Preparation method and application of bromelain composition

Abstract

The invention provides a preparation method and application of a bromelain composition, and belongs to the technical field of biology. The composition contains bromelain, superoxide dismutase, amino acid and compound polysaccharide extracted from aloe, sturgeon collagen peptide and witch hazel extract. The aloe amino acid polysaccharide, the sturgeon collagen peptide and the witch hazel extract are high-efficacy products obtained by purification after special enzymolysis or fermentation. All the components are matched for use, dead skin cells can be removed, wrinkles can be removed, the skin can be kept tight and elastic, the whitening and anti-wrinkle effects are good, the number of the components is small, absorption burden cannot be caused to the skin, and safety and high efficiency are achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a preparation method and application of a bromelain composition. Background Art

[0002] In today's era of pursuit of beauty, consumers' demands for the efficacy of cosmetics are becoming increasingly diverse and demanding. Removing dead skin cells, reducing wrinkles, tightening the skin and improving elasticity, as well as achieving whitening and anti-wrinkle have become core demands. Although the traditional physical scrub exfoliation method can remove dead skin, it is easy to damage the skin barrier. New enzyme technologies have emerged. For example, proteases extracted from plants can accurately decompose keratin in the aging stratum corneum on the surface of the skin, gently remove dead skin cells, and do not destroy the normal tissue structure of the skin, laying a good foundation for subsequent skin care effects. In terms of anti-wrinkle and firming, peptide ingredients have become a research hotspot. Peptides can block the release of neurotransmitters, relax muscles, and reduce the formation of expression lines; at the same time, they can also activate signal pathways in cells, promote the synthesis of collagen and elastin, enhance the firmness and elasticity of the skin, and fundamentally improve the sagging of the skin. For whitening needs, researchers have turned their attention to a variety of natural and synthetic active ingredients. Various plant extracts have the effect of inhibiting tyrosinase activity, reducing the production of melanin from the source, and achieving whitening effects.

[0003] At present, cosmetics with various functions have various ingredients and their effects vary. Too many ingredients will cause skin absorption disorder, excessive nutrition, and easily lead to allergic side effects; while too few ingredients will not have obvious effects and cannot meet the use requirements of consumers. Therefore, it is necessary to develop effective ingredients that are both safe and efficient, give full play to the synergistic effect, bring consumers a more significant and lasting skin care experience, meet their needs for removing dead skin, firming and anti-wrinkle, whitening, etc., and reduce the burden of skin absorption. Summary of the invention

[0004] The existing cosmetics have various ingredients and uneven effects. Too many types of ingredients will increase the absorption burden on the skin, while too few types of ingredients will result in unclear skin care effects. The present invention provides a preparation method and application of a bromelain composition, the composition contains specific ingredients of bromelain, superoxide dismutase, aloe amino acid polysaccharide, sturgeon collagen peptide and witch hazel extract, wherein the sturgeon collagen peptide and witch hazel extract are obtained by special enzymolysis and purification, and the combination of the ingredients has good dead skin removal, whitening and anti-wrinkle effects, and the number of ingredients is small, which will not cause absorption burden on the skin, and is safe and efficient. The specific technical scheme is as follows: A method for preparing a bromelain composition comprises the following steps: S1, extraction of aloe amino acid polysaccharide: comprising peeling the aloe and beating the pulp, adding water to homogenize the pulp, enzymolyzing the aloe with cellulase and pectinase, and then enzymolyzing the aloe with trypsin, centrifuging the hydrolyzate to collect supernatant A and precipitate A; inoculating the precipitate A with lactic acid bacteria and yeast for fermentation, centrifuging the fermentation product, collecting supernatant B, adding ethanol for precipitation, and collecting the precipitate to obtain precipitate B; mixing the supernatant A and the precipitate B, and then concentrating and drying to obtain an extract containing amino acids and complex polysaccharides, namely, aloe amino acid polysaccharide; S2, preparation of sturgeon collagen peptide: comprising beating the fish skin, adding water to mix, adding trypsin for enzymatic hydrolysis, and then co-enzymatic hydrolysis with serrapeptase and bromelain, centrifuging the enzymatic hydrolyzate, collecting the supernatant C and ultrafiltration with an ultrafiltration membrane with a molecular weight of 4KDa to 6KDa, taking the ultrafiltrate for resin column adsorption, and then eluting, ultrafiltration with an ultrafiltration membrane with a molecular weight of 0.5KDa to 1.5KDa, selecting the ultrafiltration retentate, and drying to obtain sturgeon collagen peptide; S3, preparation of witch hazel extract: comprising crushing witch hazel, adding water to mix, adding bromelain for enzymolysis, then adding nattokinase for enzymolysis, centrifuging the enzymolysis product to collect supernatant D, ultrafiltering the supernatant D using an ultrafiltration membrane with a molecular weight of 4KDa to 6KDa, taking the ultrafiltrate for resin column adsorption, and then eluting, ultrafiltering the eluate using an ultrafiltration membrane with a molecular weight of 0.5KDa to 1.5KDa, selecting the ultrafiltration retentate, and drying to obtain witch hazel extract; S4, mixing: according to the mass ratio of bromelain:superoxide dismutase:aloe amino acid polysaccharide:sturgeon collagen peptide:witch hazel extract = (0.3-0.5):(0.3-0.5):(1-3):(3-5):(3-5), bromelain, superoxide dismutase, aloe amino acid polysaccharide, sturgeon collagen peptide and witch hazel extract are mixed to obtain a composition.

[0005] In S1 of the above preparation method, the extraction of aloe amino acid polysaccharide comprises: The peeled aloe leaf pulp is beaten to obtain a slurry, and distilled water of 8 to 15 times the mass of the slurry is added to homogenize; the pH value is adjusted to 4.5 to 5.5, and 0.5% to 1.5% of cellulase and 0.3% to 1.0% of pectinase are added to the slurry by mass, and after mixing, enzymolysis is performed at 40°C to 50°C for 2h to 4h, and then the mixture is cooled to room temperature, and the pH value is adjusted to 8.0 to 8.5, and 0.5% to 1.5% of trypsin by mass of the slurry is added to perform enzymolysis at 35°C to 38°C for 2h to 4h, and the enzyme is inactivated in a boiling water bath, and after cooling to room temperature, the mixture is centrifuged to collect supernatant A and precipitate A; precipitate A is added to precipitate A. 3 to 5 times the mass of distilled water are inoculated with 3% to 5% of lactic acid bacteria and 1% to 3% of yeast of the mass of precipitate A, and anaerobically fermented at 30°C to 35°C for 24h to 48h, sterilized, and fermented liquid; the fermented liquid is centrifuged, and supernatant B is collected; 3 to 5 times the volume of ethanol of supernatant B is added to supernatant B, the mixture is allowed to stand at 4°C to 8°C for 12h to 24h, and then centrifuged to collect precipitate B; supernatant A and precipitate B are mixed, concentrated under reduced pressure below 60°C, and dried to constant weight below 60°C to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide.

[0006] In S2 of the above preparation method, the preparation of sturgeon collagen peptide comprises: The purified fish skin slices are beaten to obtain skin pulp, distilled water of 8 to 10 times the mass of the skin pulp is added, the pH value is adjusted to 8.0 to 8.5, trypsin of 0.5% to 1.5% by mass of the pulp is added, and enzymolysis is carried out at 35°C to 38°C for 1h to 2h, and the enzyme is inactivated in a boiling water bath. After cooling to room temperature, the pH value is adjusted to 6.5 to 7.0, serrapeptase of 0.5% to 1.5% by mass of the pulp and bromelain of 0.5% to 1.5% by mass of the pulp are added, and enzymolysis is carried out at 45°C to 50°C for 1h to 2h. h, inactivate the enzyme in a boiling water bath, cool to room temperature, centrifuge, collect supernatant C, use a 5kDa ultrafiltration membrane for ultrafiltration, take the ultrafiltrate below 5kDa, load the ultrafiltrate through a resin column for adsorption, use 2 to 3 resin column volumes of sodium chloride aqueous solution at a flow rate of 1mL / min to 2mL / min, collect all the eluate, use a 1kDa ultrafiltration membrane for ultrafiltration, retain the components above 1kDa, take the components above 1kDa for lyophilization, and obtain sturgeon collagen peptide.

[0007] In S3 of the above preparation method, the preparation of witch hazel extract comprises: Take witch hazel flowers, grind them to obtain a grind, add water 8 to 10 times the weight of the grind, adjust the pH value to 6.0 to 8.0, add 0.5% to 1.5% bromelain by weight of the grind, hydrolyze at 50 to 60°C for 1 h to 1.5 h, inactivate the enzyme in a boiling water bath, cool to room temperature, adjust the pH value to 6.5 to 7.0, add 0.5% to 1.5% nattokinase by weight of the grind, hydrolyze at 50 to 55°C for 1 h to 1.5 h, inactivate the enzyme in a boiling water bath enzyme, cool to room temperature, centrifuge, collect supernatant D, use 5kDa ultrafiltration membrane for ultrafiltration, take ultrafiltrate below 5kDa, load the ultrafiltrate through a resin column for adsorption, use 2 to 3 resin column volumes of sodium chloride aqueous solution at a flow rate of 1mL / min to 2mL / min, collect all the eluate, use 1kDa ultrafiltration membrane for ultrafiltration, retain components above 1kDa, take components above 1kDa for lyophilization, and obtain witch hazel extract.

[0008] In S1 of the above preparation method, the rotation speed of the homogenization is 10000 r / min to 15000 r / min, and the homogenization time is 3 min to 5 min.

[0009] In S1 of the above preparation method, the sterilization temperature is 121° C. to 130° C., and the sterilization time is 20 min to 30 min.

[0010] In S2 of the above preparation method, the preparation method of the purified fish skin slice comprises the following steps: taking freshly cleaned sturgeon skin, slicing it to obtain fish skin slices with a side length of less than 1 cm, adding anhydrous ethanol 3 to 5 times the mass of the fish skin slices, stirring and degreasing at room temperature for 2 hours to 3 hours, filtering and replacing the anhydrous ethanol once every 30 minutes with a 100-mesh to 250-mesh sieve, finally filtering with a 100-mesh to 250-mesh sieve, taking the sieve A, adding a 0.5 mol / L to 1.0 mol / L hydrochloric acid aqueous solution with a mass of 5 to 8 times the mass of the sieve A, stirring and decalcifying at 4° C. to 8° C. for 12 hours to 24 hours, filtering with a 100-mesh to 250-mesh sieve, taking the sieve B, rinsing with distilled water until neutral, draining the water, and obtaining the purified fish skin slices.

[0011] In S2 and S3 of the above preparation method, the resin of the resin column is an anion exchange resin.

[0012] In S2 of the above preparation method, the concentration of the sodium chloride aqueous solution is 0.3 mol / L to 0.6 mol / L.

[0013] In S3 of the above preparation method, the concentration of the sodium chloride aqueous solution is 0.7 mol / L to 1.0 mol / L.

[0014] In the above preparation method, the time for inactivating the enzyme in boiling water bath is 10 min to 20 min.

[0015] In the above preparation method, the centrifugal speed is 5000r / min to 8000r / min, and the centrifugal time is 10min to 20min.

[0016] The bromelain composition is used in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0017] The preparation method and application of a bromelain composition provided by the present invention have the following beneficial effects: 1. Bromelain can decompose the protein components in the dead skin cells on the surface of the skin, promote the shedding of dead skin cells, and achieve the effect of removing dead skin. It works by regulating the metabolism of skin cells and reducing the deposition of melanin. It promotes cell metabolism, helps repair damaged skin tissue, and reduces wrinkles.

[0018] Second, superoxide dismutase, as an antioxidant, can remove free radicals in the skin. Free radicals can damage skin cells and cause wrinkles, dullness and other problems. By removing free radicals, it delays skin aging, exerts anti-wrinkle and whitening effects, and also helps maintain the normal metabolism of skin cells, indirectly helping to remove dead skin.

[0019] 3. The amino acids in aloe amino acid polysaccharide provide nutrients for the skin and nourish the skin. Polysaccharide has good moisturizing properties and can form a moisturizing film on the skin surface to prevent dry skin. When the skin is well hydrated, the stratum corneum is more likely to fall off naturally, which helps to remove dead skin. Moisturizing makes the skin plump and reduces the appearance of wrinkles. At the same time, the nutrients are also beneficial to the repair and regeneration of skin cells, achieving a whitening effect.

[0020] 4. Sturgeon collagen peptides replenish collagen peptides in the skin, increase skin elasticity, and reduce wrinkles. Filling between skin cells makes the skin tighter, supports the skin from the inside, reduces wrinkles, and achieves anti-wrinkle effects. By promoting skin metabolism, new cells can better replace aging dead skin cells. By improving skin microcirculation, the skin becomes more radiant and pigmentation is reduced.

[0021] 5. Witch hazel extract contains some active ingredients that can regulate the secretion of skin oil, making it easier for the aged stratum corneum on the surface of the skin to fall off, thus removing dead skin. The active ingredients can inhibit the activity of melanocytes, reduce melanin production, stimulate the production of skin collagen, enhance skin elasticity, and reduce wrinkles.

[0022] 6. In the preparation method of sturgeon collagen peptide, firstly, it is subjected to defatting and decalcification treatment, which can effectively remove impurities such as fat and calcium in the fish skin, so that the subsequent enzymatic hydrolysis can act more accurately on the collagen component and improve the purity of the collagen peptide. During the enzymatic hydrolysis process, trypsin is first used for enzymatic hydrolysis at a suitable temperature and pH value to preliminarily decompose the collagen, and then further enzymatic hydrolysis is performed by combining serrapeptase and bromelain. The synergistic action of multiple enzymes decomposes the collagen more fully into specific small molecule peptide segments. Specific small molecule collagen peptides have a more efficient effect and are easily absorbed by the skin, thereby better replenishing the collagen lost by the skin, enhancing skin elasticity, accelerating skin cell renewal in terms of removing dead skin, and improving the effect of skin microcirculation in whitening. It also has a more significant synergistic effect on anti-wrinkle. After a series of fine separation and purification steps such as ultrafiltration and resin column adsorption elution, impurities are further removed to obtain high-purity, high-quality collagen peptides, which are more conducive to exerting its dead skin removal, whitening and anti-wrinkle effects in cosmetics.

[0023] 7. In the preparation method of witch hazel extract, after the witch hazel flowers are crushed, bromelain is first used for enzymatic hydrolysis at a suitable pH value and temperature. Bromelain can decompose the protein components in the flower tissue to better release the effective ingredients therein. Then nattokinase is used for further enzymatic hydrolysis. The double enzymatic hydrolysis process can more fully destroy the cell structure and release more active ingredients with the effects of removing dead skin, whitening and anti-wrinkle. Subsequent operations such as centrifugation, ultrafiltration, resin column adsorption elution and re-ultrafiltration can effectively separate and enrich these active ingredients, remove impurities, and obtain an extract with a higher concentration of active ingredients. When used in cosmetics, it can more effectively regulate skin oil secretion, promote the shedding of dead skin, inhibit melanin production and stimulate collagen production, thereby playing a synergistic advantage in the effects of removing dead skin, whitening and anti-wrinkle.

[0024] 8. Sturgeon collagen peptide can mainly supplement skin collagen, increase skin elasticity, reduce wrinkles, promote skin metabolism, help remove dead skin, improve microcirculation and help whiten. Witch hazel extract mainly regulates skin oil secretion to promote dead skin shedding, inhibits melanin production to achieve whitening, and stimulates collagen production to reduce wrinkles. The two have a synergistic effect: in terms of removing dead skin, sturgeon collagen peptide promotes skin cell renewal, and witch hazel extract regulates oil secretion to make dead skin easier to fall off. They work together to more effectively remove dead skin cells on the surface of the skin, making the skin smoother and more delicate. In terms of whitening, sturgeon collagen peptide improves skin microcirculation to make the skin shiny and reduce pigmentation, and witch hazel extract inhibits melanin production. The two work together to more significantly reduce melanin and make the skin whiter and more translucent. In terms of anti-wrinkle, sturgeon collagen peptide increases skin elasticity, fills skin tissue and reduces wrinkles, while witch hazel extract stimulates collagen production. The two work together to enhance skin firmness and elasticity, more effectively reduce the formation of wrinkles and delay skin aging. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0026] Example 1 A method for preparing a bromelain composition comprises the following steps: S1, Extraction of Aloe Amino Acid Polysaccharide: The peeled aloe leaf pulp was taken and pulped to obtain a slurry, and distilled water 12 times the mass of the slurry was added, and the slurry was homogenized at 12000 r / min for 4 minutes; the pH value was adjusted to 5.0, and 1.0% of cellulase and 0.6% of pectinase by mass of the slurry were added, and after mixing, the mixture was enzymatically hydrolyzed at 45°C for 3 hours, and then cooled to room temperature, the pH value was adjusted to 8.2, and 1.0% of trypsin by mass of the slurry was added, and the mixture was enzymatically hydrolyzed at 37°C for 3 hours, and the enzyme was inactivated in a boiling water bath for 15 minutes. After cooling to room temperature, the mixture was centrifuged at 6000 r / min for 15 minutes, and the supernatant A and the precipitate A were collected; distilled water 4 times the mass of the precipitate A was added to the precipitate A; water, inoculated with 4% lactic acid bacteria and 2% yeast of the mass of sediment A, anaerobically fermented at 32°C for 30h, sterilized at 125°C for 25min, to obtain a fermentation liquid; centrifuged the fermentation liquid at 6000r / min for 15min, and collected the supernatant B; added ethanol 4 times the volume of supernatant B to the supernatant B, mixed, allowed to stand at 6°C for 18h, then centrifuged at 6000r / min for 15min, and collected the sediment B; mixed the supernatant A and the sediment B, concentrated under reduced pressure at 50°C, and dried at 50°C to constant weight, to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide; S2, Preparation of sturgeon collagen peptide: Take freshly cleaned sturgeon skin, slice it to obtain a fish skin sheet with a side length less than 1 cm, add anhydrous ethanol with 4 times the mass of the fish skin sheet, stir and defat for 2.5 h at room temperature, adopt 150 mesh sieves to filter and replace anhydrous ethanol once every 30 min, finally filter through 150 mesh sieves, take oversize A, add 0.8 mol / L concentration of hydrochloric acid aqueous solution with 6 times the mass of oversize A, stir and decalcify for 18 h at 6 ℃, adopt 150 mesh sieves to filter, take oversize B, rinse with distilled water to neutrality, drain the water, and obtain purified fish skin sheet; The purified fish skin slices were beaten to obtain skin pulp, distilled water of 9 times the mass of the skin pulp was added, the pH value was adjusted to 8.2, trypsin (1.0% by mass of the pulp) was added, the mixture was hydrolyzed at 37°C for 1.5h, the enzyme was inactivated in a boiling water bath for 15min, and the pH value was adjusted to 6.8 after it was cooled to room temperature, serrapeptase (1.0% by mass of the pulp) and bromelain (1.0% by mass of the pulp) were added, the mixture was hydrolyzed at 48°C for 1.5h, the enzyme was inactivated in a boiling water bath for 15min, and the mixture was cooled to room temperature and the speed was 6000r / min. Centrifuge at a speed of 15 minutes, collect the supernatant C, perform ultrafiltration using a 5 kDa ultrafiltration membrane, take the ultrafiltrate below 5 kDa, load the ultrafiltrate through a resin column for adsorption, elute with 2 volumes of a resin column with a concentration of 0.5 mol / L sodium chloride aqueous solution at a flow rate of 1.5 mL / min, collect all the eluate, perform ultrafiltration using a 1 kDa ultrafiltration membrane, retain components above 1 kDa, take the components above 1 kDa for lyophilization, and obtain sturgeon collagen peptide; S3, preparation of witch hazel extract: Take witch hazel flowers, crush them to obtain crushed materials, add 9 times the mass of water to the crushed materials, adjust the pH value to 7.1, add 1.0% bromelain by mass of the crushed materials, enzymolyze at 55°C for 1 hour, inactivate the enzyme in a boiling water bath for 15 minutes, cool to room temperature, adjust the pH value to 6.8, add 1.0% nattokinase by mass of the crushed materials, enzymolyze at 52°C for 1 hour, inactivate the enzyme in a boiling water bath for 15 minutes, cool to room temperature, centrifuge at 6000r / min for 15 minutes, collect supernatant D, perform ultrafiltration using a 5kDa ultrafiltration membrane, take ultrafiltrate below 5kDa, load the ultrafiltrate through a resin column for adsorption, elute with 2 resin column volumes of a sodium chloride aqueous solution with a concentration of 0.8mol / L at a flow rate of 1.5mL / min, collect all the eluates, perform ultrafiltration using a 1kDa ultrafiltration membrane, retain components above 1kDa, take components above 1kDa for freeze-drying, and obtain witch hazel extract; S4, Mixed: According to the mass ratio of bromelain:superoxide dismutase:aloe amino acid polysaccharide:sturgeon collagen peptide:witch hazel extract=0.4:0.4:2:3.5:4, bromelain, superoxide dismutase, aloe amino acid polysaccharide, sturgeon collagen peptide and witch hazel extract are mixed to obtain a composition.

[0027] The bromelain composition is used in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0028] Example 2 A method for preparing a bromelain composition comprises the following steps: S1, Extraction of Aloe Amino Acid Polysaccharide: The peeled aloe leaf pulp is taken and beaten to obtain a slurry, and distilled water 8 times the mass of the slurry is added, and the slurry is homogenized at a speed of 10000 r / min for 3 minutes; the pH value is adjusted to 4.5, and 0.5% of cellulase and 0.3% of pectinase by mass of the slurry are added, and after mixing, enzymatic hydrolysis is carried out at 40°C for 2 hours, and then the mixture is cooled to room temperature, and the pH value is adjusted to 8.0, and 0.5% of trypsin by mass of the slurry is added, and the mixture is enzymatic hydrolysis is carried out at 35°C for 2 hours, and the enzyme is inactivated in a boiling water bath for 10 minutes. After the mixture is cooled to room temperature, the mixture is centrifuged at a speed of 5000 r / min for 10 minutes, and the supernatant A and the precipitate A are collected; distilled water 3 times the mass of the precipitate A is added to the precipitate A; water, inoculate 3% lactic acid bacteria and 1% yeast of the mass of sediment A, perform anaerobically fermentation at 30°C for 24h, sterilize at 121°C for 20min, and obtain fermentation liquid; centrifuge the fermentation liquid at 5000r / min for 10min, and collect supernatant B; add ethanol 3 times the volume of supernatant B to supernatant B, mix, let stand at 4°C for 12h, then centrifuge at 5000r / min for 10min, and collect sediment B; mix supernatant A and sediment B, concentrate under reduced pressure at 40°C, and dry at 40°C to constant weight to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide; S2, Preparation of sturgeon collagen peptide: Take freshly cleaned sturgeon skin, slice it to obtain a fish skin sheet with a side length less than 1 cm, add anhydrous ethanol 3 times the mass of the fish skin sheet, stir and defat for 2 h at room temperature, adopt 100 mesh sieve to filter and replace anhydrous ethanol once every 30 min, finally filter through 100 mesh sieve, take oversize A, add 0.5 mol / L concentration of hydrochloric acid aqueous solution 5 times the mass of oversize A, stir and decalcify for 12 h at 4 ℃, adopt 100 mesh sieve to filter, take oversize B, rinse with distilled water to neutrality, drain the water, and obtain purified fish skin sheet; The purified fish skin slices were beaten to obtain skin pulp, distilled water 8 times the mass of the skin pulp was added, the pH value was adjusted to 8.0, 0.5% of the mass of trypsin was added, and the mixture was hydrolyzed at 35°C for 1h, and the enzyme was inactivated in a boiling water bath for 10min. After the mixture was cooled to room temperature, the pH value was adjusted to 6.5, 0.5% of the mass of serrapeptase and 0.5% of the mass of bromelain were added, and the mixture was hydrolyzed at 45°C for 1h, and the enzyme was inactivated in a boiling water bath for 10min. After the mixture was cooled to room temperature, the mixture was centrifuged at a speed of 5000r / min for 10min, and the supernatant C was collected. The mixture was ultrafiltered using a 5kDa ultrafiltration membrane, and the ultrafiltrate below 5kDa was taken. The ultrafiltrate was loaded and adsorbed by a resin column, and eluted with a sodium chloride aqueous solution with a concentration of 0.3mol / L for 2 resin column volumes at a flow rate of 1mL / min. All the eluates were collected, and ultrafiltered using a 1kDa ultrafiltration membrane to intercept components above 1kDa, and the components above 1kDa were freeze-dried to obtain sturgeon collagen peptide; S3, preparation of witch hazel extract: Take witch hazel flowers, crush them to obtain crushed materials, add water 8 times the mass of the crushed materials, adjust the pH value to 6.0, add 0.5% bromelain by mass of the crushed materials, enzymolyze at 50°C for 1 hour, inactivate the enzyme in a boiling water bath for 10 minutes, cool to room temperature, adjust the pH value to 6.5, add 0.5% nattokinase by mass of the crushed materials, enzymolyze at 50°C for 1 hour, inactivate the enzyme in a boiling water bath for 10 minutes, cool to room temperature, centrifuge at 5000r / min for 10 minutes, collect supernatant D, perform ultrafiltration using a 5kDa ultrafiltration membrane, take ultrafiltrate below 5kDa, load the ultrafiltrate through a resin column for adsorption, elute with 2 resin column volumes of a sodium chloride aqueous solution with a concentration of 0.7mol / L at a flow rate of 1mL / min, collect all the eluates, perform ultrafiltration using a 1kDa ultrafiltration membrane, retain components above 1kDa, take components above 1kDa for freeze-drying, and obtain witch hazel extract; S4, Mixed: According to the mass ratio of bromelain:superoxide dismutase:aloe amino acid polysaccharide:sturgeon collagen peptide:witch hazel extract=0.35:0.3:1.5:3:5, bromelain, superoxide dismutase, aloe amino acid polysaccharide, sturgeon collagen peptide and witch hazel extract are mixed to obtain a composition.

[0029] The bromelain composition is used in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0030] Example 3 A method for preparing a bromelain composition comprises the following steps: S1, Extraction of Aloe Amino Acid Polysaccharide: The peeled aloe leaf pulp was taken and beaten to obtain a slurry, and distilled water 8 times the mass of the slurry was added, and the slurry was homogenized at 15000 r / min for 3 min; the pH value was adjusted to 5.5, and 0.5% of cellulase and 1.0% of pectinase by mass of the slurry were added, and after mixing, the mixture was enzymatically hydrolyzed at 40°C for 4 h, and then cooled to room temperature, the pH value was adjusted to 8.0, and 1.5% of trypsin by mass of the slurry was added, and the mixture was enzymatically hydrolyzed at 35°C for 4 h, and the enzyme was inactivated in a boiling water bath for 10 min. After cooling to room temperature, the mixture was centrifuged at 8000 r / min for 10 min, and the supernatant A and the precipitate A were collected; distilled water 5 times the mass of the precipitate A was added to the precipitate A; water, inoculate 3% lactic acid bacteria and 3% yeast of the mass of sediment A, perform anaerobically fermentation at 30°C for 48h, sterilize at 121°C for 30min, and obtain fermentation liquid; centrifuge the fermentation liquid at 5000r / min for 20min, and collect supernatant B; add ethanol 3 times the volume of supernatant B to supernatant B, mix, let stand at 8°C for 12h, then centrifuge at 8000r / min for 10min, and collect sediment B; mix supernatant A and sediment B, concentrate under reduced pressure at 60°C, and dry at 40°C to constant weight to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide; S2, Preparation of sturgeon collagen peptide: Take freshly cleaned sturgeon skin, slice it to obtain a fish skin sheet with a side length less than 1 cm, add anhydrous ethanol with 5 times the mass of the fish skin sheet, stir and defat for 2 h at room temperature, adopt 200 mesh sieve to filter and replace anhydrous ethanol once every 30 min, finally filter through 200 mesh sieve, take oversize A, add 0.5 mol / L concentration of hydrochloric acid aqueous solution with 8 times the mass of oversize A, stir and decalcify for 12 h at 8 ℃, adopt 200 mesh sieve to filter, take oversize B, rinse with distilled water to neutrality, drain the water, and obtain purified fish skin sheet; The purified fish skin slices were beaten to obtain skin pulp, distilled water 8 times the mass of the skin pulp was added, the pH value was adjusted to 8.5, 0.5% of the mass of trypsin was added, and the mixture was hydrolyzed at 38°C for 1 hour, and the enzyme was inactivated in a boiling water bath for 20 minutes. After the mixture was cooled to room temperature, the pH value was adjusted to 6.5, 1.5% of the mass of serrapeptase and 0.5% of the mass of bromelain were added, and the mixture was hydrolyzed at 50°C for 1 hour, and the enzyme was inactivated in a boiling water bath for 20 minutes. After the mixture was cooled to room temperature, the mixture was centrifuged at a speed of 5000 r / min for 20 minutes, and the supernatant C was collected. The mixture was ultrafiltered using a 5 kDa ultrafiltration membrane, and the ultrafiltrate below 5 kDa was taken. The ultrafiltrate was loaded and adsorbed by a resin column, and eluted with a sodium chloride aqueous solution with a concentration of 0.6 mol / L for 2 resin column volumes at a flow rate of 1 mL / min. All the eluates were collected, and ultrafiltered using a 1 kDa ultrafiltration membrane to intercept components above 1 kDa, and components above 1 kDa were freeze-dried to obtain sturgeon collagen peptide; S3, preparation of witch hazel extract: Take witch hazel flowers, crush them to obtain crushed materials, add water 10 times the mass of the crushed materials, adjust the pH value to 6.0, add 1.5% bromelain by mass of the crushed materials, enzymolyze at 50°C for 1.5 hours, inactivate the enzyme in a boiling water bath for 10 minutes, cool to room temperature, adjust the pH value to 7.0, add 0.5% nattokinase by mass of the crushed materials, enzymolyze at 55°C for 1 hour, inactivate the enzyme in a boiling water bath for 20 minutes, cool to room temperature, centrifuge at 5000r / min for 20 minutes, collect supernatant D, perform ultrafiltration using a 5kDa ultrafiltration membrane, take ultrafiltrate below 5kDa, load the ultrafiltrate through a resin column for adsorption, elute with 2 resin column volumes of a sodium chloride aqueous solution with a concentration of 1.0mol / L at a flow rate of 1mL / min, collect all the eluates, perform ultrafiltration using a 1kDa ultrafiltration membrane, retain components above 1kDa, take components above 1kDa for freeze-drying, and obtain witch hazel extract; S4, Mixed: According to the mass ratio of bromelain:superoxide dismutase:aloe amino acid polysaccharide:sturgeon collagen peptide:witch hazel extract=0.5:0.3:3:3:5, bromelain, superoxide dismutase, aloe amino acid polysaccharide, sturgeon collagen peptide and witch hazel extract are mixed to obtain a composition.

[0031] The bromelain composition is used in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0032] Example 4 A method for preparing a bromelain composition comprises the following steps: S1, Extraction of Aloe Amino Acid Polysaccharide: The peeled aloe leaf pulp is beaten to obtain a slurry, and distilled water 15 times the mass of the slurry is added, and the slurry is homogenized at 15000 r / min for 5 minutes; the pH value is adjusted to 5.5, and 1.5% of cellulase and 1.0% of pectinase by mass of the slurry are added, and after mixing, enzymatic hydrolysis is carried out at 50°C for 4 hours, and then the mixture is cooled to room temperature, and the pH value is adjusted to 8.5, and 1.5% of trypsin by mass of the slurry is added, and the mixture is enzymatic hydrolyzed at 38°C for 4 hours, and the enzyme is inactivated in a boiling water bath for 20 minutes. After the mixture is cooled to room temperature, the mixture is centrifuged at 8000 r / min for 20 minutes, and the supernatant A and the precipitate A are collected; distilled water 5 times the mass of the precipitate A is added to the precipitate A; water, inoculate 5% lactic acid bacteria and 3% yeast of the mass of sediment A, perform anaerobically fermentation at 35°C for 48h, sterilize at 130°C for 30min, and obtain fermentation liquid; centrifuge the fermentation liquid at 8000r / min for 20min, and collect supernatant B; add ethanol 5 times the volume of supernatant B to supernatant B, mix, let stand at 8°C for 24h, then centrifuge at 8000r / min for 20min, and collect sediment B; mix supernatant A and sediment B, concentrate under reduced pressure at 60°C, and dry at 60°C to constant weight to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide; S2, Preparation of sturgeon collagen peptide: Take freshly cleaned sturgeon skin, slice it to obtain a fish skin sheet with a side length less than 1 cm, add anhydrous ethanol with 5 times the mass of the fish skin sheet, stir and defat for 3 h at room temperature, adopt 250 mesh sieves to filter and replace anhydrous ethanol once every 30 min, finally filter through 250 mesh sieves, take oversize A, add 1.0 mol / L concentration of hydrochloric acid aqueous solution with 8 times the mass of oversize A, stir and decalcify for 24 h at 8 ℃, adopt 250 mesh sieves to filter, take oversize B, rinse with distilled water to neutrality, drain the water, and obtain purified fish skin sheet; The purified fish skin slices were beaten to obtain skin pulp, distilled water 10 times the mass of the skin pulp was added, the pH value was adjusted to 8.5, trypsin (1.5% by mass of the pulp) was added, the mixture was enzymolyzed at 38°C for 2h, the enzyme was inactivated in a boiling water bath for 20min, the pH value was adjusted to 7.0 after it was cooled to room temperature, serrapeptase (1.5% by mass of the pulp) and bromelain (1.5% by mass of the pulp) were added, the mixture was enzymolyzed at 50°C for 2h, the enzyme was inactivated in a boiling water bath for 20min, the mixture was cooled to room temperature, and the speed was increased to 8000r / min. Centrifuge at a speed of 20 minutes, collect the supernatant C, perform ultrafiltration using a 5 kDa ultrafiltration membrane, take the ultrafiltrate below 5 kDa, load the ultrafiltrate through a resin column for adsorption, elute with 3 volumes of a 0.6 mol / L sodium chloride aqueous solution at a flow rate of 2 mL / min, collect all the eluate, perform ultrafiltration using a 1 kDa ultrafiltration membrane, retain components above 1 kDa, take the components above 1 kDa for freeze-drying, and obtain sturgeon collagen peptide; S3, preparation of witch hazel extract: Take witch hazel flowers, crush them to obtain crushed materials, add water 10 times the mass of the crushed materials, adjust the pH value to 8.0, add 1.5% bromelain by mass of the crushed materials, enzymolyze at 60°C for 1.5 hours, inactivate the enzyme in a boiling water bath for 20 minutes, cool to room temperature, adjust the pH value to 7.0, add 1.5% nattokinase by mass of the crushed materials, enzymolyze at 55°C for 1.5 hours, inactivate the enzyme in a boiling water bath for 20 minutes, cool to room temperature, centrifuge at 8000r / min for 20 minutes, collect supernatant D, perform ultrafiltration using a 5kDa ultrafiltration membrane, take ultrafiltrate below 5kDa, load the ultrafiltrate through a resin column for adsorption, elute with 3 volumes of a sodium chloride aqueous solution with a concentration of 1.0mol / L at a flow rate of 2mL / min, collect all the eluates, perform ultrafiltration using a 1kDa ultrafiltration membrane, intercept components above 1kDa, take components above 1kDa for freeze-drying, and obtain witch hazel extract; S4, Mixed: According to the mass ratio of bromelain:superoxide dismutase:aloe amino acid polysaccharide:sturgeon collagen peptide:witch hazel extract=0.3:0.5:2:4.5:3, bromelain, superoxide dismutase, aloe amino acid polysaccharide, sturgeon collagen peptide and witch hazel extract are mixed to obtain a composition.

[0033] The bromelain composition is used in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0034] Example 5 A method for preparing a bromelain composition comprises the following steps: S1, Extraction of Aloe Amino Acid Polysaccharide: The peeled aloe leaf pulp is taken and beaten to obtain a slurry, and distilled water 15 times the mass of the slurry is added, and the slurry is homogenized at a speed of 10000 r / min for 5 minutes; the pH value is adjusted to 4.5, and 1.5% of cellulase and 0.3% of pectinase by mass of the slurry are added, and after mixing, enzymatic hydrolysis is carried out at 50°C for 2 hours, and then the mixture is cooled to room temperature, and the pH value is adjusted to 8.5, and 0.5% of trypsin by mass of the slurry is added, and the mixture is enzymatic hydrolysis is carried out at 38°C for 2 hours, and the enzyme is inactivated in a boiling water bath for 20 minutes. After the mixture is cooled to room temperature, the mixture is centrifuged at a speed of 5000 r / min for 20 minutes, and the supernatant A and the precipitate A are collected; distilled water 3 times the mass of the precipitate A is added to the precipitate A; water, inoculate 5% lactic acid bacteria and 1% yeast of the mass of sediment A, perform anaerobically fermentation at 35°C for 24h, sterilize at 130°C for 20min, and obtain fermentation liquid; centrifuge the fermentation liquid at 8000r / min for 10min, and collect supernatant B; add ethanol 5 times the volume of supernatant B to supernatant B, mix, let stand at 4°C for 24h, then centrifuge at 5000r / min for 20min, and collect sediment B; mix supernatant A and sediment B, concentrate under reduced pressure at 40°C, and dry at 60°C to constant weight to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide; S2, Preparation of sturgeon collagen peptide: Take freshly cleaned sturgeon skin, slice it to obtain a fish skin sheet with a side length less than 1 cm, add anhydrous ethanol 3 times the mass of the fish skin sheet, stir and defat for 3 h at room temperature, adopt 150 mesh sieves to filter and replace anhydrous ethanol once every 30 min, finally filter through 150 mesh sieves, take oversize A, add 1.0 mol / L concentration of hydrochloric acid aqueous solution 5 times the mass of oversize A, stir and decalcify for 24 h at 4 ℃, adopt 150 mesh sieves to filter, take oversize B, rinse with distilled water to neutrality, drain the water, and obtain purified fish skin sheet; The purified fish skin slices were beaten to obtain skin pulp, distilled water 10 times the mass of the skin pulp was added, the pH value was adjusted to 8.0, trypsin 1.5% by mass of the pulp was added, and the mixture was enzymatically hydrolyzed at 35°C for 2h, and the enzyme was inactivated in a boiling water bath for 10min. After the mixture was cooled to room temperature, the pH value was adjusted to 7.0, serrapeptase 0.5% by mass of the pulp and bromelain 1.5% by mass of the pulp were added, the mixture was enzymatically hydrolyzed at 45°C for 2h, and the enzyme was inactivated in a boiling water bath for 10min. After the mixture was cooled to room temperature, the mixture was rotated at 8000r / min. Centrifuge at a speed of 10 minutes, collect the supernatant C, perform ultrafiltration using a 5 kDa ultrafiltration membrane, take the ultrafiltrate below 5 kDa, load the ultrafiltrate through a resin column for adsorption, elute with 3 volumes of a resin column with a concentration of 0.3 mol / L sodium chloride aqueous solution at a flow rate of 2 mL / min, collect all the eluate, perform ultrafiltration using a 1 kDa ultrafiltration membrane, retain components above 1 kDa, take the components above 1 kDa for lyophilization, and obtain sturgeon collagen peptide; S3, preparation of witch hazel extract: Take witch hazel flowers, crush them to obtain crushed materials, add water 8 times the mass of the crushed materials, adjust the pH value to 8.0, add 0.5% bromelain by mass of the crushed materials, enzymolyze at 60°C for 1 hour, inactivate the enzyme in a boiling water bath for 20 minutes, cool to room temperature, adjust the pH value to 6.5, add 1.5% nattokinase by mass of the crushed materials, enzymolyze at 50°C for 1.5 hours, inactivate the enzyme in a boiling water bath for 10 minutes, cool to room temperature, centrifuge at 8000r / min for 10 minutes, collect supernatant D, perform ultrafiltration using a 5kDa ultrafiltration membrane, take ultrafiltrate below 5kDa, load the ultrafiltrate through a resin column for adsorption, elute with 3 volumes of a sodium chloride aqueous solution with a concentration of 0.7mol / L at a flow rate of 2mL / min, collect all the eluates, perform ultrafiltration using a 1kDa ultrafiltration membrane, intercept components above 1kDa, take components above 1kDa for freeze-drying, and obtain witch hazel extract; S4, Mixed: According to the mass ratio of bromelain:superoxide dismutase:aloe amino acid polysaccharide:sturgeon collagen peptide:witch hazel extract=0.3:0.5:1:5:3, bromelain, superoxide dismutase, aloe amino acid polysaccharide, sturgeon collagen peptide and witch hazel extract are mixed to obtain a composition.

[0035] The bromelain composition is used in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0036] In the above embodiments, the resin used in the resin column is an anion exchange resin, model D201MB, which comes from Tianjin Jinda Zhengtong Environmental Protection Technology Co., Ltd.

[0037] In the above embodiments, the sources of enzymes and bacteria: cellulase is derived from Shandong Jinchengda Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g. Pectinase is derived from Henan Anrui Biotechnology Co., Ltd., with an enzyme activity of 30,000 U / g. Trypsin is derived from Zhejiang Fuxuan Biotechnology Co., Ltd., with an enzyme activity of 4000 U / g. Serrapeptase is derived from Shenzhen Lefu Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g. Bromelain is derived from Zhongpeptide Biotechnology (Shanxi) Co., Ltd., with an enzyme activity of 100,000 U / g. Nattokinase is derived from Shaanxi Saien Biotechnology Co., Ltd., with an enzyme activity of 20,000 U / g. Lactic acid bacteria are Lactobacillus acidophilus CICC6081, with a bacterial activity of 20 billion CFU / g. Yeast is Saccharomyces cerevisiae CICC 1423, with a bacterial activity of 20 billion CFU / g.

[0038] Comparative Example 1 No precipitate B was added to the aloe amino acid polysaccharide, and other parameters and methods were the same as in Example 1.

[0039] Comparative Example 2 In the preparation of sturgeon collagen peptide, serrapeptase and bromelain were not used for enzymatic hydrolysis, and other parameters and methods were the same as those in Example 1.

[0040] Comparative Example 3 In the preparation of sturgeon collagen peptide, serrapeptase was not used for enzymatic hydrolysis, and other parameters and methods were the same as in Example 1.

[0041] Comparative Example 4 In the preparation of sturgeon collagen peptide, serrapeptase was not used for enzymatic hydrolysis, and resin column was not used for adsorption purification. The ultrafiltration fraction between 1 kDa and 5 kDa was intercepted and directly freeze-dried to obtain sturgeon collagen peptide. Other parameters and methods were the same as those in Example 1.

[0042] Comparative Example 5 In the preparation of witch hazel extract, nattokinase was not used for enzymatic hydrolysis, and other parameters and methods were the same as those in Example 1.

[0043] Comparative Example 6 In the preparation of the witch hazel extract, nattokinase is not used for enzymatic hydrolysis, and resin columns are not used for adsorption purification. Ultrafiltration components between 1 kDa and 5 kDa are intercepted and directly freeze-dried to obtain the witch hazel extract. Other parameters and methods are the same as in Example 1.

[0044] Comparative Example 7 In the composition, no sturgeon collagen peptide was added, and other parameters and methods were the same as in Example 1.

[0045] Comparative Example 8 In the composition, no witch hazel extract was added, and other parameters and methods were the same as in Example 1.

[0046] Comparative Example 9 In the composition, sturgeon collagen peptide and witch hazel extract are not added at the same time, and other parameters and methods are the same as in Example 1.

[0047] The compositions of the above-mentioned embodiments and comparative examples were tested for safety and efficacy.

[0048] 1. Safety Testing (1) Cytotoxicity test: A number of human immortalized epidermal cells (HaCaT cells) in the logarithmic growth phase were seeded in a 96-well plate at a density of about 5,000 cells per well. After culturing for 24 hours in a DMEM high-glucose medium containing 10 wt% fetal bovine serum and 1 wt% penicillin-streptomycin, the medium was replaced with a DMEM high-glucose medium containing a bromelain composition, the concentration of the composition being 0.5 mg / ml. A blank control group (cultured only in a DMEM high-glucose medium) was set up at the same time. The culture was continued for 24 hours. The cell viability was detected by the MTT method, and the absorbance OD value was measured at 490 nm using an ELISA reader to calculate the relative cell survival rate, which was (absorbance OD value of the test group / absorbance OD value of the blank control group) × 100%. The results are shown in Table 1 below.

[0049] (2) Skin irritation test: 140 healthy adult subjects (18-55 years old) with no history of skin allergies or skin diseases and normal skin barrier function were selected and randomly divided into 14 groups (10 subjects in each group). Four test areas of equal area (2 cm × 2 cm) were marked on the inner side of the forearm of the volunteers with a marker pen, and the areas were separated by 1 cm. 0.05 mL of the test sample (concentration 0.5 mg / ml) containing the bromelain composition was accurately aspirated with a micropipette and applied to the corresponding test areas. The samples were gently applied evenly to cover the entire area, and then covered and fixed with breathable, non-irritating medical tape. Observe the reaction of the skin in the test area 4 hours, 24 hours, and 48 hours after application, and score according to the skin irritation scoring standard established by the International Contact Dermatitis Research Group (ICDRG): 0 points for no erythema and no edema; 1 point for mild erythema and no edema; 2 points for moderate erythema and mild edema; 3 points for severe erythema and moderate edema; 4 points for severe erythema, edema, accompanied by papules, blisters, etc. The average score of each sample among all volunteers was calculated to evaluate its skin irritation degree. The lower the average score, the higher the safety. The results are shown in Table 1 below.

[0050] Table 1 Safety test results

[0051] 2. Efficacy Testing (1) Dead skin removal effect test: 150 adult subjects (25-48 years old) with dry and wrinkled facial skin, certain keratin accumulation and dull skin color were selected and randomly divided into 15 groups (10 people in each group), of which 1 group was a blank control group, using purified water without active ingredients as a placebo, and the remaining 14 groups were experimental groups. Each group used an aqueous solution containing the composition (the mass fraction of the composition was 3wt%, 97wt% purified water) once in the morning and evening. Facial skin touch test was performed before using the product and 7 days after using the product: Initial tactile evaluation before using the product: Gently touch the test site skin with the index and middle fingertips. Note that the touch force should be moderate and even, and feel the roughness of the skin surface, whether there is a grainy feeling, and the elasticity of the skin. After using the product for 7 days, re-evaluate the touch: Use the same technique and force as the initial tactile evaluation to touch the test site skin again. Focus on feeling whether the skin roughness and graininess are reduced, whether the elasticity has changed, and whether the skin has become softer and smoother. The results are shown in Table 2 below.

[0052] Touch evaluation score: 1-2 points: The entire face feels extremely rough, with a noticeable gritty feel, like touching sandpaper. A large amount of dead skin can be easily felt on the cheeks, forehead, nose and chin, and the skin texture is messy and thick. 3-4 points: The face has a relatively obvious roughness. When you touch it gently with your fingers, you can feel the granular dead skin, especially on both sides of the nose, around the lips and in the center of the forehead. The skin lacks elasticity as a whole, feels a bit hard and not soft enough, and it feels like there is a thick, uneven layer of material on the surface of the skin. 5-6 points: There is a certain degree of roughness, but the granularity is not particularly strong. When you touch it carefully, such as gently circling the cheek with your fingertips, you will find a small amount of small particles, mainly in places where the skin texture is more obvious. The skin has a certain elasticity, but it is relatively weak. It is relatively soft, but not that delicate to the touch. 7-8 points: The face has only a slight sense of roughness and almost no granularity. Only when you touch it very carefully or touch specific areas (such as the edge of the nose) can you find very small signs of dead skin. The skin has good elasticity and is relatively soft, giving a smooth feeling. 9-10 points: The facial skin feels extremely smooth, with no roughness or graininess at all, just like the surface of a finely polished jade. The skin has excellent elasticity, is soft and shiny, and no dead skin can be felt. It feels very delicate from the forehead to the chin.

[0053] (2) Skin chromaticity measurement: While testing the effect of removing dead skin, the skin chroma meter (Minolta Chroma Meter CR-400) was used to measure the L* (brightness) value on the left and right cheeks of the subjects before and after 4 weeks of use. Each area was measured 3 times, and the average value of each area was taken as the chromaticity value. △L (L value after use - L value before use), the larger the positive value of △L, the better the skin whitening effect. The results are shown in Table 2 below.

[0054] (3) Anti-wrinkle effect test: Anti-wrinkle effect test was performed at the same time as skin color measurement. Skin images of the cheeks were collected using a skin three-dimensional imager (Visiometer® VC98) before and 4 weeks after using the product. The collected skin images were processed and analyzed using the matching image analysis software to calculate skin roughness (Ra), wrinkle depth (Rz), and wrinkle volume (Vv). The changes in the values ​​of each parameter before and after using the sample were compared. The greater the decrease in the parameter value, the better the anti-wrinkle effect. Ra / Rz / Vv decrease rate = (Ra / Rz / Vv before use - Ra / Rz / Vv after use) / Ra / Rz / Vv before use × 100%. The results are shown in Table 2 below.

[0055] Table 2 Efficacy test results (average value)

[0056] It can be seen from the above experimental results that the bromelain compositions prepared in Examples 1 to 5 have safe ingredients, no cytotoxicity and no skin irritation, and can meet the needs of removing dead skin, whitening, firming and anti-wrinkle, etc.

[0057] From the results of Comparative Example 1, it can be seen that aloe amino acid polysaccharide has good moisturizing properties, can inhibit the activity of melanocytes, and stimulate fibroblasts to produce collagen, thereby achieving exfoliating, whitening and anti-wrinkle effects. Without adding precipitate B to aloe amino acid polysaccharide, the effective ingredients of the product are reduced, and the exfoliating, whitening and anti-wrinkle effects are reduced.

[0058] From the results of Comparative Examples 2 to 4, it can be seen that sturgeon collagen peptide, as a hydrolyzate of collagen, has the effects of exfoliating, whitening and anti-wrinkle. In the preparation method of sturgeon collagen peptide, serrapeptase and bromelain are not used for enzymatic hydrolysis, or serrapeptase is not used for enzymatic hydrolysis, or serrapeptase is not used for enzymatic hydrolysis and resin column is not used for adsorption purification at the same time, the effective ingredients of the product are reduced, and the exfoliating, whitening and anti-wrinkle effects are reduced.

[0059] From the results of Comparative Examples 5 to 6, it can be seen that the witch hazel extract contains a variety of antioxidant ingredients, has the effects of astringency and inhibition of tyrosinase, thereby achieving the effects of exfoliating, whitening and anti-wrinkle. In the preparation method of the witch hazel extract, nattokinase is not used for enzymatic hydrolysis, or nattokinase is not used for enzymatic hydrolysis and resin column is not used for adsorption and purification, the effective ingredients of the product are reduced, and the exfoliating, whitening and anti-wrinkle effects are reduced.

[0060] From the results of Comparative Example 7, it can be seen that without adding sturgeon collagen peptide to the composition, the exfoliating, whitening and anti-wrinkle effects are reduced; from the results of Comparative Example 8, it can be seen that without adding witch hazel extract to the composition, the exfoliating, whitening and anti-wrinkle effects are reduced; from the results of Comparative Example 9, it can be seen that without adding sturgeon collagen peptide and witch hazel extract to the composition at the same time, the exfoliating, whitening and anti-wrinkle effects are significantly reduced, and the two have a synergistic effect.

Claims

1. A method for preparing a bromelain composition, characterized in that: The steps include: S1, extraction of aloe amino acid polysaccharide: comprising peeling and beating the aloe, adding water to homogenize, adding cellulase and pectinase for enzymolysis, adding trypsin for enzymolysis, and collecting supernatant A and precipitate A by centrifugation; inoculating lactic acid bacteria and yeast into the precipitate A for fermentation, and then centrifuging, collecting supernatant B and adding ethanol for precipitation, and collecting precipitate B; mixing the supernatant A and the precipitate B, concentrating, and drying to obtain an extract containing amino acids and complex polysaccharides, namely, aloe amino acid polysaccharide; S2, preparation of sturgeon collagen peptide: comprising beating the fish skin, adding water to mix, adding trypsin to hydrolyze, adding serrapeptase and bromelain to hydrolyze, centrifuging to collect the supernatant C and ultrafiltration using a 4KDa-6KDa molecular weight ultrafiltration membrane, adsorbing the ultrafiltrate on a resin column, eluting, ultrafiltration of the eluate using a 0.5KDa-1.5KDa molecular weight ultrafiltration membrane, taking the ultrafiltration retentate and drying it to obtain sturgeon collagen peptide; S3, preparation of witch hazel extract: comprising crushing witch hazel, adding water to mix, adding bromelain for enzymolysis, adding nattokinase for enzymolysis, collecting supernatant D by centrifugation, ultrafiltration of supernatant D using 4KDa-6KDa molecular weight ultrafiltration membrane, adsorption of ultrafiltrate by resin column, elution, ultrafiltration of eluate using 0.5KDa-1.5KDa molecular weight ultrafiltration membrane, drying the ultrafiltration retentate, and obtaining witch hazel extract; S4, mixing: bromelain, superoxide dismutase, aloe amino acid polysaccharide, sturgeon collagen peptide, and witch hazel extract are mixed in a mass ratio of (0.3-0.5):(0.3-0.5):(1-3):(3-5):(3-5) to obtain a composition.

2. The method for preparing a bromelain composition according to claim 1, wherein The extraction of aloe amino acid polysaccharide comprises: taking peeled aloe leaf pulp, beating it to obtain slurry, adding distilled water of 8 to 15 times the mass of the slurry, and homogenizing; adjusting the pH value to 4.5 to 5.5, adding 0.5% to 1.5% of cellulase by mass of the slurry and 0.3% to 1.0% of pectinase by mass of the slurry, mixing, enzymolyzing at 40°C to 50°C for 2h to 4h, cooling to room temperature, adjusting the pH value to 8.0 to 8.5, adding 0.5% to 1.5% of trypsin by mass of the slurry, enzymolyzing at 35°C to 38°C for 2h to 4h, inactivating the enzyme in a boiling water bath, cooling to room temperature, centrifuging, and collecting supernatant A and precipitate A; and Add distilled water 3 to 5 times the mass of precipitate A, inoculate 3% to 5% of lactic acid bacteria and 1% to 3% of yeast by mass of precipitate A, perform anaerobically fermentation at 30°C to 35°C for 24 to 48 hours, sterilize, and obtain fermentation liquid; centrifuge the fermentation liquid, and collect supernatant B; add ethanol 3 to 5 times the volume of supernatant B to supernatant B, mix, let stand at 4°C to 8°C for 12 to 24 hours, then centrifuge, and collect precipitate B; mix supernatant A and precipitate B, concentrate under reduced pressure below 60°C, and dry to constant weight below 60°C to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide.

3. The method for preparing a bromelain composition according to claim 1, wherein The preparation of sturgeon collagen peptide comprises the following steps: beating purified fish skin slices to obtain skin pulp, adding distilled water of 8 to 10 times the mass of the skin pulp, adjusting the pH value to 8.0 to 8.5, adding trypsin of 0.5 to 1.5% by mass of the pulp, performing enzymolysis at 35 to 38° C. for 1 to 2 hours, inactivating the enzyme in a boiling water bath, cooling to room temperature, adjusting the pH value to 6.5 to 7.0, adding serrapeptase of 0.5 to 1.5% by mass of the pulp and bromelain of 0.5 to 1.5% by mass of the pulp, and boiling at 45 to 50° C. The enzyme was hydrolyzed for 1 h to 2 h, and the enzyme was inactivated in a boiling water bath. After cooling to room temperature, the supernatant C was collected and ultrafiltered using a 5 kDa ultrafiltration membrane. The ultrafiltrate below 5 kDa was taken, and the ultrafiltrate was loaded onto a resin column for adsorption. The solution was eluted with 2 to 3 resin column volumes of a sodium chloride aqueous solution at a flow rate of 1 mL / min to 2 mL / min. All the eluates were collected and ultrafiltered using a 1 kDa ultrafiltration membrane. Components above 1 kDa were retained, and the components above 1 kDa were freeze-dried to obtain sturgeon collagen peptides.

4. The method for preparing a bromelain composition according to claim 1, wherein The preparation of witch hazel extract comprises: taking witch hazel flowers, crushing them to obtain crushed materials, adding water 8 to 10 times the mass of the crushed materials, adjusting the pH value to 6.0 to 8.0, adding 0.5% to 1.5% of bromelain by mass of the crushed materials, performing enzymolysis at 50 to 60° C. for 1 h to 1.5 h, inactivating the enzyme in a boiling water bath, cooling to room temperature, adjusting the pH value to 6.5 to 7.0, adding 0.5% to 1.5% of nattokinase by mass of the crushed materials, performing enzymolysis at 50 to 55° C. for 1 h to 1.5 h h, inactivate the enzyme in a boiling water bath, cool to room temperature, centrifuge, collect the supernatant D, use a 5 kDa ultrafiltration membrane for ultrafiltration, take the ultrafiltrate below 5 kDa, load the ultrafiltrate through a resin column for adsorption, use 2 to 3 resin column volumes of sodium chloride aqueous solution at a flow rate of 1 mL / min to 2 mL / min, collect all the eluate, use a 1 kDa ultrafiltration membrane for ultrafiltration, retain the components above 1 kDa, take the components above 1 kDa for lyophilization, and obtain the witch hazel extract.

5. The method for preparing a bromelain composition according to claim 1, wherein In S2 and S3, the resin of the resin column is an anion exchange resin.

6. The method for preparing a bromelain composition according to claim 3, wherein: During the preparation of sturgeon collagen peptide, the concentration of the sodium chloride aqueous solution is 0.3 mol / L to 0.6 mol / L.

7. The method for preparing a bromelain composition according to claim 4, wherein: In the preparation process of witch hazel extract, the concentration of the sodium chloride aqueous solution is 0.7 mol / L to 1.0 mol / L.

8. A method for preparing a bromelain composition according to claim 2, 3 or 4, characterized in that: The time for inactivating the enzyme in a boiling water bath is 10 to 20 minutes.

9. The method for preparing a bromelain composition according to claim 1, wherein: The centrifugal speed is 5000r / min to 8000r / min, and the centrifugal time is 10min to 20min.

10. An application of a bromelain composition, wherein the composition is prepared by the preparation method of a bromelain composition according to claim 1, characterized in that: The invention discloses an application of the composition in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

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