material: mixing fish skin and water in a container B according to a mass ratio of less than 1:5, and heating at 60°C to 90°C for 1 hour to 6 hours; cooking and enzymolysis: adding an alkaline protease, a neutral protease, and a collagenase to the container B to extract the collagen peptide; enzymatic inactivation: after the enzymolysis is completed, heating in a boiling water bath to inactivate the enzymes; and separating the collagen peptide.

3. The preparation method of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts according to claim 2, wherein in the pretreatment of a raw material, the mass ratio of the fish skin to the water is 1:4.

4. The preparation method of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts according to claim 3, wherein in the pretreatment of a raw material, the heating is conducted at 70°C for 2 hours.

5. The preparation method of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts according to claim 2, wherein the alkaline protease and the neutral protease are derived from Bacillus subtilis (B. subtilis).

6. The preparation method of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts according to claim 5, wherein in the step of cooking and enzymolysis, a total addition amount of the enzymes accounts for 0.3% to 0.9% of a total mass of the fish skin; and the enzymolysis is conducted for 1 hour to 6 hours at a temperature of 35°C to 65°C and a pH of 7 to 7.5.

7. The preparation method of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts according to claim 2, wherein the cooking and enzymolysis comprises the LUS01617 following steps: first cooking: placing the pretreated mixed raw material in the container B, heating to 35°C to 45°C, adding the alkaline protease, the neutral protease, and the collagenase, and cooking for 1 hour to 2 hours to obtain a first collagen; second cooking: further heating some of the first collagen to 45°C to 55°C, and cooking for 1 hour to 2 hours to obtain a second collagen; and third cooking: further heating some of the second collagen to 55°C to 65°C, and cooking for 1 hour to 2 hours to obtain a third collagen.

8. The preparation method of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts according to claim 7, wherein addition amounts of the alkaline protease, the neutral protease, and the collagenase respectively account for 0.1% to 0.3%, 0.1% to 0.3%, and 0.1% to 0.3% of the total mass of the fish skin.

9. The preparation method of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts according to claim 7, wherein the first collagen, the second collagen, and the third collagen obtained in the step of cooking and enzymolysis have a mass ratio of (50-70):(20-30):(10-20).

10. The preparation method of the collagen peptide capable of activating AQP3 mRNA in dermal fibroblasts according to claim 2, wherein the separating the collagen peptide comprises the following steps: primary filtration; ultrafiltration; activated carbon filtration; secondary filtration; vacuum concentration; and spray drying.