A preparation method of a moisturizing antioxidant complex and its product and application

Abstract

The present invention relates to a preparation method of a moisturizing and antioxidant complex, and its products and applications. The preparation method comprises mixing silk colloid protein, budding short pedunculate polysaccharide, glycerol, complex enzyme and water, and then performing enzyme reaction to obtain an enzyme reaction mixture, heating to kill the enzyme, and centrifuging to obtain the supernatant to obtain the complex; the complex enzyme comprises a combination of microbial transglutaminase and laccase. Wherein, microbial transglutaminase and laccase are used in combination, and the two enzymes can interact with each other, positively affect each other, and fully improve the reaction efficiency. The prepared product is mild and non-irritating, has good stability, has excellent moisturizing and antioxidant effects and good film-forming properties, and can be directly or prepared into a composite film and applied to cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of cosmetics and relates to a preparation method of a moisturizing and antioxidant complex and a product and application thereof. Background Art

[0002] As the outermost protective layer of the human body, the skin is in direct contact with the external environment and is easily damaged and stimulated by ultraviolet rays, air pollution, etc. Long-term ultraviolet radiation will produce a large number of free radicals in the skin, which will oxidize the skin and damage the collagen in the skin, causing the skin to age and loosen. At the same time, dehydrated cells will become sunken, and dry and wrinkled skin will also cause cell aging and loosening.

[0003] There are two kinds of proteins in silk cocoon, namely sericin and fibroin. Sericin is located on the periphery of fibroin fibers. Sericin is a globulin composed of 18 kinds of amino acids, accounting for about 20% to 30% of the total weight of silk. When silkworms make cocoons, sericin plays an adhesive role, wrapping two monofilaments together to form cocoon silk. Sericin contains a large number of amino acids with hydrophilic groups on the side chains, such as serine and aspartic acid, which has multiple functions such as moisture absorption and moisturizing, antibacterial, antioxidant, promoting mineral deposition, promoting cell growth and anti-coagulation. Therefore, sericin has broad application prospects in food, cosmetics, biomedicine and other fields.

[0004] Budding Aureobasidium pullulans are extracellular polysaccharides produced by the fermentation of Aureobasidium pullulans. They have a variety of unique physical, chemical and biological properties, low viscosity, strong plasticity, good film-forming properties, and good water solubility, moisture absorption and moisturizing properties, as well as antibacterial and antibacterial functions. They can be used as additives for functional cosmetics to enhance the product's moisturizing, skin tone improvement, anti-aging and antibacterial effects, and can also remove excess heavy metal ions in the skin. Summary of the invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing a moisturizing antioxidant complex, a product thereof and applications thereof, and in particular to provide a method for preparing a moisturizing antioxidant complex, a product thereof and applications thereof in skin care products.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a moisturizing antioxidant complex, the preparation method comprising the following steps:

[0008] The silkworm sericin, budding pedunculate polysaccharide, glycerol, complex enzyme and water are mixed and subjected to enzyme reaction to obtain an enzyme reaction mixture, which is heated to inactivate the enzyme, and the supernatant is collected by centrifugation to obtain the complex.

[0009] In the present invention, sericin and budding short-stalk enzyme polysaccharide are molecularly cross-linked by biological enzymes to form a protein-polysaccharide macromolecular complex. The complex not only has the moisturizing and antioxidant effects of sericin, but also has good film-forming properties and will not cause adverse reactions to the skin.

[0010] The complex enzyme comprises a combination of microbial glutamine transaminase and laccase.

[0011] In the present invention, it is unexpectedly found that the combined use of microbial glutamine transaminase and laccase can synergistically enhance the efficiency, further improve the reaction cross-linking efficiency, and improve the related efficacy of subsequent products.

[0012] Preferably, the mass ratio of the microbial glutamine transaminase to laccase is (3-5):(1-3);

[0013] The above “3-5” may be, for example, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, etc.;

[0014] The above-mentioned "1-3" can be, for example, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0015] Preferably, the mass ratio of the silkworm sericin, budding pedunculate polysaccharide, glycerol and complex enzyme is (0.05-0.3):(1-5):(0.1-2):(0.1-1);

[0016] The above “0.05-0.3” may be, for example, 0.05, 0.08, 0.1, 0.12, 0.15, 0.18, 0.2, 0.22, 0.25, 0.28, 0.3, etc.;

[0017] The above-mentioned “1-5” may be, for example, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 3, 3.5, 4, 4.5, 5, etc.;

[0018] The above-mentioned “0.1-2” may be, for example, 0.1, 0.2, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, etc.;

[0019] The above-mentioned "0.1~1" can be, for example, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0020] In the present invention, when the raw material ratio is within the above specific ratio range, the product prepared therefrom has better moisturizing and anti-oxidation effects.

[0021] Preferably, the mass concentration of the silk fibroin in the enzyme reaction system is 0.05%-0.3%, for example, it can be 0.05%, 0.075%, 0.1%, 0.125%, 0.15%, 0.175%, 0.2%, 0.225%, 0.25%, 0.275%, 0.3%, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0022] In the present invention, when sericin with a specific mass concentration is used for the relevant enzymatic reaction, the obtained relevant composite product has more excellent moisturizing and anti-wrinkle effects.

[0023] Preferably, the temperature of the enzyme reaction is 40-65°C, for example, it can be 40°C, 42°C, 45°C, 48°C, 50°C, 52°C, 55°C, 58°C, 60°C, 62°C, 65°C, etc.; the time is 0.5-3h, for example, it can be 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, etc.; the pH is 5-8, for example, it can be 5, 5.5, 6, 6.2, 6.5, 6.8, 7, 7.2, 8, etc., and other specific point values ​​within this numerical range can be selected, which will not be repeated here.

[0024] In the present invention, the enzyme reaction is carried out under specific temperature, time and pH conditions, which can improve the efficiency of the enzymatic reaction, promote the cross-linking of silkworm sericin and budding short-stalk enzyme polysaccharide, and prepare a complex with better moisturizing and antioxidant effects and excellent film-forming properties.

[0025] Preferably, the temperature for heating to inactivate the enzyme is 70-100°C, for example, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, etc.; the time is 10-30min, for example, 10min, 15min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0026] Preferably, the centrifugal speed is 8000-12000rpm, for example, it can be 8000rpm, 8500rpm, 9000rpm, 9500rpm, 10000rpm, 10500rpm, 11000rpm, 11500rpm, 12000rpm, etc.; the time is 10-30min, for example, it can be 10min, 12min, 15min, 18min, 20min, 22min, 25min, 28min, 30min, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0027] In a second aspect, the present invention provides a moisturizing and antioxidant complex, wherein the complex is prepared by the preparation method described in the first aspect.

[0028] In a third aspect, the present invention provides a use of the moisturizing and antioxidant complex as described in the first aspect in a skin care product.

[0029] In a fourth aspect, the present invention provides a use of the moisturizing and antioxidant complex as described in the first aspect in preparing a composite film for cosmetics.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] In the present invention, the sericin protein and the budding short pedunculate polysaccharide are molecularly cross-linked by biological enzymes to form a protein-polysaccharide macromolecular complex, which not only has the moisturizing effect of sericin protein, but also has good film-forming properties and will not produce adverse reactions on the skin. In addition, it was unexpectedly found that the combination of microbial glutamine aminotransferase and laccase can synergistically enhance the efficiency of the reaction cross-linking, and enhance the moisturizing and antioxidant effects of the obtained product. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a picture of the moisturizing antioxidant complex prepared in Example 1;

[0033] Figure 2 This is a picture of an eye mask made from the moisturizing antioxidant complex prepared in Example 1. DETAILED DESCRIPTION

[0034] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0035] Preparation Example

[0036] Weigh 100 g of silk cocoons, cut them into 1-2 mm, wash them with clean water for 3 times, drain the water, add 10 L of water, put them in a sterilizer, heat them to 110 ° C and keep them warm for 60 min, cool the treated liquid to 25 ° C and filter it with gauze, collect the filtrate, sterilize it at 100 ° C for 20 min, and then concentrate it to 1 / 3 of the original volume through a 20000 Da membrane to obtain a silk colloid protein solution with a mass concentration of 0.75%, which is stored at 4 ° C for later use.

[0037] Example 1

[0038] This embodiment provides a moisturizing and antioxidant complex, and the preparation method thereof is as follows:

[0039] Take 30 parts of the sericin solution prepared in the preparation example, 3 parts of budding short-stalk enzyme polysaccharide, 1 part of glycerol, and 0.5 parts of complex enzyme (0.4 parts of microbial glutamine transaminase and 0.1 parts of laccase), add water to make up to 100 parts, mix thoroughly, adjust the pH to 6.0, and then stir the reaction at 55°C water bath and 150rpm for 2h, heat at 90°C for 20min to inactivate the enzyme in the system, and then centrifuge at 10000rpm for 20min, take the supernatant and freeze-dry to obtain the moisturizing antioxidant complex.

[0040] Example 2

[0041] This embodiment provides a moisturizing and antioxidant complex, and the preparation method thereof is as follows:

[0042] Take 7 parts of the sericin solution prepared in the preparation example, 1 part of budding short-stalk enzyme polysaccharide, 0.2 parts of glycerol, and 0.1 parts of complex enzyme (0.07 parts of microbial glutamine transaminase and 0.03 parts of laccase), add water to make up to 100 parts, mix thoroughly, adjust the pH to 5.0, and then stir the reaction at 45°C water bath and 150rpm for 1h, heat at 70°C for 30min to inactivate the enzymes in the system, and then centrifuge at 8000rpm for 30min, take the supernatant and freeze-dry to obtain the moisturizing antioxidant complex.

[0043] Example 3

[0044] This embodiment provides a moisturizing and antioxidant complex, and the preparation method thereof is as follows:

[0045] Take 40 parts of the sericin solution prepared in the preparation example, 5 parts of budding short-stalk enzyme polysaccharide, 2 parts of glycerol, and 1 part of complex enzyme (0.6 parts of microbial glutamine transaminase and 0.4 parts of laccase), add water to make up to 100 parts, mix thoroughly, adjust the pH to 8.0, and then stir the reaction for 3 hours in a 65°C water bath at 150rpm, heat at 100°C for 10 minutes to inactivate the enzyme in the system, and then centrifuge at 12000rpm for 10 minutes, take the supernatant and freeze-dry it to obtain the moisturizing antioxidant complex.

[0046] Example 4

[0047] This embodiment provides a moisturizing antioxidant complex, and the preparation method thereof is different from that of embodiment 2 only in that the sericin solution is 5 parts, the contents of the other components (except water) are the same as those of embodiment 2, and water is added to make up to 100 parts.

[0048] Example 5

[0049] This embodiment provides a moisturizing antioxidant complex, and the preparation method thereof is different from that of embodiment 3 only in that the sericin solution is 50 parts, and the other components (except water) are the same as those of embodiment 3, and water is added to 100 parts.

[0050] Comparative Example 1

[0051] This comparative example provides a moisturizing antioxidant complex, the preparation method of which is different from that of Example 1 in that the content of budding pullulanase polysaccharide is 0 parts, and the remaining components (except water) are the same as those of Example 1, and water is added to 100 parts.

[0052] Comparative Example 2

[0053] This comparative example provides a moisturizing antioxidant complex, and the difference between its preparation method and that of Example 1 is that the sericin solution is 0 parts, and the other components (except water) are the same as those of Example 1, and water is added to 100 parts.

[0054] Comparative Example 3

[0055] This comparative example provides a moisturizing antioxidant complex, and the difference between its preparation method and that of Example 1 is that glycerin is 0 parts, and the remaining components (except water) are the same as those of Example 1, and water is added to 100 parts.

[0056] Comparative Example 4

[0057] This comparative example provides a moisturizing antioxidant complex, the preparation method of which is different from that of Example 1 in that the complex enzyme in Example 1 is replaced by 0.5 parts of microbial glutamine transaminase, and the remaining components (except water) are the same as those in Example 1, and water is added to 100 parts.

[0058] Comparative Example 5

[0059] This comparative example provides a moisturizing antioxidant complex, the preparation method of which is different from that of Example 1 in that the complex enzyme in Example 1 is replaced by 0.5 parts of laccase, and the remaining components (except water) are the same as those in Example 1, and water is added to 100 parts.

[0060] Comparative Example 6

[0061] This comparative example provides a moisturizing antioxidant complex, the preparation method of which is different from that of Example 1 only in that it does not contain a complex enzyme, and the remaining components (except water) are the same as those of Example 1, and water is added to 100 parts.

[0062] Test Example 1

[0063] The film-forming solution prepared in Example 1 (solution before freeze-drying) was placed in an eye mask, and then placed in a constant temperature and humidity chamber at 37°C and 50% for 10 hours to obtain a sericin-budding stalk enzyme polysaccharide composite film, which has the following appearance: Figure 1 shown.

[0064] Test Example 2

[0065] This test example tests the moisturizing effect of the composites prepared in Examples 1-5 and Comparative Examples 1-6.

[0066] (1) Sample preparation

[0067] The products obtained in Examples 1-5, Comparative Example 2 and Comparative Examples 4-6 were prepared into aqueous solutions with a mass concentration of 5%; the product obtained in Comparative Example 1 was prepared into aqueous solutions with a mass concentration of 1.4% and the product obtained in Comparative Example 3 was prepared into aqueous solutions with a mass concentration of 3.3%, respectively, as samples to be tested.

[0068] Among them, since the sericin content in the freeze-dried powder prepared in Comparative Examples 1 and 3 is much higher than that in other embodiments and comparative examples, in order to maintain the sericin content in the test samples of other embodiments and comparative examples within a relatively low difference range, the sample concentrations of the samples to be tested configured here are different.

[0069] (2) 72 healthy Chinese subjects (15 males and 57 females) aged 24 to 40 years old who met the voluntary inclusion and exclusion criteria were selected and divided into 12 groups, with 6 subjects in each group.

[0070] (3) After cleansing and using toner in the morning and evening, the subjects took an appropriate amount of the complex solution and evenly applied it to the face. Massage gently until absorbed. Use twice a day, once in the morning and once in the evening, for 28 consecutive days.

[0071] (4) The relative moisture content of the skin surface was detected using a skin moisture content tester. The relative moisture content of the skin surface was recorded before using the sample, 14 days after using the sample, and 28 days after using the sample. The change rate of the relative moisture content of the skin surface relative to that before using the sample was calculated. At the same time, a group that only used lotion after cleansing was set as a blank control group. The cleansers and lotions used in each group were the same commercially available products. The results are shown in Table 1.

[0072] Table 1

[0073]

[0074]

[0075] From the data in the table, we can see that:

[0076] (1) It can be seen from Examples 1 to 3 that the moisturizing and antioxidant complex provided by the present invention has an excellent moisturizing effect and can significantly increase the moisture content of the skin after use.

[0077] (2) By comparing Examples 2-3 with Examples 4-5, it can be seen that when the sericin content in Examples 4-5 is too high or too low, the moisturizing effect of the composite product prepared therefrom is worse than that of Examples 2-3. It can be seen that the present invention can further improve the moisturizing effect of the obtained product by using a specific amount and concentration of sericin.

[0078] (3) By comparing Example 1 with Comparative Examples 1-3, it can be seen that when the components do not contain any of sericin, Pullulan or glycerol, the moisturizing effect of the prepared composite is worse than that of Example 1. It can be seen that the addition of sericin, Pullulan or glycerol has an important influence on the moisturizing effect of the obtained product.

[0079] (4) By comparing Example 1 with Comparative Examples 4-6, it can be seen that when the composite enzymes used in Comparative Examples 4-6 do not contain any one or both of microbial glutamine transaminase or laccase, the moisturizing effect of the composite products prepared therefrom is reduced compared with that of Example 1. It can be seen that the composite enzyme composed of microbial glutamine transaminase and laccase in the present invention can effectively promote the reaction efficiency between sericin and budding pullulanase polysaccharide, and improve the moisturizing effect of the prepared product.

[0080] Test Example 3

[0081] This test example tests the antioxidant effect of the composites prepared in Examples 1-5 and Comparative Examples 1-6.

[0082] (1) Sample preparation: The products prepared in Examples 1-5, Comparative Example 2 and Comparative Examples 4-6 were prepared into aqueous solutions with a mass concentration of 1%; the product prepared in Comparative Example 1 was prepared into aqueous solutions with a mass concentration of 0.25% and the product prepared in Comparative Example 3 was prepared into aqueous solutions with a mass concentration of 0.67%, respectively, and these solutions were used as samples to be tested. A 0.5 g / mL aqueous solution of sericin was used as a control group.

[0083] (2) Add 2.0 mL of 30 μmol / L DPPH test solution and 2.0 mL of the sample to be tested to the test tube, shake well, and react for 30 min in the dark. Adjust the concentration to zero with anhydrous ethanol and measure the absorbance at 517 nm as A. i ; In the same way, take 2.0mL of 30μmol / / L DPPH test solution and 2.0mL of anhydrous ethanol, shake well, react in the dark for 30 minutes, and measure the absorbance as A0; then take 2mL of anhydrous ethanol and 2.0mL of the sample to be tested, shake well, react in the dark for 30 minutes, and measure the absorbance as A j , and its free radical scavenging rate was calculated according to the following formula.

[0084] Clearance rate (%) = [1-(A i -A j ) / A0]×100%;

[0085] Among them, A j The contribution of the reaction sample itself to the absorbance; A i The absorbance value of the sample after the action of DPPH; A0 is the absorption of DPPH itself at the measurement wavelength.

[0086] The greater the DPPH scavenging rate, the stronger the free radical scavenging antioxidant ability. The results are shown in Table 3 (each group of samples was measured 5 times in parallel, and the data are presented as average values).

[0087] Table 3

[0088]

[0089]

[0090] From the data in the table, we can see that:

[0091] (1) It can be seen from Examples 1 to 3 that the moisturizing and antioxidant complex provided by the present invention has a significant DPPH free radical scavenging effect.

[0092] (2) By comparing Example 1 with Examples 4-5, it can be seen that when the amount of sericin used in the examples is too high or too low, the DPPH free radical scavenging ability of the related products prepared therefrom is not as good as that of Example 1.

[0093] (3) By comparing Example 1 with Comparative Examples 1-3, it can be seen that when any one of the three components of sericin, budding pullulan polysaccharide, and glycerol is missing in the examples, the antioxidant capacity of the obtained products is not as good as that of Example 1.

[0094] (4) By comparing Example 1 with Comparative Examples 4-6, it can be seen that when the composite enzymes used in Comparative Examples 4-6 do not contain any one or both of microbial glutamine transaminase and laccase, the anti-wrinkle effects of the composite products prepared are all lower than those of Example 1. It can be seen that the two enzymes of microbial glutamine transaminase and laccase used in the present invention can synergistically enhance the efficiency of the reaction between silk colloid protein and budding pedunculate polysaccharide, and improve the relevant efficacy of the prepared product.

[0095] Test Example 4

[0096] This test example tests the product safety of the composites prepared in Examples 1-5.

[0097] The products prepared in Examples 1-5 were prepared into samples with a concentration of 1% for later use. According to the inclusion criteria for the subjects, 35 women and 15 men were selected to participate in the test, and randomly divided into 5 groups (3 men and 7 women in each group) to receive the trial. 0.025 mL of the sample was taken with a syringe and placed in the chamber of the spot test tape. The spot tester with the sample was applied to the skin of the curved side of the forearm of the subject with medical tape for 24 hours. After removing the test spot tester for 30 minutes, observe the skin reaction after the indentation disappears. For those with negative results, observe again 24 hours and 48 hours after the test, and record the skin reaction results.

[0098] The results show that the products prepared in Examples 1-5 showed negative reactions 24h and 48h after the test, and there was no irritation or erythema. It can be seen that the moisturizing antioxidant complex product prepared by the present invention has no irritation to the skin and no positive reaction, which proves that the product is highly safe, less irritating, and milder.

[0099] In summary, the present invention uses biological enzymes to cross-link sericin with budding short pedunculate polysaccharide to form a protein-polysaccharide macromolecular complex, which not only has the moisturizing and antioxidant effects of sericin, but also has good film-forming properties and will not produce adverse reactions on the skin. In addition, it was unexpectedly discovered that the combination of microbial glutamine aminotransferase and laccase can synergistically enhance the reaction cross-linking efficiency and enhance the moisturizing and antioxidant effects of the product.

[0100] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0101] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0102] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a moisturizing antioxidant complex, characterized in that: The preparation method comprises the following steps: mixing silkworm sericin, budding short-stalk enzyme polysaccharide, glycerol, complex enzyme and water, and performing enzyme reaction to obtain an enzyme reaction mixture, heating to inactivate the enzyme, and centrifuging to obtain a supernatant to obtain the complex; The complex enzyme comprises a combination of microbial glutamine transaminase and laccase.

2. The preparation method according to claim 1, characterized in that The mass ratio of the microbial glutamine transaminase to laccase is (3-5):(1-3).

3. The preparation method according to claim 1, characterized in that: The mass ratio of the silkworm sericin, budding pullulanase polysaccharide, glycerol and complex enzyme is (0.05-0.3):(1-5):(0.1-2):(0.1-1).

4. The preparation method according to claim 1, characterized in that: The mass concentration of the silk fibroin in the enzyme reaction system is 0.05%-0.3%.

5. The preparation method according to claim 1, characterized in that: The temperature of the enzyme reaction is 40-65° C., the time is 0.5-3 h, and the pH is 5-8.

6. The preparation method according to claim 1, characterized in that: The temperature of the heating to inactivate the enzyme is 70-100° C. and the time is 10-30 minutes.

7. The preparation method according to claim 1, characterized in that: The centrifugal speed is 8000-12000 rpm, and the time is 10-30 min.

8. A moisturizing and antioxidant complex, characterized in that: The composite is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the moisturizing and antioxidant complex according to claim 8 in skin care products.

10. Use of the moisturizing and antioxidant complex according to claim 8 in preparing a composite film for cosmetics.

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