Preparation process of animal collagen peptide for improving skin

Abstract

The invention relates to the technical field of protein peptide preparation, and discloses a preparation process of animal collagen peptide for improving skin, which comprises the following steps: S1, raw material pretreatment; s2, carrying out enzyme-electrolyte synergistic treatment; s3, in the hydrolysis process of intelligent pH control; s4, molecular sieve and ultrafiltration combined treatment; s5, efficient spray freeze drying; according to the preparation method provided by the invention, the introduction of weak electrolyte and the synergy of the enzymolysis process can be realized, the conversion efficiency of the collagen and the extraction rate of a target product are remarkably improved, the stability and the reuse times of the enzyme can be remarkably improved by immobilizing the enzyme on the carrier, the interaction among enzyme molecules can be reduced by virtue of immobilization, and the yield of the collagen is improved. The environment tolerance is improved.

Description

Technical Field

[0001] The invention relates to the field of protein peptide preparation, and more specifically, to a preparation process of animal collagen peptide for improving skin. Background Art

[0002] Currently, with the rapid growth of the market demand for beauty and health products, collagen has received great attention as an important functional food supplement and skin care ingredient. Fish collagen peptides are considered to have significant effects in skin improvement due to their low molecular weight and superior biological activity, especially due to their good water solubility and bioavailability.

[0003] Collagen is a major structural protein in the skin, responsible for maintaining the elasticity and strength of the skin. Fish collagen, especially fish skin, is rich in type I collagen, and its excellent biocompatibility makes it an ideal source of raw materials for a variety of biomedical products.

[0004] The current preparation process of fish collagen is relatively complicated and the preparation cost is high. Especially in the long-term production process, the activity of the enzyme will gradually decrease, affecting the hydrolysis efficiency and the consistency of the final product. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a process for preparing animal collagen peptides for improving skin.

[0006] The present invention provides a process for preparing animal collagen peptides for improving skin, comprising the following steps:

[0007] S1: Raw material pretreatment;

[0008] The raw materials were subjected to low temperature cleaning and ultrasonic treatment to remove lipid and non-collagen contamination;

[0009] S2: enzyme-electrolyte co-treatment;

[0010] Immobilized enzymes are used to enzymolyze the raw materials, and weak electrolytes are added for auxiliary treatment;

[0011] S3: during hydrolysis with intelligent pH control;

[0012] The enzymatic hydrolysis process is placed in an intelligent pH-controlled reactor equipped with a continuous monitoring system to maintain an acid-base environment with a pH of 6.5-7.5;

[0013] S4: molecular sieve combined with ultrafiltration treatment;

[0014] Use molecular sieves combined with ultrafiltration to purify the target molecular weight product and improve its biological activity;

[0015] S5: High-efficiency spray freeze drying;

[0016] The collagen peptide solution is processed by high-efficiency spray freeze drying to retain its biological activity and nutritional value and encapsulate the collagen peptide.

[0017] Preferably: in step S1, the raw materials are fish skin and fish scales rich in type I and type III collagen.

[0018] Preferably: in step S2, the weak electrolyte is a NaCl solution. The electrolyte enhances the affinity of the enzyme and the solubility of the collagen fibers by changing the surface charge distribution.

[0019] Preferably: in step S2, the immobilized enzyme is trypsin immobilized on a carrier, and the carrier is chitosan microbeads and / or mesoporous silica.

[0020] Preferably, the carrier is mesoporous silica, and the preparation process of the immobilized enzyme is as follows:

[0021] S21: carrier pretreatment;

[0022] The mesoporous silicon is cleaned with a hydrochloric acid solution to remove impurities and contaminants;

[0023] S22: Carrier surface functionalization:

[0024] The carrier is stirred in an APTES (3-aminopropyltriethoxysilane) anhydrous ethanol solution to achieve modified amination;

[0025] S23: Enzyme immobilization:

[0026] Trypsin is mixed with a carrier, and glutaraldehyde is used as a cross-linking agent to achieve covalent bonding between the enzyme and the carrier;

[0027] S24: Post-processing:

[0028] The support was washed with PBS (phosphate buffer) solution to remove unbound enzyme and obtain immobilized enzyme.

[0029] Preferably: in step S21, after removing impurities and pollutants, the mixture is neutralized with deionized water and then dried.

[0030] Preferably: in step S21, the pore size in the mesoporous silicon is 5-15 nanometers.

[0031] Preferably: in step S22, the stirring time is 6 hours.

[0032] Preferably, in step S23, trypsin is mixed with the carrier and reacted at 25° C. and pH 7.5 for 12 hours.

[0033] Preferably: in step S24, after the immobilized enzyme is obtained, it is refrigerated and stored for later use.

[0034] The beneficial effect of the present invention is that the animal collagen peptide preparation process for improving skin proposed in the present invention can achieve the synergy of the introduction of weak electrolytes and the enzymatic hydrolysis process, and significantly improve the conversion efficiency of collagen and the extraction rate of the target product.

[0035] By fixing the enzyme on a carrier, the stability and number of times the enzyme can be reused can be significantly improved. This immobilization can reduce the interaction between enzyme molecules and improve its tolerance to the environment.

[0036] Improved enzyme activity: Through covalent binding and appropriate pore size setting, the active site occlusion phenomenon is reduced and higher enzyme activity is maintained.

[0037] Enhanced mass transfer performance: The high specific surface area and pore structure of mesoporous silica are conducive to material transfer, ensuring effective contact between substrate and enzyme and increasing the reaction rate.

[0038] Enhanced carrier stability: Mesoporous silica has good mechanical strength and chemical stability, allowing the immobilized enzyme to retain good activity during multiple cycles of use.

[0039] Improved economic benefits: The high stability and reusability of immobilized enzymes reduce operating costs and improve enzyme utilization efficiency and industrial applicability. DETAILED DESCRIPTION

[0040] The subject matter described herein will now be discussed with reference to example embodiments. It should be understood that the discussion of these embodiments is only to enable those skilled in the art to better understand and implement the subject matter described herein, and the functions and arrangements of the elements discussed may be changed without departing from the scope of protection of the contents of this specification. Each example may omit, replace or add various processes or components as needed. In addition, the features described relative to some examples may also be combined in other examples.

[0041] Example 1

[0042] In this embodiment, a preparation process of animal collagen peptide for improving skin is proposed, which comprises the following steps:

[0043] S1: Raw material pretreatment;

[0044] The raw materials are fish skin and scales rich in type I and type III collagen, and are low-temperature cleaned and ultrasonically treated to remove lipid and non-collagen contamination.

[0045] S2: enzyme-electrolyte co-treatment;

[0046] Immobilized enzymes are used to enzymolyze the raw materials, and weak electrolytes are added for auxiliary treatment. The weak electrolyte is NaCl solution; the electrolyte enhances the affinity of the enzyme and the solubility of the collagen fibers by changing the surface charge distribution.

[0047] The immobilized enzyme is trypsin immobilized on a carrier, and the carrier may be chitosan microbeads or mesoporous silica or a combination of chitosan microbeads and mesoporous silica. In this embodiment, chitosan microbeads and mesoporous silica are preferred.

[0048] S3: during hydrolysis with intelligent pH control;

[0049] The enzymatic hydrolysis process is placed in an intelligent pH-controlled reactor equipped with a continuous monitoring system to maintain an acid-base environment with a pH of 6.5-7.5.

[0050] S4: molecular sieve combined with ultrafiltration treatment;

[0051] Molecular sieves combined with ultrafiltration are used to purify the target molecular weight product and improve its biological activity.

[0052] S5: High-efficiency spray freeze drying;

[0053] The collagen peptide solution is processed by high-efficiency spray freeze drying to retain its biological activity and nutritional value and encapsulate the collagen peptide.

[0054] Example 2

[0055] On the basis of Example 1, the carrier is mesoporous silica. In this example, a method for preparing an immobilized enzyme is proposed. The specific process is as follows:

[0056] S21: carrier pretreatment;

[0057] The mesoporous silicon is cleaned with hydrochloric acid solution, the pore size of the mesoporous silicon is 5-15 nanometers, impurities and pollutants are removed, and then neutralized with deionized water and dried. The pore size of the mesoporous silicon can be selected to be 5 nanometers, 10 nanometers or 15 nanometers, and 10 nanometers is preferred in this embodiment.

[0058] S22: Carrier surface functionalization:

[0059] The carrier was stirred in an APTES (3-aminopropyltriethoxysilane) anhydrous ethanol solution for 6 hours to achieve modified amination.

[0060] S23: Enzyme immobilization:

[0061] Trypsin was mixed with the carrier, and the mixture was reacted for 12 hours at 25°C and pH 7.5, and glutaraldehyde was used as a cross-linking agent to achieve covalent bonding between the enzyme and the carrier;

[0062] S24: Post-processing:

[0063] The carrier was washed with PBS (phosphate) buffer solution to remove unbound enzymes, thereby obtaining immobilized enzymes, which were then refrigerated and stored for later use.

[0064] Example 3

[0065] This example is used to verify that immobilized enzymes improve enzyme stability and reusability.

[0066] By fixing the enzyme on chitosan microbeads and mesoporous silica to form an immobilized enzyme system, the disordered collision and denaturation of the enzyme active site can be reduced, and the stability and reusability of the enzyme can be improved. This experiment aims to verify the effect of improving the stability of the enzyme and increasing the number of reuses after immobilization, using free enzyme as a control.

[0067] Experimental materials and equipment:

[0068] Enzyme: Trypsin. Carrier material: Chitosan microbeads, mesoporous silica. Reagents: Glutaraldehyde (for immobilization and cross-linking), buffer (PBS, pH 7.4). Others: Standard protein solution (substrate), dye reagent (for catalytic reaction analysis), UV-Vis spectrophotometer (for activity determination).

[0069] Experimental steps:

[0070] Step 1: Support pretreatment and enzyme immobilization

[0071] 1.1 Carrier processing:

[0072] The chitosan microbeads and mesoporous silica were washed to remove surface contaminants and dried at 60 °C.

[0073] 1.2 Surface functionalization (only for mesoporous silica):

[0074] The mesoporous silica was immersed in 3-aminopropyltriethoxysilane (APTES) ethanol solution for 6 hours, and then washed and dried.

[0075] 1.3 Enzyme immobilization:

[0076] The trypsin solution was mixed with the two carriers respectively, and the glutaraldehyde solution was added, and the mixture was gently stirred and reacted at 25° C. and pH 7.5 for 12 hours.

[0077] The immobilized enzyme particles were washed with PBS to remove free enzyme.

[0078] Step 2: Activity assay

[0079] 2.1 Initial activity determination:

[0080] The initial activities of free and immobilized enzymes were measured, and the enzyme activity (μmol / min / mg enzyme) was calculated by catalyzing the substrate to produce product.

[0081] 2.2 Stability test:

[0082] Long-term stability test: The immobilized and free enzymes were stored at 30°C under the same conditions, and the enzyme activity was measured every 6 hours for 48 hours.

[0083] Step 3: Repeat the experiment

[0084] 3.1 Cyclic use determination:

[0085] A series of hydrolysis reactions were performed and the activity decay of the enzyme was measured in multiple consecutive reactions (activity was measured after 10 repetitions).

[0086] Experimental results:

[0087] Table 1: Data on enzyme activity stability and repeated use under different conditions

[0088]

[0089] Table 2: Enzyme activity retention after repeated use

[0090]

[0091] Analysis Summary

[0092] Enzyme stability: In the 48-hour stability test, the activity of the free enzyme decreased rapidly, while the activity of the enzyme immobilized on mesoporous silica or chitosan was more stable and maintained a high activity level for a long time.

[0093] Reusability: The immobilized enzyme can still maintain more than 80% of its initial activity after repeated use, while the activity of the free enzyme only remains at 20%.

[0094] Conclusion: Immobilized enzyme effectively enhances the stability and reusability of trypsin, which can significantly reduce the enzyme dosage and cost in the actual production process and improve the economic benefits and efficiency of the industrial production line.

[0095] Example 4

[0096] By immobilizing trypsin on mesoporous silica materials, the activity and stability of the enzyme, the mass transfer efficiency and the reusability of the carrier can be significantly improved. The high specific surface area and adjustable pore size structure of mesoporous silica help optimize the catalytic performance and reaction kinetics of the enzyme. In this example, it is used to achieve the actual effect in the preparation process of fish collagen peptides as described above.

[0097] Experimental materials and equipment:

[0098] Enzyme: Trypsin. Carrier material: Mesoporous silica. Reagents: Glutaraldehyde (crosslinker), PBS buffer (pH 7.4). Others: Standard protein solution (substrate), spectrophotometer, stirring device.

[0099] 1. Experimental Procedure

[0100] Step 1: Immobilized enzyme preparation

[0101] Carrier pretreatment:

[0102] The mesoporous silicon was cleaned with 1 M HCl to remove impurities and then rinsed with deionized water and dried.

[0103] Surface functionalization:

[0104] The cleaned mesoporous silicon was immersed in an APTES anhydrous ethanol solution for 6 hours.

[0105] Wash and dry in preparation for enzyme immobilization.

[0106] Enzyme immobilization:

[0107] The trypsin solution was mixed with the functionalized mesoporous silica, and glutaraldehyde was added to react at room temperature for 12 hours.

[0108] Wash with PBS to remove unbound enzyme and store in refrigerator.

[0109] Step 2: Responsiveness Test

[0110] Initial activity detection:

[0111] The initial activity of free and immobilized enzymes was measured to determine the catalytic efficiency of the enzyme (μ mol / min / m g enzyme).

[0112] Mass transfer efficiency evaluation:

[0113] The immobilized enzyme was tested for changes in reaction rate after changes in substrate concentration and the substrate dissipation rate was measured.

[0114] Reusability:

[0115] The immobilized enzyme was used for 10 cycles and the activity change after each reaction was recorded.

[0116] 2. Experimental results

[0117] Table 1: Enzyme activity and mass transfer performance

[0118] Enzyme Profile Initial activity (μmol / min / mg) Reaction rate after substrate concentration changes (μmol / min / mg) Free enzyme 100 70 Immobilized enzyme 95 90

[0119] Table 2: Activity retention after repeated use

[0120] Cycle times Activity retention rate (%) immobilized enzyme 1 100 5 92 10 86

[0121] 3. Analysis and summary

[0122] Improved activity and stability: The catalytic activity of the mesoporous silica-immobilized enzyme was close to that of the free enzyme in initial tests, indicating effective activity retention.

[0123] Higher mass transfer efficiency: The effectiveness of mesoporous silica in optimizing mass transfer is demonstrated by the relatively faster substrate consumption rate.

[0124] Excellent reusability: The mesoporous silica-immobilized enzyme still retains 86% of its initial activity after 10 cycles of use, showing good structural strength and chemical durability.

[0125] These results show that the use of mesoporous silica as an immobilization carrier significantly enhances enzyme performance and is suitable for the industrial production of fish collagen peptides.

[0126] The above describes an embodiment of the present embodiment, but the present embodiment is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present embodiment, ordinary technicians in this field can also make many forms, all of which are within the protection of the present embodiment.

Claims

1. A process for preparing animal collagen peptide for improving skin, characterized in that: The steps include: S1: Raw material pretreatment; The raw materials were subjected to low temperature cleaning and ultrasonic treatment to remove lipid and non-collagen contamination; S2: enzyme-electrolyte co-treatment; Immobilized enzymes are used to enzymolyze the raw materials, and weak electrolytes are added for auxiliary treatment; S3: during hydrolysis with intelligent pH control; The enzymatic hydrolysis process is placed in an intelligent pH-controlled reactor equipped with a continuous monitoring system to maintain an acid-base environment with a pH of 6.5-7.5; S4: molecular sieve combined with ultrafiltration treatment; Use molecular sieves combined with ultrafiltration to purify the target molecular weight product and improve its biological activity; S5: High-efficiency spray freeze drying; The collagen peptide solution is processed by high-efficiency spray freeze drying to retain its biological activity and nutritional value and encapsulate the collagen peptide.

2. The process for preparing animal collagen peptide for improving skin according to claim 1, characterized in that: In step S1, the raw materials are fish skin and fish scales rich in type I and type III collagen.

3. The process for preparing animal collagen peptide for improving skin according to claim 1, characterized in that: In step S2, the weak electrolyte is a NaCl solution, which enhances the affinity of the enzyme and the solubility of the collagen fibers by changing the surface charge distribution.

4. The process for preparing animal collagen peptide for improving skin according to claim 1, characterized in that: In step S2, the immobilized enzyme is trypsin immobilized on a carrier, and the carrier is chitosan microbeads and / or mesoporous silica.

5. The process for preparing animal collagen peptide for improving skin according to claim 4, characterized in that: The carrier is mesoporous silica, and the preparation process of the immobilized enzyme is as follows: S21: carrier pretreatment; The mesoporous silicon is cleaned with a hydrochloric acid solution to remove impurities and contaminants; S22: Carrier surface functionalization: The carrier is stirred in an APTES (3-aminopropyltriethoxysilane) anhydrous ethanol solution to achieve modified amination; S23: Enzyme immobilization: Trypsin is mixed with a carrier, and glutaraldehyde is used as a cross-linking agent to achieve covalent bonding between the enzyme and the carrier; S24: Post-processing: The support was washed with PBS (phosphate buffer) solution to remove unbound enzyme and obtain immobilized enzyme.

6. The process for preparing animal collagen peptide for improving skin according to claim 5, characterized in that: In step S21, after removing impurities and pollutants, the product is neutralized with deionized water and then dried.

7. The process for preparing animal collagen peptide for improving skin according to claim 5, characterized in that: In step S21, the pore size in the mesoporous silica is 5-15 nanometers.

8. The process for preparing animal collagen peptide for improving skin according to claim 5, characterized in that: In step S22, the stirring time is 6 hours.

9. The process for preparing animal collagen peptide for improving skin according to claim 5, characterized in that: In step S23, trypsin and the carrier are mixed and reacted at 25° C. and pH 7.5 for 12 hours.

10. The process for preparing animal collagen peptide for improving skin according to claim 5, characterized in that: In step S24, after the immobilized enzyme is obtained, it is refrigerated and stored for later use.

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