CN105648004B - Preparation method of dasyatis akajei cartilage antioxidative collagen peptide - Google Patents

Abstract

The invention discloses a preparation method of red ray cartilage antioxidant collagen peptide. The present invention uses red ray cartilage as raw material, through collagen extraction, trypsin and neutral protease double enzymatic hydrolysis to obtain enzymatic hydrolysis solution, and the enzymatic hydrolysis solution adopts ultrafiltration, ion exchange resin chromatography, gel column chromatography and reverse phase The antioxidant collagen peptide Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp was isolated and purified by high performance liquid chromatography, and the molecular weight determined by ESI-MS was 875.85 Da; Free radicals, ABTS free radicals and superoxide anion free radicals have good scavenging effects; at the same time, they also show good lipid peroxidation inhibition, and can be developed as medicines, health foods or food additives.

Description

Preparation method of red ray cartilage antioxidant collagen peptide

technical field

The invention relates to a preparation method of animal cartilage collagen peptide, in particular to a preparation method of red ray cartilage antioxidant collagen peptide.

Background technique

Collagen peptide is the product of collagen degradation by protease, with a molecular weight of about 180-3000 Da, which is considered to be more beneficial to human digestion and absorption than ordinary collagen. At the same time, existing studies have proved that collagen peptides have various biological activities, such as inhibiting angiotensin-converting enzyme activity, antioxidant activity, reducing pain in patients with osteoarthritis such as knee joints or hip joints, enhancing bone density, maintaining bone metabolism balance, etc. .

Red stingray ( Dasyatis akajei ), belonging to the class of cartilaginous fishes, Hypotheca, Rays, Stingidae, Stingrays. Red ray is a common cartilaginous fish, distributed in the coastal areas of my country. Studies have shown that red ray cartilage contains a large amount of collagen, which can be developed and utilized as a raw material for collagen peptides.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a preparation method of red ray cartilage antioxidant collagen peptide capable of scavenging free radicals and inhibiting lipid peroxidation.

The technical scheme adopted by the present invention to solve the above-mentioned technical problems is: a preparation method of red ray cartilage antioxidant collagen peptide, which comprises the following steps:

1) Preparation of red ray cartilage collagen: Sun-dried and crushed red ray cartilage, added to NaOH solution (0.1 mol/L) according to the ratio of 1 g to liquid: 15-20 mL, and soaked for 2-4 days at 1-4 °C. h, remove the non-collagen protein; after the treatment, the red ray cartilage was repeatedly washed with distilled water until neutral, drained, and the EDTA solution (0.5 mol/L) of pH 7.4 was added according to the ratio of 1 g to liquid: 5-8 mL. Soak at 4°C for 2 to 3 days (replace EDTA solution once a day), wash with distilled water 2 to 3 times, dry and pulverize to obtain decalcified red stingray cartilage powder. The decalcified red ray cartilage powder was added to 0.5 mol/L acetic acid solution according to the ratio of material to liquid of 1 g: 5-8 mL, and was dynamically soaked at 1-4 °C for 3 d, centrifuged at 10 000 g for 20-25 min, and the supernatant was taken. , and added NaCl to the final concentration of 1.0 mol/L, left standing for 60-90 min, centrifuged at 12 000 g for 25-30 min to obtain a precipitate, which was freeze-dried to obtain red ray cartilage collagen.

2) Enzymatic hydrolysis of red ray cartilage collagen: Add red ray cartilage collagen to disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 7.5, 0.2 mol/L) according to the ratio of 1 g:15-20 mL of material to liquid. , add trypsin (1.9×10 ⁴ U/g) to the solution according to 1.0-1.5% of the collagen mass, after enzymatic hydrolysis at 45-50 ℃ for 3-4 h, the solution is heated to 90-95 ℃, and After maintaining this temperature for 8-10 min, it was cooled to 45-50°C; neutral protease (1.0×10 ⁵ U/g) was added to the solution according to 1.5-1.8% of the collagen mass, and the temperature was 45-50°C. After enzymatic hydrolysis for 6 to 8 h, the solution was heated to 90 to 95 °C and kept at this temperature for 15 to 20 min, then centrifuged at 10 000 g for 20 to 25 min, and the supernatant was taken, which is the enzymatic hydrolysis product;

3) Preparation of red ray cartilage antioxidant collagen peptide: The above enzymatic hydrolysis product was subjected to ultrafiltration treatment with a 3.5 kDa ultrafiltration membrane, and the fraction with a molecular weight of less than 3.5 kDa was collected and lyophilized to obtain an ultrafiltration enzymatic hydrolysate; The substance was purified by ion exchange resin chromatography, gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) to obtain antioxidant collagen peptide.

Preferably, the red ray in the step 1) is a red ray ( Dasyatis akajei ).

Preferably, the specific process of the ion exchange resin chromatography, gel column chromatography and RP-HPLC purification in the step 4) is:

Ion exchange resin chromatography: Dissolve the above ultrafiltration enzymatic hydrolyzate in double distilled water to prepare a solution with a concentration of 25 to 30 mg/mL, pass through DEAE-52 ion exchange resin chromatography column, water, 0.5 mol/L and 1.0 mol/mL. Elute with L NaCl solution at a flow rate of 0.9-1.2 mL/min. The eluted solution is collected in a tube every 3 minutes and detected at 225 nm. The solutions in the test tube are combined according to the peak, and the scavenging activities of hydroxyl radicals of each peak are compared. The most active component is freeze-dried, that is, the ion-exchange chromatography enzymatic hydrolysate;

Gel column chromatography: Dissolve the above-mentioned ion-exchange chromatography enzymatic hydrolyzate in double distilled water to make a solution with a concentration of 25-30 mg/mL, separate it by Sephadex G-25 column chromatography, and use double distilled water for separation. Elution was carried out at a flow rate of 0.8-1.0 mL/min. The eluted solution was collected in a tube every 3 minutes and detected at 225 nm. The solutions in the test tube were combined according to the peak, and the scavenging activity of hydroxyl radicals of each peak was compared, and the strongest activity was selected. The components are freeze-dried, that is, gel chromatography enzymolysate;

RP-HPLC purification: The above gel chromatography enzymatic hydrolysate was prepared into a solution of 40-50 μg/mL with double distilled water, and purified by RP-HPLC. According to the scavenging activity of hydroxyl radicals, a highly active antibody was obtained. Oxidized collagen peptide Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW), the molecular weight determined by ESI-MS was 875.85 Da.

Further preferably, the RP-HPLC conditions are: injection volume 15-20 μL; chromatographic column Zorbax SB-C ₁₈ (250 mm×4.6 mm, 5 μm); mobile phase: 40% acetonitrile; elution rate 0.8-1.0 mL/min; UV detection wavelength 225 nm.

Compared with the prior art, the red ray cartilage antioxidant collagen peptide provided by the present invention has a good scavenging effect on DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals; at the same time, Gly-Ile-Glu-Gly-Glu -Glu-Gly-Trp(GIEGEEGW) also showed good lipid peroxidation inhibitory effect; Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp(GIEGEEGW) had safe, non-toxic side effects, strong antioxidant activity and It is easy to digest and absorb, and can be used as medicine, health food and food additive.

Description of drawings

Fig. 1 is the chromatogram of DEAE-52 ion exchange resin of the present invention.

Fig. 2 is a chromatogram of Sephadex G-25 of the present invention.

Fig. 3 RP-HPLC analysis of the digestate prepared by Sephadex G-25.

Figure 4 Mass spectrum of Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW).

Figure 5 Anti-lipid oxidation ability of Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW).

Detailed ways

The present invention will be further described in detail below with reference to the embodiments of the accompanying drawings.

Example:

A preparation method of red ray cartilage antioxidant collagen peptide, the preparation process is as follows: red ray cartilage → cartilage collagen → double enzymatic hydrolysis with trypsin and neutral protease → enzymatic hydrolysate → ultrafiltration → ion exchange chromatography → coagulation Gel filtration chromatography→preparation by high performance liquid chromatography→antioxidant collagen peptide.

1) Preparation of red ray cartilage collagen: The cartilage of red ray ( Dasyatis akajei ) was sun-dried and crushed, added to NaOH solution (0.1 mol/L) according to the ratio of material to liquid of 1 g:18 mL, and soaked at 3 °C for 3 h. Remove non-collagen protein; after treatment, red ray cartilage was repeatedly washed with distilled water until neutral, drained, and EDTA solution (0.5mol/L) with pH 7.4 was added according to the ratio of 1 g: 6 mL of material to liquid, and soaked at 4 °C for 3 days (The EDTA solution was replaced once a day), washed 3 times with distilled water, dried and crushed to obtain decalcified red ray cartilage powder. The decalcified red ray cartilage powder was added to 0.5 mol/L acetic acid solution according to the ratio of material to liquid of 1 g:6 mL, and it was dynamically soaked at 3 °C for 3 d, centrifuged at 10 000 g for 20 min, the supernatant was taken, and NaCl was added to the solution. The final concentration was 1.0 mol/L, left standing for 90 min, and centrifuged at 12 000 g for 25 min to obtain the precipitate, which was freeze-dried to obtain red ray cartilage collagen.

2) Enzymatic hydrolysis of red ray cartilage collagen: The red ray cartilage collagen was added to disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 7.5, 0.2 mol/L) according to the ratio of 1 g:15 mL of material to liquid. Trypsin (1.9×10 ⁴ U/g) was added to the solution with 1.3% of the mass of collagen. After enzymatic hydrolysis at 48°C for 3.5 h, the solution was heated to 90°C, kept at this temperature for 10 min, and then cooled to 45°C; neutral protease (1.0×10 ⁵ U/g) was added to the solution according to 1.6% of the collagen mass, enzymatically hydrolyzed at 45°C for 6 h, the solution was heated to 90°C, and kept at this temperature for 15 hours. After min, centrifuge at 10 000g for 20 min, and take the supernatant, which is the enzymatic hydrolysis product;

3) Preparation of red ray cartilage antioxidant collagen peptide: The above enzymatic hydrolysis product was subjected to ultrafiltration treatment with a 3.5 kDa ultrafiltration membrane, and the fraction with a molecular weight of less than 3.5 kDa was collected and lyophilized to obtain an ultrafiltration enzymatic hydrolysate; The substance was purified by ion exchange resin chromatography, gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) to obtain antioxidant collagen peptide.

①Ion exchange resin chromatography: Dissolve the above ultrafiltration enzymatic hydrolyzate in double distilled water to prepare a solution with a concentration of 28 mg/mL, pass through a DEAE-52 ion exchange resin chromatography column, and chromatograph with water, 0.5 mol/L and 1.0 mol/L. Elute with NaCl solution at a flow rate of 1.0-1.2 mL/min. The eluted solution is collected in a tube every 3 minutes and detected at 225 nm. The solution in the test tube is combined according to the peak, and the scavenging activity of hydroxyl radicals of each peak is compared, and the activity is selected. The strongest component is freeze-dried, namely ion exchange chromatography enzymatic hydrolyzate DCP-III (Figure 1);

②Gel column chromatography: Dissolve the above ion-exchange chromatography enzymatic hydrolysate in double distilled water to make a solution with a concentration of 25 mg/mL, and then separate it by Sephadex G-25 column chromatography, and use double distilled water for separation. Elution, the flow rate is 1.0 mL/min, the eluted solution is collected in a tube every 3 minutes and detected at 225 nm, the solutions in the test tube are combined according to the peak, the scavenging activity of hydroxyl radicals of each peak is compared, and the most active component is selected and frozen. Dry, that is, gel chromatography enzymatic hydrolysate DCP-III-3 (Figure 2);

③ RP-HPLC purification: The above gel chromatography enzymatic hydrolyzate was made into a 40 μg/mL solution with double distilled water, and purified by RP-HPLC (RP-HPLC conditions: injection volume 18 μL; chromatographic column Zorbax SB - C ₁₈ (250 mm×4.6 mm, 5 μm); mobile phase: 40% acetonitrile; elution rate 0.8 mL/min; UV detection wavelength 225 nm), according to the scavenging activity of hydroxyl radicals, a highly active antibody was obtained. The oxidized collagen peptide Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW) (Figure 3).

④Structure detection: Collect the antioxidant collagen peptide with the highest scavenging activity of DPPH free radical and hydroxyl radical, and detect it as a single peak by RP-HPLC, and use the protein/peptide sequence analyzer to determine the amino acid sequence as Gly-Ile-Glu-Gly-Glu -Glu-Gly-Trp (GIEGEEGW), with a molecular weight of 875.85 Da ([M+H] ⁺ 876.81 Da) determined by ESI-MS (Figure 4).

The obtained red ray cartilage antioxidant collagen peptide Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW) was subjected to free radical scavenging experiments and lipid peroxidation inhibition experiments. The experimental results showed that: Gly-Ile- Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW) to DPPH radical (EC ₅₀ 1.93 mg/mL), hydroxyl radical (EC ₅₀ 0.35 mg/mL), ABTS radical (EC ₅₀ 0.16 mg/mL) and superoxide anion radical (EC ₅₀ 0.24 mg/mL) has a good scavenging effect; at the same time, Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp (GIEGEEGW) also shows a good lipid peroxidation inhibitory effect (Figure 5).

Finally, it should also be noted that the above enumeration is only a specific embodiment of the present invention. Obviously, the present invention is not limited to the above embodiments, and many modifications are possible. All deformations that those of ordinary skill in the art can directly derive or associate from the disclosure of the present invention shall be considered as the protection scope of the present invention.

SEQUENCE LISTING

<110> Zhejiang Ocean University

<120> Preparation method of red ray cartilage antioxidant collagen peptide

<130> zjou-wb-201511-302

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Synthetic

<400> 1

Gly Ile Glu Gly Glu Glu Gly Trp

1 5

Claims (1)

1. The preparation method of dasyatis akajei cartilage antioxidative collagen peptide comprises the following steps:

1) preparing dasyatis akajei cartilage collagen: drying and crushing dasyatis akajei cartilage, adding the dasyatis akajei cartilage into NaOH solution according to the feed liquid ratio of 1g to 15-20 mL, soaking at 1-4 ℃ for 2-4 h, and removing non-collagen; repeatedly washing the treated dasyatis akajei cartilage with distilled water to be neutral and draining, adding an EDTA solution with the pH value of 7.4 according to the material-liquid ratio of 1g: 5-8 mL, soaking at 1-4 ℃ for 2-3 days, washing with distilled water for 2-3 times, drying and crushing to obtain decalcified dasyatis akajei cartilage powder; adding decalcified dasyatis akajei powder into 0.5mol/L acetic acid solution according to the material-liquid ratio of 1g: 5-8 mL, dynamically soaking for 3d at 1-4 ℃, centrifuging for 20-25 min at 10000g, taking supernatant, adding NaCl until the final concentration of the solution is 1.0mol/L, standing for 60-90 min, centrifuging for 25-30 min at 12000g, and freeze-drying to obtain dasyatis akajei cartilage collagen;

2) enzymolysis of dasyatis akajei cartilage collagen: adding dasyatis akajei cartilage collagen into a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution according to the material-liquid ratio of 1g: 15-20 mL, adding trypsin into the solution according to 1.0-1.5% of the mass of the collagen, performing enzymolysis for 3-4 h at the temperature of 45-50 ℃, heating the solution to 90-95 ℃, keeping the temperature for 8-10 min, and cooling to 45-50 ℃; adding neutral protease into the solution according to the mass of 1.5-1.8% of the collagen, performing enzymolysis for 6-8 hours at the temperature of 45-50 ℃, heating the solution to 90-95 ℃, keeping the temperature for 15-20 min, centrifuging for 20-25 min at 10000g, and taking supernatant, namely an enzymolysis product;

3) preparing dasyatis akajei cartilage antioxidative collagen peptide: performing ultrafiltration treatment on the enzymolysis product by using a 3.5kDa ultrafiltration membrane, collecting a part with the molecular weight less than 3.5kDa, and freeze-drying to obtain an ultrafiltration zymolyte; purifying the ultrafiltration zymolyte by ion exchange resin chromatography, gel column chromatography and reversed-phase high performance liquid chromatography to obtain antioxidant collagen peptide, wherein the prepared antioxidant collagen peptide Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp is octapeptide compound, and the molecular weight is 875.85Da in ESI-MS measurement;

wherein the dasyatis akajei in the step 1) is dasyatis akajeiDasyatis akajeiNaOH solution is 0.1 mol/L; the EDTA solution is 0.5mol/L and is replaced for 1 time every day;

the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution in the step 2) has a value of 0.2mol/L, pH of 7.5 and the trypsin has a value of 1.9 × 10⁴U/g, neutral protease 1.0 × 10⁵U/g；

The specific processes of ion exchange resin chromatography, gel column chromatography and reversed-phase high performance liquid chromatography purification in the step 3) are as follows:

ion exchange resin chromatography: dissolving the ultrafiltration zymolyte in double distilled water to prepare a solution with the concentration of 25-30 mg/mL, passing through a DEAE-52 ion exchange resin chromatographic column, eluting with water, 0.5mol/L and 1.0mol/LNaCl solutions at the flow rate of 0.9-1.2 mL/min, collecting one tube of the eluate every 3min, detecting at 225nm, merging the solutions in the test tube according to peaks, comparing the removal activity of hydroxyl radicals of each peak, and selecting the component with the strongest activity for freeze-drying to obtain the ion exchange chromatography zymolyte;

gel column chromatography: dissolving the ion exchange chromatography zymolyte in double distilled water to prepare a solution with the concentration of 25-30 mg/mL, separating by sephadex G-25 column chromatography, eluting with double distilled water at the flow rate of 0.8-1.0 mL/min, collecting one tube of the eluted solution every 3min, detecting at 225nm, merging the solutions in the test tube according to peaks, comparing the removal activity of hydroxyl radicals of each peak, selecting the component with the strongest activity, and freeze-drying to obtain the gel chromatography zymolyte;

purifying by reverse phase high performance liquid chromatography, namely preparing the gel chromatography zymolyte into a solution of 40-50 mu g/mL by using double distilled water, and purifying by using the reverse phase high performance liquid chromatography, wherein the conditions of the reverse phase high performance liquid chromatography are that the sample injection amount is 15-20 mu L, and a chromatographic column is ZorbaxSB-C with the specification of 250mm × 4.6.6 mm and 5 mu m₁₈(ii) a The mobile phase is 40% acetonitrile; the elution speed is 0.8-1.0 mL/min; ultraviolet detecting wavelength is 225nm, and molecular weight is 875.85Da when detecting 1 high-activity antioxidant collagen peptide Gly-Ile-Glu-Gly-Glu-Glu-Gly-Trp according to hydroxyl radical scavenging activity and ESI-MS.

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