Codfish skin collagen peptide for promoting wound healing and preparation method of codfish skin collagen peptide

Abstract

The invention provides cod skin collagen peptide for promoting wound healing and a preparation method of the cod skin collagen peptide, namely a protein peptide preparation method which is simple in process step, easy to operate and implement and capable of effectively utilizing a fish skin resource, and high-value development and utilization of low-value aquatic product leftovers are realized. The collagen peptide prepared by the invention has huge potential in wound healing, some polypeptides have high antioxidant, anti-inflammatory and antibacterial activities and the like, and the composition of the amino acids has potential positive influence on skin wound repair and antioxidant effects.

Description

Technical Field

[0001] The invention belongs to the field of manufacturing bioactive products, and particularly relates to a cod skin collagen peptide for promoting wound healing and a preparation method thereof. Background Art

[0002] Collagen, as the most abundant structural protein in the human body, is widely distributed in connective tissues such as skin, bones and blood vessels, and plays an important role in maintaining the mechanical strength of tissues and the integrity of the extracellular matrix. In recent years, collagen peptides, as a bioactive small molecule, have been widely used in wound healing, mucosal repair and other aspects because of their good biocompatibility, low immunogenicity and the function of promoting cell proliferation and tissue repair. Especially in the process of wound healing, collagen peptides can accelerate wound healing. The present invention provides a method for extracting and preparing collagen peptides from cod skin, which can efficiently obtain collagen peptides with good wound healing promoting effect, and the method has the advantages of simple operation and low cost, and can be widely used in the fields of wound healing and mucosal repair.

[0003] Although there have been some studies on cod skin collagen peptides in the prior art, most methods still have problems with low efficiency, complex processes and high production costs. Some existing patents use more traditional fish skin collagen extraction processes. However, these methods often have the following limitations: 1. Low raw material utilization efficiency: Existing methods often require high energy consumption, and the by-products of aquatic products are not fully utilized, resulting in resource waste and environmental pollution. 2. Low product purity: In the prior art, the extracted collagen peptides are of low purity and often contain more impurities, which reduces their biological activity and affects the effect of wound healing. 3. Lack of targeted optimization: Most current technologies lack targeted optimization of the biological activity of fish skin collagen peptides, such as screening for characteristic peptides and low molecular weight peptides that promote wound healing.

[0004] In response to the above situation, a method of producing cod skin collagen peptides to promote wound healing was developed, which can not only efficiently extract collagen peptides, but also effectively enhance their biological activity, especially in its application in wound healing. Summary of the invention

[0005] The present invention provides a cod skin collagen peptide for promoting wound healing and a preparation method thereof, that is, a protein peptide preparation method with simple process steps, easy operation and implementation, and effective utilization of fish skin resources, thereby realizing high-value development and utilization of low-value aquatic product waste.

[0006] The preparation method of the cod skin collagen peptide having the effect of promoting wound healing provided by the present invention comprises the following steps:

[0007] 1) Soak the fish skin treated by washing with water in brine to obtain cod skin with water-soluble proteins removed;

[0008] Among them, the brine, as a specific record of the example, is an aqueous NaCl solution with a temperature of 35 - 55 °C and a concentration of 0.02 - 0.1%;

[0009] 2) After draining the cod skin treated in step 1), perform acid swelling treatment with an acid solution;

[0010] Among them, the acid solution is a citric acid or sulfuric acid solution with a concentration of 0.01 - 0.035 mol / L;

[0011] Preferably, the mass-volume ratio of cod skin to acid solution is 1:6 - 1:7 g / ml;

[0012] 3) After rinsing the acid-swollen cod skin with water, adjust the pH to 5.5 - 6.5 with an alkali solution, then wash with distilled water, and crush the washed fish skin;

[0013] The alkali solution mentioned above is a sodium hydroxide solution with a concentration of 0.01 - 0.05 mol / L;

[0014] 4) Fish skin crushing: Drain, crush and homogenize the fish skin to improve the extraction rate.

[0015] 5) Adjust the initial pH of the crushed fish slices to 7.5 - 8.5, add a complex enzyme for hydrolysis, and hydrolyze at 50 - 55 °C for 3.5 - 4.5 h; after the hydrolysis is completed, inactivate the enzyme in a boiling water bath for 10 min, and centrifuge to obtain enzymatically hydrolyzed protein peptides;

[0016] The complex enzyme is a complex enzyme of Alcalase 2.4L and neutral protease, and the mass ratio is 1:1.

[0017] 6) After decolorization and deodorization treatment of the enzymatically hydrolyzed protein peptides, use a plate and frame filter press to circulate and filter under vacuum conditions, collect the clarified filtrate, perform nanofiltration for desalting, concentrate the desalted filtrate, and then spray dry to obtain cod skin collagen peptides.

[0018] The dehydration treatment mentioned above is to use powdered decolorizing activated carbon for decolorization treatment; and use macroporous adsorption resin for deodorization treatment;

[0019] The polypeptides described above include polypeptides with amino acid sequences of ASGPAGSR (SEQ ID NO:1), AGPSG (SEQ ID NO:2), LAGAS (SEQ ID NO:3), GPVG (SEQ ID NO:4), EPGAAGAR (SEQ ID NO:5), AAGPSGN (SEQ ID NO:6), LKGDGR (SEQ ID NO:7), ESGPGQ (SEQ ID NO:8), and DSGPAGPK (SEQ ID NO:9);

[0020] The cod skin collagen peptide prepared by the present invention is used for preparing products that promote wound healing.

[0021] The collagen peptide prepared by the present invention has great potential in wound healing. Some polypeptides have high antioxidant, anti-inflammatory, antibacterial and other activities. The composition of these amino acids has a potential positive impact on skin wound repair and antioxidant effects. Description of the Drawings

[0022] Figure 1 : Photographs of wound healing on the 8th day after injury;

[0023] Figure 2 : Fingerprint of collagen peptide. Detailed Embodiments

[0024] The cod skin collagen peptide with the efficacy of promoting wound healing provided by the present invention has a preparation method including the following steps:

[0025] 1) Wash the cod skin to remove impurities, sarcoplasmic proteins, fats or minced meat;

[0026] 2) Soak and wash the cleaned fish skin in warm water at 35°C - 55°C containing 0.02 - 0.1% NaCl for 35 - 55 minutes; further remove water-soluble proteins;

[0027] 3) Acid swelling treatment: After draining the fish skin, stir and soak it in 0.01 - 0.035 mol / L citric acid or sulfuric acid solution for 35 - 50 minutes, with the ratio of fish skin to acid solution being 1:6 - 1:7 (m / v); on the one hand, swell the fish skin and remove fishy and odorous substances, and on the other hand, facilitate the extraction of collagen and polypeptides.

[0028] 4) Adjust the pH: After swelling, first rinse with tap water, then with distilled water, adjust the pH to 5.5 - 6.5 with 0.01 - 0.05 mol / L sodium hydroxide solution, and then wash with distilled water to reduce the introduction of salts.

[0029] 5) Crush the fish skin: Drain and crush the fish skin into a homogeneous slurry to improve the extraction rate.

[0030] 6) Preparation of enzymatically hydrolyzed protein peptides: Adjust the initial pH to 7.5 - 8.5, add 0.5% - 2% (w / w) of a composite enzyme for hydrolysis, and carry out hydrolysis at 50 - 55 °C for 3.5 - 4.5 h. After the hydrolysis is completed, inactivate the enzyme in a boiling water bath for 10 min, and obtain collagen peptides by centrifugal separation; the composite enzyme is Alcalase 2.4L and neutral protease (1:1, w / w).

[0031] 7) Decolorization and deodorization: Add 0.02 - 0.3% powdered decolorizing activated carbon (w / v), and carry out decolorization in a water bath at 60 - 65 °C with shaking for 45 min. Finally, add 0.1 - 0.5% macroporous adsorption resin DA201 - C (w / v), and shake in a water bath at 45 - 50 °C for 30 min to remove fishy smell, off - flavor and color.

[0032] 8) Plate - frame filtration: Use a plate - frame filter press to carry out cyclic filtration under vacuum conditions until a completely clear liquid flows out, and collect the clear filtrate for the next process.

[0033] 9) Nanofiltration for desalting and concentration dehydration: After nanofiltration for desalting, inject the filtrate into a double - effect vacuum concentrator and concentrate it to 30 - 45%.

[0034] 10) Spray drying: After sterilizing the concentrated solution, dry it in a spray drying tower. The drying conditions of the spray drying tower are: the inlet air temperature is about 195 °C, and the outlet air temperature is about 95 °C.

[0035] 11) Sequence analysis and function analysis: In this invention, the peptide fingerprint map of cod - skin collagen peptides was analyzed in detail by high - resolution mass spectrometry to determine the typical fingerprint map. A wound model was established. In animal experiments, after 8 days of observation, the wound healing rate of the collagen peptide group (2000 mg / kg·bw) was significantly higher than that of the control group, showing its great potential in wound healing.

[0036] The present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings.

[0037] Example 1: Preparation of cod - skin collagen peptides

[0038] Thaw the cod skin under running water and wash it clean to remove impurities, myofibrillar proteins, part of the fat and minced meat. Soak and wash the cleaned fish skin in warm water containing 0.05% NaCl at 40 °C with continuous stirring for 40 min to further remove water-soluble proteins. After draining the fish skin, soak it in a 0.02 mol / L citric acid or sulfuric acid solution with stirring for 40 min. Swell the fish skin at a ratio of fish skin: acid solution of 1:6 (m / v) to remove fishy and off-odor substances and facilitate the extraction of collagen and polypeptides. After swelling, first rinse with tap water, then with distilled water, adjust the pH to 6.0 with 0.03 mol / L sodium hydroxide solution, and then wash with distilled water to reduce the introduction of salts. Drain and crush the fish skin into a homogeneous slurry to improve the extraction rate.

[0039] Adjust the initial pH to 7.5, add 1% (w / w) complex enzyme for hydrolysis, and enzymolyze at 50 °C for 4.0 h. After the hydrolysis is completed, inactivate the enzyme in a boiling water bath for 10 min, and centrifuge to obtain collagen peptides; the complex enzyme is Alcalase 2.4L and neutral protease (1:1, w / w). Add 0.02% powdered decolorizing activated carbon (w / v), and decolorize in a water bath at 60 °C with shaking for 45 min. Finally, add 0.2% macroporous adsorption resin DA201-C (w / v), and shake in a water bath at 45 °C for 30 min to remove fishy, off-odor and color. Use a plate and frame filter press to circulate and filter under vacuum until a completely clear liquid flows out, and collect the clear filtrate for the next process. After nanofiltration desalination, inject the filtrate into a double-effect vacuum concentrator and concentrate to 40%. After sterilizing the concentrated solution, dry it in a spray drying tower. The drying conditions of the spray drying tower are: the inlet air temperature is about 195 °C, and the outlet air temperature is about 95 °C. Finally, obtain cod skin collagen peptides.

[0040] Example 2: Effect of cod skin collagen peptides on wound healing rate

[0041] Anesthetize rats by intraperitoneal injection of sodium pentobarbital. Use an electric hair clipper to shave the hair on the back area of the rats, and disinfect the surgical area with 70% alcohol. Prepare a full-thickness skin excision wound by removing a circular skin with a diameter of 1 cm, and regard the day of making the wound as day 0. After the operation, all rats were given an intraperitoneal injection of 20,000 units of gentamicin. After the operation, the awake rats were put back into the cage and allowed to freely ingest food and drinking water. The gavage dose of the collagen peptide group was 2000 mg / kg·bw, and the model control group was gavaged with the same volume of normal saline. The rats were sacrificed on the 8th day. Use Image-Pro Plus 6.0 software to track the wound edge to measure the wound area, and the wound healing rate can be calculated by the ratio of the remaining area of the wound to the original area of the wound.

[0042] From Figure 1It can be seen that on the 8th day after injury, the wounds on the surfaces of the rats in the cod skin collagen peptide group and the model control group had scabbed over. It was visually observed that the wound area of the rats in the collagen peptide group was significantly smaller than that in the model control group. Moreover, the scabs at the wound sites in the collagen peptide group had partially fallen off, while the scab area of the wounds in the model control group was still relatively large. In terms of the wound healing rate, on the 8th day, the wound healing rate of the collagen peptide group was 10 - 15% higher than that of the model control group. The significant increase in the wound healing rate indicates that collagen peptide can effectively promote the closure of skin wounds in rats.

[0043] Example 3: Identification of Fingerprint Map of Cod Skin Collagen Peptide, Determination of Polypeptide Sequence and Effect

[0044] 1) Determination of Peptide Fingerprint Map

[0045] The fingerprint sequence of cod skin collagen peptide was determined using a TripleTOF 5600 mass spectrometer (AB SCIEX Corporation, USA). The mass spectrometry parameters were as follows: mass spectrometry scanning range: 50 - 1200 m / z; reaction mode: positive ion mode; spray voltage: 3.60 kV; capillary temperature: 320 °C.

[0046] The fingerprint map of cod collagen peptide was identified by mass spectrometry technology, and a total of 196 peptide segments were identified. Through the preliminary analysis of these peptide segments, combined with the characteristics of their molecular weights and amino acid sequences, peptide segments with antioxidant and wound healing activities were screened out. Polypeptides with amino acid sequences of ASGPAGSR, AGPSG, LAGAS, GPVG, EPGAAGAR, AAGPSGN, LKGDGR, ESGPGQ, and DSGPAGPK have high activities such as antioxidant, anti - inflammatory, antibacterial, and chelating. The composition of these amino acids has a significant promoting effect on skin wound repair and antioxidant effect.

[0047] In summary, the composition of the cod skin collagen peptide prepared in the present invention has a high similarity to the amino acid composition of collagen - rich tissues such as wound skin. The fingerprint map of cod collagen oligopeptide provides a research basis for screening characteristic peptides with antioxidant and wound healing potential.

Claims

1. A cod skin collagen peptide, characterized in that: The preparation method of cod skin collagen peptide comprises the following steps: 1) soaking the fish skin washed with water in salt water to obtain cod skin from which water-soluble protein has been removed; 2) draining the cod skin treated in step 1), and performing acid swelling treatment with an acid solution; 3) The acid-swollen cod skin is rinsed with water, the pH value is adjusted to 5.5-6.5 with alkali solution, and then washed with distilled water, and the washed fish skin is crushed; 4) Fish skin crushing: drain the fish skin and crush it into a homogenous slurry; 5) The initial pH of the crushed fish skin is adjusted to 7.5-8.5, and a composite enzyme is added for hydrolysis at 50-55°C for 3.5-4.5 hours; after the hydrolysis is completed, the enzyme is inactivated in a boiling water bath for 10 minutes, and the enzymatic protein peptides are obtained by centrifugation; 6) After decolorization and deodorization of the enzymatic protein peptide, a plate and frame filter is used for circulation filtration under vacuum conditions, and the clarified filtrate is collected for nanofiltration desalination. The desalted filtrate is concentrated and then spray-dried to obtain cod skin collagen peptide.

2. The cod skin collagen peptide according to claim 1, characterized in that The brine in 1) is a NaCl aqueous solution with a temperature of 35-55°C and a concentration of 0.02-0.1%.

3. The cod skin collagen peptide according to claim 1, characterized in that The acid solution in 2) is a citric acid or sulfuric acid solution with a concentration of 0.01-0.035 mol / L.

4. The cod skin collagen peptide according to claim 1, characterized in that The alkali solution described in 3) is a sodium hydroxide solution with a concentration of 0.01-0.05 mol / L.

5. The cod skin collagen peptide according to claim 1, characterized in that The compound enzyme described in 5) is a compound enzyme of Alcalase 2.4L and neutral protease.

6. The cod skin collagen peptide according to claim 1, characterized in that The dehydration treatment described in 6) is to use powdered decolorizing activated carbon for decolorization treatment; and to use macroporous adsorption resin for deodorization treatment.

7. The cod skin collagen peptide according to claim 1, characterized in that The amino acid sequence of the polypeptide is SEQ ID NO: 1-9.

8. Use of the cod skin collagen peptide according to claim 1 and / or claim 7 in the preparation of a product for promoting wound healing.

9. A product for promoting wound healing, characterized in that: The product contains the cod skin collagen peptide according to claim 1 and / or claim 7 at a pharmacologically effective concentration.

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