Bullfrog extracted biological collagen composition and preparation method thereof

Abstract

The invention discloses a bullfrog extracted biological collagen composition and a preparation method thereof in the technical field of collagen. The bullfrog extracted biological collagen composition comprises the following components in parts by weight: 1-1.5 parts of hydrophobic bullfrog source collagen peptide, 0.6-0.8 part of hydrophobic bullfrog source elastin peptide, 1-1.5 parts of polyglutamic acid and 1-1.5 parts of polylysine. Collagen and elastin in bullfrog skin and bullfrog cartilage are extracted in a combined mode through a three-enzyme co-production method, and after bullfrog source collagen peptide and bullfrog source elastin peptide are subjected to hydrophobic modification, polyglutamic acid and polylysine are added to form a collagen combination. The stability, the medicine carrying capacity and the slow release capacity of the composition can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of collagen, and specifically refers to a bullfrog extracted biological collagen composition and a preparation method thereof. Background Art

[0002] Collagen is a natural protein composed of a triple helix structure. It is the main component of connective tissue and is widely found in the skin, bones, cartilage and tendons of animals. Collagen has a wide range of applications in the fields of medicine, cosmetics and food due to its excellent biocompatibility, biodegradability and low immunogenicity, especially in skin repair, anti-aging, wound healing and health care product development. In recent years, the market demand for collagen has increased significantly. Traditional sources of collagen (such as cowhide, pig skin and fish skin) have gradually shown some limitations. Although collagen from aquatic products such as fish skin has good biological activity, its source is limited, the extraction process is complex and the cost is high. In addition, due to the influence of animal diseases (such as mad cow disease and foot-and-mouth disease) and environmental pollution (such as heavy metals and microplastics), consumers have concerns about the safety of collagen from these traditional sources. Therefore, the development of new sources of collagen and extraction technology has become a hot spot and difficulty in the current research field. Bullfrog (Rana catesbeiana) is a common amphibian with rich collagen resources in its tissues, especially skin, bone and muscle tissues. Bullfrogs have the following advantages: (1) rapid reproduction, short growth cycle, and stable raw material source; (2) heavy metals and pollutants are not easily accumulated in the organism, and have high ecological safety; (3) bullfrog waste byproducts (such as skin and cartilage) are often wasted during processing, and developing their added value is conducive to the comprehensive utilization of resources. Therefore, bullfrogs have become a source of collagen extraction with great development potential. In terms of extraction technology, existing collagen extraction methods mainly include acid-base treatment, enzymatic hydrolysis, and hot water extraction. Although these methods are mature, they may cause the molecular structure of collagen to be destroyed under certain conditions, thereby affecting its functional properties. In addition, the existing technology is not thorough enough in removing impurities (such as fat and pigment) in animal tissues, and the extraction efficiency is low, which is difficult to meet the needs of large-scale industrial applications. Summary of the invention

[0003] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a bullfrog extracted biological collagen composition and a preparation method thereof, wherein collagen and elastin from bullfrog skin and bullfrog cartilage are jointly extracted by a three-enzyme co-production method, and after the bullfrog-derived collagen peptides and bullfrog-derived elastin peptides are hydrophobically modified, polyglutamic acid and polylysine are added to form a collagen combination, which can improve the stability, drug loading capacity and sustained-release capacity of the composition.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows: the present invention proposes a bullfrog extracted biological collagen composition, the collagen composition comprises the following components in parts by weight: 1-1.5 parts of hydrophobized bullfrog source collagen peptide, 0.6-0.8 parts of hydrophobized bullfrog source elastin peptide, 1-1.5 parts of polyglutamic acid, and 1-1.5 parts of polylysine;

[0005] Preferably, the hydrophobized bullfrog-derived collagen peptide and the hydrophobized bullfrog-derived elastin peptide are derived from bullfrog protein;

[0006] Preferably, the method for extracting bullfrog protein comprises the following steps:

[0007] S1, take a male bullfrog, peel and remove the muscle tissue of the bullfrog, retain the bullfrog skeleton and bullfrog skin, add deionized water, adjust the pH to 8-9, wash the bullfrog skeleton and skin for 20-30min, rinse the bullfrog skeleton and skin with deionized water until neutral, place at 35 ° C. and dry for 12h, grind and crush, and pass through an 80-mesh sieve to obtain a pre-treated bullfrog powder;

[0008] Preferably, in step S1, the material-liquid ratio of the bullfrog skeleton and skin to deionized water is 0.1-0.3 g / mL;

[0009] S2, dispersing the pretreated bullfrog powder prepared in step S1 in a PBS buffer solution, adjusting the pH to 10-11, adding alkaline protease, the enzymatic hydrolysis temperature is 40-50 ° C, and performing enzymatic hydrolysis for 4-5 hours, raising the temperature to 90-95 ° C, performing enzyme inactivation for 10 minutes, placing it at 6000-8000 rpm for centrifugation for 5-10 minutes, collecting the supernatant, and freeze-drying it to obtain enzymatic crude protein ①;

[0010] Preferably, in step S2, the mass concentration of the pretreated bullfrog powder in PBS buffer is 0.1-0.12 g / mL;

[0011] Preferably, in step S2, the amount of alkaline protease added (E / S) is 2%-4%;

[0012] S3, dissolving the enzymatic crude protein ① prepared in step S2 in a PBS buffer solution, adjusting the pH to 7.4-7.6, adding clostridial peptidase A, adjusting the enzymatic temperature to 35-40°C, performing enzymatic treatment for 3-4h, raising the temperature to 90-95°C, performing enzyme inactivation treatment for 10min, centrifuging at 6000-8000rpm for 5-10min, separating the supernatant and the precipitate, collecting the supernatant, freeze-drying, obtaining bullfrog-derived collagen peptides, and collecting the precipitate as enzymatic crude protein ② for standby use;

[0013] Preferably, in step S3, the mass concentration of the enzymatically hydrolyzed crude protein ① in the PBS buffer solution is 0.08-0.12 g / mL;

[0014] Preferably, in step S3, the PBS buffer contains zinc ions at a mass concentration of 0.2-0.3 g / L;

[0015] Preferably, in step S3, the added enzyme amount (E / S) of the clostridial peptidase A is 1%-2%;

[0016] S4, dissolving the enzymatic crude protein ② prepared in step S3 in a PBS buffer solution, adjusting the pH to 7.5-8.5, adding elastase, setting the enzymatic hydrolysis temperature to 35-40°C, performing enzymatic hydrolysis for 1-2h, raising the temperature to 90-95°C, performing enzyme inactivation for 10min, centrifuging at 6000-8000rpm for 5-10min, collecting the supernatant, and freeze-drying to obtain bullfrog-derived elastin peptide;

[0017] Preferably, in step S4, the mass concentration of the enzymatically hydrolyzed crude protein ② dissolved in the PBS buffer solution is 0.06-0.08 g / mL;

[0018] Preferably, in step S4, the amount of elastase added (E / S) is 4%-6%;

[0019] The present invention provides a method for preparing a biocollagen composition extracted from bullfrog, which specifically comprises the following steps:

[0020] T1. Mix MBHA resin and DMF, introduce nitrogen for blowing, and after swelling treatment for 40-60 minutes, add piperidine solution, continue to introduce flowing nitrogen for blowing, and after 15-20 minutes, remove the piperidine solution by suction filtration, and wash with DMF and anhydrous methanol to obtain activated MBHA resin;

[0021] Preferably, in step T1, the mass concentration of the MBHA resin in DMF is 0.1-0.2 g / mL;

[0022] Preferably, in step T1, in the piperidine solution, the volume fraction of piperidine in DMF is 20%-25%, and the mass volume ratio between the MBHA resin and the piperidine solution is 0.5-1 g / mL;

[0023] T21, dissolving the bullfrog collagen peptide in DMF and transferring it to the activated MBHA resin described in step T1, adding 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and 1-hydroxybenzotriazole (HOBt) to the reaction system, mixing evenly, adding N,N-diisopropylethylamine to the reaction system, stirring and mixing at a speed of 150-180 rpm for 5-8 minutes, placing it in a dark environment, passing nitrogen gas for 8-12 hours, vacuum filtering to remove the reaction solvent, washing with DMF and anhydrous methanol to obtain MBHA resin-bullfrog collagen peptide;

[0024] Preferably, in step T21, the mass concentration of the bullfrog-derived collagen peptide in DMF is 0.1-0.15 g / mL;

[0025] Preferably, in step T21, the mass ratio of the bullfrog-derived collagen peptide, HATU and HOBt is 1:0.15-0.18:0.05-0.1;

[0026] Preferably, in step T21, the mass volume ratio between the bullfrog-derived collagen peptide and N,N-diisopropylethylamine is 12.6-13.6 g / mL;

[0027] Preferably, in step T21, the mass ratio between the bullfrog-derived collagen peptide and the MBHA resin before activation described in step T1 is 1-1.5:1;

[0028] T22, dissolving the bullfrog elastin peptide in DMF and transferring it to a beaker containing the activated MBHA resin described in step T1, adding 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and 1-hydroxybenzotriazole (HOBt) to the reaction system, mixing evenly, adding N,N-diisopropylethylamine to the reaction system, stirring and mixing at a speed of 150-180 rpm for 5-8 minutes, placing it in a dark environment, passing nitrogen gas for 8-12 hours, vacuum filtering out the reaction solvent, washing with DMF and anhydrous methanol, and obtaining MBHA resin-bullfrog elastin peptide;

[0029] Preferably, in step T22, the mass concentration of the bullfrog-derived elastin peptide in DMF is 0.1-0.15 g / mL;

[0030] Preferably, in step T22, the mass ratio of the bullfrog-derived elastin peptide, HATU and HOBt is 1:0.2-0.25:0.075-0.14;

[0031] Preferably, in step T22, the mass volume ratio between the bullfrog derived elastin peptide and N,N-diisopropylethylamine is 12.5-18.5 g / mL;

[0032] Preferably, in step T22, the mass ratio between the bullfrog-derived elastin peptide and the MBHA resin before activation described in step T1 is 1:0.8;

[0033] T31, dissolving palmitic acid in DMF, adding EDC and NHS, mixing evenly, stirring at 150-180 rpm for 3-5 min, adding to the MBHA resin-bullfrog collagen peptide prepared in step T21, placing in a dark environment, passing nitrogen gas for 3-4 h, vacuum filtering to remove the reaction solvent, washing with DMF and anhydrous methanol, to obtain a hydrophobic MBHA resin-bullfrog collagen peptide;

[0034] Preferably, in step T31, the mass concentration of palmitic acid in DMF is 10-15 g / L;

[0035] Preferably, in step T31, the mass ratio of palmitic acid, EDC and NHS is 1:0.6-0.9:0.45-1.4;

[0036] T32, dissolving palmitic acid in DMF, adding EDC and NHS, mixing evenly, adding to the MBHA resin-bullfrog derived elastin peptide prepared in step T22, mixing evenly, adding N,N-diisopropylethylamine to the reaction system, stirring and mixing at a speed of 150-180 rpm for 3-5 minutes, placing in a dark environment, passing nitrogen gas for 8-12 hours, vacuum filtering out the reaction solvent, washing with DMF and anhydrous methanol, and drying to obtain a hydrophobic MBHA resin-bullfrog derived elastin peptide;

[0037] Preferably, in step T32, the mass concentration of palmitic acid in DMF is 10-15 g / L;

[0038] Preferably, in step T32, the mass ratio of palmitic acid, EDC and NHS is 1:0.6-0.9:0.45-1.3;

[0039] T4, take the hydrophobized MBHA resin-bullfrog collagen peptide prepared in step T31 and the hydrophobized MBHA resin-bullfrog elastin peptide cleavage reagent prepared in step T32, stir at 200-250rpm at room temperature and in the dark, stir and react for 4-5h, collect the filtrate by suction, add ice ether to the filtrate, place it at -20°C, stand it for 24-36h, centrifuge it at 5000-7000rpm for 3-5min, collect the precipitate, wash it thoroughly with ether and deionized water, and freeze-dry it to obtain hydrophobized bullfrog collagen peptide and hydrophobized bullfrog elastin peptide;

[0040] Preferably, in step T4, the cleavage reagent comprises the following components: trifluoroacetic acid, β-mercaptoethanol, methyl phenyl sulfide, deionized water, and phenol;

[0041] Preferably, in step T4, the amount of the cutting agent is calculated according to the total amount of the MBHA resin in step T21 and step T22, and the mass volume ratio of the total amount of the MBHA resin in step T21 and step T22 to the cutting agent in step T4 is 0.05-0.08 g / mL;

[0042] T51, dissolving polyglutamic acid in DMF solvent, adding EDC and NHS and stirring, mixing evenly, adjusting the pH of the reaction system to 5.5-6.0 with acetic acid, adding hydrophobized bullfrog-derived elastin peptide, raising the reaction temperature to 35-40° C., stirring and mixing at a speed of 150-180 rpm, reacting for 3-5 hours, removing the solvent by rotary evaporation, washing with DMF and deionized water, and freeze-drying to obtain polyglutamic acid-hydrophobized bullfrog-derived elastin peptide;

[0043] Preferably, in step T51, the mass concentration of the polyglutamic acid in DMF is 10-20 g / L;

[0044] Preferably, in step T51, the added mass of EDC is 5-7% of the mass of polyglutamic acid, and the added mass of NHS is 3-5% of the mass of polyglutamic acid;

[0045] T52, dissolving polylysine in DMF solvent, adding EDC and NHS and stirring, after mixing evenly, adding hydrophobized bullfrog collagen peptide, raising the reaction temperature to 35-40°C, stirring and mixing at a speed of 150-180rpm, reacting for 3-5h, rotary evaporating to remove the solvent, washing with DMF and deionized water, and freeze-drying to obtain polylysine-hydrophobized bullfrog collagen peptide;

[0046] Preferably, in step T52, the mass concentration of the polylysine in DMF is 10-20 g / L;

[0047] Preferably, in step T52, the added mass of EDC is 6-8% of the mass of polylysine, and the added mass of NHS is 4-5% of the mass of polylysine;

[0048] T6, dissolving the polyglutamic acid-hydrophobized bullfrog-derived elastin peptide prepared in step T51 and the polylysine-hydrophobized bullfrog-derived collagen peptide prepared in step T52 in DMSO, dripping into pure water at a speed of 0.5-1 mL / min, stirring at a speed of 180-200 rpm, reacting for 4-6 hours, and concentrating by ultrafiltration to obtain a biocollagen composition;

[0049] Preferably, in step T6, the mass concentration of the polyglutamic acid-hydrophobized bullfrog-derived elastin peptide in DMSO is 30-40 mg / mL;

[0050] Preferably, in step T6, the mass concentration of the poly-lysine-hydrophobized bullfrog-derived collagen peptide in DMSO is 40-50 mg / mL.

[0051] The beneficial effects achieved by the present invention are as follows:

[0052] The invention provides a bullfrog extracted biological collagen composition and a preparation method thereof. The collagen and elastin from bullfrog skin and bullfrog cartilage are jointly extracted by a three-enzyme co-production method. After bullfrog-derived collagen peptides and bullfrog-derived elastin peptides are hydrophobically modified, polyglutamic acid and polylysine are added to form a collagen combination, which can improve the stability, drug loading capacity and sustained release capacity of the composition. In the invention, alkaline protease, clostridial peptidase A and elastase are used to treat bullfrog skin and bullfrog cartilage, and after multiple protein extractions, bullfrog-derived collagen peptides and bullfrog-derived elastin peptides are obtained. The collagen peptides and elastin are grafted The hydrophobic modification of the collagen peptide and the elastin peptide with palmitic acid can improve the stability of the collagen composition in the gastrointestinal environment. The collagen peptide and the elastin peptide are modified with polyglutamic acid and polylysine respectively. The ends of the collagen peptide and the elastin peptide are combined through the electrostatic effect brought about by the opposite charges of the carboxyl and amine groups on polyglutamic acid and polylysine to form an amphiphilic collagen composition. The cross-linking density of the collagen combination is increased by combining the collagen with high cross-linking properties and the elastin peptide with low cross-linking properties, thereby forming different sustained-release effects on different drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the SDS-PAGE image of the bullfrog-derived collagen peptide and elastin peptide prepared in Examples 1-3 of the present invention;

[0054] Figure 2 This is a graph showing the embedding efficiency of curcumin by the collagen composition described in Examples 4-6 of the present invention and Comparative Examples 1-3;

[0055] Figure 3 The release results of the collagen compositions prepared in Examples 4-6 of the present invention and Comparative Examples 1-3 in the gastrointestinal environment are shown in FIG.

[0056] Figure 4 The graph shows the encapsulation and sustained-release performance results of the collagen compositions prepared in Examples 4-6 of the present invention and Comparative Examples 1-3.

[0057] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0058] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0059] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0060] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials and test strains used in the following examples are purchased from commercial channels unless otherwise specified.

[0061] The sources of the materials described in the present invention are as follows:

[0062] Alkaline protease was purchased from Angel Yeast Co., Ltd., and the specification of alkaline protease was 200U / mg;

[0063] Clostridial peptidase A was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with the product number C754928. The specification of Clostridial peptidase A was 125 CDU / mg;

[0064] The polyglutamic acid described in the present invention is poly-L-glutamic acid sodium salt, which is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with the product number P477788.

[0065] The polylysine described in the present invention is ε-polylysine hydrochloride, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with a product number of P192512.

[0066] Example 1

[0067] This embodiment provides a method for extracting bullfrog protein, which specifically comprises the following steps:

[0068] S1, take a male bullfrog, peel and remove the muscle tissue of the bullfrog, retain the bullfrog skeleton and bullfrog skin, add deionized water according to the solid-liquid ratio of 0.1g / mL, adjust the pH to 9, wash the bullfrog skeleton and skin for 20min, rinse the bullfrog skeleton and skin with deionized water until neutral, place it at 35°C and dry it for 12h, grind it, and pass it through an 80-mesh sieve to obtain a pre-treated bullfrog powder;

[0069] S2. Accurately weigh 50 g of pre-treated bullfrog powder and place it in a conical flask. Add 500 mL of PBS buffer and mix well. Adjust the pH of the reaction system to 10. Add 2 g of alkaline protease and mix well. Raise the temperature to 40 ° C for enzymatic hydrolysis. After 4 h of enzymatic hydrolysis, raise the temperature to 95 ° C for 10 min to inactivate the enzyme. After the reaction is cooled to room temperature, centrifuge at 8000 rpm for 5 min, collect the supernatant, and freeze-dry to obtain enzymatic crude protein ①.

[0070] S3, take 16g of the enzymatic crude protein ① prepared in step S2, prepare 200mL PBS buffer solution, add 40mg of ZnCl2 to the PBS buffer solution, mix with the enzymatic crude protein ①, adjust the pH to 7.5, add 0.24g of Clostridial peptidase A to the reaction system, raise the temperature to 37°C, perform enzymatic hydrolysis, after 4h of enzymatic hydrolysis, raise the temperature to 95°C to inactivate the enzyme for 10min, centrifuge at 8000rpm for 5min, separate the supernatant and the precipitate, collect the supernatant and place it in a 3000Da dialysis bag for dialysis with deionized water, after 48h, freeze-dry to obtain bullfrog source collagen peptide; collect the precipitate as enzymatic crude protein ②, for standby use;

[0071] S4. Disperse the enzymatic crude protein ② prepared in step S3 in 220 mL PBS buffer solution, adjust the pH to 8, add 0.5 g elastase, adjust the enzymatic temperature to 40 ° C, and after enzymatic treatment for 2 hours, raise the temperature to 95 ° C to inactivate the enzyme. After 10 minutes, centrifuge at 8000 rpm for 5 minutes, collect the supernatant, and dialyze it in a 3000Da dialysis bag with deionized water. After 48 hours, freeze-dry to obtain bullfrog-derived elastin peptide.

[0072] Example 2

[0073] This embodiment provides a method for extracting bullfrog protein, which specifically comprises the following steps:

[0074] S1, take a male bullfrog, peel and remove the muscle tissue of the bullfrog, retain the bullfrog skeleton and bullfrog skin, add deionized water according to the solid-liquid ratio of 0.3g / mL, adjust the pH to 9, wash the bullfrog skeleton and skin for 30min, rinse the bullfrog skeleton and skin with deionized water until neutral, place it at 35°C and dry it for 12h, grind it, and pass it through an 80-mesh sieve to obtain a pre-treated bullfrog powder;

[0075] S2. Accurately weigh 50 g of pre-treated bullfrog powder and place it in a conical flask. Add 500 mL of PBS buffer and mix well. Adjust the pH of the reaction system to 11. Add 1 g of alkaline protease and mix well. Raise the temperature to 50 ° C for enzymatic hydrolysis. After 5 h of enzymatic hydrolysis, raise the temperature to 90 ° C for 10 min to inactivate the enzyme. After the reaction is cooled to room temperature, centrifuge at 6000 rpm for 10 min, collect the supernatant, and freeze-dry to obtain enzymatic crude protein ①.

[0076] S3, take 15g of the enzymatic crude protein ① prepared in step S2, prepare 150mL PBS buffer solution, add 45mg ZnCl2 to the PBS buffer solution, mix with the enzymatic crude protein ①, adjust the pH to 7.6, add 0.15g clostridial peptidase A to the reaction system, raise the temperature to 40°C, perform enzymatic hydrolysis, after 5h of enzymatic hydrolysis, raise the temperature to 90°C to inactivate the enzyme for 10min, centrifuge at 6000rpm for 10min, separate the supernatant and the precipitate, collect the supernatant and place it in a 3000Da dialysis bag for dialysis with deionized water, after 48h, freeze-dry to obtain bullfrog source collagen peptide; collect the precipitate as enzymatic crude protein ②, set aside;

[0077] S4. Disperse the enzymatically hydrolyzed crude protein ② prepared in step S3 in 175 mL PBS buffer solution, adjust the pH to 8.5, add 0.6 g elastase, adjust the enzymatic hydrolysis temperature to 37 ° C, and after enzymatic hydrolysis for 1 hour, raise the temperature to 90 ° C to inactivate the enzyme. After 10 minutes, centrifuge at 7000 rpm for 8 minutes, collect the supernatant, and dialyze it in a 3000 Da dialysis bag with deionized water. After 48 hours, freeze-dry it to obtain bullfrog-derived elastin peptide.

[0078] Example 3

[0079] This embodiment provides a method for extracting bullfrog protein, which specifically comprises the following steps:

[0080] S1, take a male bullfrog, peel and remove the muscle tissue of the bullfrog, retain the bullfrog skeleton and bullfrog skin, add deionized water according to the solid-liquid ratio of 0.2g / mL, adjust the pH to 8.5, wash the bullfrog skeleton and skin for 30min, rinse the bullfrog skeleton and skin with deionized water until neutral, place it at 35°C and dry it for 12h, grind it, and pass it through an 80-mesh sieve to obtain a pre-treated bullfrog powder;

[0081] S2. Accurately weigh 50 g of pre-treated bullfrog powder and place it in a conical flask. Add 500 mL of PBS buffer and mix well. Adjust the pH of the reaction system to 11. Add 1.5 g of alkaline protease and mix well. Raise the temperature to 40 ° C for enzymatic hydrolysis. After 5 h of enzymatic hydrolysis, raise the temperature to 90 ° C for 10 min to inactivate the enzyme. After the reaction is cooled to room temperature, centrifuge at 6000 rpm for 10 min, collect the supernatant, and freeze-dry to obtain enzymatic crude protein ①.

[0082] S3, take 16g of the enzymatic crude protein ① prepared in step S2, prepare 130mL PBS buffer solution, add 26mg of ZnCl2 to the PBS buffer solution, mix with the enzymatic crude protein ①, adjust the pH to 7.4, add 0.32g of Clostridial peptidase A to the reaction system, raise the temperature to 35°C, perform enzymatic hydrolysis, after 5h of enzymatic hydrolysis, raise the temperature to 95°C to inactivate the enzyme for 10min, centrifuge at 7000rpm for 8min, separate the supernatant and the precipitate, collect the supernatant and place it in a 3000Da dialysis bag for dialysis with deionized water, after 48h, freeze-dry to obtain bullfrog source collagen peptide; collect the precipitate as enzymatic crude protein ②, set aside;

[0083] S4. Disperse the enzymatic crude protein ② prepared in step S3 in 170 mL PBS buffer solution, adjust the pH to 7.5, add 0.8 g elastase, adjust the enzymatic temperature to 35 ° C, and after enzymatic treatment for 2 hours, raise the temperature to 95 ° C to inactivate the enzyme. After 10 minutes, centrifuge at 6000 rpm for 10 minutes, collect the supernatant, and dialyze it in a 3000 Da dialysis bag with deionized water. After 48 hours, freeze-dry to obtain bullfrog-derived elastin peptide.

[0084] Example 4

[0085] The present invention provides a bullfrog extracted biological collagen composition, comprising the following components in parts by weight: 1.2 parts of hydrophobized bullfrog-derived collagen peptide, 0.72 parts of hydrophobized bullfrog-derived elastin peptide, 1 part of polyglutamic acid, and 1 part of polylysine;

[0086] This embodiment provides a method for preparing a biocollagen composition extracted from bullfrog, which specifically comprises the following steps:

[0087] T1. Take 10 g of MBHA resin and place it in a synthesis tube. Add 100 mL of DMF to mix. Then blow nitrogen gas. After 40 minutes, swell the MBHA resin. Prepare a 20 vol% piperidine / DMF solution. Take 10 mL and add it to the reaction system. After blowing with flowing nitrogen gas for 15 minutes, remove the Fmoc amino protective agent on the MBHA resin. Remove the piperidine / DMF solution after suction filtration. Wash it three times with DMF and anhydrous methanol alternately. Then, obtain an activated MBHA resin.

[0088] T21. Accurately weigh 1 g of bullfrog collagen peptide and place it in a dry centrifuge tube. Add 10 mL of DMF to dissolve the bullfrog collagen peptide. Add 0.15 g of HATU and 54 mg of HOBt to a beaker and mix well. Accurately weigh 76 μL of N,N-diisopropylethylamine and add it to the reaction system. Mix at 180 rpm for 5 min, transfer it to a synthesis tube, add 0.6 g of MBHA resin activated in step T1 to the synthesis tube, introduce nitrogen gas for 8 h at room temperature and in the dark, vacuum filter, remove the reaction solution, wash the MBHA resin three times with DME and anhydrous methanol alternately, and determine whether the bullfrog collagen peptide is successfully coupled with the activated MBHA resin by ninhydrin. The result shows colorless, indicating that the bullfrog collagen peptide is successfully coupled with the activated MBHA resin to obtain MBHA resin-bullfrog collagen peptide;

[0089] T22, accurately weigh 1 g of bullfrog elastin peptide and place it in a dry beaker, add 10 mL of DMF to dissolve the bullfrog elastin peptide, add 0.2 g of HATU and 75 mg of HOBt in the beaker, mix well, accurately weigh 54 μL of N,N-diisopropylethylamine and add it to the reaction system, mix at 180 rpm for 5 min, transfer to a synthesis tube, add 0.8 g of MBHA resin activated in step T1 to the synthesis tube, introduce nitrogen gas for 8 h at room temperature and in the dark, vacuum filter, remove the reaction solution, and wash the MBHA resin alternately with DME and anhydrous methanol for three times, and determine whether the bullfrog elastin peptide is successfully coupled with the activated MBHA resin by ninhydrin. The result shows colorless, indicating that the bullfrog elastin peptide is successfully coupled with the activated MBHA resin, and MBHA resin-bullfrog elastin peptide is obtained;

[0090] T31, accurately weigh 1.0 g of palmitic acid and place it in 100 mL of DMF, stir until the palmitic acid is completely dissolved, add 0.62 g of EDC (155 g / mol) and 0.46 g of NHS, heat in a water bath to 40° C. until the reactants are completely dissolved in DMF, mix evenly, stir at 180 rpm for 5 min, add to the MBHA resin-bullfrog collagen peptide prepared in step T21, pass nitrogen gas for 3 h at room temperature and in the dark, then perform vacuum filtration to remove the reaction solvent, and wash repeatedly with DMF and anhydrous methanol three times alternately to obtain MBHA resin-bullfrog collagen peptide;

[0091] T32, accurately weigh 0.72 g of palmitic acid and place it in 60 mL of DMF, stir until the palmitic acid is completely dissolved, add 0.43 g of EDC and 0.32 g of NHS, heat in a water bath to 40° C. until the reactants are completely dissolved in DMF, mix well, stir at 180 rpm for 5 min, add to the MBHA resin-bullfrog derived elastin peptide prepared in step T22, introduce nitrogen gas for 3 h at room temperature and in the dark, then perform vacuum filtration to remove the reaction solvent, and wash repeatedly with DMF and anhydrous methanol three times in sequence to obtain MBHA resin-bullfrog derived elastin peptide;

[0092] T4, take the hydrophobic MBHA resin-bullfrog collagen peptide prepared in step T31 and the hydrophobic MBHA resin-bullfrog elastin peptide prepared in step T32, wash them alternately with DMF and anhydrous methanol, remove DMF and anhydrous methanol by vacuum filtration, and transfer them to flasks after nitrogen drying. The amount of MBHA resin in step T21 is 0.6 g, and the amount of MBHA resin in step T22 is 0.8 g. Prepare 200 mL of cutting reagent according to 160 mL of trifluoroacetic acid, 10 mL of β-mercaptoethanol, 10 mL of methyl phenyl sulfide, 14 mL of deionized water, and 6 mL of phenol, take 120mL was added to a flask containing hydrophobized MBHA resin-bullfrog collagen peptide, 80mL was added to a flask containing hydrophobized MBHA resin-bullfrog elastin peptide, and stirred at 250rpm under room temperature and light-proof conditions. After stirring and reacting for 5h, the filtrate was filtered and the filtrate was collected. 100mL of ice ether was added to the filtrate, and the filtrate was placed at -20°C. After standing for 36h, the precipitate was collected after centrifugation at 5000rpm for 5min. The precipitate was fully washed with ether and deionized water, and freeze-dried to obtain hydrophobized bullfrog collagen peptide and hydrophobized bullfrog elastin peptide, respectively;

[0093] T51, accurately weigh 100 mg of polyglutamic acid and place it in 10 mL of DMF, mix until the polyglutamic acid is completely dissolved, add 5 mg of EDC and 4 mg of NHS to activate the polyglutamic acid, stir and mix evenly, adjust the pH of the reaction system to 5.5 with acetic acid, add 72 mg of hydrophobized bullfrog elastin peptide, increase the reaction temperature to 37° C., stir and mix at a speed of 150 rpm, react for 5 hours, remove the solvent by rotary evaporation, wash repeatedly with DMF and deionized water three times in sequence, and freeze-dry to obtain polyglutamic acid-hydrophobized bullfrog elastin peptide;

[0094] T52, accurately weigh 100 mg of polylysine and place it in 10 mL of DMF, mix until polyglutamic acid is completely dissolved, add 6 mg of EDC and 4.5 mg of NHS to activate the polylysine, stir and mix evenly, add 120 mg of hydrophobized bullfrog collagen peptide, increase the reaction temperature to 37° C., stir and mix at a speed of 150 rpm, react for 5 hours, remove the solvent by rotary evaporation, wash repeatedly with DMF and deionized water three times in sequence, and freeze-dry to obtain polyglutamic acid-hydrophobized bullfrog elastin peptide;

[0095] T6. Dissolve the polyglutamic acid-hydrophobized bullfrog-derived elastin peptide prepared in step T51 in 4.5 mL DMSO, and dissolve the polylysine-hydrophobized bullfrog-derived collagen peptide prepared in step T52 in 4.25 mL DMSO. After mixing evenly, add dropwise into pure water at a rate of 0.5 mL / min, stir at a speed of 200 rpm, react for 4 hours, and concentrate by ultrafiltration to obtain a biocollagen composition.

[0096] Example 5

[0097] The present invention provides a bullfrog extracted biological collagen composition, comprising the following components in parts by weight: 1 part of hydrophobized bullfrog-derived collagen peptide, 0.6 parts of hydrophobized bullfrog-derived elastin peptide, 1.2 parts of polyglutamic acid, and 1.5 parts of polylysine;

[0098] This embodiment provides a method for preparing a biocollagen composition extracted from bullfrog, which specifically comprises the following steps:

[0099] T1. Take 10 g of MBHA resin and place it in a synthesis tube. Add 100 mL of DMF to mix. Then blow nitrogen gas. After 40 minutes, swell the MBHA resin. Prepare a 23 vol% piperidine / DMF solution. Take 5 mL and add it to the reaction system. After blowing with flowing nitrogen gas for 15 minutes, remove the Fmoc amino protective agent on the MBHA resin. Remove the piperidine / DMF solution after suction filtration. Wash it three times with DMF and anhydrous methanol alternately. Then, obtain an activated MBHA resin.

[0100] T21. Accurately weigh 1.5 g of bullfrog collagen peptide and place it in a dry beaker. Add 100 mL of DMF to dissolve the bullfrog collagen peptide. Add 0.27 g of HATU and 0.16 g of HOBt to the beaker and mix well. Accurately weigh 0.11 mL of N,N-diisopropylethylamine and add it to the reaction system. Mix at 160 rpm for 7 min, transfer it to a synthesis tube, add 0.85 g of MBHA resin activated in step T1 to the synthesis tube, introduce nitrogen gas for 12 h at room temperature and in the dark, vacuum filter, remove the reaction solution, wash the MBHA resin three times with DME and anhydrous methanol alternately, and determine whether the bullfrog collagen peptide is successfully coupled with the activated MBHA resin by ninhydrin. The result shows colorless, indicating that the bullfrog collagen peptide is successfully coupled with the activated MBHA resin to obtain MBHA resin-bullfrog collagen peptide;

[0101] T22, accurately weigh 1.5g of bullfrog elastin peptide and place it in a dry beaker, add 10mL DMF to dissolve the bullfrog elastin peptide, add 0.32g HATU and 0.11g HOBt in the beaker, mix well, accurately weigh 120μL of N,N-diisopropylethylamine and add it to the reaction system, mix at 180rpm for 5min, transfer to a synthesis tube, add 1.2g MBHA resin activated in step T1 to the synthesis tube, introduce nitrogen gas for 8h at room temperature and in the dark, vacuum filter, remove the reaction solution, and wash the MBHA resin alternately with DME and anhydrous methanol three times, and determine whether the bullfrog elastin peptide is successfully coupled with the activated MBHA resin by ninhydrin. The result shows colorless, indicating that the bullfrog elastin peptide is successfully coupled with the activated MBHA resin, and MBHA resin-bullfrog elastin peptide is obtained;

[0102] T31, accurately weigh 1.0 g of palmitic acid and place it in 100 mL of DMF, stir until the palmitic acid is completely dissolved, add 0.93 g of EDC and 1.38 g of NHS, heat in a water bath to 40° C. until the reactant is completely dissolved in DMF, mix evenly, stir at 170 rpm for 6 min, add to the MBHA resin-bullfrog collagen peptide prepared in step T21, and blow nitrogen for 3.5 h at room temperature and in the dark, then perform vacuum filtration to remove the reaction solvent, and wash repeatedly with DMF and anhydrous methanol three times alternately to obtain MBHA resin-bullfrog collagen peptide;

[0103] T32, accurately weigh 0.72 g of palmitic acid and place it in 60 mL of DMF, stir until the palmitic acid is completely dissolved, add 0.65 g of EDC and 0.96 g of NHS, heat in a water bath to 40° C. until the reactants are completely dissolved in DMF, mix well, stir at 150 rpm for 8 min, add to the MBHA resin-bullfrog-derived elastin peptide prepared in step T22, introduce nitrogen gas for 4 h at room temperature and in the dark, then perform vacuum filtration to remove the reaction solvent, and wash repeatedly with DMF and anhydrous methanol three times in sequence to obtain MBHA resin-bullfrog-derived elastin peptide;

[0104] T4, take the hydrophobic MBHA resin-bullfrog collagen peptide prepared in step T31 and the hydrophobic MBHA resin-bullfrog elastin peptide prepared in step T32, wash them alternately with DMF and anhydrous methanol, remove DMF and anhydrous methanol by vacuum filtration, and transfer them to flasks after nitrogen drying. The amount of MBHA resin in step T21 is 0.85g, and the amount of MBHA resin in step T22 is 1.2g. Prepare 200mL of cutting reagent according to 160mL of trifluoroacetic acid, 10mL of β-mercaptoethanol, 10mL of methyl phenyl sulfide, 14mL of deionized water, and 6mL of phenol, take 1 20mL was added to a flask containing hydrophobized MBHA resin-bullfrog collagen peptide, and 30mL was added to a flask containing hydrophobized MBHA resin-bullfrog elastin peptide, and stirred at 200rpm under room temperature and light-proof conditions. After stirring and reacting for 4.5h, the filtrate was filtered and the filtrate was collected. 100mL of ice ether was added to the filtrate, and the filtrate was placed at -20°C. After standing for 30h, the precipitate was collected after centrifugation at 7000rpm for 3min, and the precipitate was fully washed with ether and deionized water. After freeze-drying, the hydrophobized bullfrog collagen peptide and the hydrophobized bullfrog elastin peptide were obtained respectively;

[0105] T51, accurately weigh 120 mg of polyglutamic acid and place it in 7 mL of DMF, mix until the polyglutamic acid is completely dissolved, add 7 mg of EDC and 5 mg of NHS to activate the polyglutamic acid, stir and mix evenly, adjust the pH of the reaction system to 5.8 with acetic acid, add 60 mg of hydrophobized bullfrog elastin peptide, increase the reaction temperature to 35 ° C, stir and mix at a speed of 160 rpm, react for 4 hours, remove the solvent by rotary evaporation, wash repeatedly with DMF and deionized water three times in sequence, and freeze-dry to obtain polyglutamic acid-hydrophobized bullfrog elastin peptide;

[0106] T52, accurately weigh 150 mg of polylysine and place it in 8 mL of DMF, mix until polyglutamic acid is completely dissolved, add 8 mg of EDC and 5 mg of NHS to activate the polylysine, stir and mix evenly, add 100 mg of hydrophobized bullfrog collagen peptide, increase the reaction temperature to 40°C, stir and mix at a speed of 160 rpm, react for 4 hours, remove the solvent by rotary evaporation, wash repeatedly with DMF and deionized water three times in sequence, and freeze-dry to obtain polyglutamic acid-hydrophobized bullfrog elastin peptide;

[0107] T6. Dissolve the polyglutamic acid-hydrophobized bullfrog-derived elastin peptide prepared in step T51 in 3.25 mL DMSO, and dissolve the polylysine-hydrophobized bullfrog-derived collagen peptide prepared in step T52 in 3.4 mL DMSO. After mixing evenly, add dropwise into pure water at a rate of 0.5 mL / min, stir at a speed of 200 rpm, react for 4 hours, and concentrate by ultrafiltration to obtain a biocollagen composition.

[0108] Example 6

[0109] The present invention provides a bullfrog extracted biological collagen composition, comprising the following components in parts by weight: 1.5 parts of hydrophobized bullfrog-derived collagen peptide, 0.8 parts of hydrophobized bullfrog-derived elastin peptide, 1.5 parts of polyglutamic acid, and 1.2 parts of polylysine;

[0110] This embodiment provides a method for preparing a biocollagen composition extracted from bullfrog, which specifically comprises the following steps:

[0111] T1. Take 10 g of MBHA resin and place it in a synthesis tube. Add 100 mL of DMF to mix, blow it with nitrogen, and after 40 minutes, swell the MBHA resin, prepare a 25 vol% piperidine / DMF solution, take 5 mL and add it to the reaction system, blow it with flowing nitrogen for 15 minutes, remove the Fmoc amino protective agent on the MBHA resin, remove the piperidine / DMF solution after suction filtration, and wash it repeatedly with DMF and anhydrous methanol three times alternately to obtain an activated MBHA resin;

[0112] T21. Accurately weigh 1.2 g of bullfrog collagen peptide and place it in a dry beaker. Add 10 mL of DMF to dissolve the bullfrog collagen peptide. Add 0.19 g of HATU and 0.1 mg of HOBt to the beaker and mix well. Accurately weigh 95 μL of N,N-diisopropylethylamine and add it to the reaction system. Mix at 150 rpm for 8 min, transfer it to a synthesis tube, add 0.7 g of MBHA resin activated in step T1 to the synthesis tube, introduce nitrogen gas for 10 h at room temperature and in the dark, vacuum filter, remove the reaction solution, wash the MBHA resin three times with DME and anhydrous methanol alternately, and determine whether the bullfrog collagen peptide is successfully coupled with the activated MBHA resin by ninhydrin. The result shows colorless, indicating that the bullfrog collagen peptide is successfully coupled with the activated MBHA resin to obtain MBHA resin-bullfrog collagen peptide;

[0113] T22, accurately weigh 1.2 g of bullfrog elastin peptide and place it in a dry beaker, add 10 mL of DMF to dissolve the bullfrog elastin peptide, add 0.3 g of HATU and 0.16 g of HOBt in the beaker, mix well, accurately weigh 78 μL of N,N-diisopropylethylamine and add it to the reaction system, mix at 180 rpm for 5 min, transfer to a synthesis tube, add 0.96 g of MBHA resin activated in step T1 to the synthesis tube, introduce nitrogen gas for 8 h at room temperature and in the dark, vacuum filter, remove the reaction solution, and wash the MBHA resin alternately with DME and anhydrous methanol three times, and determine whether the bullfrog elastin peptide is successfully coupled with the activated MBHA resin by ninhydrin. The result shows colorless, indicating that the bullfrog collagen peptide is successfully coupled with the activated MBHA resin to obtain MBHA resin-bullfrog elastin peptide;

[0114] T31, accurately weigh 1.0 g of palmitic acid and place it in 100 mL of DMF, stir until the palmitic acid is completely dissolved, add 0.74 g of EDC and 0.82 g of NHS, heat in a water bath to 40° C. until the reactants are completely dissolved in DMF, mix evenly, stir at 150 rpm for 8 min, add to the MBHA resin-bullfrog collagen peptide prepared in step T21, pass nitrogen gas for 4 h at room temperature and in the dark, then perform vacuum filtration to remove the reaction solvent, and wash repeatedly with DMF and anhydrous methanol three times in sequence to obtain MBHA resin-bullfrog collagen peptide;

[0115] T32, accurately weigh 0.72 g of palmitic acid and place it in 60 mL of DMF, stir until the palmitic acid is completely dissolved, add 0.52 g of EDC and 0.77 g of NHS, heat in a water bath to 40° C. until the reactants are completely dissolved in DMF, mix well, stir at 160 rpm for 7 min, add to the MBHA resin-bullfrog derived elastin peptide prepared in step T22, introduce nitrogen gas for 3.5 h at room temperature and in the dark, perform vacuum filtration, remove the reaction solvent, and wash repeatedly with DMF and anhydrous methanol three times in sequence to obtain MBHA resin-bullfrog derived elastin peptide;

[0116] T4, take the hydrophobic MBHA resin-bullfrog collagen peptide prepared in step T31 and the hydrophobic MBHA resin-bullfrog elastin peptide prepared in step T32, wash them alternately with DMF and anhydrous methanol, remove DMF and anhydrous methanol by vacuum filtration, and transfer them to flasks after nitrogen drying. The amount of MBHA resin in step T21 is 0.7 g, and the amount of MBHA resin in step T22 is 0.96 g. Prepare 200 mL of cutting reagent according to 160 mL of trifluoroacetic acid, 10 mL of β-mercaptoethanol, 10 mL of methyl phenyl sulfide, 14 mL of deionized water, and 6 mL of phenol. Take 30 mL and add it to a flask containing hydrophobized MBHA resin-bullfrog collagen peptide, take 20 mL and add it to a flask containing hydrophobized MBHA resin-bullfrog elastin peptide, stir at 220 rpm under room temperature and dark conditions, stir and react for 4 hours, filter and collect the filtrate, add 100 mL of ice ether to the filtrate, place it at -20 ° C, stand for 24 hours, centrifuge at 6000 rpm for 4 minutes, collect the precipitate, wash it thoroughly with ether and deionized water, and freeze-dry to obtain hydrophobized bullfrog collagen peptide and hydrophobized bullfrog elastin peptide, respectively;

[0117] T51, accurately weigh 150 mg of polyglutamic acid and place it in 5 mL of DMF, mix until the polyglutamic acid is completely dissolved, add 6 mg of EDC and 3 mg of NHS to activate the polyglutamic acid, stir and mix evenly, adjust the pH of the reaction system to 6 with acetic acid, add 80 mg of hydrophobized bullfrog elastin peptide, increase the reaction temperature to 40°C, stir and mix at a speed of 180 rpm, react for 3 hours, remove the solvent by rotary evaporation, wash repeatedly with DMF and deionized water three times in sequence, and freeze-dry to obtain polyglutamic acid-hydrophobized bullfrog elastin peptide;

[0118] T52, accurately weigh 120 mg of polylysine and place it in 5 mL of DMF, mix until polyglutamic acid is completely dissolved, add 7 mg of EDC and 4 mg of NHS to activate the polylysine, stir and mix evenly, add 150 mg of hydrophobized bullfrog collagen peptide, increase the reaction temperature to 35°C, stir and mix at a speed of 180 rpm, react for 3 hours, remove the solvent by rotary evaporation, wash repeatedly with DMF and deionized water three times in sequence, and freeze-dry to obtain polyglutamic acid-hydrophobized bullfrog elastin peptide;

[0119] T6. Dissolve the polyglutamic acid-hydrophobized bullfrog-derived elastin peptide prepared in step T51 in 3.7 mL DMSO, and dissolve the polylysine-hydrophobized bullfrog-derived collagen peptide prepared in step T52 in 3.8 mL DMSO. After mixing evenly, add dropwise into pure water at a rate of 0.5 mL / min, stir at a speed of 200 rpm, react for 4 hours, and concentrate by ultrafiltration to obtain a biocollagen composition.

[0120] Comparative Example 1

[0121] This comparative example provides a collagen composition and a preparation method thereof, which differs from Example 1 only in that T31 and T32 are not included in the preparation method, that is, the collagen peptide and elastin peptide are not modified with palmitic acid; the remaining components and component contents are the same as those in Example 1.

[0122] Comparative Example 2

[0123] This comparative example provides a collagen composition and a preparation method thereof, which differs from Example 1 only in that T51 and T52 are not included in the preparation method, and polyglutamic acid and polylysine are not included in the collagen composition; the remaining components and component contents are the same as those in Example 1.

[0124] Comparative Example 3

[0125] This comparative example provides a collagen composition and a preparation method thereof, which is different from Example 1 only in that T32 and T51 are not included in the preparation method, and the collagen composition contains polyglutamic acid and hydrophobized elastin peptide; the remaining components and component contents are the same as those in Example 1.

[0126] Experimental Example 1

[0127] This experimental example analyzes the amino acid components of the bullfrog collagen peptide and bullfrog elastin peptide prepared in Examples 1-3. 5009.124-2016 "National Food Safety Standard Determination of Amino Acids in Food" determines the composition and content of hydrolyzed amino acids in the bullfrog collagen peptide and bullfrog elastin peptide described in Examples 1-3; prepare a mixed amino acid standard stock solution, prepare a standard mother solution according to the concentration of 80 mg / L of a single amino acid, take 1 mL and dry it, then use 1 mL of sodium citrate buffer solution (pH = 2.2), filter through a 0.22 μm filter membrane to obtain a mixed amino acid standard stock solution; weigh 20 mg of the bullfrog collagen peptide and bullfrog elastin peptide prepared in Examples 1-3 respectively, dissolve them in 10 mL of hydrochloric acid solution (6M), add phenol solution, freeze-dry, place in a 50 mL volumetric flask, make up to volume with deionized water, take 1 mL and dry it, then re-dissolve it with 1 mL of sodium citrate buffer solution (pH = 2.2), filter through a 0.22 μm filter membrane, and test.

[0128] The following table shows the amino acid composition and content results of the bullfrog-derived collagen peptides and bullfrog-derived elastin peptides prepared in Examples 1-3 of the present invention; according to the results, the bullfrog-derived collagen peptides described in Examples 1-3 contain high levels of glutamic acid, glycine, and aspartic acid, and also include histidine, methionine, arginine, and tyrosine components, and have good antioxidant activity. The elastin peptides contain high levels of alanine, valine, proline, glycine and.

[0129]

[0130] Experimental Example 2

[0131] In this experiment, 5% concentrated gel and 12% separation gel were used to perform SDS-PAGE analysis on collagen peptides and elastin peptides. Figure 1 It is an SDS-PAGE image of the bullfrog-derived collagen peptide and elastin peptide prepared in Examples 1-3 of the present invention, wherein band M is a marker band, band a is a band of the bullfrog-derived collagen peptide prepared in Example 1, band b is a band of the bullfrog-derived collagen peptide prepared in Example 2, band c is a band of the bullfrog-derived collagen peptide prepared in Example 3, band d is a band of the bullfrog-derived elastin peptide prepared in Example 1, band e is a band of the bullfrog-derived elastin peptide prepared in Example 2, and band f is a band of the bullfrog-derived elastin peptide prepared in Example 3. The molecular weight of the bullfrog-derived collagen peptide prepared in the present invention is between 1000-2500Da, and the molecular weight of the bullfrog-derived elastin peptide is between 800-2000Da.

[0132] Experimental Example 3

[0133] The present invention tests the encapsulation and sustained release performance of the collagen composition prepared in Examples 4-6 and Comparative Examples 1-3. Taking curcumin as an example, in step T6 of preparing the collagen composition in Examples 4-6 and Comparative Examples 1-3, 10 mg / L of curcumin is added to the reaction system, and the curcumin-loaded collagen composition is obtained according to the preparation method described in step T6. The curcumin-loaded collagen composition prepared in Examples 4-6 and Comparative Examples 1-3 is placed in anhydrous ethanol, ultrasonically treated at 400 W for 30 min, and centrifuged at 5000 rpm for 5 min. The absorbance of curcumin is measured at a wavelength of 425 nm using an ultraviolet-visible spectrophotometer, the concentration of curcumin is converted by a standard curve, and the encapsulation rate (%) of curcumin is calculated according to the following formula:

[0134] Embedding rate (%) = M1 / M0 × 100%;

[0135] Wherein, M1 is the mass of curcumin in the collagen composition, in mg; M0 is the mass of the original added curcumin, in mg;

[0136] A simulated gastric fluid with a pH of 1.2 was prepared according to 3.2 g / L of pepsin (enzyme activity> 250 U / mg) and 2.0 g / L of sodium chloride, and a simulated intestinal fluid with a pH of 6.8 was prepared according to 6.8 g / L of sodium bicarbonate, 1.0 g / L of trypsin (enzyme activity> 250 U / mg), and 10.0 g / L of ox bile salt. 1 g of the collagen composition prepared in Examples 4-6 and Comparative Examples 1-3 was placed in 20 mL of simulated gastric fluid / intestinal fluid, and gastrointestinal digestion was simulated at 37° C. and 100 rpm. The cumulative release rate of curcumin was calculated according to the following formula:

[0137] Cumulative release rate (%) = (Ct•V) / M×100%;

[0138] Wherein, Ct is the concentration of curcumin sampled at time t (expressed in mg / mL); V is the total volume of the release medium (expressed in mL); M is the initial total drug amount in the collagen composition (expressed in mg);

[0139] Figure 2 The results of the embedding rate of curcumin by the collagen composition described in Examples 4-6 of the present invention and Comparative Examples 1-3 are shown. As shown in the figure, the embedding rate of curcumin by the collagen composition prepared in Examples 4-6 of the present invention is significantly higher, and the composition has a hydrophobic end, which can improve the affinity with curcumin, thereby improving the embedding rate of curcumin; at the same time, in Examples 4-6 of the present invention, collagen peptides and elastin peptides are used for compounding, and a stable coating effect is formed on curcumin through different groups on the side chains of small molecule polypeptides.

[0140] Figure 3 The release result diagram of the collagen composition prepared by Examples 4-6 of the present invention and Comparative Examples 1-3 in the gastrointestinal environment is shown in the figure. As shown in the figure, the release rate of the collagen composition prepared by Examples 1-3 in the gastric environment is maintained between 0-0.5, that is, the composition is basically not decomposed in the gastric environment, while the collagen composition prepared by Comparative Example 1 has begun to decompose in the gastric environment; in the field environment, the collagen composition prepared by Examples 1-3 can maintain a high release efficiency. In a neutral environment, the carboxyl group of polyglutamic acid and the amino group on polylysine have opposite electrical properties in the environment, and curcumin can be fixed in the collagen carrier by electrostatic force. After degradation, curcumin can be slowly released. The collagen prepared by Comparative Example 2 has obvious burst release in a long environment, and burst release occurs within 0.5h after entering the intestinal environment; in Comparative Example 3, the collagen peptide lacks bullfrog elastin peptide, and the cross-linking density of the high-content collagen peptide is too high, resulting in poor release of curcumin.

[0141] Experimental Example 4

[0142] The present invention tests the drug encapsulation and sustained release performance of the collagen composition prepared in Examples 4-6 and Comparative Examples 1-3. Taking icariin as an example, in step T6 of preparing the collagen composition in Examples 4-6 and Comparative Examples 1-3, 20 mg / L icariin is added to the reaction system, and the collagen composition loaded with curcumin is obtained according to the preparation method described in step T6. The icariin-loaded collagen composition prepared in Examples 4-6 and Comparative Examples 1-3 is placed in anhydrous ethanol, ultrasonically treated at 400 W for 30 min, and centrifuged at 5000 rpm for 5 min. The absorption value of icariin is measured at a wavelength of 270 m using an ultraviolet-visible spectrophotometer, the concentration of icariin is converted by a standard curve, and the encapsulation rate (%) of curcumin is calculated according to the following formula:

[0143] Embedding rate (%) = M1 / M0 × 100%;

[0144] Wherein, M1 is the mass of icariin in the collagen composition, in mg; M0 is the mass of the originally added icariin, in mg;

[0145] A simulated gastric fluid with a pH of 1.2 was prepared according to 3.2 g / L of pepsin (enzyme activity> 250 U / mg) and 2.0 g / L of sodium chloride, and a simulated intestinal fluid with a pH of 6.8 was prepared according to 6.8 g / L of sodium bicarbonate, 1.0 g / L of trypsin (enzyme activity> 250 U / mg), and 10.0 g / L of ox bile salt. 1 g of the collagen composition prepared in Examples 4-6 and Comparative Examples 1-3 was placed in 20 mL of simulated gastric fluid and simulated intestinal fluid, respectively. After 3 hours at 37° C. and 100 rpm, the absorption value of icariin was measured at a wavelength of 270 m using a UV-visible spectrophotometer. The concentration of icariin was converted using a standard curve, and the release rate of icariin was calculated according to the following formula:

[0146] Release rate (%) = (Ct•V) / M×100%;

[0147] Wherein, Ct is the concentration of icariin sampled at time t (expressed in mg / mL); V is the total volume of the release medium (expressed in mL); M is the initial total drug amount in icariin (expressed in mg);

[0148] Figure 4 The encapsulation and sustained-release performance results of the collagen compositions prepared by Examples 4-6 of the present invention and Comparative Examples 1-3 are shown in the figure. As shown in the figure, for the hydrophilic drug Epimedium, the collagen composition prepared by the present invention has a higher encapsulation performance. At the same time, it hardly degrades in the gastrointestinal environment, and its sustained-release performance is basically the same as that of the hydrophobic drug in the gastrointestinal environment.

[0149] While the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that many changes, modifications, substitutions and variations can be made to the embodiments without departing from the principles and spirit of the invention.

[0150] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A biocollagen composition extracted from bullfrog, characterized in that: The collagen composition comprises the following components in parts by weight: 1-1.5 parts of hydrophobized bullfrog collagen peptide, 0.6-0.8 parts of hydrophobized bullfrog elastin peptide, 1-1.5 parts of polyglutamic acid, and 1-1.5 parts of polylysine; the hydrophobized bullfrog collagen peptide and the hydrophobized bullfrog elastin peptide are derived from bullfrog protein; The method for extracting bullfrog protein specifically comprises the following steps: S1, take a male bullfrog, peel and remove the muscle tissue of the bullfrog, retain the bullfrog skeleton and bullfrog skin, according to the solid-liquid ratio of bullfrog skeleton, skin and deionized water 0.1-0.3g / mL, add deionized water, adjust the pH to 8-9, wash the bullfrog skeleton and skin for 20-30min, rinse the bullfrog skeleton and skin with deionized water to neutrality, place it at 35 ° C and dry it for 12h, grind it, and pass it through an 80-mesh sieve to obtain pre-treated bullfrog powder; S2, according to the mass concentration of the pre-treated bullfrog powder in PBS buffer solution is 0.1-0.12g / mL, the pre-treated bullfrog powder prepared in step S1 is dispersed in PBS buffer solution, the pH is adjusted to 10-11, alkaline protease is added, the enzymatic hydrolysis temperature is 40-50°C, after enzymatic hydrolysis for 4-5h, the temperature is increased to 90-95°C, the enzyme is inactivated for 10min, and the supernatant is collected after centrifugation at 6000-8000rpm for 5-10min, and freeze-dried to obtain enzymatic crude protein①; S3, according to the mass concentration of the enzymatic crude protein ① in the PBS buffer solution is 0.08-0.12g / mL, the enzymatic crude protein ① prepared in step S2 is dissolved in a PBS buffer solution, the PBS buffer solution contains a zinc ion mass concentration of 0.2-0.3g / L, the pH is adjusted to 7.4-7.6, clostridial peptidase A is added, the enzymatic hydrolysis temperature is adjusted to 35-40°C, and the enzymatic hydrolysis treatment is performed for 3-4h, and then the temperature is increased to 90-95°C, and the enzyme is inactivated for 10min, and then centrifuged at 6000-8000rpm for 5-10min, and the supernatant and the precipitate are separated, and the supernatant is collected. After freeze-drying, bullfrog-derived collagen peptides are obtained, and the precipitate is collected as the enzymatic crude protein ② for standby use; S4, according to the mass concentration of the enzymatic crude protein ② dissolved in the PBS buffer solution is 0.06-0.08g / mL, the enzymatic crude protein ② prepared in step S3 is dissolved in the PBS buffer solution, the pH is adjusted to 7.5-8.5, elastase is added, the enzymatic hydrolysis temperature is set to 35-40°C, and the enzymatic hydrolysis treatment is performed for 1-2h, and then the temperature is increased to 90-95°C, and the enzyme is inactivated for 10min, and then centrifuged at 6000-8000rpm for 5-10min, and the supernatant is collected and freeze-dried to obtain bullfrog-derived elastin peptide.

2. The biocollagen composition extracted from bullfrog according to claim 1, characterized in that: In step S2, the enzyme amount E / S of the alkaline protease is 2%-4%; in step S3, the enzyme amount E / S of the clostridial peptidase A is 1%-2%; in step S4, the enzyme amount E / S of the elastase is 4%-6%.

3. A method for preparing the bullfrog extracted biological collagen composition according to claim 1 or 2, characterized in that: The specific steps include: T1. Mix MBHA resin and DMF, introduce nitrogen for blowing, and after swelling treatment for 40-60 minutes, add piperidine solution, continue to introduce flowing nitrogen for blowing, and after 15-20 minutes, remove the piperidine solution by suction filtration, and wash with DMF and anhydrous methanol to obtain activated MBHA resin; T21, dissolving the bullfrog collagen peptide in DMF and transferring it to MBHA resin, adding HATU and HOBt to the reaction system, mixing evenly, adding N,N-diisopropylethylamine to the reaction system, stirring and mixing at a speed of 150-180 rpm for 5-8 minutes, adding the activated MBHA resin prepared in step T1, placing it in a dark environment, passing nitrogen gas for 8-12 hours, vacuum filtering to remove the reaction solvent, washing with DMF and anhydrous methanol, and obtaining MBHA resin-bullfrog collagen peptide; T22, placing the activated MBHA resin prepared in step T1 in a beaker, dissolving the bullfrog elastin peptide in DMF and transferring the mixture to the beaker containing the MBHA resin, adding HATU and HOBt to the reaction system, mixing evenly, adding N,N-diisopropylethylamine to the reaction system, stirring at 150-180 rpm for 5-8 min, adding the activated MBHA resin prepared in step T1, placing the mixture in a dark environment, passing nitrogen gas for 8-12 h, vacuum filtering out the reaction solvent, washing with DMF and anhydrous methanol, and obtaining MBHA resin-bullfrog elastin peptide; T31, dissolving palmitic acid in DMF, adding EDC and NHS, mixing evenly, stirring at 150-180 rpm for 3-5 min, adding to the MBHA resin-bullfrog collagen peptide prepared in step T21, placing in a dark environment, passing nitrogen gas for 3-4 h, vacuum filtering to remove the reaction solvent, washing with DMF and anhydrous methanol, to obtain a hydrophobic MBHA resin-bullfrog collagen peptide; T32, dissolving palmitic acid in DMF, adding EDC and NHS, mixing evenly, adding to the MBHA resin-bullfrog derived elastin peptide prepared in step T22, mixing evenly, adding N,N-diisopropylethylamine to the reaction system, stirring and mixing at a speed of 150-180 rpm for 3-5 min, placing in a dark environment, passing nitrogen gas for 3-4 h, vacuum filtering out the reaction solvent, washing with DMF and anhydrous methanol, and drying to obtain a hydrophobic MBHA resin-bullfrog derived elastin peptide; T4, take the hydrophobized MBHA resin-bullfrog collagen peptide prepared in step T31 and the hydrophobized MBHA resin-bullfrog elastin peptide cleavage reagent prepared in step T32, stir at 200-250rpm at room temperature and in the dark, stir and react for 4-5h, collect the filtrate by suction, add ice ether to the filtrate, place it at -20°C, stand it for 24-36h, centrifuge it at 5000-7000rpm for 3-5min, collect the precipitate, wash it thoroughly with ether and deionized water, and freeze-dry it to obtain hydrophobized bullfrog collagen peptide and hydrophobized bullfrog elastin peptide; T51, dissolving polyglutamic acid in DMF solvent, adding EDC and NHS and stirring, mixing evenly, adjusting the pH of the reaction system to 5.5-6.0 with acetic acid, adding hydrophobized bullfrog-derived elastin peptide, raising the reaction temperature to 35-40° C., stirring and mixing at a speed of 150-180 rpm, reacting for 3-5 hours, removing the solvent by rotary evaporation, washing with DMF and deionized water, and freeze-drying to obtain polyglutamic acid-hydrophobized bullfrog-derived elastin peptide; T52, dissolving polylysine in DMF solvent, adding EDC and NHS and stirring, after mixing evenly, adding hydrophobized bullfrog collagen peptide, raising the reaction temperature to 35-40°C, stirring and mixing at a speed of 150-180rpm, reacting for 3-5h, rotary evaporating to remove the solvent, washing with DMF and deionized water, and freeze-drying to obtain polylysine-hydrophobized bullfrog collagen peptide; T6. Dissolve the polyglutamic acid-hydrophobized bullfrog-derived elastin peptide prepared in step T51 and the polylysine-hydrophobized bullfrog-derived collagen peptide prepared in step T52 in DMSO, drip into pure water at a rate of 0.5-1 mL / min, stir at a speed of 180-200 rpm, react for 4-6 hours, and concentrate by ultrafiltration to obtain a biocollagen composition.

4. The method for preparing the biocollagen composition extracted from bullfrog according to claim 3, characterized in that: In step T1, the mass concentration of the MBHA resin in DMF is 0.1-0.2 g / mL; in the piperidine solution, the volume fraction of piperidine in DMF is 20%-25%, and the mass-to-volume ratio between the MBHA resin and the piperidine solution is 0.5-1 g / mL.

5. The method for preparing the biocollagen composition extracted from bullfrog according to claim 4, characterized in that: In step T21, the mass concentration of the bullfrog-derived collagen peptide in DMF is 0.1-0.15 g / mL; the mass ratio of the bullfrog-derived collagen peptide, HATU and HOBt is 1:0.15-0.18:0.05-0.1; the mass volume ratio of the bullfrog-derived collagen peptide and N,N-diisopropylethylamine is 12.6-13.6 g / mL; the mass ratio of the bullfrog-derived collagen peptide to the MBHA resin before activation described in step T1 is 1-1.5:

1.

6. The method for preparing the biocollagen composition extracted from bullfrog according to claim 5, characterized in that: In step T22, the mass concentration of the bullfrog-derived elastin peptide in DMF is 0.1-0.15 g / mL; the mass ratio of the bullfrog-derived elastin peptide, HATU and HOBt is 1:0.2-0.25:0.075-0.14; the mass volume ratio of the bullfrog-derived elastin peptide and N,N-diisopropylethylamine is 12.5-18.5 g / mL; the mass ratio of the bullfrog-derived elastin peptide to the MBHA resin described in step T1 is 1:0.

8.

7. The method for preparing the biocollagen composition extracted from bullfrog according to claim 6, characterized in that: In step T31, the mass concentration of palmitic acid in DMF is 10-15 g / L; the mass ratio of palmitic acid, EDC and NHS is 1:0.6-0.9:0.45-1.4; In step T32, the mass concentration of the palmitic acid in DMF is 10-15 g / L; the mass ratio of the palmitic acid, EDC and NHS is 1:0.6-0.9:0.45-1.

3.

8. The method for preparing the biocollagen composition extracted from bullfrog according to claim 7, characterized in that: In step T4, the cutting reagent includes components: trifluoroacetic acid, β-mercaptoethanol, methyl phenyl sulfide, deionized water, and phenol; the amount of the cutting reagent is calculated according to the total amount of the MBHA resin in step T21 and step T22, and the mass volume ratio of the total amount of the MBHA resin in step T21 and step T22 to the cutting reagent in step T4 is 0.05-0.08 g / mL.

9. The method for preparing the biocollagen composition extracted from bullfrog according to claim 8, characterized in that: In step T51, the mass concentration of the polyglutamic acid in DMF is 10-20 g / L; the added mass of the EDC is 5-7% of the mass of the polyglutamic acid, and the added mass of the NHS is 3-5% of the mass of the polyglutamic acid; In step T52, the mass concentration of the polylysine in DMF is 10-20 g / L; the added mass of the EDC is 6-8% of the mass of the polylysine, and the added mass of the NHS is 4-5% of the mass of the polylysine.

10. The method for preparing the biocollagen composition extracted from bullfrog according to claim 9, characterized in that: In step T6, the mass concentration of the polyglutamic acid-hydrophobized bullfrog-derived elastin peptide in DMSO is 30-40 mg / mL; the mass concentration of the polylysine-hydrophobized bullfrog-derived collagen peptide in DMSO is 40-50 mg / mL.

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