Preparation method of collagen peptide for improving facial skin quality

Abstract

The application belongs to the technical field of collagen peptide preparation methods, and particularly relates to a preparation method of collagen peptide for improving facial skin quality, which uses fish tissues as raw materials and comprises the following steps: picking and washing, cooking, two-step enzymolysis, filtration, first ultrafiltration, glycosylation reaction, second ultrafiltration, concentration, sterilization, spray drying and sieving. The glycosylation reaction comprises the process of glycosylating fish collagen peptide crude products by using plant polysaccharides and disaccharides. The collagen peptide prepared by the method has good antioxidant performance, good water absorption and moisturizing performance, and can effectively improve facial skin quality.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of collagen peptide preparation method, and particularly relates to a collagen peptide preparation method for improving facial skin quality. BACKGROUND

[0002] With the development of social economy, people are increasingly pursuing high-quality life, and not only pay attention to their own physical health, but also pay attention to skin care, which has become an important part of daily life. Paying attention to beauty care and keeping the face skin white and tender has become the skin care goal of many people, especially women. Collagen is one of the main components of human tissue structure, which is distributed in all tissues and organs of the body, such as skin, bone, cartilage, ligament, cornea, various endometrium and fascia, etc., and is the main component for maintaining the shape and structure of skin and tissue organs, and is also an important raw material for repairing damaged tissues. Directly supplementing collagen from cosmetics or skin care products can quickly supplement the lost collagen of the skin, so that the skin is more permeable and elastic.

[0003] Collagen peptide (CP) is a mixture between amino acids and proteins formed by enzymatic hydrolysis of collagen, which is a small molecular peptide with a molecular weight of several hundred to several thousand daltons, usually derived from salmon skin, fish scales, chicken breast cartilage, cowhide and oyster, etc. The molecular weight of CP is generally less than 3000 Da. Collagen is limited in its application due to its high molecular weight and difficulty in being digested and absorbed. CP has good bioavailability, antioxidant properties and low allergenicity due to its relatively small molecular weight and easy solubility in water. SUMMARY

[0004] The purpose of the present application is to provide a collagen peptide preparation method for improving facial skin quality to solve the defects pointed out in the background.

[0005] According to the first aspect of the embodiment of the present application, a collagen peptide preparation method for improving facial skin quality comprises the following steps:

[0006] Step 1a: taking fish tissues, picking out impurities from the fish tissues and washing the fish tissues;

[0007] Step 2a: putting the washed fish tissues into a reactor, adding water to the reactor and heating at 75-85℃ for 2-5h, and then standing for 1-2h after the reaction is completed to obtain a heated mixture;

[0008] Step 3a: obtaining an enzymatic hydrolysis mixture by two-step enzymatic hydrolysis of the heated mixture obtained in step 2a;

[0009] Step 4a: After the enzymatically hydrolyzed mixture obtained in step 3a is cooled to below 50° C., it is filtered using diatomaceous earth to obtain a filtered mixture;

[0010] Step 5a: subjecting the filtered mixture obtained in step 4a to a first ultrafiltration to obtain a crude fish collagen peptide;

[0011] Step 6a: mixing the crude fish collagen peptide obtained in step 5a with sodium chondroitin sulfate and a carbohydrate composition, and subjecting the crude fish collagen peptide to a glycosylation reaction to obtain a glycosylated crude fish collagen peptide;

[0012] Step 7a: The crude glycosylated fish collagen peptide obtained in step 6a is subjected to a second ultrafiltration, and then concentrated, sterilized, spray-dried and sieved to obtain the collagen peptide.

[0013] In one aspect of the embodiments of the present invention, in step 1a, the fish tissue is at least one of fish skin, fish bones, and fish scales.

[0014] In one aspect of the embodiments of the present invention, the fish skin is cod skin, tilapia skin, tuna skin, horse mackerel skin, golden pomfret skin or snakehead fish skin.

[0015] In one aspect of the embodiments of the present invention, the fish bone is a cod bone or an eel bone.

[0016] In one aspect of the embodiments of the present invention, the fish scales are sardine scales, redfin snapper scales, tilapia scales, silver carp scales, grass carp scales or seabass scales.

[0017] In one aspect of the embodiments of the present invention, specifically, the fish tissue used in step 1a is cod bone.

[0018] In one aspect of the embodiments of the present invention, the two-step enzymatic hydrolysis method in step 3a comprises:

[0019] The temperature of the heated mixture is controlled to be 55° C.-65° C. and the pH value is 7-8, papain is used to perform a first enzymatic hydrolysis, the time of the first enzymatic hydrolysis is 1-1.5 hours, and a product after the first enzymatic hydrolysis is obtained. Then, the temperature of the product after the first enzymatic hydrolysis is controlled to be 65° C.-75° C. and the pH value is 8-9, a composite protease is used to perform a second enzymatic hydrolysis, the time of the second enzymatic hydrolysis is 1-1.5 hours, and a mixture after enzymatic hydrolysis is obtained; wherein the composite protease is at least two of alkaline protease, papain, trypsin, and bromelain.

[0020] In one aspect of the embodiments of the present invention, specifically, the composite protease comprises alkaline protease and trypsin.

[0021] In one aspect of the embodiments of the present invention, in step 3a, the mass of the added papain is 0.3%-0.5% of the mass of the fish tissue obtained in step 1a.

[0022] In one aspect of the embodiments of the present invention, in step 3a, the mass of the added composite protease is 0.1%-0.2% of the mass of the fish tissue obtained in step 1a.

[0023] In one aspect of the embodiments of the present invention, in step 3a, the mass ratio of alkaline protease to trypsin in the composite protease is (1-3): 1. Specifically, the mass ratio of alkaline protease to trypsin in the composite protease is 1:1, 1.5:1, 2:1, 2.5:1 or 3:1.

[0024] In one aspect of the embodiments of the present invention, the glycosylation reaction in step 6a comprises:

[0025] The crude fish collagen peptide obtained in step 5a is dissolved in water with sodium chondroitin sulfate and a carbohydrate composition to obtain an aqueous solution, which is stirred at 65° C.-75° C. for 0.5-1 hour, and the pH value of the aqueous solution is adjusted to 8. At the same time, the temperature of the aqueous solution is lowered to below 30° C. to obtain a precipitate, and the precipitate is freeze-dried to obtain the crude glycosylated fish collagen peptide.

[0026] Specifically, the glycosylation reaction in step 6a includes:

[0027] The crude fish collagen peptide obtained in step 5a is dissolved in water with sodium chondroitin sulfate and a carbohydrate composition to obtain an aqueous solution, which is stirred at 70° C. for 45 minutes, and the pH value of the aqueous solution is adjusted to 8. At the same time, the temperature of the aqueous solution is lowered to 4° C. to obtain a precipitate. The precipitate is freeze-dried to obtain the crude glycosylated fish collagen peptide.

[0028] In one aspect of an embodiment of the present invention, the carbohydrate composition comprises plant polysaccharides and at least one disaccharide.

[0029] In one aspect of the embodiments of the present invention, the disaccharide is maltose, lactose, sucrose, trehalose or cellobiose.

[0030] In one aspect of the embodiments of the present invention, the disaccharide is trehalose.

[0031] In one aspect of an embodiment of the present invention, the plant polysaccharide comprises annona polysaccharide.

[0032] In one aspect of the embodiments of the present invention, the annona polysaccharide is prepared by the following steps:

[0033] Step 1b: taking the pulp portion of a mature sugar apple fruit and preparing sugar apple powder from the pulp portion;

[0034] Step 2b: Put the annona powder obtained in step 1b into a reactor, and then add water into the reactor, and then ultrasonic extract the annona powder at an ultrasonic temperature of 40-45℃ and an ultrasonic power of 300-400W for 0.5-1h to obtain a crude annona polysaccharide extract;

[0035] Step 3b: Decolorize and deproteinize the crude annona polysaccharide extract obtained in step 2b to obtain an annona polysaccharide extract.

[0036] Step 4b: Combine and concentrate the annona polysaccharide extract obtained in step 3b, and then add 95% ethanol until the concentration of ethanol in the final solution is 28%-32%, to obtain a precipitate and a supernatant, and then wash and dry the precipitate to obtain annona polysaccharide.

[0037] In one aspect of the embodiment of the present application, specifically, step 1b comprises: taking the pulp part of the ripe annona fruit, washing the pulp part, and then air-drying, and then drying at 55-65℃, and then crushing and passing through a 60-mesh sieve, and then defatting with petroleum ether, and then washing and air-drying again to obtain annona powder.

[0038] In one aspect of the embodiment of the present application, specifically, step 2b comprises: putting the annona powder obtained in step 1b into a reactor, and then adding water into the reactor, and then ultrasonic extract the annona powder at an ultrasonic temperature of 45℃ and an ultrasonic power of 400W for 45min to obtain a crude annona polysaccharide extract.

[0039] In one aspect of the embodiment of the present application, specifically, step 3b comprises: decolorizing the crude annona polysaccharide extract obtained in step 2b by activated carbon, and then filtering, washing and drying after decolorization is completed, and then deproteinizing the decolorized product by Sevag method to obtain an annona polysaccharide extract.

[0040] In one aspect of the embodiment of the present application, specifically, the volume ratio of chloroform to n-butanol in the Sevag method is 4:1.

[0041] In one aspect of the embodiment of the present application, specifically, step 4b comprises: combining and concentrating the annona polysaccharide extract obtained in step 3b to 2 / 3 of the original solution, and then adding 95% ethanol until the concentration of ethanol in the final solution is 30%, to obtain a precipitate and a supernatant, and then washing and drying the precipitate to obtain annona polysaccharide.

[0042] In one aspect of the embodiment of the present application, the annona polysaccharide is further acetylated.

[0043] In one aspect of the embodiment of the present application, the step of acetylation comprises:

[0044] Step 5b: the anona polysaccharide obtained in step 4b is dissolved in water, the pH value is adjusted to 9-10, 5-15 wt% of acetic anhydride of the anona polysaccharide is added every 15 minutes, the pH value is controlled to be constant, the acetic anhydride is added for 4-7 times, after the acetic anhydride is added completely, stirring is continued for 0.5-1 h, then hydrochloric acid is added until the pH value of the solution is 6-7 to terminate the reaction, 95% ethanol is added until the concentration of ethanol in the final solution is 28%-32%, a precipitate is obtained, the precipitate is washed and dried to obtain the acetylated modified anona polysaccharide.

[0045] In an aspect of the embodiment of the present application, specifically, the acetylation modification step comprises:

[0046] Step 5b: the anona polysaccharide obtained in step 4b is dissolved in water, the pH value is adjusted to 9-10, 5-15 wt% of acetic anhydride of the anona polysaccharide is added every 15 minutes, the pH value is controlled to be constant, the acetic anhydride is added for 4-7 times, after the acetic anhydride is added completely, stirring is continued for 0.5-1 h, then hydrochloric acid is added until the pH value of the solution is 6-7 to terminate the reaction, 95% ethanol is added until the concentration of ethanol in the final solution is 28%-32%, a precipitate is obtained, the precipitate is washed and dried to obtain the acetylated modified anona polysaccharide.

[0047] In an aspect of the embodiment of the present application, the saccharide composition comprises anona polysaccharide and trehalose.

[0048] In an aspect of the embodiment of the present application, in the saccharide composition, the mass ratio of anona polysaccharide to trehalose is (1.5-2.5):1.

[0049] In an aspect of the embodiment of the present application, the saccharide composition comprises acetylated modified anona polysaccharide and trehalose.

[0050] In an aspect of the embodiment of the present application, in the saccharide composition, the mass ratio of acetylated modified anona polysaccharide to trehalose is (1.5-2.5):1.

[0051] In an aspect of the embodiment of the present application, in step 6a, the mass ratio of the fish collagen peptide crude product to chondroitin sulfate sodium is 1:(0.15-0.3).

[0052] In an aspect of the embodiment of the present application, in step 6a, the mass ratio of the fish collagen peptide crude product to the saccharide composition is 1:(3-6).

[0053] In an aspect of the embodiment of the present application, the plant polysaccharide further comprises osmanthus leaf polysaccharide.

[0054] In an aspect of the embodiment of the present application, the plant polysaccharide comprises anona polysaccharide and osmanthus leaf polysaccharide.

[0055] In one aspect of the embodiments of the present application, the plant polysaccharide comprises acetylated annona polysaccharide and osmanthus leaf polysaccharide.

[0056] In one aspect of the embodiments of the present application, in the saccharide composition, the mass ratio of acetylated annona polysaccharide, osmanthus leaf polysaccharide and trehalose is (1.5-2.5):(0.4-0.7):1.

[0057] In one aspect of the embodiments of the present application, the osmanthus leaf polysaccharide is prepared by the following steps:

[0058] Step 1c: weigh osmanthus leaves, add water as the extraction agent, and add water at a mass ratio of osmanthus leaves:water of 1:(20-30), reflux extract 1-3 times at 70-80°C to obtain osmanthus leaf polysaccharide extract;

[0059] Step 2c: combine and concentrate the osmanthus leaf polysaccharide extract to 1 / 3 of the original volume;

[0060] Step 3c: add 3 times the volume of anhydrous ethanol to the concentrated osmanthus leaf polysaccharide extract, centrifuge after precipitating for 3h to obtain a precipitate, and wash and dry the precipitate to obtain the osmanthus leaf polysaccharide.

[0061] In one aspect of the embodiments of the present application, a 3000 Da ultrafiltration membrane is used for the first ultrafiltration.

[0062] In one aspect of the embodiments of the present application, a 15000 Da ultrafiltration membrane is used for the second ultrafiltration.

[0063] In one aspect of the embodiments of the present application, in step 7a, the concentration process is: pumping the material liquid after the second ultrafiltration into a single-effect concentrator and concentrating to 2 / 3 of the original volume.

[0064] In one aspect of the embodiments of the present application, in step 7a, the sterilization process is: pumping the concentrated material liquid into a sterilization tank, heating to 80-85°C, and sterilizing for 20-30 min.

[0065] In one aspect of the embodiments of the present application, in step 7a, the spray drying process is: pumping the sterilized material into a feed intermediate tank, and pumping into a spray drying tower through a filter for spray drying. Specifically, in the spray drying process, the inlet air temperature is 180-190°C, the outlet air temperature is 85-95°C, and the material liquid rotation speed is adjusted for spray drying.

[0066] In one aspect of the embodiments of the present application, in step 7a, the sieving process is as follows: according to different requirements, the spray-dried material is sieved using a screen with a corresponding mesh size, and metal impurities are adsorbed by a magnetic frame (magnetic field strength of the magnetic frame is greater than or equal to 8000 GS (800 MT)).

[0067] According to a second aspect of the embodiments of the present application, a collagen peptide is prepared by the aforementioned preparation method.

[0068] According to a third aspect of the embodiments of the present application, a cosmetic or skin care product comprises the aforementioned collagen peptide and at least one excipient.

[0069] In one aspect of the embodiments of the present application, the excipient is an antioxidant, a humectant, a thickening agent, a foaming agent, a preservative, a cooling agent, a buffering agent, a pH adjusting agent, a coloring agent, an excipient, a dispersing agent, an emulsifying agent, a co-solvent, a surfactant, an astringent, a lubricant, a stabilizer, a solvent, and a fragrance, but is not limited thereto.

[0070] Compared with the prior art, the present application has the following beneficial effects:

[0071] The collagen peptide prepared by the present application not only has good antioxidant properties, but also has good water absorption and moisturizing properties, thereby effectively improving the facial skin quality.

[0072] One of the raw materials used in the present application is cod bone, which is hard and not suitable for eating, and is usually treated as waste. Using cod bone to prepare cod bone collagen peptide can save costs and improve resource utilization.

[0073] The raw material annona polysaccharide used in the present application can reduce the apoptosis of normal human epidermal keratinocytes caused by gamma irradiation, increase the activity of antioxidant enzymes SOD and catalase CAT in cells, and reduce the level of pro-inflammatory cytokine interleukin-1β (IL-1β) and inflammatory body signaling pathway indicators, and has a significant anti-skin aging function. The annona polysaccharide is used to glycosylate fish collagen peptides, and the glycosylated fish collagen peptides obtained have strong antioxidant properties compared to other bone collagen peptide products, can effectively scavenge DPPH and ·OH free radicals, can eliminate the damage to the skin caused by excessive free radicals, and can be applied to cosmetics to delay skin aging.

[0074] And, before the fish collagen peptide is glycosylated by the annona polysaccharide, the fish collagen peptide is further acetylated, the acetylation modification of the polysaccharide refers to that the branch hydroxyl of the polysaccharide and an acetyl group occur nucleophilic substitution reaction, when the degree of substitution is high, the molecular weight of the polysaccharide is increased, the molecular conformation is more compact, and the solubility of the polysaccharide is improved due to the change of the spatial arrangement of the polysaccharide chain and more hydroxyl groups are exposed. Therefore, the fish collagen peptide is glycosylated by the trehalose and the acetylated annona polysaccharide, the wetting property of the product can be further improved, the moisturizing performance is effectively improved, and the skin moisturizing effect can be improved when the product is applied to cosmetics.

[0075] It should be understood that the general description above and the following detailed description are only exemplary and explanatory, and cannot limit the present application. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical scheme and advantages of the present application more clear, the technical scheme of the present application will be described clearly and completely below in combination with examples. Obviously, the described examples are a part of the examples of the present application, rather than all the examples. The examples described herein are illustrative in nature and are used to provide a basic understanding of the present application. The examples of the present application should not be interpreted as a limitation of the present application.

[0077] For the sake of brevity, only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with any other lower limit to form a range not explicitly recited, and likewise any upper limit can be combined with any other upper limit to form a range not explicitly recited. Further, each individual disclosed point or single numerical value can be combined with any other point or single numerical value to form a range not explicitly recited, either as a lower or upper limit, or in combination with other lower or upper limits.

[0078] In this document, the terms "comprises", "comprising", or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by "comprises... a" does not, without more constraints, exclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0079] In the description herein, "above", "below" include the number, unless otherwise specified.

[0080] Unless otherwise indicated, the terms used in the present application have the meanings that are commonly understood by a person of ordinary skill in the art. Unless otherwise indicated, the numerical values of various parameters in the present application can be measured using various measurement methods commonly used in the art (for example, can be tested according to the methods given in the examples of the present application).

[0081] The term "about" is used to describe and account for small variations. When used in connection with an event or circumstance, the term can refer to instances in which the event or circumstance occurs exactly, as well as instances in which the event or circumstance occurs with minor deviations. For example, when used in connection with a numerical value, the term can refer to a range of variation of less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. Additionally, quantities, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be taken as a literal disclosure of each distinct end point specified as well as every number or sub-range encompassed therein.

[0082] The list of items connected by "at least one of," "one or more of," or "at least one of each" or other similar phrases can mean any combination of the listed items. For example, if the items listed are A and B, the phrase "at least one of A and B" means only A; only B; or A and B. In another example, if the items listed are A, B, and C, the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and C. Item A can include a single component or multiple components. Item B can include a single component or multiple components. Item C can include a single component or multiple components.

[0083] The present application is further illustrated below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application.

[0084] Example One

[0085] The following steps are included:

[0086] A: Preparation of a saccharide composition comprising acetylated modified annona polysaccharide and trehalose

[0087] Take the pulp part of 50g of ripe annona fruit, wash the pulp part, then air dry, then dry at 60°C, crush and pass through a 60 mesh sieve, then defat with petroleum ether, then wash and dry again to obtain annona powder.

[0088] The cherimoya powder was put into a reactor, and water was added to the reactor. The cherimoya polysaccharide was extracted under the conditions of an ultrasonic temperature of 45℃ and an ultrasonic power of 400W for 45min to obtain a crude extraction solution of the cherimoya polysaccharide.

[0089] The crude extraction solution of the cherimoya polysaccharide was decolorized by using 1g of activated carbon. After the decolorization was completed, the product was subjected to vacuum filtration, washing and drying. The Sevag method was used to remove the protein from the decolorized product to obtain a cherimoya polysaccharide extraction solution.

[0090] The cherimoya polysaccharide extraction solution was concentrated to 2 / 3 of the original solution, and 95% ethanol was added until the concentration of ethanol in the final solution was 30%. The precipitate and supernatant were obtained, and the precipitate was washed and dried to obtain the cherimoya polysaccharide.

[0091] The cherimoya polysaccharide was dissolved in water, and the pH value was adjusted to 9. Every 15 minutes, 10wt% of acetic anhydride was added to the cherimoya polysaccharide, and the pH value was controlled to be constant. The number of times of adding acetic anhydride was 5. After the addition of acetic anhydride was completed, the stirring was continued for 50min. Then, hydrochloric acid was added until the pH value of the solution was 6 to terminate the reaction. Then, 95% ethanol was added until the concentration of ethanol in the final solution was 30%. The precipitate was obtained, and the precipitate was washed and dried to obtain the acetylated modified cherimoya polysaccharide.

[0092] 3g of the acetylated modified cherimoya polysaccharide prepared was mixed with 1.5g of trehalose (commercially available) to obtain the saccharide composition of Example One.

[0093] B: Preparation of crude fish collagen peptide:

[0094] The cod bones were taken, and the impurities were picked out from the cod bones and washed to obtain 150g of cod bones.

[0095] The washed cod bones were put into a reactor, and 450mL of water was added to the reactor. The mixture was heated at 80℃ for 4h. After the reaction was completed, the mixture was allowed to stand for 2h to obtain a heated mixture.

[0096] The temperature of the heated mixture was controlled to be 60℃, and the pH value was controlled to be 8. The first step of enzymatic hydrolysis was performed by using 0.4wt% of papain based on the cod bones. The time of the first step of enzymatic hydrolysis was 1.2h to obtain a first step of enzymatic hydrolysis product. Then, the temperature of the first step of enzymatic hydrolysis product was controlled to be 70℃, and the pH value was controlled to be 9. The second step of enzymatic hydrolysis was performed by using a complex protease containing alkaline protease and trypsin (wherein the mass ratio of alkaline protease to trypsin was 2:1). The time of the second step of enzymatic hydrolysis was 1.2h to obtain an enzymatic hydrolysis mixture.

[0097] The enzymatic hydrolysis mixture was cooled to 45℃, and the filtration was performed by using diatomite in a chamber filter press to obtain a filtered mixture.

[0098] The filtrated mixture was ultrafiltrated using an ultrafiltration membrane with a molecular weight cut-off of 3000 Da to obtain the crude fish collagen peptide.

[0099] C: Preparation of collagen peptide of preparation example one

[0100] 1 g of the crude fish collagen peptide was weighed out and dissolved in water together with the aforementioned saccharide composition to obtain an aqueous solution, and then 0.2 g of sodium chondroitin sulfate was added. The aqueous solution was stirred at 70°C for 40 min, and then the pH value of the aqueous solution was adjusted to 8 while the temperature of the aqueous solution was reduced to 4°C to obtain a precipitate. The precipitate was freeze-dried to obtain the glycosylated crude fish collagen peptide.

[0101] The glycosylated crude fish collagen peptide was ultrafiltrated using an ultrafiltration membrane with a molecular weight cut-off of 15000 Da, and then concentrated, sterilized, spray-dried and sieved to obtain the collagen peptide of preparation example one.

[0102] Preparation example two

[0103] The method comprises the following steps:

[0104] A: Preparation of saccharide composition comprising acetylated modified annona squamosa polysaccharide and trehalose

[0105] The pulp part of 50 g of mature annona squamosa fruits was washed, air-dried, oven-dried at 60°C, crushed, sieved through a 60-mesh sieve, degreased with petroleum ether, washed and air-dried again to obtain annona squamosa powder.

[0106] The annona squamosa powder was put into a reactor, and water was added to the reactor. The annona squamosa powder was ultrasonically extracted at an ultrasonic temperature of 45°C and an ultrasonic power of 400 W for 45 min to obtain a crude annona squamosa polysaccharide extract.

[0107] The crude annona squamosa polysaccharide extract was decolorized using 1 g of activated carbon. After decolorization, the product was filtered under reduced pressure, washed and dried. The decolorized product was deproteinized using the Sevag method to obtain an annona squamosa polysaccharide extract.

[0108] The annona squamosa polysaccharide extract was concentrated to 2 / 3 of the original solution, and 95% ethanol was added until the concentration of ethanol in the final solution was 30%. The precipitate and supernatant were obtained, and the precipitate was washed and dried to obtain annona squamosa polysaccharide.

[0109] 3 g of the prepared annona squamosa polysaccharide was weighed out and mixed with 1.5 g of trehalose to obtain the saccharide composition of preparation example two.

[0110] B: Preparation of crude fish collagen peptide:

[0111] Take cod bones, pick out impurities from the cod bones and wash the cod bones to obtain 150 g of cod bones.

[0112] Put the washed cod bones into a reactor, add 450 mL of water to the reactor and heat at 80°C for 4 h, and after the reaction is completed, stand for 2 h to obtain a heated mixture.

[0113] Control the temperature of the heated mixture to be 60°C and the pH value to be 8, use 0.4 wt% of papain of cod bones for the first step of enzymatic hydrolysis, the time of the first step of enzymatic hydrolysis is 1.2 h, to obtain the product after the first step of enzymatic hydrolysis, then control the temperature of the product after the first step of enzymatic hydrolysis to be 70°C and the pH value to be 9, use a complex protease containing alkaline protease and trypsin (wherein the mass ratio of alkaline protease to trypsin is 2:1) for the second step of enzymatic hydrolysis, the time of the second step of enzymatic hydrolysis is 1.2 h, to obtain the enzymatic hydrolysis mixture.

[0114] Cool the enzymatic hydrolysis mixture to 45°C, filter in a chamber filter press using diatomite to obtain the filtered mixture.

[0115] Use an ultrafiltration membrane with a molecular weight cut-off of 3000 Da to ultrafiltrate the filtered mixture to obtain the crude fish collagen peptide.

[0116] C: Preparation of collagen peptide of Example Two

[0117] Take 1 g of the crude fish collagen peptide, dissolve it in water together with the aforementioned saccharide composition to obtain an aqueous solution, then add 0.2 g of sodium chondroitin sulfate, stir at 70°C for 40 min, adjust the pH value of the aqueous solution to 8, and at the same time, reduce the temperature of the aqueous solution to 4°C to obtain a precipitate, and after freeze-drying the precipitate, obtain the glycosylated crude fish collagen peptide.

[0118] Use an ultrafiltration membrane with a molecular weight cut-off of 15000 Da to ultrafiltrate the glycosylated crude fish collagen peptide, then concentrate, sterilize, spray dry and sieve to obtain the collagen peptide of Example Two.

[0119] Example Three

[0120] The steps of Example Three are the same as those of Example One, except that in Example Three, 1.5 g of acetylated modified annona polysaccharide prepared is mixed with 3 g of trehalose to obtain the saccharide composition of Example Three.

[0121] Example Four

[0122] The procedure of Example Four is the same as Example One, except that in Example Four, 2.25 g of the acetylated modified annona polysaccharide prepared is weighed and mixed with 2.25 g of trehalose to obtain the saccharide composition of Example Four.

[0123] Example Five

[0124] The procedure of Example Five is the same as Example One, except that in Example Five, 4 g of the acetylated modified annona polysaccharide prepared is weighed and mixed with 0.5 g of trehalose to obtain the saccharide composition of Example Five.

[0125] Example Six

[0126] The procedure of Example Six is the same as Example One, except that in Example Six, sucrose is used instead of trehalose.

[0127] Example Seven

[0128] The procedure of Example Seven is the same as Example One, except that in Example Six, glucose is used instead of trehalose.

[0129] Example Eight

[0130] The procedure includes the following steps:

[0131] A: Preparation of a saccharide composition comprising acetylated modified annona polysaccharide, osmanthus leaf polysaccharide and trehalose

[0132] Take the pulp part of 50 g of mature annona fruit, wash the pulp part and dry in the air, then dry at 60 °C, crush and pass through a 60 mesh sieve, then degrease with petroleum ether, then wash and dry again to obtain annona powder.

[0133] Put the annona powder into a reactor, add water to the reactor, and ultrasonically extract at an ultrasonic temperature of 45 °C and an ultrasonic power of 400 W for 45 min to obtain a crude annona polysaccharide extract.

[0134] Use 1 g of activated carbon to decolorize the crude annona polysaccharide extract, after decolorization, perform vacuum suction filtration, washing and drying, and use the Sevag method to remove protein from the decolorized product to obtain an annona polysaccharide extract.

[0135] Combine and concentrate the annona polysaccharide extract to 2 / 3 of the original solution, then add 95% ethanol until the concentration of ethanol in the final solution is 30%, to obtain a precipitate and a supernatant, wash and dry the precipitate to obtain annona polysaccharide.

[0136] Dissolve the annona polysaccharide in water, adjust the pH value to 9, add 10wt% acetic anhydride every 15 minutes, and control the pH value to be constant, the number of acetic anhydride addition is 5 times, after the addition of acetic anhydride is completed, continue to stir for 50 min, then add hydrochloric acid to the solution until the pH value is 6 to terminate the reaction, then add 95% ethanol until the concentration of ethanol in the final solution is 30%, obtain the precipitate, and obtain the acetylated modified annona polysaccharide after washing and drying the precipitate.

[0137] Weigh 12g of Osmanthus fragrans leaves, use water as the extraction agent, add 25 times the mass of water, and reflux extract at 75℃ for 2 times to obtain the Osmanthus fragrans leaf polysaccharide extract.

[0138] Combine and concentrate the Osmanthus fragrans leaf polysaccharide extract to 1 / 3 of the original volume; add 3 times the volume of anhydrous ethanol to the concentrated Osmanthus fragrans leaf polysaccharide extract, centrifuge after precipitating for 3h to obtain the precipitate; wash and dry the precipitate to obtain the Osmanthus fragrans leaf polysaccharide.

[0139] Weigh 2.5g of the acetylated modified annona polysaccharide and 0.75g of the Osmanthus fragrans leaf polysaccharide prepared, and mix them with 1.25g of trehalose to obtain the saccharide composition of Example Eight.

[0140] B: Preparation of fish collagen peptide crude product:

[0141] Take cod bones, pick out impurities from the cod bones and wash the cod bones to obtain 150g of cod bones.

[0142] Put the washed cod bones into a reactor, add 450mL of water to the reactor, and heat at 80℃ for 4h, and after the reaction is completed, stand for 2h to obtain a heated mixture.

[0143] Control the temperature of the heated mixture to be 60℃ and the pH value to be 8, use 0.4wt% papain of the cod bones for the first step of enzymolysis, the time of the first step of enzymolysis is 1.2h, obtain the product after the first step of enzymolysis, then control the temperature of the product after the first step of enzymolysis to be 70℃ and the pH value to be 9, use a complex protease containing alkaline protease and trypsin (wherein the mass ratio of alkaline protease to trypsin is 2:1) for the second step of enzymolysis, the time of the second step of enzymolysis is 1.2h, and obtain the enzymolyzed mixture.

[0144] Cool the enzymolyzed mixture to 45℃, and use diatomite for filtration in a chamber filter press to obtain a filtered mixture.

[0145] Use an ultrafiltration membrane with a molecular weight cut-off of 3000Da for ultrafiltration of the filtered mixture to obtain a fish collagen peptide crude product.

[0146] C: Preparation of the collagen peptide of Example Eight

[0147] 1 g of the crude fish collagen peptide was weighed out and dissolved in water together with the aforementioned saccharide composition to obtain an aqueous solution, and then 0.2 g of sodium chondroitin sulfate was added. The aqueous solution was stirred at 70°C for 40 min, and then the pH value of the aqueous solution was adjusted to 8 while the temperature of the aqueous solution was reduced to 4°C to obtain a precipitate. The precipitate was freeze-dried to obtain the saccharide-modified crude fish collagen peptide.

[0148] The saccharide-modified crude fish collagen peptide was subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off of 15000 Da, and then was subjected to concentration, sterilization, spray drying, and sieving to obtain the collagen peptide of Example Eight.

[0149] Example Nine

[0150] The steps of Example Nine were the same as those of Example Eight, except that in Example Nine, 0.75 g of the acetylated modified annona polysaccharide and 2.5 g of the osmanthus leaf polysaccharide prepared were mixed with 1.25 g of trehalose to obtain the saccharide composition of Example Nine.

[0151] Example Ten

[0152] The steps of Example Ten were the same as those of Example One, except that in Example Ten, the saccharide-modified crude fish collagen peptide was subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off of 5000 Da, and then was subjected to concentration, sterilization, spray drying, and sieving to obtain the collagen peptide of Example Ten.

[0153] Comparative Example One

[0154] The steps included:

[0155] A: Preparation of fish collagen peptide:

[0156] Cleithrum was taken, and impurities were picked out from the cleithrum and the cleithrum was washed to obtain 150 g of cleithrum.

[0157] The washed cleithrum was put into a reactor, 450 mL of water was added to the reactor, and the reactor was heated at 80°C for 4 h. After the reaction was completed, the reactor was allowed to stand for 2 h to obtain a heated mixture.

[0158] The temperature of the heated mixture was controlled at 60°C, and the pH value was controlled at 8. Papain enzyme was used for first-step enzymolysis at a dosage of 0.4 wt% of the cleithrum, and the first-step enzymolysis was performed for 1.2 h to obtain a first-step enzymolysis product. Then, the temperature of the first-step enzymolysis product was controlled at 70°C, and the pH value was controlled at 9. Compound protease including alkaline protease and trypsin (wherein the mass ratio of the alkaline protease to the trypsin was 2:1) was used for second-step enzymolysis, and the second-step enzymolysis was performed for 1.2 h to obtain an enzymolysis mixture.

[0159] The mixture after enzymolysis was cooled to 45℃, filtered in a chamber filter press using diatomite to obtain a filtered mixture.

[0160] The filtered mixture was ultrafiltered using an ultrafiltration membrane with a molecular weight cut-off of 3000 Da to obtain the fish collagen peptide of Comparative Example 1.

[0161] Comparative Example 2

[0162] Comparative Example 2 was prepared by the same procedure as Example 1, except that Comparative Example 2 used 4.5 g of acetylated modified annona polysaccharide instead of the saccharide composition of Example 1 (3 g of acetylated modified annona polysaccharide + 1.5 g of trehalose).

[0163] Comparative Example 3

[0164] Comparative Example 3 was prepared by the same procedure as Example 1, except that Comparative Example 3 used 4.5 g of trehalose instead of the saccharide composition of Example 1 (3 g of acetylated modified annona polysaccharide + 1.5 g of trehalose).

[0165] Comparative Example 4

[0166] Comparative Example 4 was prepared by the same procedure as Example 1, except that Comparative Example 4 used 4.5 g of arabinose instead of the saccharide composition of Example 1 (3 g of acetylated modified annona polysaccharide + 1.5 g of trehalose).

[0167] Moisture retention rate test:

[0168] The same mass of the collagen peptide samples prepared in Examples 1-10 and Comparative Examples 1-4 were weighed into a weighing bottle that had been dried to a constant weight, 3 times the sample mass of water was added, stirred until uniform, and precisely weighed. The weighing bottles were each placed open in a silica gel desiccator with a relative humidity of 33%, the room temperature was kept at 20℃, and the weighing bottles were weighed at 12 h and 60 h, and the values of two parallel experiments were taken. The moisture retention rate of the sample was calculated according to the following formula: moisture retention rate (%) = W2 / W1 x 100; in the formula: W1 is the mass of water before placement; W2 is the mass of water after placement for a certain time.

[0169] Hygroscopicity test:

[0170] Saturated NaCl solution and saturated MgCl2 solution were placed in a closed desiccator to form an environment with a relative humidity of 33%, and then the desiccator was placed in a constant temperature environment. A certain amount of collagen peptide sample prepared in Examples 1 to 10 and Comparative Examples 1 to 4 dried to a constant weight was precisely weighed in a weighing bottle dried to a constant weight. The weighing bottle was placed in a desiccator with a relative humidity of 73% and 33%, respectively, and the room temperature was kept at 25°C. The weighing bottle was weighed after 12 h and 60 h, respectively, and the average of two parallel experiments was taken. The moisture absorption rate of the sample was calculated according to the following formula: moisture absorption rate (%) = (W1-W0) / W0x100; wherein: W0 is the mass of the dried sample; W1 is the mass of the sample after being placed for a certain time.

[0171] DPPH radical scavenging activity determination:

[0172] The same mass of collagen peptide samples prepared in Examples 1 to 10 and Comparative Examples 1 to 4 was weighed, and 500 μL of sample solution with a concentration of 30 mg / mL was taken in a 96-well plate. 100 μL of DPPH ethanol solution with a concentration of 0.04 mg / mL was added, and the reaction was carried out at room temperature for 30 min in the dark. In the control group, 100 μL of anhydrous ethanol was used instead of DPPH ethanol solution. In the blank group, 100 μL of deionized water was used instead of sample solution. In the positive control group, 100 μL of ascorbic acid was used instead of sample solution. The rest was consistent with the preparation method of the detection solution. The absorbance was measured at 517 nm, and three parallel experiments were performed. Scavenging rate (%) = [1[A sample A control group] / A blank group]x100%.

[0173] Hydroxyl radical scavenging ability determination:

[0174] Take 0.2 mL of FeSO4-EDTA mixed solution (10.0 mmol / L) in a test tube, add 0.5 mL of 2-deoxyribose solution (10.0 mmol / L) and 1.0 mL of 30 mg / mL solution of the samples of Examples 1 to 10 and Comparative Examples 1 to 4, and dilute with phosphate buffer (pH = 7.4, 0.1 mol / L) to 1.8 mL, then add 0.2 mL of H2O2 (10.0 mol / L), mix well and place in a 37°C constant temperature water bath for 1 h, then add 2.8% (w / w) trichloroacetic acid (TCA) solution 1.0 mL, centrifuge at 4000 r / min for 20 min, take 2.0 mL of supernatant and place in another test tube, add 1.0% (w / w) thiobarbituric acid (TBA) solution 1.0 mL, mix well and place in a boiling water bath for 15 min, cool and dilute 5 times, and measure the absorbance value at 532 nm. The clearance rate SA (%) = [(Ac-As) / (Ac-A0)]x100; in the formula: Ac is the absorbance of the mixed solution; As is the absorbance after adding the samples of Examples 1 to 10 and Comparative Examples 1 to 4; A0 is the absorbance of the reagent blank.

[0175] The values of the moisture retention rate test and the moisture absorption rate test are shown in Table 1.

[0176] Table 1

[0177]

[0178] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the application embrace any and all variations of the present application that fall within the scope of the other embodiments of the present application. Accordingly, other embodiments are intended to be encompassed by this description. Although specific embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that any arrangement which is calculated to achieve the same or similar result can be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments of the application. Therefore, it is intended that this application be construed as not limited to the examples described herein, but that it includes all those alternatives and modifications which fall within the scope of the present application.

Claims

1. A method for preparing collagen peptide for improving facial skin quality, characterized in that: The following steps are involved: Step 1a: taking fish tissue, picking out impurities from the fish tissue and cleaning the fish tissue; The fish tissue is at least one of fish skin, fish bones, and fish scales; Step 2a: putting the cleaned fish tissue into a reactor, adding water to the reactor and heating at 75° C.-85° C. for 2-5 hours. After the reaction is completed, standing for 1-2 hours to obtain a heated mixture; Step 3a: subjecting the heated mixture obtained in step 2a to a two-step enzymatic hydrolysis method to obtain an enzymatically hydrolyzed mixture; Controlling the temperature of the heated mixture to 55° C.-65° C. and the pH value to 7-8, performing a first enzymatic hydrolysis using papain for 1-1.5 hours to obtain a product after the first enzymatic hydrolysis, then controlling the temperature of the product after the first enzymatic hydrolysis to 65° C.-75° C. and the pH value to 8-9, performing a second enzymatic hydrolysis using a composite protease for 1-1.5 hours to obtain an enzymatic hydrolysis mixture; Wherein, the composite protease is at least two of alkaline protease, papain, trypsin and bromelain; Step 4a: After the enzymatically hydrolyzed mixture obtained in step 3a is cooled to below 50° C., it is filtered using diatomaceous earth to obtain a filtered mixture; Step 5a: subjecting the filtered mixture obtained in step 4a to a first ultrafiltration to obtain a crude fish collagen peptide; Step 6a: mixing the crude fish collagen peptide obtained in step 5a with sodium chondroitin sulfate and a carbohydrate composition, and subjecting the crude fish collagen peptide to a glycosylation reaction to obtain a glycosylated crude fish collagen peptide; Step 7a: subjecting the crude glycosylated fish collagen peptide obtained in step 6a to a second ultrafiltration, followed by concentration, sterilization, spray drying, and sieving to obtain the collagen peptide; Wherein, the carbohydrate composition comprises plant polysaccharides and at least one disaccharide; the disaccharide is trehalose; the plant polysaccharides comprise acetylated custard apple polysaccharide and osmanthus leaf polysaccharide; The first ultrafiltration was performed with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the second ultrafiltration was performed with an ultrafiltration membrane with a molecular weight cutoff of 15000 Da. In the carbohydrate composition, the mass ratio of acetylated annona polysaccharide, osmanthus leaf polysaccharide and trehalose is (1.5-2.5):(0.4-0.7):1; The annona polysaccharide is prepared by the following steps: Step 1b: taking the pulp portion of a mature sugar apple fruit and preparing sugar apple powder from the pulp portion; Step 2b: adding the sugar apple powder obtained in step 1b into a reactor, adding water to the reactor, and ultrasonically extracting the sugar apple powder for 0.5-1 hour at an ultrasonic temperature of 40° C.-45° C. and an ultrasonic power of 300-400 W to obtain a crude sugar apple polysaccharide extract; Step 3b: decolorizing and deproteinizing the crude sugar apple polysaccharide extract obtained in step 2b to obtain a sugar apple polysaccharide extract; Step 4b: The annona polysaccharide extracts obtained in step 3b are combined and concentrated, and 95% ethanol is added until the ethanol concentration in the final solution is 28%-32%, to obtain a precipitate and a supernatant, and the precipitate is washed and dried to obtain annona polysaccharide; The annona polysaccharide is further acetylated, and the acetylation modification step comprises: Step 5b: dissolving the sugar apple polysaccharide obtained in step 4b in water, adjusting the pH value to 9-10, adding acetic anhydride of 5-15wt% of the sugar apple polysaccharide every 15 minutes, while controlling the pH value to remain unchanged, and adding acetic anhydride 4-7 times. After the addition of acetic anhydride, stirring is continued for 0.5-1h, and then hydrochloric acid is added until the pH of the solution is 6-7 to terminate the reaction, and then 95% ethanol is added until the ethanol concentration in the final solution is 28%-32% to obtain a precipitate. The precipitate is washed and dried to obtain acetylated sugar apple polysaccharide.

2. The preparation method according to claim 1, characterized in that The glycosylation reaction in step 6a includes: The crude fish collagen peptide obtained in step 5a is dissolved in water with sodium chondroitin sulfate and a carbohydrate composition to obtain an aqueous solution, which is stirred at 65° C.-75° C. for 0.5-1 hour, and the pH value of the aqueous solution is adjusted to 8. At the same time, the temperature of the aqueous solution is lowered to below 30° C. to obtain a precipitate, and the precipitate is freeze-dried to obtain the crude glycosylated fish collagen peptide.

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