Hydrolyzed collagen as well as preparation method and application thereof

Abstract

The invention relates to the technical field of cosmetics, in particular to a preparation method of hydrolyzed collagen, according to the preparation method for preparing the hydrolyzed collagen from bullfrog skin by using saliva, gastric juice, pancreatic juice and bile fluid, the in-vivo natural digestion process is simulated, the condition is mild, and the biological activity is reserved to the maximum extent; a plurality of digestive juice synergistically act, collagen molecular chains are accurately cut, fragments with reasonable structures and excellent activity are generated, and the limitation of action of a single enzyme or a few enzymes in an enzymatic method is made up. Acid-base reagents are abandoned, the risk of chemical residues is eradicated, the product safety is guaranteed from the source, the immunogenicity is effectively reduced, and the problems that the structure is damaged and the epitope is exposed by an acid-base method are solved. Reaction conditions are easy to control on the basis of stable physiological parameters, temperature, pH value and the like of human digestive juice, so that the product quality is as stable as one; in the aspect of cost control, dependence on expensive commercial enzymes is expected to be reduced, no acid-alkali wastewater is generated, remarkable environmental protection benefits are achieved, and comprehensive upgrading of the preparation process is truly achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and more specifically, to hydrolyzed collagen and a preparation method and application thereof. Background Art

[0002] The bullfrog breeding industry is growing stronger and stronger, and its output is increasing year by year. The skin tissue of bullfrogs accounts for about 5% of their body weight, which is usually discarded during consumption, resulting in a large waste of biological resources. Extracting high-quality undenatured natural collagen from discarded bullfrog skin tissue can not only solve the problem of waste pollution, but also deeply develop high value-added products, which will generate huge environmental and social benefits.

[0003] Conventional methods for preparing bullfrog hydrolyzed collagen include enzymatic method and acid-base method. However, the enzymatic method is costly. The enzyme preparation itself is expensive and the dosage is large. The subsequent tedious separation and purification steps make the situation even worse, greatly increasing the production cost. The enzymatic hydrolysis conditions are harsh, and it is easy to have problems such as excessive hydrolysis that causes damage to the collagen structure and loss of activity, or insufficient hydrolysis that leads to low extraction rate. In addition, the potential hidden danger of enzyme residues may affect product stability and induce allergic reactions. The acid-base method is also not optimistic. The strong acid and alkali treatment environment will seriously damage the triple helix structure and key amino acid residues of collagen, causing its biological activity to drop sharply. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for preparing hydrolyzed collagen, which can solve the technical problems of high cost and low biological activity in the current preparation of bullfrog hydrolyzed collagen.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] A method for preparing hydrolyzed collagen comprises the following steps:

[0007] (1) Preliminary hydrolysis: Place the processed bullfrog skin pieces into a reaction vessel, add saliva in a ratio of 1:2 between the mass of bullfrog skin and the volume of saliva, adjust the temperature of the reaction system 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 enzyme in the saliva to preliminarily decompose some polysaccharides and loose protein structures in the bullfrog skin;

[0008] (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 of bullfrog skin mass to gastric juice volume, 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;

[0009] (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, and pancreatic juice and bile are added in a ratio of 1:1 to pancreatic juice and 1:0.5 to bile juice. 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, and the bile assists in emulsifying fats to promote overall digestion.

[0010] (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 is selected, and 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 to be 1-2 mL / min, and the eluate is collected; finally, the eluate is dried using freeze-drying technology to obtain hydrolyzed collagen.

[0011] Preferably, the freeze-drying procedure is: pre-freezing temperature -40°C to -50°C, time 2-3 hours; sublimation drying temperature -20°C to -30°C, vacuum degree 10-30Pa, 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 cutoff of 10-30 kDa is selected for ultrafiltration.

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

[0015] Another object of the present invention is to provide an application of hydrolyzed collagen, specifically: using the aforementioned hydrolyzed collagen as a raw material for preparing cosmetics; the dosage forms of the cosmetics include but are not limited to common aqueous solutions, emulsions, creams, and ointments.

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

[0017] The present invention has the following advantages over the prior art: the preparation method of hydrolyzed collagen prepared from bullfrog skin by using saliva, gastric juice, pancreatic juice and bile simulates the natural digestion process in the body, and the conditions are mild, just like performing "fine surgery" on collagen molecules, retaining biological activity to the greatest extent; multiple digestive juices work synergistically to accurately cut collagen molecular chains, generate fragments with reasonable structure and excellent activity, and make up for the limitations of single enzyme or a few enzymes in the enzymatic method. Abandon acid and alkali reagents, eliminate the risk of chemical residues, ensure product safety from the source, effectively reduce immunogenicity, and overcome the problem of acid and alkali method destroying structure and exposing antigenic epitopes. The reaction conditions are based on the stable physiological parameters of human digestive juices, and the temperature, pH value, etc. are easy to control to ensure the stability of product quality; in terms of cost control, it is expected to reduce dependence on expensive commercial enzymes, and because no acid and alkali wastewater is generated, it has significant environmental benefits and truly realizes the all-round upgrade of the preparation process. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0020] Figure 3 This is a diagram showing the effect of using Application Example 1 of the present invention;

[0021] Figure 4 This is a diagram showing the effect of using Application Example 2 of the present invention;

[0022] Figure 5 This is a diagram showing the effect of using Application Example 3 of the present invention;

[0023] Figure 6 It is a diagram showing the effect of application example 4 of the present invention. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with embodiments, but the scope of protection claimed by the present invention is not limited to the scope described in the embodiments.

[0025] Example 1

[0026] Prepare raw materials: Collect fresh bullfrog skin to ensure that there are no obvious lesions or damages. After removing surface impurities, rinse with physiological saline and cut into small pieces (about 0.5-1 cm in length) for later use. Collect fresh saliva, which can be secreted by stimulating healthy human bodies. Immediately after collection, store it in an ice bath to prevent enzyme inactivation. Filter to remove impurities before use. Obtain fresh pig gastric juice from the slaughterhouse. The pig's digestive system has certain similarities with bullfrogs, and the source is relatively stable. It is also stored in an ice bath and filtered before use. Pancreatic juice and bile are purchased from conventional biological reagent companies and stored according to the storage conditions in the instructions.

[0027] Start preparation: The preparation method of hydrolyzed collagen specifically comprises the following steps:

[0028] (1) Preliminary hydrolysis: Place the treated bullfrog skin pieces into a reaction vessel, add saliva at a ratio of 1:2 (1 gram of bullfrog skin: 2 ml of saliva), adjust the temperature of the reaction system to 36-37°C (close to the human oral temperature, providing a suitable active environment for enzymes such as salivary amylase), adjust the pH value to 6.5-7.0 (the suitable pH range for salivary amylase) with dilute hydrochloric acid or sodium hydroxide, and then stir at a low speed of 50-100 rpm for 2-3 hours to allow the enzymes in the saliva to initially decompose some polysaccharides and loose protein structures in the bullfrog skin;

[0029] (2) Gastric digestion simulation: After the saliva treatment is completed, the reaction system is heated to 38-40°C (close to the temperature in the pig stomach), and gastric juice is added at a ratio of 1:1.5 (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, and the stirring speed is increased to 150-200 rpm. The reaction is carried out for 4-6 hours to allow pepsin to fully hydrolyze the protein into polypeptide fragments;

[0030] (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 (a suitable environment for pancreatic juice and bile), and pancreatic juice and bile are added in a ratio of 1:1 between the mass of bullfrog skin and the volume of pancreatic juice and 1:0.5 between the mass of bullfrog skin and the volume of bile (1 gram of bullfrog skin, 1 ml of pancreatic juice, 0.5 ml of bile). The temperature is maintained at 37-38°C and the reaction is stirred at a speed of 200-250 rpm for 6-8 hours to allow the various enzymes in the pancreatic juice to further decompose polypeptides and fats, and the bile assists in emulsifying fats to promote overall digestion;

[0031] (4) Separation and purification: After the enzymatic hydrolysis reaction is completed, the reaction liquid is first filtered with a 100-150 mesh filter cloth to remove the undigested large pieces of bullfrog skin tissue residues, and the filtrate is collected; the ultrafiltration method is used for preliminary separation, and an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa is selected, and 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 (pH 4.5-5.5) is used as the balance solution and eluent, and the flow rate is controlled to be 1-2 mL / min, and the eluate is collected; finally, the eluate is dried using freeze-drying technology to obtain hydrolyzed collagen, wherein the freeze-drying procedure 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, and drying time 12-24 hours.

[0032] Comparative Example 1

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

[0034] Comparative Example 2

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

[0036] Comparative Example 3

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

[0038] Comparative Example 4

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

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

[0041] Test 1

[0042] The bioavailability test was performed by cell uptake experiment. The specific steps are as follows:

[0043] (1) Human skin fibroblasts were cultured to the logarithmic growth phase and digested with trypsin. 5×10 4 The cells were seeded in 24-well plates at a density of 10 cells / mL and cultured for 24 hours to allow them to adhere to the plate.

[0044] (2) An experimental group (adding the hydrolyzed collagen prepared in Example 1, prepared into a 1 mg / mL solution in serum-free culture medium, and added at 100 μL / well), a control group (adding an equal amount of hydrolyzed collagen prepared in Comparative Examples 1-4, at the same concentration) and a blank group (adding only serum-free culture medium) were set up respectively.

[0045] (5) After incubation for 4 hours, the culture medium was removed, the cells were gently washed three times with PBS buffer, 0.5 mL of trypsin was added to digest the cells, and the cell suspension was collected.

[0046] (4) The fluorescence intensity in the cells is detected by flow cytometry, and the collagen is pre-labeled with a fluorescent marker (such as FITC).

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

[0048] Test 2

[0049] The biological activity test was carried out through cell proliferation experiment. The specific steps are as follows:

[0050] (1) Referring to Test 1, human skin fibroblasts were cultured to the logarithmic growth phase with 1×10 4 The cells were seeded in 96-well plates at a density of 10 cells / mL and cultured for 24 hours.

[0051] (2) Experimental group, control group and blank group were set up, and the operation was the same as the cell uptake experiment in the bioavailability test, but the incubation time was extended to 72 hours.

[0052] (3) After the incubation, add 10 μL of MTT reagent to each well and continue incubation for 4 hours. Then carefully remove the supernatant and add 150 μL of DMSO to each well. Oscillate for 10 minutes to fully dissolve the crystals.

[0053] (4) Measure the absorbance at 570 nm using an enzyme-labeled instrument.

[0054] (5) Calculate the cell proliferation rate, cell 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.

[0055] Test 3

[0056] The low immunogenicity test was performed by lymphocyte proliferation experiment. The specific steps are as follows:

[0057] (1) Peripheral blood was collected from healthy subjects, lymphocytes were separated by density gradient centrifugation, and the cell density was adjusted to 1×10 6 Pieces / mL.

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

[0059] (3) The cell suspension was inoculated into a 96-well plate, 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 the cell proliferation experiment.

[0061] (5) Calculate the lymphocyte proliferation rate, 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 of hydrolyzed collagen obtained in Examples and Comparative Examples

[0063] Group Cellular uptake rate (%) Cell 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] It can be seen from Table 1 that the hydrolyzed collagen prepared in Example 1 of the present invention has better bioavailability, physiological activity and low immunogenicity than the hydrolyzed collagen prepared in Comparative Examples 1-4.

[0065] The hydrolyzed collagen obtained in Example 1 was sent to Shanghai Weipu Laboratory for typing and quantitative analysis (report number: SHA01-22031537-FX-01CnR2). The results are shown in Table 2 and Figure 1-2 shown.

[0066] Table 2 Hydrolyzed collagen composition

[0067] Components Mass content (%) CAS No. Total Protein 2.5 / Hyaluronic acid 2.4 9004-61-9 Total Sugar 0.4 / Peptides* margin / 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 1.4 / Calcium 0.3 / Silicon 0.2 /

[0068] Note: The ingredients marked with “*” in Table 2 are inferred from the fragment information and comprehensive experience.

[0069] In order to further expand the scope of application, the hydrolyzed collagen prepared above is made into a cosmetic with a matrix commonly used in the cosmetic field. The dosage forms of the cosmetic include but are not limited to common aqueous solutions, emulsions, creams, and ointments.

[0070] The present invention provides a collagen peptide repair lotion, which is composed of 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 lotion prepared above was used on a 6-year-old child who suffered from severe atopic dermatitis, dry and itchy skin, scratches all over the body, and could not sleep well at night. The lotion was applied thickly for 6 consecutive days. The results were as follows: Figure 3 As shown, the itching symptoms of the child's calves, thighs and buttocks are improved, the wounds are scabby, and the skin is restored to health. This application example shows that the collagen peptide repair lotion prepared by the present invention can effectively relieve atopic dermatitis, dry itching symptoms, and is conducive to wound healing.

[0073] Application Example 2

[0074] The collagen peptide repair lotion prepared above was used on a 25-year-old woman with sensitive skin. Her skin became red and hot due to sunburn caused by long-term outdoor sports. Figure 4 As shown, the redness, swelling, heat, pain and itching caused by sunburn can be relieved within one day. This application example shows that the collagen peptide repair lotion prepared by the present invention has the effect of sunburn repair.

[0075] Application Example 3

[0076] The collagen peptide repair lotion prepared above was used on a 46-year-old woman whose face was covered with rashes that were difficult to subside after the water laser treatment. The lotion was applied thickly for 1 day. The results were as follows: Figure 5 As shown, the rash caused by laser treatment gradually subsided within 1 day. This application example shows that the collagen peptide repair lotion prepared by the present invention has the effect of eliminating rash and fading redness.

[0077] Application Example 4

[0078] The collagen peptide repair lotion prepared above was given to a 5-year-old child. The child fell and got bruised while playing. After the wound was cleaned, the collagen peptide repair lotion prepared above was applied thickly. Figure 6 As shown, the wound was basically healed after 12 days. This application example shows that the collagen peptide repairing milk prepared by the present invention is beneficial to wound repair and healing.

[0079] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing hydrolyzed collagen, characterized in that: The preparation method comprises the following steps: (1) Preliminary hydrolysis: Place the processed bullfrog skin pieces into a reaction vessel, add saliva in a ratio of 1:2 between the mass of bullfrog skin and the volume of saliva, adjust the temperature of the reaction system 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 enzyme in the saliva to preliminarily decompose some 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 of bullfrog skin mass to gastric juice volume, 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, and pancreatic juice and bile are added in a ratio of 1:1 to pancreatic juice and 1:0.5 to bile juice. 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, and the bile assists in emulsifying fats to promote 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 is selected, and 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 to be 1-2 mL / min, and the eluate is collected; finally, the eluate is dried using freeze-drying technology to obtain hydrolyzed collagen.

2. The preparation method according to claim 1, characterized in that: The freeze-drying procedure is: pre-freezing temperature -40°C to -50°C, time 2-3 hours; sublimation drying temperature -20°C to -30°C, vacuum degree 10-30Pa, drying time 12-24 hours.

3. The preparation method according to claim 1, characterized in that: The pH of acetate buffer is 4.5-5.

5.

4. The preparation method according to claim 1, characterized in that: Select an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa for ultrafiltration.

5. Hydrolyzed collagen, characterized in that Prepared by the preparation method according to any one of claims 1 to 4.

6. The use of hydrolyzed collagen, characterized in that: The hydrolyzed collagen according to claim 5 is used as a raw material for preparing cosmetics.

7. The use of hydrolyzed collagen according to claim 6, characterized in that: The mass percentage of the hydrolyzed collagen added in the cosmetics is 1-30%.

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