Hydrolyzed collagen, its preparation method and application

Abstract

The present application relates to the field of cosmetic technology, and in particular to a preparation method of hydrolyzed collagen, which simulates the in-vivo natural digestion process by using saliva, gastric juice, pancreatic juice and bile to prepare hydrolyzed collagen from bullfrog skin, and has mild conditions and retains biological activity to the greatest extent; the synergistic effect of multiple digestive juices precisely cuts the collagen molecular chain to generate fragments with reasonable structure and excellent activity, making up for the limitations of single enzyme or a small number of enzymes in enzyme method. The acid-base reagent is abandoned to eliminate the risk of chemical residues, ensure product safety from the source, effectively reduce immunogenicity, and overcome the problems of structure damage and antigen epitope exposure caused by acid-base method. The reaction conditions are based on the stable physiological parameters of human digestive juice, and the temperature and pH value are easy to control, so as to ensure the stable product quality; in terms of cost control, the dependence on expensive commercial enzymes can be reduced, and no acid-base wastewater is generated, which has significant environmental benefits, and truly realizes the overall upgrading of the preparation process.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics, more particularly, it relates to hydrolyzed collagen and its preparation method and application. BACKGROUND

[0002] The bullfrog farming industry is growing rapidly, and its output is increasing year by year. The skin tissue of bullfrog accounts for about 5% of its body weight, which is usually discarded during the eating process, causing a large amount of biological resources to be wasted. Extracting high-quality undenatured natural collagen from discarded bullfrog skin tissue not only solves the pollution problem of waste, but also deeply develops high-value-added products, thereby generating huge environmental and social benefits.

[0003] The conventional method for preparing bullfrog hydrolyzed collagen includes enzymatic method and acid-base method. However, the enzymatic method has high cost, the enzyme preparation itself is expensive and has a large amount, and the subsequent complicated separation and purification steps further increase the production cost; the enzymatic hydrolysis conditions are harsh, which easily leads to over-hydrolysis, resulting in the destruction of collagen structure and the loss of activity, or insufficient hydrolysis, resulting in low extraction rate, and potential enzyme residue, which may affect the stability of the product and cause allergic reactions. The acid-base method is also not optimistic, and the treatment environment of strong acid and strong base can seriously damage the triple helix structure and key amino acid residues of collagen protein, resulting in sharp reduction of biological activity. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application aims to provide a preparation method of hydrolyzed collagen, which can solve the technical problems of high cost and low biological activity in the preparation of bullfrog hydrolyzed collagen.

[0005] To achieve the above-mentioned purpose, the present application realizes the following technical scheme:

[0006] A preparation method of hydrolyzed collagen, comprising the following steps:

[0007] (1) preliminary hydrolysis: put the treated small pieces of bullfrog skin into a reaction container, add saliva at a ratio of 1:2 of the mass of bullfrog skin to the volume of saliva, adjust the temperature of the reaction system to 36-37℃ and the pH value to 6.5-7.0, and then stir at a speed of 50-100 rpm for 2-3 h, so that the enzymes in the saliva preliminarily decompose part of the polysaccharides and loose protein structure in the bullfrog skin;

[0008] (2) gastric digestion simulation: after the saliva treatment is completed, the reaction system is warmed to 38-40℃, the gastric juice is added at a ratio of 1:1.5 of the mass of bullfrog skin to the volume of gastric juice, the pH value is adjusted to 1.5-2.0 with concentrated hydrochloric acid, and the stirring speed is increased to 150-200 rpm, and the reaction is carried out for 4-6 h, so that the pepsin fully hydrolyzes the protein into polypeptide fragments;

[0009] (3) Deep digestion: after the end of gastric digestion, the reaction system is neutralized to pH 7.5-8.0 with sodium hydroxide solution, and then pancreas juice and gall juice are added in the proportion of 1:1 and 1:0.5 respectively, based on the mass of bullfrog skin and the volume of pancreas juice and gall juice, the temperature is maintained at 37-38℃, and the reaction is stirred at a speed of 200-250 rpm for 6-8 hours, so that the enzymes in the pancreas juice further decompose the polypeptides and fats, and the gall juice helps to emulsify the fats and promote overall digestion;

[0010] (4) Separation and purification: after the end of enzymatic reaction, the reaction liquid is first filtered with 100-150 mesh filter cloth to remove undigested large pieces of bullfrog skin tissue residues, and the filtrate is collected; preliminary separation is carried out by ultrafiltration, and an ultrafiltration membrane with a target molecular weight cut-off is selected, and ultrafiltration is carried out under a pressure of 0.1-0.3 MPa, and the permeate is retained, which contains most of the target product and small molecular impurities; ion exchange chromatography separation is carried out on the permeate, weak acid cation exchange resin is selected, 0.02-0.05 mol / L acetate buffer is used as the equilibrium liquid and eluent, the flow rate is controlled at 1-2 mL / min, and the eluent is collected; finally, the eluent is dried by freeze-drying technology, and the hydrolyzed collagen is obtained.

[0011] Preferably, the freeze-drying procedure is: pre-freezing temperature -40℃ to -50℃, time 2-3 hours; sublimation drying temperature -20℃ to -30℃, vacuum degree 10-30 Pa, drying time 12-24 hours.

[0012] Preferably, the pH value of the acetate buffer is 4.5-5.5.

[0013] Preferably, an ultrafiltration membrane with a molecular weight cut-off of 10-30 kDa is selected for ultrafiltration.

[0014] Another object of the present application is to provide a hydrolyzed collagen prepared by the aforementioned preparation method.

[0015] Still another object of the present application is to provide an application of the hydrolyzed collagen, specifically: using the aforementioned hydrolyzed collagen as a raw material for preparing cosmetics; the dosage form of the cosmetics includes but is not limited to common water, emulsion, cream, and paste.

[0016] Preferably, the mass percentage of the hydrolyzed collagen added in the cosmetics is 1-30%.

[0017] The present application has the following advantages relative to the prior art: the present application simulates the in-vivo natural digestion process by using saliva, gastric juice, pancreatic juice and bile to prepare hydrolyzed collagen from bullfrog skin, and the conditions are mild, like a 'fine surgery' for collagen molecules, which maximally preserves the bioactivity; the synergistic effect of multiple digestive juices precisely cuts the collagen molecular chain, generating fragments with reasonable structure and excellent activity, which makes up for the limitations of single enzyme or a few enzymes in enzyme method. The present application abandons acid-base reagents, eliminates the risk of chemical residues, ensures product safety from the source, effectively reduces immunogenicity, and overcomes the problems of structure destruction and exposure of antigen epitopes in acid-base method. The reaction conditions are based on the stable physiological parameters of human digestive juice, and the temperature and pH value are easy to control, which ensures the stability of product quality; in terms of cost control, the present application is expected to reduce the dependence on expensive commercial enzymes, and also has significant environmental benefits due to the absence of acid-base wastewater, which truly realizes the overall upgrading of the preparation process. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the FTIR test graph of the hydrolyzed collagen prepared in Example 1 of the present application, which is speculated to contain cis peptide bonds (1394cm -1 ), β-sheet structure (1233cm -1 );

[0019] Figure 2 is the MS test graph of the hydrolyzed collagen prepared in Example 1 of the present application;

[0020] Figure 3 is the use effect graph of Application Example 1 in the present application;

[0021] Figure 4 is the use effect graph of Application Example 2 in the present application;

[0022] Figure 5 is the use effect graph of Application Example 3 in the present application;

[0023] Figure 6 is the use effect graph of Application Example 4 in the present application. DETAILED DESCRIPTION

[0024] The present application will be further described below in conjunction with examples, but the scope of protection claimed by the present application is not limited to the scope expressed by the examples.

[0025] Example 1

[0026] Preparation of raw materials: Collect fresh bullfrog skin, make sure there is no obvious lesions, injury, remove surface impurities after washing with normal saline, cut into small pieces (about 0.5-1 cm in length), ready for use. Collect fresh saliva, which can be collected by stimulating the secretion of healthy people, and immediately placed in ice bath to prevent enzyme inactivation. Before use, filter out impurities. Fresh pig stomach juice is obtained from slaughterhouse. The digestive system of pigs has some similarity to bullfrogs, and the source is relatively stable. Also stored in ice bath and filtered before use. Pancreatic juice and bile are purchased from regular biological reagent companies and stored according to the storage conditions specified in the instructions.

[0027] Start preparation: the preparation method of hydrolyzed collagen, specifically including the following steps:

[0028] (1) Preliminary hydrolysis: Put the treated bullfrog skin pieces into the reaction container, add saliva according to the ratio of 1:2 of the mass of bullfrog skin to the volume of saliva (1 gram of bullfrog skin, 2 ml of saliva), adjust the temperature of the reaction system to 36-37°C (close to the temperature of the human oral cavity, to provide a suitable active environment for saliva amylase and other enzymes), adjust the pH value to 6.5-7.0 (the suitable pH range for saliva amylase) with dilute hydrochloric acid or sodium hydroxide, and then stir at a low speed of 50-100 rpm for 2-3 hours to preliminarily decompose part of the polysaccharides and loose protein structure in the bullfrog skin by the enzymes in the saliva;

[0029] (2) Gastric digestion simulation: After the saliva treatment is completed, the reaction system is warmed to 38-40°C (close to the temperature inside the pig stomach), and the gastric juice is added according to the ratio of 1:1.5 of the mass of bullfrog skin to the volume of gastric juice (1 gram of bullfrog skin, 1.5 ml of gastric juice), the pH value is adjusted to 1.5-2.0 (the most suitable pH for pepsin) with concentrated hydrochloric acid, the stirring speed is increased to 150-200 rpm, and the reaction is carried out for 4-6 hours to fully hydrolyze the protein into polypeptide fragments by pepsin;

[0030] (3) Deep digestion: After the gastric juice digestion is completed, the reaction system is neutralized to a pH value of 7.5-8.0 (the suitable environment for pancreatic juice and bile) with sodium hydroxide solution, and the pancreatic juice and bile are added according to the ratio of 1:1 of the mass of bullfrog skin to the volume of pancreatic juice and 1:0.5 of the mass of bullfrog skin to the volume of bile (1 gram of bullfrog skin, 1 ml of pancreatic juice, and 0.5 ml of bile), the temperature is maintained at 37-38°C, and the stirring is carried out at a speed of 200-250 rpm for 6-8 hours to further decompose the polypeptides and fats in the pancreatic juice, and the bile assists in emulsifying the fats to promote overall digestion;

[0031] (4) Separation and purification: after the enzymatic reaction is completed, the reaction solution is first filtered with 100-150 mesh filter cloth to remove undigested large pieces of bullfrog skin tissue residues, and the filtrate is collected; preliminary separation is performed by ultrafiltration, a ultrafiltration membrane with a molecular weight cut-off of 10-30 kDa is selected, and ultrafiltration is performed under a pressure of 0.1-0.3 MPa, and the permeate is retained, which contains most of the target product and small molecular impurities; ion exchange chromatography separation is performed on the permeate, a weak acid cation exchange resin is selected, and 0.02-0.05 mol / L acetate buffer (pH 4.5-5.5) is used as the equilibration liquid and eluent, the flow rate is controlled at 1-2 mL / min, and the eluate is collected; finally, the eluate is dried by freeze-drying technology, and the hydrolyzed collagen is obtained, wherein the freeze-drying program is: pre-freezing temperature -40°C to -50°C, time 2-3 hours; sublimation drying temperature -20°C to -30°C, vacuum degree 10-30 Pa, drying time 12-24 hours.

[0032] Comparative Example 1

[0033] Compared with Example 1, the only difference is that the temperature of the reaction system is adjusted from 36-37°C to 38-40°C during the saliva action stage.

[0034] Comparative Example 2

[0035] Compared with Example 1, the only difference is that the pH value is adjusted from 6.5-7.0 to 7.5-8.0 during the saliva action stage; the pH value is adjusted from 1.5-2.0 to 2.0-2.5 during the gastric juice action stage; and the pH value is adjusted from 7.5-8.0 to 8.5-9.0 during the pancreatic juice and bile action stage.

[0036] Comparative Example 3

[0037] Compared with Example 1, the only difference is that the stirring speed is adjusted from 50-100 rpm to 200-250 rpm during the saliva action stage.

[0038] Comparative Example 4

[0039] The hydrolyzed collagen is prepared according to the preparation method described in Example 1 of the patent document with publication number CN102276716A.

[0040] The hydrolyzed collagens prepared in the above examples and comparative examples are subjected to bioavailability test, physiological activity test and low immunogenicity test.

[0041] Test 1

[0042] The bioavailability test is performed by cell uptake experiment, and the specific steps are as follows:

[0043] (1) Cultured human skin fibroblasts to the logarithmic growth phase, trypsinized and seeded at a density of 5 x 105cells per well in a 24-well plate and incubated for 24 hours to allow them to adhere. 4

[0044] (2) Set up an experimental group (added the hydrolyzed collagen prepared in Example 1, prepared as a 1 mg / mL solution using serum-free medium, at 100 μL / well), a control group (added the same amount of hydrolyzed collagen prepared in Comparative Examples 1 to 4, at the same concentration), and a blank group (added only serum-free medium).

[0045] (3) After 4 hours of incubation, the medium was removed and the cells were gently washed three times with PBS buffer, and 0.5 mL of trypsin was added to digest the cells and collect the cell suspension.

[0046] (4) The fluorescence intensity in the cells was measured by flow cytometry, and the collagen was labeled with a fluorescent marker (e.g., FITC) in advance.

[0047] (5) The average fluorescence intensity of the cells in the experimental and control groups was recorded, and the cell uptake rate was calculated. Cell uptake rate = (experimental group average fluorescence intensity - blank group average fluorescence intensity) / (control group average fluorescence intensity - blank group average fluorescence intensity) x 100%. The results are shown in Table 1.

[0048] Test 2

[0049] The biological activity was tested by a cell proliferation test, and the specific steps were as follows:

[0050] (1) Referring to Test 1, human skin fibroblasts were cultured to the logarithmic growth phase and seeded at a density of 1 x 105cells per well in a 96-well plate and incubated for 24 hours. 4

[0051] (2) Set up an experimental group, a control group, and a blank group, and perform the cell uptake test as in the bioavailability test, but extend the incubation time to 72 hours.

[0052] (3) After the incubation was complete, 10 μL of MTT reagent was added to each well, and incubation was continued for 4 hours, after which the supernatant was carefully removed, 150 μL of DMSO was added to each well, and the mixture was shaken for 10 minutes to fully dissolve the crystals.

[0053] (4) The absorbance value at 570 nm was measured using a microplate reader.

[0054] (5) The cell proliferation rate was calculated. Cell proliferation rate = (experimental group absorbance value - blank group absorbance value) / (control group absorbance value - blank group absorbance value) x 100%. The results are shown in Table 1.

[0055] Test 3

[0056] ​​The low immunogenicity test was carried out by a lymphocyte proliferation experiment, and the specific steps were as follows:

[0057] (1) The peripheral blood of healthy people was collected, and the lymphocytes were separated by density gradient centrifugation, and the cell density was adjusted to 1×10 6 / mL by using RPMI 1680 medium.

[0058] (2) The experimental group, the control group and the blank group were set up, the hydrolyzed collagen prepared in Example 1 was added to the experimental group (prepared into a solution of 1 mg / mL with serum-free medium, added at 100 μL / well), the control group (added with the same amount of hydrolyzed collagen prepared in Comparative Examples 1-4, the same concentration) and the blank group (only added with serum-free medium).

[0059] (3) The cell suspension was inoculated in a 96-well plate at 200 μL per well, and cultured for 72 hours.

[0060] (4) After the culture was completed, 10 μL of MTT reagent was added to each well, and the subsequent operation was the same as that of the cell proliferation experiment.

[0061] (5) The lymphocyte proliferation rate was calculated, and the lymphocyte proliferation rate = (absorbance value of the experimental group-absorbance value of the blank group) / (absorbance value of the control group-absorbance value of the blank group)×100%. The results are shown in Table 1.

[0062] Table 1 Performance data table of hydrolyzed collagen prepared in examples and comparative examples

[0063] Group Cellular uptake rate (%) Cellular proliferation rate (%) Lymphocyte proliferation rate (%) Example 1 76 162 5 Comparative Example 1 64 140 10 Comparative Example 2 32 108 28 Comparative Example 3 47 132 11 Comparative Example 4 55 136 22

[0064] As can be seen from Table 1, the hydrolyzed collagen prepared in Example 1 has better bioavailability, physiological activity and low immunogenicity compared with the hydrolyzed collagen prepared in Comparative Examples 1-4.

[0065] The hydrolyzed collagen prepared in Example 1 was sent to Shanghai Weipu Laboratory for qualitative and quantitative analysis (report number: SHA01-22031537-FX-01CnR2), and the results are shown in Table 2 and Figures 1-2

[0066] Table 2 Composition table of hydrolyzed collagen

[0067] Component Mass content (%) CAS No. Total protein 2.5 / Hyaluronic acid 2.4 9004-61-9 Total sugar 0.4 / Peptidic substances* Balance / Isoleucine 0.1 73-32-5 Phenylalanine 0.1 673-31-4 Threonine 0.1 72-19-5 Methionine 0.07 63-68-3 Proline 0.04 609-36-9 Leucine 0.03 3588-60-1 Tyrosine 0.002 70642-86-3 Sodium element 1.4 / Calcium element 0.3 / Silicon element 0.2 /

[0068] Note: The components marked with “*” in Table 2 are inferred by fragment information and experience.

[0069] In order to further expand the application range, the hydrolyzed collagen prepared above and the commonly used matrix in the cosmetic field were made into a kind of cosmetic, and the dosage form of the cosmetic included but was not limited to common water agent, emulsion, cream and paste.

[0070] ​The collagen peptide repair cream provided by the present application is prepared from the following raw materials in percentage by mass: 10% of the hydrolyzed collagen prepared in Example 1, 0.15% of carbomer, 1% of propylene glycol, 3% of butylene glycol, 0.05% of disodium edetate, 0.15% of triethanolamine, 0.3% of potassium sorbate, 5% of water-soluble azone, 0.3% of aloe vera gel essence, 0.75% of 305 emulsifier, and pure water to make up to 100%.

[0071] Application Example 1

[0072] The collagen peptide repair cream prepared above was used on a 6-year-old child who suffered from severe atopic dermatitis, dry and itchy skin, and constantly scratched wounds on the body, and could not sleep well at night. The cream was applied in a thick layer for 6 consecutive days, and the results are shown in Table 1. The itchy symptoms of the child's lower leg, thigh, and buttocks were improved, the wounds scabbed, and the skin recovered. This application example shows that the collagen peptide repair cream prepared by the present application can effectively relieve the symptoms of atopic dermatitis and dry and itchy skin, and is beneficial to wound healing. Figure 3

[0073] Application Example 2

[0074] The collagen peptide repair cream prepared above was used on a 25-year-old woman who had sensitive skin and was sunburned due to long-term outdoor exercise. The cream was applied for 1 day, and the results are shown in Table 2. The redness, swelling, heat, pain, and itching caused by sunburn were relieved within one day. This application example shows that the collagen peptide repair cream prepared by the present application has the effect of repairing sunburn. Figure 4

[0075] Application Example 3

[0076] The collagen peptide repair cream prepared above was used on a 46-year-old woman whose face was full of red rashes after water light laser treatment and could not subside. The cream was applied in a thick layer for 1 day, and the results are shown in Table 3. The red rashes caused by laser treatment gradually subsided within one day. This application example shows that the collagen peptide repair cream prepared by the present application has the effect of subsiding rashes and fading redness. Figure 5

[0077] Application Example 4

[0078] The collagen peptide repair cream prepared above was used on a 5-year-old child who fell and got a bruise on the nose and face while playing. After the wound was cleaned, the collagen peptide repair cream prepared above was applied in a thick layer, and the results are shown in Table 4. The wound was basically healed after 12 days. This application example shows that the collagen peptide repair cream prepared by the present application is beneficial to wound repair and healing. Figure 6

[0079] ​​​​The above merely describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above-described embodiments. Any technical solution falling within the concept of the present application shall fall within the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application shall also be considered as falling within the protection scope of the present application.

Claims

1. A method for preparing hydrolyzed collagen, characterized by, The preparation method comprises the following steps: (1) Preliminary hydrolysis: Place the processed bullfrog skin pieces into a reaction vessel, add saliva at a ratio of 1:2 between the mass of bullfrog skin and the volume of saliva, and adjust the reaction system temperature to 36-37°C and the pH value to 6.5-7.

0. Then stir at 50-100 rpm for 2-3 hours to allow the enzymes in the saliva to initially decompose some of the polysaccharides and loose protein structures in the bullfrog skin. (2) Gastric digestion simulation: After the saliva treatment is completed, the reaction system is heated to 38-40°C, gastric juice is added at a ratio of 1:1.5 between the mass of bullfrog skin and the volume of gastric juice, the pH value is adjusted to 1.5-2.0 with concentrated hydrochloric acid, the stirring speed is increased to 150-200 rpm, and the reaction is carried out for 4-6 hours to allow pepsin to fully hydrolyze the protein into polypeptide fragments; (3) Deep digestion: After the gastric juice digestion is completed, the reaction system is neutralized with sodium hydroxide solution to a pH value of 7.5-8.

0. Pancreatic juice and bile are added in a ratio of 1:1 between the weight of bullfrog skin and the volume of pancreatic juice and 1:0.5 between the weight of bullfrog skin and the volume of bile. The temperature is maintained at 37-38 ° C and the reaction is stirred at 200-250 rpm for 6-8 hours to allow the enzymes in the pancreatic juice to further decompose polypeptides and fats. The bile assists in emulsifying fat and promotes overall digestion. (4) Separation and purification: After the enzymatic hydrolysis reaction is completed, the reaction solution is first filtered with a 100-150 mesh filter cloth to remove the undigested large pieces of bullfrog skin tissue residue, and the filtrate is collected; the ultrafiltration method is used for preliminary separation, and an ultrafiltration membrane with a target molecular weight cutoff of 10-30 kDa is selected. Ultrafiltration is performed at a pressure of 0.1-0.3 MPa, and the permeate is retained. The permeate contains most of the target product and small molecular impurities; the permeate is subjected to ion exchange chromatography separation, and a weakly acidic cation exchange resin is selected. 0.02-0.05 mol / L acetate buffer is used as the balance solution and eluent, and the flow rate is controlled at 1-2 mL / min, and the eluate is collected; finally, the eluate is dried using freeze-drying technology to obtain hydrolyzed collagen; The freeze-drying procedure is as follows: pre-freezing temperature -40°C to -50°C, time 2-3 hours; sublimation drying temperature -20°C to -30°C, vacuum degree 10-30 Pa, drying time 12-24 hours; The pH of acetate buffer is 4.5-5.

5.

2. Hydrolyzed collagen, characterized in that Prepared by the preparation method according to claim 1.

3. Use of hydrolyzed collagen, characterized in that, The hydrolyzed collagen according to claim 2 is used as a raw material for preparing cosmetics.

4. Use of hydrolysed collagen according to claim 3, characterised in that The mass percentage of the hydrolyzed collagen added to the cosmetic is 1-30%.

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