High-activity SOD enzyme compound raw material, its preparation method and application

Abstract

The present application provides a high-activity SOD enzyme compound raw material, its preparation method and application in the preparation of cosmetics. The compound raw material of the present application is obtained by preparing SOD enzyme, sugar and optionally zinc aspartate, providing good stability at normal temperature and high temperature, and significantly reducing the requirements for the transportation, storage and use conditions of SOD enzyme, a commonly used raw material in cosmetics.

Description

Technical Field

[0001] This application belongs to the fields of cosmetics and protein preparations. Specifically, this application provides a high-activity SOD enzyme compound raw material, its preparation method, and application. Background Art

[0002] Superoxide dismutase (SOD) is widely distributed in animals, plants, and microorganisms. It is the most important component of the biological antioxidant enzyme system, catalyzing the dismutation reaction of superoxide anion radicals to scavenge superoxide anions, and thus playing a role in anti-DNA damage and lipid peroxidation.

[0003] Superoxide dismutase has a wide range of applications in drugs, health products, and cosmetics. In actual production and application, whether obtained by extraction from animals or plants or by recombinant expression, SOD faces serious inactivation problems during raw material storage or product storage (factors such as temperature, pH, and salt can cause its instability). Although its stability can be enhanced by means such as stabilizers and low temperature, these methods have deficiencies in terms of safety and subsequent processability, or have high requirements for storage conditions.

[0004] Therefore, there is a need in the art for a high-activity and high-stability SOD enzyme compound raw material. Summary of the Invention

[0005] To solve the above problems, on the one hand, this application provides a high-activity SOD enzyme compound raw material, which is obtained by SOD enzyme, sugar, and optionally zinc aspartate.

[0006] Further, the SOD enzyme is Rosa roxburghii Tratt SOD enzyme or bovine blood SOD enzyme.

[0007] Further, the sugar is glucose, sucrose, or raffinose.

[0008] Further, the preparation method of the SOD enzyme compound raw material includes:

[0009] (1) Prepare an aqueous solution containing 500 - 1000 mg / L of SOD enzyme and 10 - 30 mg / L of sugar;

[0010] (2) Treat the aqueous solution in a water bath at 55 - 70 °C for 60 - 100 minutes, and then treat it in a water bath at 90 - 100 °C for 30 - 45 minutes.

[0011] Further, the preparation method of the SOD enzyme compound raw material includes:

[0012] (1) preparing an aqueous solution containing 750-850 mg / L of roxburghii SOD enzyme, 20-25 mg / L of raffinose, and 80-100 mg / L of zinc aspartate;

[0013] (2) Treat the aqueous solution in a 60-65°C water bath for 80-100 minutes, and then in a 90-95°C water bath for 30-40 minutes.

[0014] Furthermore, the preparation method of the SOD enzyme compound raw material comprises:

[0015] (1) preparing an aqueous solution containing 750-850 mg / L of bovine blood SOD enzyme, 20-25 mg / L of raffinose, and 80-100 mg / L of zinc aspartate;

[0016] (2) Treat the aqueous solution in a 60-65°C water bath for 80-100 minutes, and then in a 90-95°C water bath for 30-40 minutes.

[0017] Furthermore, the SOD enzyme compound raw material is in liquid or solid form.

[0018] According to the needs of storage and application, the SOD enzyme compound raw material of the present application can be stored and used in liquid form with or without appropriate concentration; or it can be prepared into solid form through appropriate concentration and drying methods for storage and application. Excipients and fillers such as maltodextrin can be added during drying. The loss of enzyme activity that may be caused by drying can also be anticipated by technical personnel in this field and avoided as much as possible.

[0019] Furthermore, the method for preparing the SOD enzyme compound raw material also includes a freeze-drying or spray-drying step.

[0020] On the other hand, the present application provides the use of the above-mentioned SOD enzyme compound raw material in the preparation of cosmetics.

[0021] Furthermore, the cosmetics are cosmetics with anti-oxidation function.

[0022] In the present application, SOD, SOD enzyme, and superoxide dismutase can be used interchangeably to represent the same meaning. Various plant, animal, or recombinantly expressed SODs can be used in the present application.

[0023] On the other hand, the present application provides cosmetics comprising the above-mentioned SOD enzyme compound raw material.

[0024] The above-mentioned cosmetics can be in the form of essence, toner, cream, facial mask, facial cleanser, etc.

[0025] In this application, stable SOD nanoparticles are formed through the Maillard reaction of SOD enzyme, sugar, and zinc aspartate in appropriate proportions, which can effectively extend their storage time at high and normal temperatures. Raffinose and zinc aspartate are both ingredients approved for use in cosmetics and have no obvious adverse effects on subsequent processing and product performance. Description of the Drawings

[0026] Figure 1 High-temperature stability of Maillard reaction products of SOD and different sugars;

[0027] Figure 2 Normal-temperature stability of Maillard reaction products of SOD and different sugars;

[0028] Figure 3 High-temperature stability of Maillard reaction products of SOD, different sugars, and zinc aspartate;

[0029] Figure 4 Normal-temperature stability of Maillard reaction products of SOD, different sugars, and zinc aspartate;

[0030] Figure 5 Zeta potential change during normal-temperature storage of the product of the preferred technical solution. Detailed Description of the Invention

[0031] Main Materials and Methods

[0032] Main Raw Materials:

[0033] SOD: In the experimental part, self-made SOD from Rosa roxburghii Tratt was used. The basic steps were as follows: Wash the Rosa roxburghii Tratt blocks in ice water; Juice, filter, and centrifuge with a spiral low-speed juicer to obtain Rosa roxburghii Tratt juice; Ultrafilter with a 50 kDa ultrafiltration membrane and concentrate by reverse osmosis; Spray-dry to obtain a powdered solid SOD product; Detect multiple batches, and the SOD enzyme activity was at the level of 26,000 - 27,000 U / g. Subsequently, commercially available SOD from bovine blood was used for verification, with a nominal SOD enzyme activity of 30,000 U / g, and the actual detection was basically in line.

[0034] Zinc glycinate, zinc aspartate, glucose, galactose, sucrose, fructose, gentiobiose, and raffinose are all of food / cosmetic grade: purchased from Shandong Aicai Biotechnology Co., Ltd., Guangdong Huayu Biotechnology Co., Ltd., Shanghai Yuanye Biotechnology Co., Ltd., etc.

[0035] Method for Measuring SOD Enzyme Activity:

[0036] For the detection of a large number of samples during formula screening, a commercially available WST-1 method SOD activity detection kit (Saichuang, CLS-A001-3-96T) was selected, and the detection steps were carried out according to the instructions.

[0037] Particle Size and Zeta Potential Analysis:

[0038] Detection using NANOTRAC WAVE II of Microtrac MRB.

[0039] Example 1 Maillard reaction of sugar and SOD and its effects

[0040] The applicant attempted to use different sugars to produce Maillard reaction with SOD to improve the stability of SOD:

[0041] Prepare an aqueous solution containing 800 mg / L SOD and 15 mg / L monosaccharide or 20 mg / L disaccharide / trisaccharide; treat the aqueous solution in a water bath at 65 °C for 90 minutes, and then treat it in a water bath at 90 °C for 30 minutes. The SOD activities of the initial solution and the obtained product solution are shown in Table 1:

[0042] Table 1 Changes in SOD activity of the solution before and after Maillard reaction

[0043]

[0044] Fructose <![CDATA[2.08×10 4 > <![CDATA[1.13±0.07×10 3 > Gentiobiose <![CDATA[2.08×10 4 > <![CDATA[0.85±0.02×10 3 > Raffinose <![CDATA[2.08×10 4 > <![CDATA[2.76±0.05×10 3 >

[0045] Detect the particle size and Zeta potential of each product solution, and the results are shown in Table 2

[0046] Table 2 Particle size and Zeta potential of Maillard reaction products

[0047] Sugar type Average particle size (nm) Zeta potential (mV) Glucose 168.22±1.12 -18.98±0.28 Galactose N.D. N.D. Sucrose 173.29±0.53 -19.27±0.15 Fructose N.D. N.D. Gentiobiose N.D. N.D. Raffinose 184.46±0.87 -21.15±0.32

[0048] The results show that after Maillard reaction of various oligosaccharides and SOD, the SOD activity loses more than 90%; after Maillard reaction of glucose, sucrose, and raffinose, nanoparticles are formed and the SOD activity remains slightly better; after Maillard reaction of galactose, fructose, and gentiobiose, no nanoparticles are formed (accurate and stable particle size cannot be measured), and the SOD activity remains worse than that of glucose, sucrose, and raffinose.

[0049] After ultraviolet sterilization packaging of the reaction product and SOD solution (100 mg / L), place them at 25 °C and in a water bath at 90 °C, and observe the percentage of remaining enzyme activity after a certain period of time. The results are as Figure 1 and Figure 2 shown.

[0050] The results show that at high temperature and normal temperature, the Maillard reaction products forming nanoparticles have a better activity preservation rate than untreated SOD and SOD treated with other sugars.

[0051] Example 2 Further introduction of zinc aspartate

[0052] Although Example 1 demonstrated that the Maillard reaction of adding glucose, sucrose, and raffinose formed SOD-sugar nanoparticles and brought good stability, as shown in Table 1, most of the SOD activity was lost during the Maillard reaction (about 85% was lost even for raffinose with the best effect). The activity of the final SOD product can be adjusted by appropriate concentration and other methods, but on the one hand, the large loss of activity will lead to the waste of relatively expensive raw materials, and on the other hand, denatured proteins and their Maillard reaction products may also have potential impacts on the stability and even safety of the product.

[0053] The applicant tried to add buffer solutions, supplement copper ions, zinc ions, etc. to protect the SOD activity during the Maillard reaction, but the effects were not ideal: the addition of the buffer system and copper ions basically could not protect the SOD activity, and the addition of 1 mM zinc ions seemed to have a little protective effect on the SOD activity (the inactivation rate could be slightly lower than 85%), but the Zeta potential of the product system was only about 13 mV, and the stability was significantly reduced.

[0054] When the applicant compounded the cosmetic raw materials containing SOD, it was found that adding a certain amount of zinc aspartate (aspartic acid and zinc ions are helpful for skin nutrition and antioxidant) to the solution before the Maillard reaction not only did not affect the formation of nanoparticles, but also brought further positive changes to the stability of the product:

[0055] Prepare an aqueous solution containing 800 mg / L SOD, 15 mg / L glucose or 20 mg / L sucrose / raffinose, and 100 mg / L zinc aspartate; treat the aqueous solution in a water bath at 65 °C for 90 minutes, and then treat it in a water bath at 90 °C for 30 minutes. The SOD activities of the initial solution and the obtained product solution are shown in Table 2, and the particle sizes and Zeta potentials of each product solution are detected, and the results are shown in Table 2:

[0056] Table 3 Changes in SOD activity of the solution before and after the Maillard reaction

[0057]

[0058] Table 4 Particle size and Zeta potential of the Maillard reaction product

[0059] Sugar type Average particle size (nm) Zeta potential (mV) Glucose 173.22±0.72 -19.78±0.13 Sucrose 185.29±0.29 -21.48±0.25 Raffinose 203.31±0.44 -28.35±0.27

[0060] The results showed that adding zinc aspartate during the Maillard reaction significantly increased the proportion of the remaining SOD activity after the Maillard reaction, especially more significantly in the case of raffinose, and the SOD activity retention rate was close to 50%, and the feasibility in production was significantly improved. In terms of the product, the particle size of the product increased slightly, and the absolute value of the Zeta potential also approached the stable limit of 30 mV.

[0061] Prepare the SOD enzyme compounding raw materials according to the preferred method: prepare an aqueous solution containing 800 mg / L SOD, 20 mg / L raffinose, and 100 mg / L zinc aspartate; treat the aqueous solution in a water bath at 65 °C for 90 minutes, and then treat it in a water bath at 90 °C for 30 minutes. As Figure 5 shown, when the product is stored at room temperature for 60 days, the zeta potential and particle size change little and remain basically stable.

[0062] Verification of Example 3 on other SODs

[0063] Verify the method of Example 2 using commercially available bovine blood-derived SOD. The results of the single-factor experiment show that except for slightly different dosages of suitable sugar and zinc aspartate (the single-factor experiment results show that the most suitable solution contains 800 mg / L SOD, 25 mg / L raffinose, and 80 mg / L zinc aspartate), its activity remains above 83% after being treated at 90 °C for 90 minutes, and can maintain about 90% of its activity when stored at room temperature for 60 days.

Claims

1. A high-activity SOD enzyme compounding raw material, characterized in that, The preparation method of the SOD enzyme compound raw material includes: (1) Prepare an aqueous solution containing 750 - 850 mg / L of SOD enzyme from Rosa roxburghii Tratt, 20 - 25 mg / L of raffinose, and 80 - 100 mg / L of zinc aspartate; (2) Treat the aqueous solution obtained in step (1) in a water bath at 60 - 65 °C for 80 - 100 minutes, and then treat it in a water bath at 90 - 95 °C for 30 - 40 minutes.

2. The SOD enzyme compound raw material according to claim 1, wherein the SOD enzyme compound raw material is in liquid or solid form.

3. The preparation method of the SOD enzyme compound raw material according to claim 1 further includes a freeze-drying or spray-drying step.

4. A high-activity SOD enzyme compound raw material, characterized in that, The preparation method of the SOD enzyme compound raw material includes: (1) Prepare an aqueous solution containing 750 - 850 mg / L of SOD enzyme from bovine blood, 20 - 25 mg / L of raffinose, and 80 - 100 mg / L of zinc aspartate; (2) Treat the aqueous solution obtained in step (1) in a water bath at 60 - 65 °C for 80 - 100 minutes, and then treat it in a water bath at 90 - 95 °C for 30 - 40 minutes.

5. The SOD enzyme compound raw material according to claim 4, wherein the SOD enzyme compound raw material is in liquid or solid form.

6. The preparation method of the SOD enzyme compound raw material according to claim 4 further includes a freeze-drying or spray-drying step.

7. Use of the SOD enzyme compound raw material according to any one of claims 1 - 6 in the preparation of cosmetics.

8. The use according to claim 7, wherein the cosmetics are cosmetics with antioxidant function.

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