A method for preparing and using a bromelain composition

Abstract

The application provides a preparation method and application of a bromelain composition, and belongs to the technical field of biotechnology.The composition contains bromelain, superoxide dismutase, amino acids and complex polysaccharides extracted from aloe, sturgeon collagen peptide and hamamelis extract; the aloe amino acid polysaccharide, sturgeon collagen peptide and hamamelis extract are high-efficiency products obtained through special enzymolysis or fermentation and purification. The various components are used in combination, can remove dead skin cells, remove wrinkles, keep the skin firm and elastic, have good whitening and anti-wrinkle effects, and have few component types, do not cause absorption burden to the skin, and are safe and efficient.

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 beauty, consumers are increasingly demanding and diverse cosmetic product benefits. Removing dead skin cells, reducing wrinkles, firming the skin, improving elasticity, and achieving whitening and anti-wrinkle effects are key demands. While traditional physical exfoliation methods can remove dead skin cells, they can also damage the skin barrier. This is why new enzyme technologies are emerging. For example, proteases extracted from plants can precisely break down keratin in the stratum corneum of the skin's aged surface, gently removing dead skin cells without disrupting the skin's normal tissue structure, laying a solid foundation for subsequent skincare benefits. For anti-wrinkle and firming, peptides are a hot topic. Peptides can block the release of neurotransmitters, relax muscles, and reduce the appearance of expression lines. They can also activate intracellular signaling pathways, promoting collagen and elastin synthesis, enhancing skin firmness and elasticity, and fundamentally improving sagging. To address this whitening need, researchers are turning to a variety of natural and synthetic active ingredients. Various plant extracts have been shown to inhibit tyrosinase activity, reducing melanin production at the source and achieving whitening benefits.

[0003] Currently, cosmetics with various functional properties have a wide variety of ingredients, with varying degrees of effectiveness. Excessive ingredients can disrupt skin absorption, leading to nutritional overload and potentially allergic side effects. However, too few ingredients can lead to ineffective results, failing to meet consumer expectations. Therefore, it is crucial to develop safe and effective ingredients that maximize synergistic effects, providing consumers with a more significant and long-lasting skincare experience, satisfying their diverse needs for exfoliation, firming, anti-wrinkle, and whitening, while also minimizing the burden on skin absorption. Summary of the Invention

[0004] The existing cosmetics contain a variety of ingredients with varying effects. Too many ingredients will increase the absorption burden on the skin, while too few ingredients will result in an unclear skin care effect. The present invention provides a preparation method and application of a bromelain composition, which contains specific ingredients of bromelain, superoxide dismutase, aloe amino acid polysaccharide, sturgeon collagen peptide and witch hazel extract. The sturgeon collagen peptide and witch hazel extract are obtained by special enzymatic hydrolysis and purification. The combination of the ingredients has good dead skin removal, whitening and anti-wrinkle effects, and the few types of ingredients will not cause absorption burden on the skin, making it safe and efficient. The specific technical scheme is as follows:

[0005] A method for preparing a bromelain composition comprises the following steps:

[0006] S1. Extraction of aloe amino acid polysaccharide: comprising peeling the aloe vera, beating the pulp, adding water to the pulp, enzymatically hydrolyzing the aloe vera with cellulase and pectinase, and then enzymatically hydrolyzing the aloe vera 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, and then adding ethanol for precipitation, collecting the precipitate to obtain precipitate B; mixing the supernatant A and precipitate B, and then concentrating and drying to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide;

[0007] S2, preparation of sturgeon collagen peptide: comprising beating fish skin, adding water to mix, adding trypsin for enzymatic hydrolysis, then co-enzymatic hydrolysis with serrapeptase and bromelain, centrifuging the enzymatic hydrolyzate, collecting supernatant C and ultrafiltration using an ultrafiltration membrane with a molecular weight of 4KDa to 6KDa, taking the ultrafiltrate for resin column adsorption, and then eluting, ultrafiltration using an ultrafiltration membrane with a molecular weight of 0.5KDa to 1.5KDa, selecting the ultrafiltration retentate, and drying to obtain sturgeon collagen peptide;

[0008] S3, preparing a witch hazel extract: comprising crushing the witch hazel, mixing with water, adding bromelain for enzymatic hydrolysis, then adding nattokinase for enzymatic hydrolysis, centrifuging the enzymatic hydrolyzate to collect a supernatant D, ultrafiltering the supernatant D using an ultrafiltration membrane with a molecular weight of 4 kDa to 6 kDa, adsorbing the ultrafiltrate on a resin column, and then eluting the eluate, ultrafiltering the eluate using an ultrafiltration membrane with a molecular weight of 0.5 kDa to 1.5 kDa, selecting the ultrafiltration retentate, and drying to obtain the witch hazel extract;

[0009] 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.

[0010] In S1 of the above preparation method, the extraction of aloe amino acid polysaccharide includes:

[0011] The peeled aloe vera leaf pulp is beaten to obtain a slurry, and distilled water 8 to 15 times the mass of the slurry is added to homogenize the slurry; 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 weight. After mixing, the mixture is enzymatically hydrolyzed at 40°C to 50°C for 2 to 4 hours, and then cooled to room temperature. The pH value is adjusted to 8.0 to 8.5, and 0.5% to 1.5% of trypsin by weight of the slurry is added to hydrolyze at 35°C to 38°C for 2 to 4 hours. The enzyme is inactivated in a boiling water bath, and after cooling to room temperature, the mixture is centrifuged to collect the supernatant A and the precipitate A; and the precipitate A is added to the precipitate A. 3 to 5 times the mass of distilled water are inoculated with 3% to 5% of the mass of precipitate A in terms of lactic acid bacteria and 1% to 3% of the mass of yeast, and the mixture is anaerobically fermented at 30°C to 35°C for 24 to 48 hours, sterilized, and a fermentation liquid is obtained; the fermentation liquid is centrifuged, and a supernatant B is collected; 3 to 5 times the volume of ethanol of the supernatant B is added to the supernatant B, the mixture is mixed, and the mixture is allowed to stand and settle at 4°C to 8°C for 12 to 24 hours, and then centrifuged to collect the precipitate B; the supernatant A and the precipitate B are mixed, concentrated under reduced pressure below 60°C, and dried below 60°C to a constant weight to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide.

[0012] In S2 of the above preparation method, the preparation of sturgeon collagen peptide includes:

[0013] The purified fish skin slices were beaten to obtain skin pulp, and distilled water 8 to 10 times the mass of the skin pulp was added to adjust the pH value to 8.0 to 8.5, and trypsin was added at 0.5% to 1.5% of the mass of the pulp, and the mixture was enzymatically hydrolyzed at 35°C to 38°C for 1h to 2h. The enzyme was inactivated in a boiling water bath, and the mixture was cooled to room temperature, and the pH value was adjusted to 6.5 to 7.0. Serrapeptase 0.5% to 1.5% of the mass of the pulp and bromelain 0.5% to 1.5% of the mass of the pulp were added, and the mixture was enzymatically hydrolyzed 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 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, and elute with 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, 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.

[0014] In S3 of the above preparation method, the preparation of the witch hazel extract includes:

[0015] Take witch hazel flowers, grind them to obtain a grind, add 8 to 10 times the weight of water to the grind, adjust the pH value to 6.0 to 8.0, add 0.5% to 1.5% of bromelain by weight of the grind, hydrolyze at 50 to 60 degrees Celsius for 1 hour to 1.5 hours, 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% of nattokinase by weight of the grind, hydrolyze at 50 to 55 degrees Celsius for 1 hour to 1.5 hours, inactivate the enzyme in a boiling water bath The enzyme was cooled to room temperature and centrifuged to collect the supernatant D, which was ultrafiltered using a 5 kDa ultrafiltration membrane, and the ultrafiltrate below 5 kDa was taken. The ultrafiltrate was loaded onto a resin column for adsorption, and 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 eluate was collected and ultrafiltered using a 1 kDa ultrafiltration membrane to retain the components above 1 kDa. The components above 1 kDa were lyophilized to obtain the witch hazel extract.

[0016] In S1 of the above preparation method, the rotation speed of the homogenizer is 10000 r / min to 15000 r / min, and the homogenization time is 3 min to 5 min.

[0017] 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.

[0018] In S2 of the above preparation method, the preparation method of the purified fish skin sheet comprises the following steps: taking freshly cleaned sturgeon skin, slicing to obtain fish skin sheets with a side length of less than 1 cm, adding anhydrous ethanol 3 to 5 times the mass of the fish skin sheets, stirring and degreasing at room temperature for 2 to 3 hours, filtering and replacing the anhydrous ethanol once every 30 minutes through a 100-mesh to 250-mesh sieve, finally filtering through a 100-mesh to 250-mesh sieve, taking an oversize material A, adding an aqueous hydrochloric acid solution with a concentration of 0.5 mol / L to 1.0 mol / L that is 5 to 8 times the mass of the oversize material A, stirring and decalcifying at 4° C. to 8° C. for 12 to 24 hours, filtering through a 100-mesh to 250-mesh sieve, taking an oversize material B, rinsing with distilled water until neutral, draining the water, and obtaining the purified fish skin sheet.

[0019] In S2 and S3 of the above preparation method, the resin of the resin column is an anion exchange resin.

[0020] 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.

[0021] 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.

[0022] In the above preparation method, the time for inactivating the enzyme in the boiling water bath is 10 min to 20 min.

[0023] In the above preparation method, the centrifugal speed is 5000r / min to 8000r / min, and the centrifugal time is 10min to 20min.

[0024] The invention relates to an application of the bromelain composition in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0025] The preparation method and application of a bromelain composition provided by the present invention have the following beneficial effects:

[0026] First, bromelain breaks down the protein in dead skin cells on the skin's surface, promoting their shedding and removing dead skin cells. It works by regulating skin cell metabolism and reducing melanin deposition. This boosts cell metabolism, helps repair damaged skin tissue, and reduces wrinkles.

[0027] Second, superoxide dismutase, an antioxidant, can scavenge free radicals in the skin. Free radicals can damage skin cells and cause wrinkles, dullness, and other problems. By scavenging free radicals, it delays skin aging, exerts anti-wrinkle and whitening effects, and also helps maintain normal skin cell metabolism, indirectly helping to remove dead skin.

[0028] The amino acids in aloe amino acid polysaccharides provide nutrients and nourish the skin. The polysaccharides have excellent moisturizing properties, forming a moisturizing film on the skin's surface to prevent dryness. When the skin is well-hydrated, the stratum corneum naturally sheds more easily, helping to remove dead skin cells. Moisturizing plumps the skin and reduces the appearance of wrinkles. The nutrients also promote the repair and regeneration of skin cells, achieving a whitening effect.

[0029] Fourth, Sturgeon Collagen Peptides replenish the skin's collagen peptides, increasing skin elasticity and reducing wrinkles. They fill the spaces between skin cells, tightening the skin and supporting it from within, reducing wrinkles and achieving anti-wrinkle effects. By promoting skin metabolism, new cells can better replace aging and dead skin cells. By improving skin microcirculation, the skin becomes more radiant and pigmentation is reduced.

[0030] 5. Witch hazel extract contains active ingredients that can regulate skin oil secretion, making it easier to shed the aging stratum corneum on the skin's surface, thereby removing dead skin cells. The active ingredients can inhibit the activity of melanocytes, reduce melanin production, stimulate the production of skin collagen, enhance skin elasticity, and reduce wrinkles.

[0031] 6. The sturgeon collagen peptide preparation method begins with defatting and decalcifying the skin to effectively remove impurities such as fat and calcium. This allows the subsequent enzymatic hydrolysis to more precisely target the collagen components and improve the purity of the collagen peptide. During the enzymatic hydrolysis process, trypsin is first used at an appropriate temperature and pH to initially decompose the collagen. This is then further hydrolyzed with serrapeptase and bromelain. The synergistic action of these enzymes more fully decomposes the collagen into specific small-molecule peptide fragments. These specific small-molecule collagen peptides are more effective and easily absorbed by the skin, thereby better replenishing collagen lost from the skin, enhancing skin elasticity, accelerating skin cell renewal for dead skin removal, and significantly improving skin microcirculation for whitening, with a more pronounced synergistic effect on anti-wrinkle effects. A series of meticulous separation and purification steps, including ultrafiltration and resin column adsorption elution, further remove impurities, resulting in high-purity, high-quality collagen peptides, which are more conducive to their dead skin removal, whitening, and anti-wrinkle benefits in cosmetics.

[0032] 7. In the preparation method of witch hazel extract, after the witch hazel flowers are crushed, they are first enzymatically hydrolyzed using bromelain at an appropriate pH and temperature. Bromelain can break down the protein components in the flower tissue, better releasing the active ingredients. Nattokinase is then used for further enzymatic hydrolysis. This dual enzymatic hydrolysis process can more fully disrupt the cell structure, releasing more active ingredients with dead skin removal, whitening, and anti-wrinkle effects. 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 dead skin exfoliation, inhibit melanin production, and stimulate collagen production, thereby achieving synergistic advantages in dead skin removal, whitening, and anti-wrinkle effects.

[0033] 8. Sturgeon collagen peptides primarily replenish skin collagen, increasing skin elasticity and reducing wrinkles. They also promote skin metabolism, help remove dead skin cells, and improve microcirculation for whitening. Witch hazel extract primarily regulates skin oil secretion, promoting dead skin exfoliation, inhibits melanin production for whitening, and stimulates collagen production to reduce wrinkles. The two work together to create a synergistic effect: Sturgeon collagen peptides promote skin cell renewal, while witch hazel extract regulates oil secretion for easier shedding. Together, they effectively remove dead skin cells from the skin's surface, leaving skin smoother and more refined. Regarding whitening, sturgeon collagen peptides improve microcirculation for a radiant complexion and reduced pigmentation, while witch hazel extract inhibits melanin production. Together, the two significantly reduce melanin, resulting in fairer and more translucent skin. 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

[0034] The present invention will be further described below with reference to specific implementation cases, but the present invention is not limited to these embodiments.

[0035] Example 1

[0036] A method for preparing a bromelain composition comprises the following steps:

[0037] S1, extraction of aloe amino acid polysaccharide:

[0038] The aloe leaf pulp is removed, and is beaten to obtain a slurry, 12 times the mass of distilled water is added, and the slurry is homogenized at a speed of 12000 r / min for 4 min; the pH value is adjusted to 5.0, 1.0% of the mass of the slurry of cellulase and 0.6% of the mass of the slurry of pectinase are added, after mixing, enzymolysis is carried out at 45℃ for 3h, the temperature is reduced to room temperature, the pH value is adjusted to 8.2, 1.0% of the mass of the slurry of trypsin is added, and enzymolysis is carried out at 37℃ for 3h, and the enzyme is inactivated by boiling in a water bath for 15 min; after the temperature is reduced to room temperature, the supernatant A and the precipitate A are collected by centrifugation at a speed of 6000 r / min for 15 min; 4 times the mass of distilled water is added to the precipitate A, and 4% of the mass of the precipitate A of lactic acid bacteria and 2% of the mass of the precipitate A of yeast are added, and anaerobic fermentation is carried out at 32℃ for 30h, and sterilization is carried out at 125℃ for 25 min to obtain a fermentation liquor; the fermentation liquor is centrifuged at a speed of 6000 r / min for 15 min, and the supernatant B is collected; 4 times the volume of ethanol is added to the supernatant B, and after mixing, the mixture is precipitated at 6℃ for 18h, and then centrifuged at a speed of 6000 r / min for 15 min, and the precipitate B is collected; the supernatant A and the precipitate B are mixed, concentrated under reduced pressure at 50℃, dried at 50℃ to a constant weight, and an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide, is obtained;

[0039] S2, preparation of sturgeon collagen peptide:

[0040] Fresh and washed sturgeon skin is sliced to obtain skin pieces with a side length of less than 1 cm, 4 times the mass of anhydrous ethanol is added, and the skin pieces are stirred to remove fat at room temperature for 2.5h, the anhydrous ethanol is replaced every 30 min by filtering through a 150-mesh screen, and finally the skin pieces are filtered through a 150-mesh screen to obtain the sieve residue A, 6 times the mass of the sieve residue A of 0.8 mol / L hydrochloric acid aqueous solution is added, and the mixture is stirred to remove calcium at 6℃ for 18h, and then filtered through a 150-mesh screen, and the sieve residue B is washed with distilled water until neutral, and then drained to obtain the purified skin pieces;

[0041] The purified fish skin slices were beaten to obtain skin pulp, and distilled water 9 times the mass of the skin pulp was added to adjust the pH value to 8.2, and 1.0% trypsin by mass of the pulp was added, and the mixture was enzymatically hydrolyzed at 37°C for 1.5h, and the enzyme was inactivated in a boiling water bath for 15min. After cooling to room temperature, the pH value was adjusted to 6.8, and 1.0% serrapeptase by mass of the pulp and 1.0% bromelain by mass of the pulp were added. The mixture was enzymatically hydrolyzed at 48°C for 1.5h, and the enzyme was inactivated in a boiling water bath for 15min. After cooling to room temperature, the mixture was rotated at 6000r / min. Centrifuge at a speed of 15 minutes, collect the supernatant C, perform ultrafiltration using a 5kDa ultrafiltration membrane, take the ultrafiltrate below 5kDa, load the ultrafiltrate through a resin column for adsorption, and elute with a 0.5 mol / L sodium chloride aqueous solution with a concentration of 2 resin column volumes at a flow rate of 1.5 mL / min. Collect all the eluates, perform ultrafiltration using a 1kDa ultrafiltration membrane, retain the components above 1kDa, and take the components above 1kDa for lyophilization to obtain sturgeon collagen peptide;

[0042] S3, preparation of witch hazel extract:

[0043] Take witch hazel flowers, crush them to obtain a crushed material, add 9 times the mass of water to the crushed material, adjust the pH value to 7.1, add 1.0% bromelain by mass of the crushed material, 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 material, enzymolyze at 52°C for 1 hour, inactivate the enzyme in a boiling water bath for 15 minutes, cool to room temperature, centrifuge at 6000 r / min for 15 minutes, collect the supernatant D, perform ultrafiltration using a 5 kDa ultrafiltration membrane, take the ultrafiltrate below 5 kDa, load the ultrafiltrate onto a resin column for adsorption, elute with 2 resin column volumes of a 0.8 mol / L sodium chloride aqueous solution at a flow rate of 1.5 mL / min, collect all the eluates, perform ultrafiltration using a 1 kDa ultrafiltration membrane, retain components above 1 kDa, and freeze-dry the components above 1 kDa to obtain a witch hazel extract;

[0044] S4, Mixed:

[0045] 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.

[0046] The invention relates to an application of the bromelain composition in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0047] Example 2

[0048] A method for preparing a bromelain composition comprises the following steps:

[0049] S1, extraction of aloe amino acid polysaccharide:

[0050] The peeled aloe vera 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 a speed of 10000 r / min for 3 minutes; the pH value was adjusted to 4.5, and 0.5% of cellulase and 0.3% of pectinase by mass of the slurry were added. After mixing, the mixture was enzymatically hydrolyzed at 40°C for 2 hours, cooled to room temperature, and the pH value was adjusted to 8.0. 0.5% of trypsin by mass of the slurry was added, and the mixture was enzymatically hydrolyzed at 35°C for 2 hours. The enzyme was inactivated in a boiling water bath for 10 minutes, cooled to room temperature, and centrifuged at a speed of 5000 r / min for 10 minutes, and the supernatant A and the precipitate A were collected; distilled water 3 times the mass of the precipitate A was added to the precipitate A. water, inoculated with 3% lactic acid bacteria and 1% yeast of the mass of sediment A, anaerobically fermented at 30°C for 24 hours, and sterilized at 121°C for 20 minutes to obtain a fermentation liquid; the fermentation liquid was centrifuged at 5000 r / min for 10 minutes, and the supernatant B was collected; ethanol 3 times the volume of supernatant B was added to the supernatant B, mixed, and allowed to stand at 4°C for 12 hours, and then centrifuged at 5000 r / min for 10 minutes to collect the precipitate B; the supernatant A and the precipitate B were mixed, concentrated under reduced pressure at 40°C, and dried at 40°C to constant weight to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide;

[0051] S2, Preparation of sturgeon collagen peptide:

[0052] Get freshly cleaned sturgeon fish-skin, slice, obtain the fish-skin sheet that length of side is less than 1cm, add the dehydrated alcohol of 3 times of quality of fish-skin sheet, at room temperature stir degreasing 2h, every 30min adopts 100 mesh screens to filter and change a dehydrated alcohol, finally filter through 100 mesh screens, get oversize A, add the aqueous hydrochloric acid of 0.5mol / L concentration of 5 times of oversize A quality, stir decalcification 12h at 4 ℃, adopt 100 mesh screens to filter, get oversize B, rinse with distilled water to neutral, drain the water, obtain purifying fish-skin sheet;

[0053] 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 was adjusted to 8.0, 0.5% of the slurry mass of trypsin was added, and enzymatic hydrolysis was carried out at 35°C for 1h, and the enzyme was inactivated in a boiling water bath for 10min. After cooling to room temperature, the pH was adjusted to 6.5, 0.5% of the slurry mass of serrapeptase and 0.5% of the slurry mass of bromelain were added, and enzymatic hydrolysis was carried out at 45°C for 1h, and the enzyme was inactivated in a boiling water bath for 10min. After cooling to room temperature, the supernatant C was collected and ultrafiltered using a 5kDa ultrafiltration membrane. The ultrafiltrate below 5kDa was taken, and the ultrafiltrate was loaded and adsorbed by a resin column. The mixture was eluted at a flow rate of 1mL / min with a sodium chloride aqueous solution having a concentration of 0.3mol / L for 2 resin column volumes. All eluates were collected and ultrafiltered using a 1kDa ultrafiltration membrane. The components above 1kDa were retained, and the components above 1kDa were lyophilized to obtain sturgeon collagen peptide.

[0054] S3, preparation of witch hazel extract:

[0055] Take witch hazel flowers, crush them to obtain a crushed material, add 8 times the mass of water to the crushed material, adjust the pH value to 6.0, add 0.5% bromelain by mass of the crushed material, enzymatically hydrolyze 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 material, enzymatically hydrolyze at 50°C for 1 hour, inactivate the enzyme in a boiling water bath for 10 minutes, cool to room temperature, centrifuge at 5000 r / min for 10 minutes, collect the supernatant D, perform ultrafiltration using a 5 kDa ultrafiltration membrane, take the ultrafiltrate below 5 kDa, load the ultrafiltrate onto a resin column for adsorption, elute with 2 resin column volumes of a 0.7 mol / L sodium chloride aqueous solution at a flow rate of 1 mL / min, collect all the eluates, perform ultrafiltration using a 1 kDa ultrafiltration membrane, retain components above 1 kDa, and freeze-dry the components above 1 kDa to obtain a witch hazel extract;

[0056] S4, Mixed:

[0057] 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.

[0058] The invention relates to an application of the bromelain composition in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0059] Example 3

[0060] A method for preparing a bromelain composition comprises the following steps:

[0061] S1, extraction of aloe amino acid polysaccharide:

[0062] The peeled aloe vera 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 minutes; 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. After mixing, the mixture was enzymatically hydrolyzed at 40°C for 4 hours, cooled to room temperature, and the pH value was adjusted to 8.0. 1.5% of trypsin by mass of the slurry was added, and the mixture was enzymatically hydrolyzed at 35°C for 4 hours. The enzyme was inactivated in a boiling water bath for 10 minutes, cooled to room temperature, and centrifuged at 8000 r / min for 10 minutes, 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, inoculated with 3% lactic acid bacteria and 3% yeast of the mass of sediment A, anaerobically fermented at 30°C for 48 hours, and sterilized at 121°C for 30 minutes to obtain a fermentation liquid; the fermentation liquid was centrifuged at 5000 r / min for 20 minutes, and the supernatant B was collected; ethanol in an amount 3 times the volume of supernatant B was added to the supernatant B, mixed, and allowed to stand at 8°C for 12 hours, and then centrifuged at 8000 r / min for 10 minutes to collect the precipitate B; the supernatant A and the precipitate B were mixed, concentrated under reduced pressure at 60°C, and dried at 40°C to constant weight to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide;

[0063] S2, Preparation of sturgeon collagen peptide:

[0064] Get freshly cleaned sturgeon fish-skin, slice, obtain the fish-skin sheet that length of side is less than 1cm, add the dehydrated alcohol of 5 times of quality of fish-skin sheet, at room temperature stir degreasing 2h, every 30min adopts 200 mesh screens to filter and change a dehydrated alcohol, finally filter through 200 mesh screens, get oversize A, add the aqueous hydrochloric acid of 0.5mol / L concentration of 8 times of oversize A quality, stir decalcification 12h at 8 ℃, adopt 200 mesh screens to filter, get oversize B, rinse with distilled water to neutral, drain the water, obtain purifying fish-skin sheet;

[0065] 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 was adjusted to 8.5, 0.5% of the slurry mass of trypsin was added, and enzymatic hydrolysis was carried out at 38°C for 1h, and the enzyme was inactivated in a boiling water bath for 20min. After cooling to room temperature, the pH was adjusted to 6.5, 1.5% of the slurry mass of serrapeptase and 0.5% of the slurry mass of bromelain were added, and enzymatic hydrolysis was carried out at 50°C for 1h, and the enzyme was inactivated in a boiling water bath for 20min. After cooling to room temperature, the supernatant C was collected and ultrafiltered using a 5kDa ultrafiltration membrane. The ultrafiltrate below 5kDa was taken, and the ultrafiltrate was loaded and adsorbed by a resin column. The mixture was eluted at a flow rate of 1mL / min with a sodium chloride aqueous solution at a concentration of 0.6mol / L for 2 resin column volumes. All eluates were collected and ultrafiltered using a 1kDa ultrafiltration membrane to intercept components above 1kDa. The components above 1kDa were lyophilized to obtain sturgeon collagen peptide.

[0066] S3, preparation of witch hazel extract:

[0067] Take witch hazel flowers, crush them to obtain a crushed material, add 10 times the mass of water to the crushed material, adjust the pH value to 6.0, add 1.5% bromelain by mass of the crushed material, enzymatically hydrolyze 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 material, enzymatically hydrolyze at 55°C for 1 hour, inactivate the enzyme in a boiling water bath for 20 minutes, cool to room temperature, centrifuge at 5000 r / min for 20 minutes, collect the supernatant D, perform ultrafiltration using a 5 kDa ultrafiltration membrane, take the ultrafiltrate below 5 kDa, load the ultrafiltrate onto a resin column for adsorption, elute with 2 resin column volumes of a 1.0 mol / L sodium chloride aqueous solution at a flow rate of 1 mL / min, collect all the eluates, perform ultrafiltration using a 1 kDa ultrafiltration membrane, retain components above 1 kDa, and freeze-dry the components above 1 kDa to obtain a witch hazel extract;

[0068] S4, Mixed:

[0069] 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.

[0070] The invention relates to an application of the bromelain composition in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0071] Example 4

[0072] A method for preparing a bromelain composition comprises the following steps:

[0073] S1, extraction of aloe amino acid polysaccharide:

[0074] The peeled aloe vera leaf pulp was beaten to obtain a slurry, distilled water 15 times the mass of the slurry was added, and the slurry was homogenized at 15000 r / min for 5 minutes; the pH value was adjusted to 5.5, 1.5% of cellulase by mass of the slurry and 1.0% of pectinase by mass of the slurry were added, mixed, and enzymatically hydrolyzed at 50°C for 4 hours, cooled to room temperature, the pH value was adjusted to 8.5, 1.5% of trypsin by mass of the slurry was added, and enzymatically hydrolyzed at 38°C for 4 hours, and the enzyme was inactivated in a boiling water bath for 20 minutes. After cooling to room temperature, 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, inoculated with 5% lactic acid bacteria and 3% yeast of the mass of sediment A, anaerobically fermented at 35°C for 48 hours, and sterilized at 130°C for 30 minutes to obtain a fermentation liquid; the fermentation liquid was centrifuged at 8000 r / min for 20 minutes, and the supernatant B was collected; ethanol 5 times the volume of supernatant B was added to the supernatant B, mixed, and allowed to stand at 8°C for 24 hours, and then centrifuged at 8000 r / min for 20 minutes to collect the sediment B; the supernatant A and the sediment B were mixed, concentrated under reduced pressure at 60°C, and dried at 60°C to constant weight to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide;

[0075] S2, Preparation of Sturgeon Collagen Peptide:

[0076] Get freshly cleaned sturgeon fish-skin, slice, obtain the fish-skin sheet that length of side is less than 1cm, add the dehydrated alcohol of 5 times of quality of fish-skin sheet, at room temperature stir degreasing 3h, every 30min adopts 250 mesh screens to filter and change a dehydrated alcohol, finally filter through 250 mesh screens, get oversize A, add the aqueous hydrochloric acid of 1.0mol / L concentration of 8 times of oversize A quality, stir decalcification 24h at 8 ℃, adopt 250 mesh screens to filter, get oversize B, rinse with distilled water to neutral, drain the water, obtain purifying fish-skin sheet;

[0077] 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, 1.5% of trypsin by mass of the pulp was added, and the mixture was enzymatically hydrolyzed at 38°C for 2h, and the enzyme was inactivated in a boiling water bath for 20min. After cooling to room temperature, the pH value was adjusted to 7.0, 1.5% of serrapeptase by mass of the pulp and 1.5% of bromelain by mass of the pulp were added, the mixture was enzymatically hydrolyzed at 50°C for 2h, and the enzyme was inactivated in a boiling water bath for 20min. After cooling to room temperature, the mixture was rotated at 8000r / min. The supernatant C was collected and ultrafiltered using a 5 kDa ultrafiltration membrane. The ultrafiltrate below 5 kDa was taken and loaded onto a resin column for adsorption. The mixture was eluted with a 0.6 mol / L sodium chloride aqueous solution at a flow rate of 2 mL / min using 3 resin column volumes. All the eluates were collected and ultrafiltered using a 1 kDa ultrafiltration membrane. The components above 1 kDa were retained and freeze-dried to obtain sturgeon collagen peptide.

[0078] S3, preparation of witch hazel extract:

[0079] Take witch hazel flowers, crush them to obtain a crushed material, add 10 times the mass of water to the crushed material, adjust the pH value to 8.0, add 1.5% bromelain by mass of the crushed material, enzymatically hydrolyze 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 material, enzymatically hydrolyze at 55°C for 1.5 hours, inactivate the enzyme in a boiling water bath for 20 minutes, cool to room temperature, centrifuge at 8000 r / min for 20 minutes, collect the supernatant D, perform ultrafiltration using a 5 kDa ultrafiltration membrane, take the ultrafiltrate below 5 kDa, load the ultrafiltrate onto a resin column for adsorption, elute with 3 resin column volumes of a 1.0 mol / L sodium chloride aqueous solution at a flow rate of 2 mL / min, collect all the eluates, perform ultrafiltration using a 1 kDa ultrafiltration membrane, retain components above 1 kDa, and freeze-dry the components above 1 kDa to obtain a witch hazel extract;

[0080] S4, Mixed:

[0081] 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.

[0082] The invention relates to an application of the bromelain composition in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0083] Example 5

[0084] A method for preparing a bromelain composition comprises the following steps:

[0085] S1, extraction of aloe amino acid polysaccharide:

[0086] The peeled aloe vera leaf pulp was beaten to obtain a slurry, distilled water 15 times the mass of the slurry was added, and the slurry was homogenized at 10000 r / min for 5 minutes; the pH value was adjusted to 4.5, 1.5% of the mass of cellulase and 0.3% of the mass of pectinase were added, mixed, and enzymatically hydrolyzed at 50°C for 2 hours, cooled to room temperature, the pH value was adjusted to 8.5, 0.5% of the mass of trypsin was added, and enzymatically hydrolyzed at 38°C for 2 hours, and the enzyme was inactivated in a boiling water bath for 20 minutes. After cooling to room temperature, the supernatant A and the precipitate A were collected; distilled water 3 times the mass of precipitate A was added to the precipitate A. water, inoculated with 5% lactic acid bacteria and 1% yeast of the mass of sediment A, anaerobically fermented at 35°C for 24 hours, and sterilized at 130°C for 20 minutes to obtain a fermentation liquid; the fermentation liquid was centrifuged at 8000 r / min for 10 minutes, and the supernatant B was collected; ethanol 5 times the volume of supernatant B was added to the supernatant B, mixed, and allowed to stand at 4°C for 24 hours, and then centrifuged at 5000 r / min for 20 minutes to collect the sediment B; the supernatant A and the sediment B were mixed, concentrated under reduced pressure at 40°C, and dried at 60°C to constant weight to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide;

[0087] S2, Preparation of sturgeon collagen peptide:

[0088] Get freshly cleaned sturgeon fish-skin, slice, obtain the fish-skin sheet that length of side is less than 1cm, add the dehydrated alcohol of 3 times of quality of fish-skin sheet, at room temperature stir degreasing 3h, every 30min adopts 150 mesh screens to filter and change a dehydrated alcohol, finally filter through 150 mesh screens, get oversize A, add the aqueous hydrochloric acid of 1.0mol / L concentration of 5 times of oversize A quality, stir decalcification 24h at 4 ℃, adopt 150 mesh screens to filter, get oversize B, rinse with distilled water to neutral, drain the water, obtain purifying fish-skin sheet;

[0089] 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, 1.5% trypsin 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 cooling to room temperature, the pH value was adjusted to 7.0, 0.5% serrapeptase by mass of the pulp and 1.5% bromelain 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 cooling 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 5kDa ultrafiltration membrane, take the ultrafiltrate below 5kDa, load the ultrafiltrate through a resin column for adsorption, and elute with a 0.3 mol / L sodium chloride aqueous solution with a flow rate of 2 mL / min for 3 resin column volumes. Collect all the eluates and perform ultrafiltration using a 1kDa ultrafiltration membrane to retain components above 1kDa. Take the components above 1kDa for freeze-drying to obtain sturgeon collagen peptide;

[0090] S3, preparation of witch hazel extract:

[0091] Take witch hazel flowers, crush them to obtain a crushed material, add 8 times the mass of water to the crushed material, adjust the pH value to 8.0, add 0.5% bromelain by mass of the crushed material, enzymatically hydrolyze 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 material, enzymatically hydrolyze at 50°C for 1.5 hours, inactivate the enzyme in a boiling water bath for 10 minutes, cool to room temperature, centrifuge at 8000 r / min for 10 minutes, collect the supernatant D, perform ultrafiltration using a 5 kDa ultrafiltration membrane, take the ultrafiltrate below 5 kDa, load the ultrafiltrate onto a resin column for adsorption, elute with 3 resin column volumes of a 0.7 mol / L sodium chloride aqueous solution at a flow rate of 2 mL / min, collect all the eluates, perform ultrafiltration using a 1 kDa ultrafiltration membrane, retain components above 1 kDa, and freeze-dry the components above 1 kDa to obtain a witch hazel extract;

[0092] S4, Mixed:

[0093] 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.

[0094] The invention relates to an application of the bromelain composition in preparing cosmetics with the effects of removing dead skin, whitening and anti-wrinkle.

[0095] In the above embodiments, the resin used in the resin column is an anion exchange resin, model D201MB, which is sourced from Tianjin Jinda Zhengtong Environmental Protection Technology Co., Ltd.

[0096] In the above embodiments, the sources of enzymes and bacteria are: 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 is 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.

[0097] Comparative Example 1

[0098] No precipitate B was added to the aloe amino acid polysaccharide, and other parameters and methods were the same as in Example 1.

[0099] Comparative Example 2

[0100] 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.

[0101] Comparative Example 3

[0102] 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.

[0103] Comparative Example 4

[0104] In the preparation of sturgeon collagen peptide, no serrapeptase was used for enzymatic hydrolysis, and no resin column was used for adsorption purification. The ultrafiltration fraction between 1 kDa and 5 kDa was intercepted and directly lyophilized to obtain sturgeon collagen peptide. Other parameters and methods were the same as those in Example 1.

[0105] Comparative Example 5

[0106] In the preparation of the witch hazel extract, nattokinase was not used for enzymatic hydrolysis, and other parameters and methods were the same as in Example 1.

[0107] Comparative Example 6

[0108] In the preparation of the witch hazel extract, nattokinase was not used for enzymatic hydrolysis, and resin columns were not used for adsorption purification. The ultrafiltration fraction between 1 kDa and 5 kDa was intercepted and directly freeze-dried to obtain the witch hazel extract. Other parameters and methods were the same as those in Example 1.

[0109] Comparative Example 7

[0110] No sturgeon collagen peptide was added to the composition, and other parameters and methods were the same as in Example 1.

[0111] Comparative Example 8

[0112] In the composition, no witch hazel extract was added, and other parameters and methods were the same as in Example 1.

[0113] Comparative Example 9

[0114] In the composition, sturgeon collagen peptide and witch hazel extract were not added at the same time, and other parameters and methods were the same as in Example 1.

[0115] Safety and efficacy tests were performed on the compositions of the above embodiments and comparative examples.

[0116] 1. Safety Testing

[0117] (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 approximately 5,000 cells per well. After culturing for 24 hours in DMEM high-glucose medium containing 10 wt% fetal bovine serum and 1 wt% penicillin-streptomycin, the cells were replaced with DMEM high-glucose medium containing a bromelain composition at a concentration of 0.5 mg / ml. A blank control group (cultured only with DMEM high-glucose medium) was set up. The cells were cultured for another 24 hours. The cell viability was detected by the MTT method. The absorbance OD value was measured at 490 nm using a microplate reader. The relative cell survival rate was calculated as: relative cell survival rate = (absorbance OD value of the experimental group / absorbance OD value of the blank control group) × 100%. The results are shown in Table 1 below.

[0118] (2) Skin irritation test: 140 healthy adult subjects (aged 18-55 years) 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 divided on the inner side of the forearm of the volunteers with a marker pen, with a 1 cm interval between the areas. 0.05 mL of the test sample containing the bromelain composition (concentration 0.5 mg / ml) was accurately aspirated with a micropipette and applied to the corresponding test area. The sample was gently applied evenly to cover the entire area, and then covered and fixed with breathable, non-irritating medical tape. The skin reaction in the test area was observed 4, 24, and 48 hours after application and scored according to the skin irritation scoring system developed by the International Contact Dermatitis Research Group (ICDRG): 0 indicates no erythema or edema; 1 indicates mild erythema and no edema; 2 indicates moderate erythema and mild edema; 3 indicates severe erythema and moderate edema; and 4 indicates severe erythema, edema, and associated papules, blisters, etc. The average score for each sample was calculated across all volunteers to assess its skin irritation level. Lower average scores indicate higher safety. The results are shown in Table 1 below.

[0119] Table 1 Safety test results

[0120]

[0121] 2. Efficacy Testing

[0122] (1) Dead skin removal effect test: 150 adult subjects (aged 25-48) with dry and wrinkled facial skin, a certain amount of keratin accumulation, and a dull complexion were selected and randomly divided into 15 groups (10 subjects in each group). One group was a blank control group, which used purified water without active ingredients as a placebo. 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:

[0123] Initial tactile evaluation before product use: Gently touch the test area with the pads of your index and middle fingers. Use moderate and even pressure to feel the skin's surface roughness, graininess, and elasticity. Seven days after using the product, perform a second tactile evaluation: Using the same technique and pressure as in the initial tactile evaluation, touch the test area again. Focus on detecting a reduction in roughness and graininess, any changes in elasticity, and whether the skin has become softer and smoother. The results are shown in Table 2 below.

[0124] Touch evaluation score:

[0125] 1-2: The entire face feels extremely rough, with a noticeable gritty feel, like sandpaper. Dead skin can be easily felt on various areas, including the cheeks, forehead, nose, and chin, resulting in a rough, thick texture. 3-4: The face has a noticeable roughness. Grains of dead skin can be felt with a gentle touch, particularly on the sides of the nose, around the lips, and in the center of the forehead. The skin lacks overall elasticity and feels somewhat hard and less supple, with a sense of a thick, uneven surface. 5-6: Some roughness is present, but the graininess is not particularly pronounced. Careful touch, such as gently rubbing the cheeks with the fingertips, reveals a small number of small grains, primarily in areas with pronounced skin texture. The skin has some elasticity, but it's subtle and relatively soft, but not delicate to the touch. 7-8: The face has only a slight sense of roughness, with almost no graininess. Only very slight signs of dead skin are detectable with very careful touch or in specific areas, such as the edge of the nose. The skin has good elasticity and is relatively soft, giving it a smooth feel. 9-10: Facial skin feels incredibly smooth, completely free of roughness or graininess, like the surface of finely polished jade. The skin is incredibly elastic, soft, and radiant, with no trace of dead skin. From forehead to chin, it feels incredibly delicate.

[0126] (2) Skin color measurement: While testing the dead skin removal effect, the L* (lightness) value was measured on the left and right cheeks of the subjects using a skin colorimeter (Minolta Chroma Meter CR-400) before and after 4 weeks of use. Each area was measured three times, and the average value of each area was taken as the color value. △L (L value after use - L value before use) indicates that the larger the positive △L value, the better the skin whitening effect. The results are shown in Table 2 below.

[0127] (3) Anti-wrinkle effect testing: Anti-wrinkle effect testing was performed simultaneously with skin colorimetry. A skin 3D imager (Visiometer® VC98) was used to capture cheek skin images before and four weeks after product use. The captured skin images were processed and analyzed using the accompanying image analysis software. Skin roughness (Ra), wrinkle depth (Rz), and wrinkle volume (Vv) were calculated. Changes in the values ​​of these parameters before and after sample use 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.

[0128] Table 2 Efficacy test results (average value)

[0129]

[0130] From the above experimental results, it can be seen that the bromelain compositions prepared in Examples 1 to 5 have safe ingredients, are non-cytotoxic and non-irritating to the skin, and can meet the needs of removing dead skin, whitening, firming and anti-wrinkle, etc.

[0131] 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 exfoliation, whitening and anti-wrinkle effects. Without adding precipitate B to aloe amino acid polysaccharide, the effective ingredients of the product are reduced, and the exfoliation, whitening and anti-wrinkle effects are reduced.

[0132] 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 exfoliation, 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 no resin column is used for adsorption purification. The effective ingredients of the product are reduced, and the exfoliation, whitening and anti-wrinkle effects are reduced.

[0133] 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 exfoliation, 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 a resin column is not used for adsorption purification. The effective ingredients of the product are reduced, and the exfoliation, whitening and anti-wrinkle effects are reduced.

[0134] From the results of Comparative Example 7, it can be seen that when sturgeon collagen peptide is not added to the composition, the exfoliating, whitening and anti-wrinkle effects are reduced; from the results of Comparative Example 8, it can be seen that when witch hazel extract is not added to the composition, the exfoliating, whitening and anti-wrinkle effects are reduced; from the results of Comparative Example 9, it can be seen that when both sturgeon collagen peptide and witch hazel extract are not added to the composition, 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 vera, homogenizing the pulp with water, enzymatically hydrolyzing the aloe vera with cellulase and pectinase, and then enzymatically hydrolyzing the aloe vera with trypsin, and collecting supernatant A and precipitate A by centrifugation; inoculating the precipitate A with lactic acid bacteria and yeast for fermentation, then centrifuging the precipitate A, collecting supernatant B, adding ethanol for precipitation, and collecting precipitate B; mixing the supernatant A and precipitate B, concentrating, and drying to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide; S2, preparation of sturgeon collagen peptide: comprising beating fish skin, adding water to mix, adding 0.5% to 1.5% of trypsin by weight of the slurry for enzymatic hydrolysis, adding 0.5% to 1.5% of serrapeptase by weight of the slurry and 0.5% to 1.5% of bromelain by weight of the slurry for enzymatic hydrolysis, collecting supernatant C by centrifugation and ultrafiltration using a 4KDa to 6KDa molecular weight ultrafiltration membrane, adsorbing the ultrafiltrate on a resin column, the resin being an anion exchange resin model D201MB, eluting with 2 to 3 resin column volumes of a sodium chloride aqueous solution, ultrafiltration of the eluate using a 0.5KDa to 1.5KDa molecular weight ultrafiltration membrane, and drying the ultrafiltration retentate to obtain sturgeon collagen peptide; S3, preparation of witch hazel extract: comprising crushing witch hazel, mixing with water, adding bromelain for enzymatic hydrolysis, adding nattokinase for enzymatic hydrolysis, collecting supernatant D by centrifugation, ultrafiltration of supernatant D using a 4 kDa to 6 kDa molecular weight ultrafiltration membrane, adsorbing the ultrafiltrate on a resin column using a D201MB anion exchange resin, eluting with 2 to 3 resin column volumes of a sodium chloride aqueous solution, ultrafiltration of the eluate using a 0.5 kDa to 1.5 kDa molecular weight ultrafiltration membrane, and drying the ultrafiltration retentate to obtain the 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 preparation method of a bromelain composition according to claim 1, wherein The extraction of aloe amino acid polysaccharide includes: taking peeled aloe leaf pulp, beating it to obtain a slurry, adding distilled water 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, 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, and sterilize to obtain a fermentation liquid; centrifuge the fermentation liquid and collect the supernatant B; add ethanol 3 to 5 times the volume of supernatant B to the supernatant B, mix, let stand at 4°C to 8°C for 12 to 24 hours, and then centrifuge to collect the precipitate B; mix the supernatant A and precipitate B, concentrate under reduced pressure below 60°C, and dry below 60°C to constant weight to obtain an extract containing amino acids and complex polysaccharides, i.e., aloe amino acid polysaccharide.

3. The preparation method of 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 8 to 10 times the mass of the skin pulp, adjusting the pH value to 8.0 to 8.5, adding trypsin in an amount of 0.5% to 1.5% by mass of the pulp, performing enzymatic hydrolysis 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 in an amount of 0.5% to 1.5% by mass of the pulp and bromelain in an amount of 0.5% to 1.5% by mass of the pulp, and boiling the mixture at 45 to 50° C. The enzyme was hydrolyzed for 1 to 2 hours, 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. The components above 1 kDa were retained and freeze-dried to obtain sturgeon collagen peptide.

4. The preparation method of a bromelain composition according to claim 1, wherein The preparation of witch hazel extract includes: 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 enzymatic hydrolysis at 50 to 60° C. for 1 hour to 1.5 hours, 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, and performing enzymatic hydrolysis at 50 to 55° C. for 1 hour to 1.5 hours. h, inactivate the enzyme in a boiling water bath, cool to room temperature, centrifuge, collect the supernatant D, perform ultrafiltration using a 5 kDa ultrafiltration membrane, take the ultrafiltrate below 5 kDa, load the ultrafiltrate through a resin column for adsorption, and elute with 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, perform ultrafiltration using a 1 kDa ultrafiltration membrane, retain components above 1 kDa, and lyophilize the components above 1 kDa to obtain a witch hazel extract.

5. The preparation method of a bromelain composition according to claim 3, wherein In S2, the concentration of the sodium chloride aqueous solution is 0.3 mol / L to 0.6 mol / L.

6. The method for preparing a bromelain composition according to claim 4, wherein In S3, the concentration of the sodium chloride aqueous solution is 0.7 mol / L to 1.0 mol / L.

7. The preparation method of a bromelain composition according to claim 2, 3 or 4, wherein The time for inactivating the enzyme in the boiling water bath is 10 to 20 minutes.

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

9. Use of a composition prepared by the method for preparing a bromelain composition according to claim 1 in preparing cosmetics having the effects of removing dead skin, whitening and anti-wrinkle.

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