An anti-aging composition containing sea cucumber peptide and a preparation method thereof

Abstract

This invention provides an anti-aging composition containing sea cucumber peptides and its preparation method, belonging to the field of cosmetic technology. Sea cucumber peptide complex, fermented snow lotus fruit extract, grape seed extract, and clam peptides are mixed at 23-25℃ for 10-15 min to obtain a mixture; xanthan gum and sodium carboxymethyl cellulose are dispersed in purified water at 85-90℃ and stirred for 20-30 min to obtain mixture A; mixture A, sodium hyaluronate, butylene glycol, hydroxyethyl urea, stearyl alcohol polyether-21, and glyceryl glucoside are mixed at 50-55℃ for 10-15 min to obtain mixture B; mixture B and the mixture are mixed at 40-45℃ for 20-30 min to obtain the anti-aging composition. In this invention, sea cucumber peptides and clam peptides synergistically stimulate collagen synthesis, achieving good anti-aging and moisturizing effects through the synergistic effect of multiple components.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to an anti-aging composition containing sea cucumber peptides and its preparation method. Background Technology

[0002] With the accelerating aging of the global population and the continuous improvement of people's pursuit of quality of life, anti-aging has become a popular research direction in the fields of beauty, skincare, and health care. Consumers' demands for anti-aging products are no longer limited to a single effect, but rather they expect products to achieve goals such as delaying aging, improving skin condition, and enhancing bodily functions from multiple dimensions. Traditional anti-aging compositions mostly rely on chemically synthesized ingredients or common natural extracts. While they can meet market demand to a certain extent, long-term use may pose safety risks, and the lasting and comprehensive effects are gradually failing to meet the growing needs of consumers.

[0003] Despite some progress in the research of anti-aging compositions, many challenges remain. In terms of ingredient extraction, many substances with potential anti-aging effects have limited sources, and the extraction process is complex, costly, and difficult to implement on a large scale. Even if extraction is successful, the active ingredients are easily damaged during the extraction process, leading to a reduction or even loss of their anti-aging activity.

[0004] In the formulation design stage, the interactions between different anti-aging ingredients are highly complex. Due to insufficient research on the synergistic or antagonistic effects between ingredients, many anti-aging compositions lack scientific rigor. This not only fails to fully leverage the advantages of each ingredient but may also trigger adverse reactions such as allergies due to improper ingredient combinations. Furthermore, existing anti-aging compositions generally suffer from insufficient stability, being significantly affected by environmental factors such as temperature, light, and humidity. During storage and transportation, active ingredients are prone to degradation or deterioration, leading to a shortened shelf life and a substantial reduction in anti-aging efficacy.

[0005] Chinese patent application CN109223651A discloses a skincare composition with whitening effects and its preparation method. The composition uses Scutellaria baicalensis to achieve whitening by inhibiting chronic inflammation, and Centella asiatica to increase skin elasticity and make the skin appear brighter. It works synergistically with rose and Phyllanthus emblica to inhibit tyrosinase activity, thus achieving antioxidant and whitening effects. However, in actual use, only a small portion of these active ingredients can penetrate the stratum corneum to reach the deeper layers of the skin, resulting in the active ingredients not being fully utilized.

[0006] Therefore, providing an anti-aging composition that can enhance the skincare effects of active ingredients is an important problem that urgently needs to be solved in this field. Summary of the Invention

[0007] This invention addresses the problems existing in the prior art by providing an anti-aging composition containing sea cucumber peptides and its preparation method, which can improve the skin care effect of active ingredients and exhibits good anti-aging and moisturizing properties.

[0008] Specifically, the technical solution of the present invention includes the following:

[0009] A method for preparing an anti-aging composition containing sea cucumber peptides, the method comprising the following steps:

[0010] Sea cucumber peptide complex, fermented snow lotus fruit extract, grape seed extract and clam peptide were mixed at 23~25℃ for 10~15 min to obtain a mixture;

[0011] Xanthan gum and sodium carboxymethyl cellulose were dispersed in purified water at 85-90℃ and stirred for 20-30 minutes to obtain mixed system A;

[0012] Mixture system A, sodium hyaluronate, butanediol, hydroxyethyl urea, stearyl alcohol polyether-21 and glyceryl glucoside are mixed at 50~55℃ for 10~15min to obtain mixture system B;

[0013] The anti-aging composition is obtained by mixing system B and the mixture at 40-45°C for 20-30 minutes.

[0014] Furthermore, the preparation method of the sea cucumber peptide complex includes the following steps:

[0015] After mixing sea cucumber fragments and primary phosphate buffer at a weight ratio of 1:8~10, homogenize the mixture at 2~4℃ for 10~15 minutes to obtain a homogenate.

[0016] Neutral protease was added to the homogenate for a single enzymatic hydrolysis to obtain a single hydrolysate.

[0017] Flavor protease and aminopeptidase are added to the primary enzymatic hydrolysate for secondary enzymatic hydrolysis to obtain a secondary enzymatic hydrolysate;

[0018] The secondary enzymatic hydrolysate and ammonium sulfate were mixed at a weight ratio of 10:2~3 and allowed to stand. The supernatant was collected to obtain sea cucumber peptides.

[0019] Sea cucumber peptide and maleimide-PEG2-succinimide ester were reacted at a weight ratio of 3~5:0.01~0.02 at 23~25℃ for 3~4h to obtain an intermediate;

[0020] The intermediate and 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol were reacted at a weight ratio of 7~9:0.05~0.07 at 23~25℃ for 8~12 h to obtain modified sea cucumber peptide;

[0021] A lipid membrane was obtained by rotary evaporation after mixing modified sea cucumber peptides, soybean lecithin, and cholesterol in a weight ratio of 1.5~2:12~20:3~4.5.

[0022] First phosphate buffer was added to the lipid membrane and swollen at 45-50℃ for 2-4 hours to obtain a liposome suspension.

[0023] Chitosan oligosaccharide was dispersed in phosphate buffer at a material-to-liquid ratio of 8-10:50 to obtain a chitosan oligosaccharide solution.

[0024] The sea cucumber peptide complex was prepared by reacting liposome suspension and chitosan oligosaccharide solution at a weight ratio of 10:10~15 at 23~25℃ for 3~4h.

[0025] Furthermore, the pH of the first phosphate buffer solution is 7.4.

[0026] Furthermore, the amount of the neutral protease used is 0.2% to 0.3% of the weight of the sea cucumber fragments.

[0027] Furthermore, the conditions for the first enzymatic hydrolysis include a hydrolysis pH of 6.8 to 7.2, a hydrolysis temperature of 45 to 50°C, and a hydrolysis time of 4 to 6 hours.

[0028] Furthermore, the amount of the flavor protease used is 0.4% to 0.5% of the weight of the sea cucumber fragments.

[0029] Furthermore, the amount of aminopeptidase used is 0.15% to 0.2% of the weight of the crushed sea cucumber.

[0030] Furthermore, the conditions for the secondary enzymatic hydrolysis include a hydrolysis pH of 7.2 to 7.6, a hydrolysis temperature of 50 to 55°C, and a hydrolysis time of 8 to 12 hours.

[0031] Furthermore, the conditions for mixing the secondary enzymatic hydrolysate and ammonium sulfate include a mixing temperature of 2-4°C and a mixing time of 1-2 hours.

[0032] Furthermore, the conditions for settling include a settling temperature of 2-4°C and a settling time of 10-12 hours.

[0033] Furthermore, the pH of the second phosphate buffer solution is 5.5.

[0034] Furthermore, the preparation method of the fermented yacon extract includes the following steps:

[0035] Yacon pulp fragments were extracted by soaking in 80% ethanol at a material-to-liquid ratio of 1:10-15 at 50-60℃ for 1.5-2.5 hours. The extract was then filtered and concentrated to obtain an extract. The extract was diluted with purified water to obtain a diluted solution, which was then fermented with lactic acid bacteria to obtain the fermented yacon extract.

[0036] Furthermore, the diluent is 15 to 20 times the weight of the extract.

[0037] Furthermore, the lactic acid bacteria include Lactobacillus fermentum or Lactobacillus plantarum.

[0038] Furthermore, the amount of lactic acid bacteria used is 1% to 5% of the weight of the chopped snow lotus fruit pulp.

[0039] Furthermore, the fermentation conditions include a treatment temperature of 37~40℃ and a treatment time of 72~96h.

[0040] Further, the weight ratio of the sea cucumber peptide complex, fermented snow lotus fruit extract, grape seed extract, clam peptide, xanthan gum, sodium carboxymethyl cellulose, sodium hyaluronate, butylene glycol, hydroxyethyl urea, stearyl alcohol polyether-21, glyceryl glucoside and purified water is 3~5:1~2:1~3:0.5~1.5:0.6~1.2:0.3~1:0.2~0.5:2~4:0.5~1:0.8~1.5:0.3~1:65~85.

[0041] An anti-aging composition prepared by a method for preparing an anti-aging composition containing sea cucumber peptides.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] (1) In this invention, neutral protease, flavor protease and aminopeptidase are used to synergistically process sea cucumber peptides with small molecular weight. The sea cucumber peptides are then modified to have phospholipid groups to obtain modified sea cucumber peptides. The modified sea cucumber peptides, soybean lecithin and cholesterol react to obtain liposomes that encapsulate the sea cucumber peptides. Subsequently, chitosan oligosaccharide is used to encapsulate the liposomes to obtain sea cucumber peptide complexes. The modified sea cucumber peptides can protect the peptide segments from skin enzyme degradation and improve the utilization rate of active ingredients. The chitosan oligosaccharide on the outer layer of the liposomes prolongs the residence time through electrostatic adsorption. The liposomes can carry the modified sea cucumber peptides into the lipid interstitial space of the stratum corneum and deliver them to the deep epidermis through fusion-release of small molecule peptides. This enhances the skin care effect of active ingredients and enhances the anti-aging and moisturizing properties of the anti-aging composition.

[0044] (2) In this invention, the sea cucumber peptide complex can provide small molecule active peptides that are easily absorbed by the skin, promote collagen synthesis, repair damaged cells, and enhance skin elasticity; the hydrophilic peptide segments in the sea cucumber peptide lock in water, which can repair the barrier and reduce water loss; the yacon extract has enhanced bioactivity after being fermented by lactic acid bacteria, which further enhances the moisturizing effect; sodium hyaluronate, butylene glycol and hydroxyethyl urea have moisture-absorbing and water-locking effects; glyceryl glucoside can stimulate the synthesis of endogenous moisturizing factors; chitosan oligosaccharide and liposomes can repair and strengthen the skin barrier; the fermented yacon extract has enhanced bioactivity, which further enhances the moisturizing effect; and the above substances work together in multiple dimensions to achieve a long-lasting moisturizing effect.

[0045] (3) In this invention, sea cucumber peptide and clam peptide work together to stimulate collagen synthesis, grape seed extract effectively removes free radicals and has a strong antioxidant effect, strengthens capillary walls and improves microcirculation; glycerol glucoside can activate the self-repair mechanism of skin cells and enhance cell vitality, etc. Through the synergistic effect of multiple components, a good anti-aging effect is achieved. Detailed Implementation

[0046] The technical solution of the present invention will be clearly and completely described below through embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Unless otherwise stated, all raw materials and reagents used in this invention are commercially available or can be prepared by known methods.

[0048] Preparation Example 1:

[0049] The preparation method of sea cucumber peptide complex includes the following steps:

[0050] After washing the fresh sea cucumber with clean water, remove the internal organs, take the body wall, clean the body wall with distilled water, and grind it in a mincer for 3 minutes to obtain sea cucumber fragments. Mix 10 parts by weight of sea cucumber fragments with 80 parts by weight of first phosphate buffer solution with pH 7.4, and homogenize at 2℃ for 10 minutes to obtain a homogenate.

[0051] 0.02 parts by weight of neutral protease were added to the homogenate, the pH was adjusted to 6.8, and the temperature was raised to 45°C. The mixture was stirred for 4 hours for the first enzymatic hydrolysis. After the first enzymatic hydrolysis, the temperature was raised to 90°C for 10 minutes to inactivate the enzyme and obtain the first enzymatic hydrolysate. The temperature of the first enzymatic hydrolysate was adjusted to 50°C, 0.04 parts by weight of flavor protease and 0.015 parts by weight of aminopeptidase were added, and the pH was adjusted to 7.2. The mixture was stirred for 8 hours for the second enzymatic hydrolysis. After the second enzymatic hydrolysis, the temperature was raised to 90°C for 10 minutes to inactivate the enzyme and obtain the second enzymatic hydrolysate.

[0052] After cooling 100 parts by weight of the secondary enzymatic hydrolysate to 2°C, 20 parts by weight of ammonium sulfate were added while stirring. The reaction was carried out at 2°C for 1 hour. After the reaction was completed, the mixture was allowed to stand at 2°C for 10 hours. The supernatant was collected by centrifugation at 8000 r / min for 20 min. The supernatant was filtered through a 1000 Da molecular sieve ultrafiltration membrane to collect the peptides and obtain sea cucumber peptides.

[0053] Three parts by weight of sea cucumber peptide and 0.01 parts by weight of maleimide-PEG2-succinimide ester were dispersed in 50 parts by weight of pH 7.4 first phosphate buffer. The reaction was carried out under nitrogen protection at 23°C for 3 hours with stirring. After the reaction was completed, the reaction was terminated by ultrafiltration and unreacted small molecules were removed to obtain the intermediate.

[0054] Seven parts by weight of the intermediate and 0.05 parts by weight of 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol were dispersed in 50 parts by weight of pH 7.4 first phosphate buffer. The reaction was carried out under nitrogen protection at 23°C for 8 h with stirring. After the reaction was completed, the solution was dialyzed for 24 h using a 3500 Da dialysis bag to obtain the dialysate. The dialysate was then sterilized by passing through a 0.22 μm filter membrane and freeze-dried to obtain the modified sea cucumber peptide.

[0055] 1.5 parts by weight of modified sea cucumber peptide, 12 parts by weight of soybean lecithin and 3 parts by weight of cholesterol were dispersed in 500 parts by weight of ethanol, transferred to a flask and heated to 40°C for rotary evaporation to obtain a lipid membrane; 500 parts by weight of pH 7.4 first phosphate buffer preheated to 45°C were added, the lipid membrane detached when the flask was rotated, and allowed to swell at 45°C for 2 hours. After swelling, the liposome suspension was obtained by sonication.

[0056] Eight parts by weight of chitosan oligosaccharide were dispersed in 500 parts by weight of pH 5.5 second phosphate buffer to obtain a chitosan oligosaccharide solution; 10 parts by weight of liposome suspension and 10 parts by weight of chitosan oligosaccharide solution were mixed and stirred at 23°C for 3 hours. After the reaction was completed, the sea cucumber peptide complex was obtained by dialysis and freeze-drying.

[0057] Preparation Example 2:

[0058] The preparation method of sea cucumber peptide complex includes the following steps:

[0059] After washing the fresh sea cucumber with clean water, remove the internal organs, take the body wall, clean the body wall with distilled water, and grind it in a mincer for 3.5 minutes to obtain sea cucumber fragments. Mix 10 parts by weight of sea cucumber fragments with 85 parts by weight of pH 7.4 first phosphate buffer, and homogenize at 2.5℃ for 11 minutes to obtain a homogenate.

[0060] 0.022 parts by weight of neutral protease were added to the homogenate, the pH was adjusted to 6.9 and the temperature was raised to 46°C. The mixture was stirred and reacted for 4.5 h for the first enzymatic hydrolysis. After the first enzymatic hydrolysis, the temperature was raised to 91°C for 11 min to inactivate the enzyme and obtain the first enzymatic hydrolysate. The temperature of the first enzymatic hydrolysate was adjusted to 51°C, 0.042 parts by weight of flavor protease and 0.016 parts by weight of aminopeptidase were added, and the pH was adjusted to 7.3. The mixture was stirred and reacted for 9 h for the second enzymatic hydrolysis. After the second enzymatic hydrolysis, the temperature was raised to 91°C for 11 min to inactivate the enzyme and obtain the second enzymatic hydrolysate.

[0061] After cooling 100 parts by weight of the secondary enzymatic hydrolysate to 2.5℃, 22 parts by weight of ammonium sulfate were added while stirring. The reaction was carried out at 2.5℃ for 1.2 h. After the reaction was completed, the mixture was allowed to stand at 2.5℃ for 10.5 h. The supernatant was collected by centrifugation at 8000 r / min for 20 min. The supernatant was filtered through a 500 Da molecular sieve ultrafiltration membrane to collect the peptides and obtain sea cucumber peptides.

[0062] 3.5 parts by weight of sea cucumber peptide and 0.012 parts by weight of maleimide-PEG2-succinimide ester were dispersed in 50 parts by weight of pH 7.4 first phosphate buffer and reacted at 24°C for 3.2 h under nitrogen protection. After the reaction was completed, the reaction was terminated by ultrafiltration and unreacted small molecules were removed to obtain the intermediate.

[0063] 7.5 parts by weight of the intermediate and 0.055 parts by weight of 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol were dispersed in 50 parts by weight of pH 7.4 first phosphate buffer and stirred at 24°C for 9 h under nitrogen protection. After the reaction, the solution was dialyzed for 30 h using a 3500 Da dialysis bag to obtain the dialysate. The dialysate was then sterilized by passing through a 0.22 μm filter membrane and freeze-dried to obtain the modified sea cucumber peptide.

[0064] 1.6 parts by weight of modified sea cucumber peptide, 14 parts by weight of soybean lecithin and 3.4 parts by weight of cholesterol were dispersed in 500 parts by weight of ethanol, transferred to a flask and heated to 43°C for rotary evaporation to obtain a lipid membrane; 500 parts by weight of phosphate buffer (pH 7.4) preheated to 46°C were added, and the lipid membrane detached when the flask was rotated, and the membrane was allowed to swell at 46°C for 2.5 h. After swelling, the membrane was homogenized by sonication to obtain a liposome suspension.

[0065] 8.5 parts by weight of chitosan oligosaccharide were dispersed in 500 parts by weight of pH 5.5 phosphate buffer to obtain a chitosan oligosaccharide solution; 10 parts by weight of liposome suspension and 11 parts by weight of chitosan oligosaccharide solution were mixed and stirred at 24°C for 3.2 h. After the reaction was completed, the sea cucumber peptide complex was obtained by dialysis and freeze drying.

[0066] Preparation Example 3:

[0067] The preparation method of sea cucumber peptide complex includes the following steps:

[0068] After washing the fresh sea cucumber with clean water and removing the internal organs, take its body wall, clean the body wall with distilled water, and grind it in a mincer for 4 minutes to obtain sea cucumber fragments. Mix 10 parts by weight of sea cucumber fragments with 90 parts by weight of first phosphate buffer solution with pH 7.4, and homogenize at 3℃ for 12 minutes to obtain a homogenate.

[0069] 0.024 parts by weight of neutral protease were added to the homogenate, the pH was adjusted to 7.0, and the temperature was raised to 47°C. The mixture was stirred for 5 hours for the first enzymatic hydrolysis. After the first enzymatic hydrolysis, the temperature was raised to 92°C for 12 minutes to inactivate the enzyme and obtain the first enzymatic hydrolysate. The temperature of the first enzymatic hydrolysate was adjusted to 52°C, 0.044 parts by weight of flavor protease and 0.017 parts by weight of aminopeptidase were added, and the pH was adjusted to 7.4. The mixture was stirred for 10 hours for the second enzymatic hydrolysis. After the second enzymatic hydrolysis, the temperature was raised to 92°C for 12 minutes to inactivate the enzyme and obtain the second enzymatic hydrolysate.

[0070] After cooling 100 parts by weight of the secondary enzymatic hydrolysate to 3°C, 24 parts by weight of ammonium sulfate were added while stirring. The reaction was carried out at 3°C ​​for 1.4 h. After the reaction was completed, the mixture was allowed to stand at 3°C ​​for 11 h. The supernatant was collected by centrifugation at 8000 r / min for 20 min. The supernatant was filtered through a 600 Da molecular sieve ultrafiltration membrane to collect the peptides and obtain sea cucumber peptides.

[0071] Four parts by weight of sea cucumber peptide and 0.014 parts by weight of maleimide-PEG2-succinimide ester were dispersed in 50 parts by weight of pH 7.4 first phosphate buffer and reacted at 24°C for 3.4 h under nitrogen protection. After the reaction was completed, the reaction was terminated by ultrafiltration and unreacted small molecules were removed to obtain the intermediate.

[0072] Eight parts by weight of the intermediate and 0.06 parts by weight of 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol were dispersed in 50 parts by weight of pH 7.4 first phosphate buffer. The mixture was stirred and reacted at 24°C for 10 h under nitrogen protection. After the reaction, the mixture was dialyzed for 36 h using a 3500 Da dialysis bag to obtain the dialysate. The dialysate was then sterilized by passing it through a 0.22 μm filter membrane and freeze-dried to obtain the modified sea cucumber peptide.

[0073] 1.7 parts by weight of modified sea cucumber peptide, 16 parts by weight of soybean lecithin and 3.7 parts by weight of cholesterol were dispersed in 500 parts by weight of ethanol, transferred to a flask and heated to 46°C for rotary evaporation to obtain a lipid membrane; 500 parts by weight of phosphate buffer (pH 7.4) preheated to 47°C were added, and the lipid membrane detached when the flask was rotated, and the membrane was allowed to swell at 47°C for 3 hours. After swelling, the membrane was homogenized by sonication to obtain a liposome suspension.

[0074] Nine parts by weight of chitosan oligosaccharide were dispersed in 500 parts by weight of pH 5.5 phosphate buffer to obtain a chitosan oligosaccharide solution; 10 parts by weight of liposome suspension and 12 parts by weight of chitosan oligosaccharide solution were mixed and stirred at 24°C for 3.4 h. After the reaction was completed, the sea cucumber peptide complex was obtained by dialysis and freeze-drying.

[0075] Preparation Example 4:

[0076] The preparation method of sea cucumber peptide complex includes the following steps:

[0077] After washing the fresh sea cucumber with clean water, remove the internal organs, take the body wall, clean the body wall with distilled water, and grind it in a mincer for 4.5 minutes to obtain sea cucumber fragments. Mix 10 parts by weight of sea cucumber fragments with 95 parts by weight of pH 7.4 first phosphate buffer, and homogenize at 3.5℃ for 13 minutes to obtain a homogenate.

[0078] 0.027 parts by weight of neutral protease were added to the homogenate, the pH was adjusted to 7.1 and the temperature was raised to 48°C. The mixture was stirred for 5.5 hours for the first enzymatic hydrolysis. After the first enzymatic hydrolysis, the temperature was raised to 93°C for 14 minutes to inactivate the enzyme and obtain the first enzymatic hydrolysate. The temperature of the first enzymatic hydrolysate was adjusted to 54°C, 0.047 parts by weight of flavor protease and 0.018 parts by weight of aminopeptidase were added, and the pH was adjusted to 7.5. The mixture was stirred for 11 hours for the second enzymatic hydrolysis. After the second enzymatic hydrolysis, the temperature was raised to 94°C for 13 minutes to inactivate the enzyme and obtain the second enzymatic hydrolysate.

[0079] After cooling 100 parts by weight of the secondary enzymatic hydrolysate to 3.5℃, 27 parts by weight of ammonium sulfate were added while stirring. The reaction was carried out at 3℃ for 1.7h. After the reaction was completed, the mixture was allowed to stand at 3℃ for 11.5h. The supernatant was collected by centrifugation at 8000r / min for 20min. The supernatant was filtered through a 700Da molecular sieve ultrafiltration membrane to collect the peptides and obtain sea cucumber peptides.

[0080] 4.5 parts by weight of sea cucumber peptide and 0.017 parts by weight of maleimide-PEG2-succinimide ester were dispersed in 50 parts by weight of pH 7.4 first phosphate buffer and reacted at 25°C for 3.7 h under nitrogen protection. After the reaction was completed, the reaction was terminated by ultrafiltration and unreacted small molecules were removed to obtain the intermediate.

[0081] 8.5 parts by weight of the intermediate and 0.065 parts by weight of 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol were dispersed in 50 parts by weight of pH 7.4 first phosphate buffer. The reaction was carried out under nitrogen protection at 25°C for 11 h with stirring. After the reaction was completed, the solution was dialyzed through a 3500 Da dialysis bag for 42 h to obtain the dialysate. The dialysate was then sterilized by passing through a 0.22 μm filter membrane and freeze-dried to obtain the modified sea cucumber peptide.

[0082] 1.8 parts by weight of modified sea cucumber peptide, 18 parts by weight of soybean lecithin and 4.2 parts by weight of cholesterol were dispersed in 500 parts by weight of ethanol, transferred to a flask and heated to 48°C for rotary evaporation to obtain a lipid membrane; 500 parts by weight of pH 7.4 first phosphate buffer preheated to 47°C were added, and the lipid membrane detached when the flask was rotated, and the membrane was allowed to swell at 47°C for 3.5 hours. After swelling, the membrane was homogenized by sonication to obtain a liposome suspension.

[0083] 9.5 parts by weight of chitosan oligosaccharide were dispersed in 500 parts by weight of pH 5.5 phosphate buffer to obtain a chitosan oligosaccharide solution; 10 parts by weight of liposome suspension and 13 parts by weight of chitosan oligosaccharide solution were mixed and stirred at 25°C for 3.7 h. After the reaction was completed, the sea cucumber peptide complex was obtained by dialysis and freeze drying.

[0084] Preparation Example 5:

[0085] The preparation method of sea cucumber peptide complex includes the following steps:

[0086] After washing the fresh sea cucumber with clean water, remove the internal organs, take the body wall, clean the body wall with distilled water, and grind it in a mincer for 5 minutes to obtain sea cucumber fragments. Mix 10 parts by weight of sea cucumber fragments with 100 parts by weight of first phosphate buffer solution with pH 7.4, and homogenize at 4℃ for 15 minutes to obtain a homogenate.

[0087] 0.03 parts by weight of neutral protease were added to the homogenate, the pH was adjusted to 7.2, and the temperature was raised to 50°C. The mixture was stirred for 6 hours for the first enzymatic hydrolysis. After the first enzymatic hydrolysis, the temperature was raised to 95°C for 15 minutes to inactivate the enzyme and obtain the first enzymatic hydrolysate. The temperature of the first enzymatic hydrolysate was adjusted to 55°C, 0.05 parts by weight of flavor protease and 0.02 parts by weight of aminopeptidase were added, and the pH was adjusted to 7.6. The mixture was stirred for 12 hours for the second enzymatic hydrolysis. After the second enzymatic hydrolysis, the temperature was raised to 95°C for 15 minutes to inactivate the enzyme and obtain the second enzymatic hydrolysate.

[0088] After cooling 100 parts by weight of the secondary enzymatic hydrolysate to 4°C, 30 parts by weight of ammonium sulfate were added while stirring. The reaction was carried out at 4°C for 2 hours. After the reaction was completed, the mixture was allowed to stand at 4°C for 12 hours. The supernatant was collected by centrifugation at 8000 r / min for 20 min. The supernatant was filtered through a 1000 Da molecular sieve ultrafiltration membrane to collect the peptides and obtain sea cucumber peptides.

[0089] Five parts by weight of sea cucumber peptide and 0.02 parts by weight of maleimide-PEG2-succinimide ester were dispersed in 50 parts by weight of first phosphate buffer at pH 7.4. The reaction was stirred at 25°C for 4 hours under nitrogen protection. After the reaction was completed, the reaction was terminated by ultrafiltration and unreacted small molecules were removed to obtain the intermediate.

[0090] Nine parts by weight of the intermediate and 0.07 parts by weight of 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol were dispersed in 50 parts by weight of pH 7.4 first phosphate buffer. The reaction was carried out under nitrogen protection at 25°C for 12 h with stirring. After the reaction was completed, the solution was dialyzed through a 3500 Da dialysis bag for 48 h to obtain the dialysate. The dialysate was then sterilized by passing through a 0.22 μm filter membrane and freeze-dried to obtain the modified sea cucumber peptide.

[0091] Two parts by weight of modified sea cucumber peptide, 20 parts by weight of soybean lecithin and 4.5 parts by weight of cholesterol were dispersed in 500 parts by weight of ethanol, transferred to a flask and heated to 50°C for rotary evaporation to obtain a lipid membrane; 500 parts by weight of phosphate buffer (pH 7.4) preheated to 50°C were added, and the lipid membrane detached when the flask was rotated, and the membrane was allowed to swell at 50°C for 4 hours. After swelling, the membrane was homogenized by sonication to obtain a liposome suspension.

[0092] Ten parts by weight of chitosan oligosaccharide were dispersed in 500 parts by weight of pH 5.5 phosphate buffer to obtain a chitosan oligosaccharide solution; ten parts by weight of liposome suspension and 15 parts by weight of chitosan oligosaccharide solution were mixed and stirred at 25°C for 4 hours. After the reaction was completed, the sea cucumber peptide complex was obtained by dialysis and freeze-drying.

[0093] Preparation Example 6:

[0094] The preparation method of sea cucumber peptide complex includes the following steps:

[0095] After washing the fresh sea cucumber with clean water, remove the internal organs, take the body wall, clean the body wall with distilled water, and grind it in a mincer for 5 minutes to obtain sea cucumber fragments. Mix 10 parts by weight of sea cucumber fragments with 100 parts by weight of first phosphate buffer solution with pH 7.4, and homogenize at 4℃ for 15 minutes to obtain a homogenate.

[0096] 0.03 parts by weight of neutral protease were added to the homogenate, the pH was adjusted to 7.2, and the temperature was raised to 50°C. The mixture was stirred for 6 hours for the first enzymatic hydrolysis. After the first enzymatic hydrolysis, the temperature was raised to 95°C for 15 minutes to inactivate the enzyme and obtain the first enzymatic hydrolysate. The temperature of the first enzymatic hydrolysate was adjusted to 55°C, 0.05 parts by weight of flavor protease and 0.02 parts by weight of aminopeptidase were added, and the pH was adjusted to 7.6. The mixture was stirred for 12 hours for the second enzymatic hydrolysis. After the second enzymatic hydrolysis, the temperature was raised to 95°C for 15 minutes to inactivate the enzyme and obtain the second enzymatic hydrolysate.

[0097] After cooling 100 parts by weight of the secondary enzymatic hydrolysate to 4°C, 30 parts by weight of ammonium sulfate were added while stirring. The reaction was carried out at 4°C for 2 hours. After the reaction was completed, the mixture was allowed to stand at 4°C for 12 hours. The supernatant was collected by centrifugation at 8000 r / min for 20 min. The supernatant was filtered through a 1000 Da molecular sieve ultrafiltration membrane to collect the peptides and obtain sea cucumber peptides.

[0098] Two parts by weight of sea cucumber peptide, 20 parts by weight of soybean lecithin and 4.5 parts by weight of cholesterol were dispersed in 500 parts by weight of ethanol, transferred to a flask and heated to 50°C for rotary evaporation to obtain a lipid membrane; 500 parts by weight of phosphate-buffered saline (PBS) preheated to 50°C and pH 7.4 were added, and the lipid membrane detached when the flask was rotated, and the membrane was allowed to swell at 50°C for 4 hours. After swelling, the membrane was homogenized by sonication to obtain a liposome suspension.

[0099] Ten parts by weight of chitosan oligosaccharide were dispersed in 500 parts by weight of pH 5.5 phosphate buffer to obtain a chitosan oligosaccharide solution; ten parts by weight of liposome suspension and 15 parts by weight of chitosan oligosaccharide solution were mixed and stirred at 25°C for 4 hours. After the reaction was completed, the sea cucumber peptide complex was obtained by dialysis and freeze-drying.

[0100] Preparation Example 7:

[0101] The preparation method of sea cucumber peptide complex includes the following steps:

[0102] In Preparation Example 5, 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol was replaced with distearylphosphatidylethanolamine-polyethylene glycol-thiol, and all other operations were the same as in Preparation Example 5.

[0103] Preparation Example 8:

[0104] The preparation method of sea cucumber peptide complex includes the following steps:

[0105] After washing the fresh sea cucumber with clean water, remove the internal organs, take the body wall, clean the body wall with distilled water, and grind it in a mincer for 5 minutes to obtain sea cucumber fragments. Mix 10 parts by weight of sea cucumber fragments with 100 parts by weight of first phosphate buffer solution with pH 7.4, and homogenize at 4℃ for 15 minutes to obtain a homogenate.

[0106] 0.03 parts by weight of neutral protease were added to the homogenate, the pH was adjusted to 7.2, and the temperature was raised to 50°C. The mixture was stirred for 6 hours for the first enzymatic hydrolysis. After the first enzymatic hydrolysis, the temperature was raised to 95°C for 15 minutes to inactivate the enzyme and obtain the first enzymatic hydrolysate. The temperature of the first enzymatic hydrolysate was adjusted to 55°C, 0.05 parts by weight of flavor protease and 0.02 parts by weight of aminopeptidase were added, and the pH was adjusted to 7.6. The mixture was stirred for 12 hours for the second enzymatic hydrolysis. After the second enzymatic hydrolysis, the temperature was raised to 95°C for 15 minutes to inactivate the enzyme and obtain the second enzymatic hydrolysate.

[0107] After cooling 100 parts by weight of the secondary enzymatic hydrolysate to 4°C, 30 parts by weight of ammonium sulfate were added while stirring. The reaction was carried out at 4°C for 2 hours. After the reaction was completed, the mixture was allowed to stand at 4°C for 12 hours. The supernatant was collected by centrifugation at 8000 r / min for 20 min. The supernatant was filtered through a 1000 Da molecular sieve ultrafiltration membrane to collect the peptides and obtain sea cucumber peptides.

[0108] Five parts by weight of sea cucumber peptide and 0.02 parts by weight of maleimide-PEG2-succinimide ester were dispersed in 50 parts by weight of first phosphate buffer at pH 7.4. The reaction was stirred at 25°C for 4 hours under nitrogen protection. After the reaction was completed, the reaction was terminated by ultrafiltration and unreacted small molecules were removed to obtain the intermediate.

[0109] Nine parts by weight of the intermediate and 0.07 parts by weight of 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol were dispersed in 50 parts by weight of pH 7.4 first phosphate buffer. The reaction was carried out under nitrogen protection at 25°C for 12 h with stirring. After the reaction was completed, the solution was dialyzed through a 3500 Da dialysis bag for 48 h to obtain the dialysate. The dialysate was then sterilized by passing through a 0.22 μm filter membrane and freeze-dried to obtain the modified sea cucumber peptide.

[0110] Two parts by weight of modified sea cucumber peptide, 20 parts by weight of soybean lecithin and 4.5 parts by weight of cholesterol were dispersed in 500 parts by weight of ethanol, transferred to a flask and heated to 50°C for rotary evaporation to obtain a lipid membrane; 100 parts by weight of first phosphate buffer with pH 7.4 preheated to 50°C were added, and the lipid membrane detached when the flask was rotated, and the membrane was allowed to swell at 50°C for 4 hours. After swelling, the membrane was homogenized by ultrasound to obtain the sea cucumber peptide complex.

[0111] Preparation Example 9:

[0112] The preparation method of sea cucumber peptides includes the following steps:

[0113] After washing the fresh sea cucumber with clean water, remove the internal organs, take the body wall, clean the body wall with distilled water, and grind it in a mincer for 5 minutes to obtain sea cucumber fragments. Mix 10 parts by weight of sea cucumber fragments with 100 parts by weight of first phosphate buffer solution with pH 7.4, and homogenize at 4℃ for 15 minutes to obtain a homogenate.

[0114] 0.03 parts by weight of neutral protease were added to the homogenate, the pH was adjusted to 7.2, and the temperature was raised to 50°C. The mixture was stirred for 6 hours for the first enzymatic hydrolysis. After the first enzymatic hydrolysis, the temperature was raised to 95°C for 15 minutes to inactivate the enzyme and obtain the first enzymatic hydrolysate. The temperature of the first enzymatic hydrolysate was adjusted to 55°C, 0.05 parts by weight of flavor protease and 0.02 parts by weight of aminopeptidase were added, and the pH was adjusted to 7.6. The mixture was stirred for 12 hours for the second enzymatic hydrolysis. After the second enzymatic hydrolysis, the temperature was raised to 95°C for 15 minutes to inactivate the enzyme and obtain the second enzymatic hydrolysate.

[0115] After cooling 100 parts by weight of the secondary enzymatic hydrolysate to 4°C, 30 parts by weight of ammonium sulfate were added while stirring. The mixture was stirred at 4°C for 2 hours. After the reaction was completed, the mixture was allowed to stand at 4°C for 12 hours. The supernatant was collected by centrifugation at 8000 r / min for 20 min. The supernatant was filtered through a 1000 Da molecular sieve ultrafiltration membrane to collect the peptides and obtain sea cucumber peptides.

[0116] Preparation Example 10:

[0117] The preparation method of fermented yacon extract includes the following steps:

[0118] Yacon was washed, sliced, and the resulting yacon pulp was minced. Ten parts by weight of the yacon pulp minced were soaked in 80% ethanol at a material-to-liquid ratio of 1:10 at 50°C for 1.5 hours. The extract was collected by filtration and the extract was concentrated under reduced pressure to recover the ethanol until no more ethanol was distilled off, resulting in an extract. The extract was dispersed and diluted with purified water to 15 times its weight, and 0.1 parts by weight of Lactobacillus fermentation was added. The temperature was adjusted to 37°C and fermented for 72 hours. After fermentation, the temperature was raised to 95°C and kept at that temperature for 10 minutes, then lowered to 23°C. The extract was collected by ultrafiltration using a 3000 Da ultrafiltration membrane. The extract was then freeze-dried and pulverized to obtain the fermented yacon extract.

[0119] Preparation Example 11:

[0120] The preparation method of fermented yacon extract includes the following steps:

[0121] Yacon was washed, sliced, and the resulting yacon pulp was extracted. Ten parts by weight of the yacon pulp were soaked in 80% ethanol at a material-to-liquid ratio of 1:11 at 52°C for 1.7 hours. The extract was collected by filtration and concentrated under reduced pressure to recover the ethanol until no more ethanol was distilled off, yielding an extract. The extract was dispersed and diluted with purified water to 16 times its weight, and 0.2 parts by weight of *Lactobacillus plantarum* were added. The temperature was adjusted to 38°C and fermented for 78 hours. After fermentation, the temperature was raised to 96°C and held for 11 minutes, then lowered to 24°C. The extract was collected by ultrafiltration using a 3000 Da ultrafiltration membrane. The extract was then freeze-dried and pulverized to obtain the fermented yacon extract.

[0122] Preparation Example 12:

[0123] The preparation method of fermented yacon extract includes the following steps:

[0124] Yacon was washed, sliced, and the resulting yacon pulp was extracted. Ten parts by weight of the yacon pulp were soaked in 80% ethanol at a material-to-liquid ratio of 1:12 at 54°C for 1.9 hours. The extract was collected by filtration and concentrated under reduced pressure to recover the ethanol until no more ethanol was distilled off, yielding an extract. The extract was dispersed and diluted with purified water to 17 times its weight, and 0.3 parts by weight of Lactobacillus fermentation was added. The temperature was adjusted to 39°C and fermented for 84 hours. After fermentation, the temperature was raised to 97°C and held for 12 minutes, then lowered to 24°C. The extract was collected by ultrafiltration using a 3000 Da ultrafiltration membrane. The extract was then freeze-dried and pulverized to obtain the fermented yacon extract.

[0125] Preparation Example 13:

[0126] The preparation method of fermented yacon extract includes the following steps:

[0127] Yacon was washed, sliced, and the resulting yacon pulp was extracted. Ten parts by weight of the yacon pulp were soaked in 80% ethanol at a material-to-liquid ratio of 1:13 at 57°C for 2.3 hours. The extract was collected by filtration and concentrated under reduced pressure to recover the ethanol until no more ethanol was distilled off, yielding an extract. The extract was dispersed and diluted with purified water to 19 times its weight, and 0.4 parts by weight of *Lactobacillus plantarum* were added. The temperature was adjusted to 39°C and fermented for 90 hours. After fermentation, the temperature was raised to 98°C and held for 14 minutes, then lowered to 25°C. The extract was collected by ultrafiltration using a 3000 Da ultrafiltration membrane. The fermented yacon extract was then obtained by freeze-drying and pulverizing.

[0128] Preparation Example 14:

[0129] The preparation method of fermented yacon extract includes the following steps:

[0130] Yacon was washed, sliced, and the resulting yacon pulp was extracted. Ten parts by weight of the yacon pulp were soaked in 80% ethanol at a ratio of 1:15 at 60°C for 2.5 hours. The extract was collected by filtration and concentrated under reduced pressure to recover the ethanol until no more ethanol was distilled off, yielding an extract. The extract was dispersed and diluted with purified water to 20 times its weight, and 0.5 parts by weight of Lactobacillus fermentation was added. The temperature was adjusted to 40°C and fermented for 96 hours. After fermentation, the temperature was raised to 100°C and kept at that temperature for 15 minutes, then lowered to 25°C. The extract was collected by ultrafiltration using a 3000 Da ultrafiltration membrane. The extract was then freeze-dried and pulverized to obtain the fermented yacon extract.

[0131] Preparation Example 15:

[0132] The preparation method of yacon extract includes the following steps:

[0133] Yacon was washed, sliced, and minced. Ten parts by weight of yacon were soaked in 80% ethanol at 60°C for 2.5 hours at a material-to-liquid ratio of 1:15. The extract was collected by filtration and concentrated under reduced pressure to recover the ethanol until no more ethanol was distilled off, thus obtaining yacon extract.

[0134] Example 1:

[0135] A method for preparing an anti-aging composition containing sea cucumber peptides includes the following steps:

[0136] Three parts by weight of the sea cucumber peptide complex prepared in Preparation Example 1, one part by weight of the fermented snow lotus fruit extract prepared in Preparation Example 10, one part by weight of grape seed extract, and 0.5 parts by weight of clam peptide were mixed for 10 min to obtain a mixture.

[0137] 65 parts by weight of purified water were heated to 85°C, and 0.6 parts by weight of xanthan gum and 0.3 parts by weight of sodium carboxymethyl cellulose were added and stirred for 20 minutes to obtain mixture A;

[0138] Mixture A was cooled to 50°C and 0.2 parts by weight of sodium hyaluronate, 2 parts by weight of butanediol, 0.5 parts by weight of hydroxyethyl urea, 0.8 parts by weight of stearyl alcohol polyether-21 and 0.3 parts by weight of glycerol glucoside were added. The mixture was stirred for 10 minutes to obtain mixture B.

[0139] Mixture system B is then cooled to 40°C, the above mixture is added, and the pH is adjusted to 5.5 with lactic acid. The mixture is stirred for 20 minutes to obtain the anti-aging composition.

[0140] Example 2:

[0141] A method for preparing an anti-aging composition containing sea cucumber peptides includes the following steps:

[0142] 3.5 parts by weight of the sea cucumber peptide complex prepared in Preparation Example 2, 1.2 parts by weight of the fermented yacon extract prepared in Preparation Example 11, 1.5 parts by weight of grape seed extract and 0.7 parts by weight of clam peptide were mixed for 11 min to obtain a mixture;

[0143] 70 parts by weight of purified water were heated to 86°C, and 0.8 parts by weight of xanthan gum and 0.5 parts by weight of sodium carboxymethyl cellulose were added and stirred for 22 minutes to obtain mixture A;

[0144] Mixture A was cooled to 51°C and 0.25 parts by weight of sodium hyaluronate, 2.5 parts by weight of butanediol, 0.6 parts by weight of hydroxyethyl urea, 0.9 parts by weight of stearyl alcohol polyether-21 and 0.5 parts by weight of glyceryl glucoside were added. The mixture was stirred for 11 min to obtain mixture B.

[0145] Mixture system B is then cooled to 41°C, the above mixture is added, and the pH is adjusted to 5.7 with lactic acid. The mixture is stirred for 22 minutes to obtain the anti-aging composition.

[0146] Example 3:

[0147] A method for preparing an anti-aging composition containing sea cucumber peptides includes the following steps:

[0148] 4 parts by weight of the sea cucumber peptide complex prepared in Preparation Example 3, 1.4 parts by weight of the fermented yacon extract prepared in Preparation Example 12, 2 parts by weight of grape seed extract and 0.9 parts by weight of clam peptide were mixed for 13 min to obtain a mixture.

[0149] 75 parts by weight of purified water were heated to 87°C, and 0.9 parts by weight of xanthan gum and 0.7 parts by weight of sodium carboxymethyl cellulose were added and stirred for 24 min to obtain mixture A;

[0150] Mixture A was cooled to 52°C and 0.3 parts by weight of sodium hyaluronate, 3 parts by weight of butanediol, 0.8 parts by weight of hydroxyethyl urea, 1.1 parts by weight of stearyl alcohol polyether-21 and 0.7 parts by weight of glycerol glucoside were added. The mixture was stirred for 12 minutes to obtain mixture B.

[0151] Mixture system B is then cooled to 42°C, the above mixture is added, and the pH is adjusted to 5.8 with lactic acid. The mixture is stirred for 24 minutes to obtain the anti-aging composition.

[0152] Example 4:

[0153] A method for preparing an anti-aging composition containing sea cucumber peptides includes the following steps:

[0154] 4.5 parts by weight of the sea cucumber peptide complex prepared in Preparation Example 4, 1.8 parts by weight of the fermented yacon extract prepared in Preparation Example 13, 2.5 parts by weight of grape seed extract and 1.2 parts by weight of clam peptide were mixed for 14 min to obtain a mixture;

[0155] 80 parts by weight of purified water were heated to 88°C, and 1.1 parts by weight of xanthan gum and 0.9 parts by weight of sodium carboxymethyl cellulose were added and stirred for 28 minutes to obtain mixture A;

[0156] Mixture A was cooled to 54℃ and 0.4 parts by weight of sodium hyaluronate, 3.5 parts by weight of butanediol, 0.9 parts by weight of hydroxyethyl urea, 1.3 parts by weight of stearyl alcohol polyether-21 and 0.8 parts by weight of glycerol glucoside were added. The mixture was stirred for 14 min to obtain mixture B.

[0157] Mixture system B is then cooled to 44°C, the above mixture is added, and the pH is adjusted to 5.9 with lactic acid. The mixture is stirred for 28 minutes to obtain the anti-aging composition.

[0158] Example 5:

[0159] A method for preparing an anti-aging composition containing sea cucumber peptides includes the following steps:

[0160] Five parts by weight of the sea cucumber peptide complex prepared in Preparation Example 5, two parts by weight of the fermented snow lotus fruit extract prepared in Preparation Example 14, three parts by weight of grape seed extract and one and a half parts by weight of clam peptide were mixed for 15 minutes to obtain a mixture.

[0161] 85 parts by weight of purified water were heated to 90°C, and 1.2 parts by weight of xanthan gum and 1 part by weight of sodium carboxymethyl cellulose were added and stirred for 30 minutes to obtain mixture A;

[0162] Mixture A was cooled to 55°C and 0.5 parts by weight of sodium hyaluronate, 4 parts by weight of butanediol, 1 part by weight of hydroxyethyl urea, 1.5 parts by weight of stearyl alcohol polyether-21 and 1 part by weight of glycerol glucoside were added. The mixture was stirred for 15 minutes to obtain mixture B.

[0163] Mixture system B is then cooled to 45°C, the above mixture is added, and the pH is adjusted to 6.0 with lactic acid. The mixture is stirred for 30 minutes to obtain the anti-aging composition.

[0164] Comparative Example 1:

[0165] A method for preparing an anti-aging composition containing sea cucumber peptides includes the following steps:

[0166] The sea cucumber peptide complex prepared in Example 5 was replaced with the sea cucumber peptide complex prepared in Example 6, and all other operations were the same as in Example 5.

[0167] Comparative Example 2:

[0168] A method for preparing an anti-aging composition containing sea cucumber peptides includes the following steps:

[0169] The sea cucumber peptide complex prepared in Example 5 was replaced with the sea cucumber peptide complex prepared in Example 7, and all other operations were the same as in Example 5.

[0170] Comparative Example 3:

[0171] A method for preparing an anti-aging composition containing sea cucumber peptides includes the following steps:

[0172] The sea cucumber peptide complex prepared in Example 5 was replaced with the sea cucumber peptide complex prepared in Example 8, and all other operations were the same as in Example 5.

[0173] Comparative Example 4:

[0174] A method for preparing an anti-aging composition containing sea cucumber peptides includes the following steps:

[0175] The sea cucumber peptide complex prepared in Example 5 was replaced with the sea cucumber peptide prepared in Example 9, and all other operations were the same as in Example 5.

[0176] Comparative Example 5:

[0177] A method for preparing an anti-aging composition containing sea cucumber peptides includes the following steps:

[0178] The fermented yacon extract prepared in Preparation Example 14 of Example 5 was replaced with the yacon extract prepared in Preparation Example 15, and all other operations were the same as in Example 5.

[0179] Comparative Example 6:

[0180] A method for preparing an anti-aging composition containing sea cucumber peptides includes the following steps:

[0181] The fermented yacon extract obtained in Preparation Example 14 of Example 5 was removed, and other operations were the same as in Example 5.

[0182] Skin irritation test:

[0183] Test Method: 220 volunteers were selected and divided into groups of 20. The anti-aging compositions prepared in Examples 1-5 and Comparative Examples 1-6 were tested respectively. Each volunteer used the inner side of their left and right arms as controls. 0.5 mL of the anti-aging composition prepared in Examples 1-5 and Comparative Examples 1-6 was applied to the inner side of the left arm each time, while the right arm was left untreated as a control. The treatment was carried out twice a day, and the skin reaction was recorded after 24 hours and 72 hours.

[0184] The results showed that after applying the anti-aging compositions prepared in Examples 1-5 and Comparative Examples 1-6 of the present invention for 24 hours and 72 hours, no skin redness or erythema or other allergic reactions were observed, indicating that the anti-aging compositions prepared in the present invention are safe to use in cosmetics without adverse effects.

[0185] Anti-aging effect test:

[0186] The scavenging effect of the anti-aging compositions prepared in Examples 1-5 and Comparative Examples 1-6 on hydroxyl radical scavenging and anti-inflammatory and anti-aging properties were tested. The test method is as follows: 10 μL of 1.25 mg / mL salicylic acid solution and 1.5 mg / mL ferrous sulfate solution were added to an ELISA plate, followed by 150 μL of pure water, then 50 μL of the anti-aging composition prepared in this invention, and finally 10 μL of 0.03% hydrogen peroxide. After reacting for 10 min, the absorbance was measured using an ELISA reader and compared with the blank control sample (50 μL of anti-aging composition replaced with 50 μL of pure water). The scavenging rate of the anti-aging compositions prepared in Examples 1-5 and Comparative Examples 1-6 on strong free radicals was calculated. The test results are shown in Table 1.

[0187] Table 1. Hydroxyl radical scavenging rate

[0188]

[0189] Moisturizing test:

[0190] Test environment: Temperature 25±0.5℃, Humidity 30±0.5%;

[0191] Forty-four volunteers were selected, half male and half female, and divided into 11 groups, with two males and two females in each group. Each group used the anti-aging compositions prepared in Examples 1-5 and Comparative Examples 1-6, respectively. 2g of each composition was evenly applied to the forearm. The hydration content of the stratum corneum of the skin was measured at different times before and after use. The test results are shown in Table 2.

[0192] Table 2. Hydration content of the stratum corneum of the skin

[0193]

[0194] The data in Tables 1 and 2 show that the anti-aging compositions prepared in Examples 1-5 of this invention have good anti-aging and long-lasting moisturizing properties. Table 1 shows that the anti-aging compositions prepared in Examples 1-5 of this invention can effectively scavenge strong free radicals, exhibiting excellent anti-aging effects and giving the skin a healthy state. Table 2 shows that the anti-aging compositions prepared in Examples 1-5 of this invention still exhibit good moisturizing properties 10 hours after application. The decline in anti-aging and moisturizing performance in Comparative Example 1 may be due to the lack of modification of sea cucumber peptides, resulting in low utilization and poor anti-aging and moisturizing effects, thus affecting the performance of the anti-aging composition. The decline in anti-aging and moisturizing performance in Comparative Example 2 may be due to the use of distearate phosphatidylethanolamine-polyethylene glycol-thiol in the modification of sea cucumber peptides, leading to excessively large molecular weights and affecting their utilization. The decline in performance in Comparative Example 3 may be due to the lack of chitosan oligosaccharides, resulting in reduced residence time and affecting the performance of the anti-aging composition. The decline in performance in Comparative Example 4 may be due to the lack of modification and encapsulation of sea cucumber peptides, failing to improve their utilization and residence time on the skin surface, thus affecting the performance of the anti-aging composition. The decline in performance in Comparative Example 5 may be due to the lack of fermentation treatment of the fermented yacon extract, failing to enhance its bioactivity and affecting the performance of the anti-aging composition. The decline in performance in Comparative Example 6 may be due to the significant reduction in moisturizing components after the removal of the fermented yacon extract, thus affecting the performance of the anti-aging composition.

[0195] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for preparing an anti-aging composition containing sea cucumber peptides, characterized in that, The preparation method includes the following steps: Sea cucumber peptide complex, fermented snow lotus fruit extract, grape seed extract and clam peptide were mixed at 23~25℃ for 10~15 min to obtain a mixture; Xanthan gum and sodium carboxymethyl cellulose were dispersed in purified water at 85-90℃ and stirred for 20-30 minutes to obtain mixed system A; Mixture system A, sodium hyaluronate, butanediol, hydroxyethyl urea, stearyl alcohol polyether-21 and glyceryl glucoside are mixed at 50~55℃ for 10~15min to obtain mixture system B; The anti-aging composition is obtained by mixing system B and the mixture at 40-45°C for 20-30 minutes. The preparation method of the sea cucumber peptide complex includes the following steps: After mixing sea cucumber fragments and primary phosphate buffer at a weight ratio of 1:8~10, homogenize the mixture at 2~4℃ for 10~15 minutes to obtain a homogenate. Neutral protease was added to the homogenate for a single enzymatic hydrolysis to obtain a single hydrolysate. Flavor protease and aminopeptidase are added to the primary enzymatic hydrolysate for secondary enzymatic hydrolysis to obtain a secondary enzymatic hydrolysate; The secondary enzymatic hydrolysate and ammonium sulfate were mixed at a weight ratio of 10:2~3 and allowed to stand. The supernatant was collected to obtain sea cucumber peptides. Sea cucumber peptide and maleimide-PEG2-succinimide ester were reacted at a weight ratio of 3~5:0.01~0.02 at 23~25℃ for 3~4h to obtain an intermediate; The intermediate and 1,2-dipalmitoyl-sn-glycerol-3-phosphate thioethanol were reacted at a weight ratio of 7~9:0.05~0.07 at 23~25℃ for 8~12 h to obtain modified sea cucumber peptide; A lipid membrane was obtained by rotary evaporation after mixing modified sea cucumber peptides, soybean lecithin, and cholesterol in a weight ratio of 1.5~2:12~20:3~4.

5. First phosphate buffer was added to the lipid membrane and swollen at 45-50℃ for 2-4 hours to obtain a liposome suspension. Chitosan oligosaccharide was dispersed in phosphate buffer at a material-to-liquid ratio of 8-10:50 to obtain a chitosan oligosaccharide solution. The sea cucumber peptide complex was prepared by reacting liposome suspension and chitosan oligosaccharide solution at a weight ratio of 10:10~15 at 23~25℃ for 3~4h. The preparation method of the fermented yacon extract includes the following steps: Yacon pulp fragments were soaked in 80% ethanol at a material-to-liquid ratio of 1:10-15 at 50-60℃ for 1.5-2.5 hours. The extract was then filtered and concentrated to obtain an extract. The extract was diluted with purified water to obtain a diluted solution, which was then fermented with lactic acid bacteria to obtain the fermented yacon extract. The lactic acid bacteria include Lactobacillus fermentum or Lactobacillus plantarum.

2. The method for preparing an anti-aging composition containing sea cucumber peptides as described in claim 1, characterized in that, The amount of neutral protease used is 0.2% to 0.3% of the weight of the sea cucumber fragments.

3. The method for preparing an anti-aging composition containing sea cucumber peptides as described in claim 1, characterized in that, The amount of the flavor protease used is 0.4% to 0.5% of the weight of the sea cucumber pieces.

4. The method for preparing an anti-aging composition containing sea cucumber peptides as described in claim 1, characterized in that, The amount of aminopeptidase used is 0.15% to 0.2% of the weight of the crushed sea cucumber.

5. The method for preparing an anti-aging composition containing sea cucumber peptides as described in claim 1, characterized in that, The amount of lactic acid bacteria used is 1% to 5% of the weight of the chopped snow lotus fruit pulp.

6. The method for preparing an anti-aging composition containing sea cucumber peptides as described in claim 1, characterized in that, The weight ratio of the sea cucumber peptide complex, fermented snow lotus fruit extract, grape seed extract, clam peptide, xanthan gum, sodium carboxymethyl cellulose, sodium hyaluronate, butylene glycol, hydroxyethyl urea, stearyl alcohol polyether-21, glyceryl glucoside and purified water is 3~5:1~2:1~3:0.5~1.5:0.6~1.2:0.3~1:0.2~0.5:2~4:0.5~1:0.8~1.5:0.3~1:65~85.

7. An anti-aging composition containing sea cucumber peptides, characterized in that, It is prepared by the preparation method of the anti-aging composition containing sea cucumber peptide as described in any one of claims 1 to 6.

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