Preparation method of collagen peptide for improving facial skin

Abstract

The invention belongs to the technical field of preparation methods of collagen peptides, and particularly relates to a preparation method of a collagen peptide for improving facial skin, fish tissues are used as raw materials, and the preparation method comprises the steps of picking and washing, cooking, two-step enzymolysis, filtration, primary ultrafiltration, glycosylation reaction, secondary ultrafiltration, concentration, sterilization, spray drying and sieving. The glycosylation reaction involved in the invention comprises a process of performing glycosylation on a fish collagen peptide crude product by using plant polysaccharide and disaccharide. The collagen peptide prepared by the preparation method disclosed by the invention not only has good oxidation resistance, but also has good water absorption and moisturizing properties, so that the facial skin can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation methods of collagen peptides, and specifically relates to a preparation method of collagen peptides for improving facial skin quality. Background Art

[0002] With the development of social economy, people are increasingly pursuing a high-quality life. They not only pay attention to their own physical health, but also skin care has become an important part of daily life. Paying attention to beauty maintenance and keeping the facial skin fair and tender has become the skin care goal pursued by many people, especially women. Collagen is one of the main components of human tissue structure. It is distributed throughout the body's various tissues and organs, such as skin, bones, cartilage, ligaments, cornea, various inner membranes and fascia. It is the main component to maintain the morphology and structure of skin and tissue organs, and is also an important raw material for repairing damaged tissues. Supplementing collagen directly from cosmetics or skin care products can quickly replenish the collagen lost in the skin, making the skin more transparent and elastic.

[0003] Collagen peptide (CP) is a mixture between amino acids and proteins formed by enzymatic hydrolysis of collagen. It is a small molecule peptide with a molecular weight between several hundred and several thousand Daltons, usually derived from salmon skin, fish scales, chicken sternum cartilage, cowhide and oysters. The molecular weight of CP is generally less than 3000Da. Collagen is difficult to digest and absorb due to its high molecular weight, which limits its application. CP has good biological properties such as bioavailability, antioxidant and hypoallergenicity due to its relatively small molecular weight and easy solubility in water. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing collagen peptides for improving facial skin quality, so as to solve the defects pointed out in the above background technology.

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

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

[0007] 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, and standing for 1-2 hours after the reaction is completed to obtain a heated mixture;

[0008] Step 3a: subjecting the heated mixture obtained in step 2a to a two-step enzymatic hydrolysis method to obtain an enzymatically hydrolyzed mixture;

[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 the first enzymolysis, the time of the first enzymolysis is 1-1.5 hours, and a product after the first enzymolysis is obtained; then the temperature of the product after the first enzymolysis is controlled to be 65°C-75°C and the pH value is 8-9, a composite protease is used to perform the second enzymolysis, the time of the second enzymolysis is 1-1.5 hours, and a mixture after enzymolysis 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 embodiment 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, and the solution is stirred at 65° C.-75° C. for 0.5-1 h, and the pH value of the aqueous solution is adjusted to 8, and the temperature of the aqueous solution is reduced to below 30° C. to obtain a precipitate, and the precipitate is freeze-dried to obtain the glycosylated crude 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, and the mixture is stirred at 70° C. for 45 minutes. The pH value of the aqueous solution is adjusted to 8, and the temperature of the aqueous solution is reduced to 4° C. to obtain a precipitate. The precipitate is freeze-dried to obtain the glycosylated crude 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 of a ripe custard apple fruit and preparing custard apple powder from the pulp;

[0034] Step 2b: putting the sugar apple powder obtained in step 1b into a reactor, adding water into the reactor, and ultrasonically extracting for 0.5-1h under the conditions of ultrasonic temperature of 40°C-45°C and ultrasonic power of 300-400W to obtain a crude extract of sugar apple polysaccharide;

[0035] Step 3b: decolorizing and deproteinizing the crude custard apple polysaccharide extract obtained in step 2b to obtain a custard apple polysaccharide extract;

[0036] Step 4b: The annona polysaccharide extracts obtained in step 3b are combined and concentrated, and 95% ethanol is added until the concentration of ethanol 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.

[0037] In one aspect of an embodiment of the present invention, specifically, step 1b includes: taking the pulp part of a mature sugar apple fruit, washing the pulp part and air-drying it, then drying it at 55°C-65°C, crushing it and passing it through a 60-mesh sieve, defatting it with petroleum ether, and then washing and air-drying it again to obtain sugar apple powder.

[0038] In one aspect of an embodiment of the present invention, specifically, step 2b comprises: putting the sugar apple powder obtained in step 1b into a reactor, adding water to the reactor, and ultrasonically extracting for 45 minutes under the conditions of an ultrasonic temperature of 45° C. and an ultrasonic power of 400 W to obtain a crude extract of sugar apple polysaccharide.

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

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

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

[0042] In one aspect of the embodiments of the present invention, the annona polysaccharide is further modified by acetylation.

[0043] In one aspect of the embodiments of the present invention, the acetylation modification step comprises:

[0044] Step 5b: dissolving the annona polysaccharide obtained in step 4b in water, adjusting the pH value to 9-10, adding 5-15wt% acetic anhydride of the annona polysaccharide every 15 minutes, while controlling the pH value to remain unchanged, the number of times the acetic anhydride is added is 4-7 times, and after the acetic anhydride is added, stirring is continued for 0.5-1h, and then hydrochloric acid is added until the pH value of the solution is 6-7 to terminate the reaction, and then 95% ethanol is added until the concentration of ethanol in the final solution is 28%-32% to obtain a precipitate, and the precipitate is washed and dried to obtain acetylated annona polysaccharide.

[0045] In one aspect of the embodiments of the present invention, specifically, the acetylation modification step comprises:

[0046] Step 5b: dissolving the annona polysaccharide obtained in step 4b in water, adjusting the pH value to 9, adding 10wt% acetic anhydride of the annona polysaccharide every 15 minutes, while controlling the pH value to remain unchanged, adding acetic anhydride 5 times, and after the acetic anhydride is added, stirring is continued for 50 minutes, and then hydrochloric acid is added until the pH value of the solution is 6 to terminate the reaction, and then 95% ethanol is added until the concentration of ethanol in the final solution is 30%, to obtain a precipitate, and the precipitate is washed and dried to obtain acetylated annona polysaccharide.

[0047] In one aspect of an embodiment of the present invention, the carbohydrate composition comprises annona polysaccharide and trehalose.

[0048] In one aspect of the embodiments of the present invention, in the carbohydrate composition, the mass ratio of annona polysaccharide to trehalose is (1.5-2.5):1.

[0049] In one aspect of the embodiments of the present invention, the carbohydrate composition comprises acetylated-modified annona polysaccharide and trehalose.

[0050] In one aspect of an embodiment of the present invention, in the carbohydrate composition, the mass ratio of acetylated annona polysaccharide to trehalose is (1.5-2.5):1.

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

[0052] In one aspect of the embodiments of the present invention, in step 6a, the mass ratio of the crude fish collagen peptide to the carbohydrate composition is 1:(3-6).

[0053] In one aspect of the embodiments of the present invention, the plant polysaccharide further comprises osmanthus leaf polysaccharide.

[0054] In one aspect of the embodiments of the present invention, the plant polysaccharide comprises annona polysaccharide and osmanthus leaf polysaccharide.

[0055] In one aspect of the embodiments of the present invention, the plant polysaccharide comprises acetylated Annona mume polysaccharide and Osmanthus fragrans leaf polysaccharide.

[0056] In one aspect of the embodiments of the present invention, 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.

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

[0058] Step 1c: weigh osmanthus leaves, use water as an extractant, add water in a mass ratio of osmanthus leaves: water of 1: (20-30), reflux extract at 70° C.-80° C. for 1-3 times, and obtain an osmanthus leaf polysaccharide extract;

[0059] Step 2c: combining and concentrating the osmanthus leaf polysaccharide extracts 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, precipitate for 3 hours and then centrifuge to obtain a precipitate, wash and dry the precipitate to obtain the osmanthus leaf polysaccharide.

[0061] In one aspect of the embodiment of the present invention, an ultrafiltration membrane with a molecular weight cutoff of 3000 Da is used in the first ultrafiltration.

[0062] In one aspect of the embodiment of the present invention, an ultrafiltration membrane with a molecular weight cutoff of 15000 Da is used in the second ultrafiltration.

[0063] In one aspect of the embodiment of the present invention, in step 7a, the concentration process is: the feed liquid after the second ultrafiltration is pumped into a single-effect concentrator and concentrated to 2 / 3 of the original volume.

[0064] In one aspect of the embodiment of the present invention, in step 7a, the sterilization process is: the concentrated liquid is pumped into a sterilization tank, heated to 80°C-85°C, and kept warm for 20-30 minutes for sterilization.

[0065] In one aspect of the embodiment of the present invention, in step 7a, the spray drying process is: the sterilized material is pumped into a feed transfer tank, and then pumped into a spray drying tower through a filter for spray drying. Specifically, during the spray drying process, the inlet air temperature is 180°C-190°C, the outlet air temperature is 85°C-95°C, and the spray drying is performed after adjusting the feed liquid rotation speed.

[0066] In one aspect of an embodiment of the present invention, in step 7a, the screening process is: according to different requirements, the spray-dried material is screened using a sieve of corresponding mesh size, and the metal impurities are adsorbed by a magnetic frame (magnetic frame magnetic field strength ≥ 8000GS (800MT)).

[0067] According to a second aspect of an embodiment of the present invention, a collagen peptide is provided. The collagen peptide is prepared by the aforementioned preparation method.

[0068] According to a third aspect of an embodiment of the present invention, a cosmetic or skin care product comprises the aforementioned collagen peptide and at least one auxiliary material.

[0069] In one aspect of an embodiment of the present invention, the auxiliary materials are antioxidants, humectants, thickeners, foaming agents, preservatives, cooling agents, buffers, pH regulators, colorants, excipients, dispersants, emulsifiers, solubilizers, surfactants, astringents, lubricants, stabilizers, solvents and fragrances, but are not limited thereto.

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

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

[0072] One of the raw materials used in the present invention is cod bones, which are hard in texture and not suitable for consumption and are usually treated as waste. The use of cod bones to prepare cod bone collagen peptides can save costs and improve resource utilization.

[0073] The raw material custard apple polysaccharide used in the present invention can reduce the cell apoptosis caused by normal human epidermal keratin under γ irradiation, increase the activity of intracellular antioxidant enzymes SOD and catalase CAT, reduce the level of proinflammatory cytokine interleukin-1β (IL-1β) and inflammasome signaling pathway indicators, and has significant anti-skin aging function. The present invention uses custard apple polysaccharide to glycosylate fish collagen peptides, and the obtained glycosylated fish collagen peptides have strong antioxidant properties compared to other collagen peptide products, can effectively remove DPPH and ·OH free radicals, can eliminate the damage caused to the skin by excessive free radicals, and can be applied to cosmetics to delay skin aging.

[0074] In addition, before using sugar apple polysaccharide to glycosylate fish collagen peptide, the present invention further acetylates it. The acetylation modification of polysaccharide refers to the nucleophilic substitution reaction between the branched hydroxyl group of polysaccharide and the acetyl group. When the degree of substitution is high, the molecular weight of the polysaccharide is increased, and its molecular weight increases with the increase of the degree of acetylation, making the molecular conformation more compact; due to the change in the spatial arrangement of the polysaccharide chain, more hydroxyl groups are exposed, and the solubility of the polysaccharide is also improved. Therefore, the present invention uses trehalose and acetylated sugar apple polysaccharide to glycosylate fish collagen peptide, which can further improve the wettability of the product, effectively improve its moisture retention performance, and can be used in cosmetics to improve the effect of skin hydration and water retention.

[0075] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. The relevant embodiments described herein are illustrative and are used to provide a basic understanding of the present invention. The embodiments of the present invention should not be interpreted as limiting the present invention.

[0077] For simplicity, only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and any upper limit can be combined with any other upper limit to form an unspecified range. In addition, each separately disclosed point or single value can itself be combined as a lower limit or upper limit with any other point or single value or with other lower limits or upper limits to form an unspecified range.

[0078] In this article, the terms "comprises", "comprising" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions, an element defined by the sentence "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device that includes the element.

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

[0080] Unless otherwise specified, the terms used in the present invention have the commonly known meanings generally understood by those skilled in the art. Unless otherwise specified, the numerical values ​​of the various parameters mentioned in the present invention can be measured using various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the embodiments of the present invention).

[0081] The term "about" is used to describe and illustrate small changes. When used in conjunction with an event or situation, the term may refer to an example in which the event or situation occurs precisely and an example in which the event or situation occurs very approximately. For example, when used in conjunction with a numerical value, the term may 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%. In addition, amounts, ratios, and other numerical values ​​are sometimes presented in this article in a range format. It should be understood that such a range format is for convenience and simplicity, and should be flexibly understood to include not only numerical values ​​explicitly designated as range limits, but also all individual numerical values ​​or subranges encompassed within the range, as if each numerical value and subrange were explicitly designated.

[0082] A list of items connected by the terms "at least one of," "at least one of," "at least one of," or other similar terms may mean any combination of the listed items. For example, if items A and B are listed, the phrase "at least one of A and B" means only A; only B; or A and B. In another example, if items A, B, and C are listed, 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 may contain a single component or multiple components. Item B may contain a single component or multiple components. Item C may contain a single component or multiple components.

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

[0084] Embodiment 1

[0085] The following steps are involved:

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

[0087] 50 g of the pulp of a ripe custard apple fruit was taken, the pulp was washed and air-dried, then dried at 60° C., crushed and passed through a 60-mesh sieve, defatted with petroleum ether, and then washed and air-dried again to obtain the custard apple powder.

[0088] Put the sugar apple powder into the reactor, add water into the reactor, and perform ultrasonic extraction for 45 minutes under the conditions of ultrasonic temperature of 45° C. and ultrasonic power of 400 W to obtain a crude extract of sugar apple polysaccharide.

[0089] The crude extract of custard apple polysaccharide 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 the custard apple polysaccharide extract.

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

[0091] The sugar apple polysaccharide is dissolved in water, and the pH value is adjusted to 9. 10 wt % acetic anhydride of the sugar apple polysaccharide is added every 15 minutes while the pH value is kept constant. The acetic anhydride is added 5 times. After the acetic anhydride is added, stirring is continued for 50 minutes. Then, hydrochloric acid is added until the pH value of the solution is 6 to terminate the reaction. Then, 95% ethanol is added until the concentration of ethanol in the final solution is 30% to obtain a precipitate. The precipitate is washed and dried to obtain acetylated sugar apple polysaccharide.

[0092] 3 g of the prepared acetylated annona polysaccharide was weighed and mixed with 1.5 g of trehalose (commercially available) to obtain the carbohydrate composition of Example 1.

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

[0094] Take cod bones, pick out impurities from the cod bones and clean the cod bones to obtain 150g of cod bones.

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

[0096] The temperature of the heated mixture is controlled to be 60°C and the pH value is 8, and the first step of enzymolysis is carried out using 0.4wt% papain from cod bones. The time of the first step of enzymolysis is 1.2h to obtain a product after the first step of enzymolysis. Then, the temperature of the product after the first step of enzymolysis is controlled to be 70°C and the pH value is 9. A second step of enzymolysis is carried out using a composite protease comprising alkaline protease and trypsin (wherein the mass ratio of alkaline protease to trypsin is 2:1). The time of the second step of enzymolysis is 1.2h to obtain a mixture after enzymolysis.

[0097] The enzymatically hydrolyzed mixture was cooled to 45° C. and filtered using diatomaceous earth in a chamber filter press to obtain a filtered mixture.

[0098] The filtered mixture was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to obtain a crude fish collagen peptide.

[0099] C: Preparation of collagen peptides from Example 1

[0100] Weigh 1 g of crude fish collagen peptide and dissolve it in water with the aforementioned carbohydrate composition to obtain an aqueous solution, then add 0.2 g of sodium chondroitin sulfate, stir at 70°C for 40 minutes, adjust the pH value of the aqueous solution to 8, and reduce the temperature of the aqueous solution to 4°C to obtain a precipitate. After freeze-drying the precipitate, obtain a glycosylated crude fish collagen peptide.

[0101] The crude glycosylated fish collagen peptide was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 15,000 Da, and then concentrated, sterilized, spray-dried and sieved to obtain the collagen peptide of Example 1.

[0102] Embodiment 2

[0103] The following steps are involved:

[0104] A: Preparation of a carbohydrate composition comprising acetylated annona polysaccharide and trehalose

[0105] 50 g of the pulp of a ripe custard apple fruit was taken, the pulp was washed and air-dried, then dried at 60° C., crushed and passed through a 60-mesh sieve, defatted with petroleum ether, and then washed and air-dried again to obtain the custard apple powder.

[0106] Put the sugar apple powder into the reactor, add water into the reactor, and perform ultrasonic extraction for 45 minutes under the conditions of ultrasonic temperature of 45° C. and ultrasonic power of 400 W to obtain a crude extract of sugar apple polysaccharide.

[0107] The crude extract of custard apple polysaccharide 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 the custard apple polysaccharide extract.

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

[0109] 3 g of the prepared annona polysaccharide was weighed and mixed with 1.5 g of trehalose to obtain the carbohydrate composition of Example 2.

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

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

[0112] The cleaned cod bones were put into a reactor, 450 mL of water was added into the reactor, and the mixture was heated at 80° C. for 4 h. After the reaction was completed, the mixture was allowed to stand for 2 h to obtain a heated mixture.

[0113] The temperature of the heated mixture is controlled to be 60°C and the pH value is 8, and the first step of enzymolysis is carried out using 0.4wt% papain from cod bones. The time of the first step of enzymolysis is 1.2h to obtain a product after the first step of enzymolysis. Then, the temperature of the product after the first step of enzymolysis is controlled to be 70°C and the pH value is 9. A second step of enzymolysis is carried out using a composite protease comprising alkaline protease and trypsin (wherein the mass ratio of alkaline protease to trypsin is 2:1). The time of the second step of enzymolysis is 1.2h to obtain a mixture after enzymolysis.

[0114] The enzymatically hydrolyzed mixture was cooled to 45° C. and filtered using diatomaceous earth in a chamber filter press to obtain a filtered mixture.

[0115] The filtered mixture was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to obtain a crude fish collagen peptide.

[0116] C: Preparation of collagen peptides from Example 2

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

[0118] The crude glycosylated fish collagen peptide was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 15,000 Da, and then concentrated, sterilized, spray-dried and sieved to obtain the collagen peptide of Example 2.

[0119] Embodiment 3

[0120] The steps of Example 3 are the same as those of Example 1, except that, in Example 3, 1.5 g of the prepared acetylated custard apple polysaccharide is weighed and mixed with 3 g of trehalose to obtain the carbohydrate composition of Example 3.

[0121] Embodiment 4

[0122] The steps of Example 4 are the same as those of Example 1, except that, in Example 4, 2.25 g of the prepared acetylated custard apple polysaccharide is weighed and mixed with 2.25 g of trehalose to obtain the carbohydrate composition of Example 4.

[0123] Embodiment 5

[0124] The steps of Example 5 are the same as those of Example 1, except that, in Example 5, 4 g of the prepared acetylated custard apple polysaccharide is weighed and mixed with 0.5 g of trehalose to obtain the carbohydrate composition of Example 5.

[0125] Embodiment 6

[0126] The steps of Example 6 are the same as those of Example 1, except that in Example 6, sucrose is used instead of trehalose.

[0127] Embodiment 7

[0128] The steps of Example 7 are the same as those of Example 1, except that in Example 6, glucose is used instead of trehalose.

[0129] Embodiment 8

[0130] The following steps are involved:

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

[0132] 50 g of the pulp of a ripe custard apple fruit was taken, the pulp was washed and air-dried, then dried at 60° C., crushed and passed through a 60-mesh sieve, defatted with petroleum ether, and then washed and air-dried again to obtain the custard apple powder.

[0133] Put the sugar apple powder into the reactor, add water into the reactor, and perform ultrasonic extraction for 45 minutes under the conditions of ultrasonic temperature of 45° C. and ultrasonic power of 400 W to obtain a crude extract of sugar apple polysaccharide.

[0134] The crude extract of custard apple polysaccharide 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 the custard apple polysaccharide extract.

[0135] The annona polysaccharide extracts were combined and concentrated to 2 / 3 of the original solution, and then 95% ethanol was added until the ethanol concentration in the final solution was 30%, to obtain a precipitate and a supernatant, and the precipitate was washed and dried to obtain the annona polysaccharide.

[0136] The sugar apple polysaccharide is dissolved in water, and the pH value is adjusted to 9. 10 wt % acetic anhydride of the sugar apple polysaccharide is added every 15 minutes while the pH value is kept constant. The acetic anhydride is added 5 times. After the acetic anhydride is added, stirring is continued for 50 minutes. Then, hydrochloric acid is added until the pH value of the solution is 6 to terminate the reaction. Then, 95% ethanol is added until the concentration of ethanol in the final solution is 30% to obtain a precipitate. The precipitate is washed and dried to obtain acetylated sugar apple polysaccharide.

[0137] Weigh 12 g of osmanthus leaves, use water as the extractant, add 25 times the mass of water, and reflux extract twice at 75° C. to obtain an osmanthus leaf polysaccharide extract.

[0138] The osmanthus leaf polysaccharide extracts are combined and concentrated to 1 / 3 of the original volume; 3 times the volume of anhydrous ethanol is added to the concentrated osmanthus leaf polysaccharide extract, and the mixture is precipitated for 3 hours and then centrifuged to obtain a precipitate; the precipitate is washed and dried to obtain osmanthus leaf polysaccharide.

[0139] Weigh 2.5 g of the prepared acetylated annona polysaccharide and 0.75 g of osmanthus leaf polysaccharide, and mix them with 1.25 g of trehalose to obtain the carbohydrate composition of Example 8.

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

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

[0142] The cleaned cod bones were put into a reactor, 450 mL of water was added into the reactor, and the mixture was heated at 80° C. for 4 h. After the reaction was completed, the mixture was allowed to stand for 2 h to obtain a heated mixture.

[0143] The temperature of the heated mixture is controlled to be 60°C and the pH value is 8, and the first step of enzymolysis is carried out using 0.4wt% papain from cod bones. The time of the first step of enzymolysis is 1.2h to obtain a product after the first step of enzymolysis. Then, the temperature of the product after the first step of enzymolysis is controlled to be 70°C and the pH value is 9. A second step of enzymolysis is carried out using a composite protease comprising alkaline protease and trypsin (wherein the mass ratio of alkaline protease to trypsin is 2:1). The time of the second step of enzymolysis is 1.2h to obtain a mixture after enzymolysis.

[0144] The enzymatically hydrolyzed mixture was cooled to 45° C. and filtered using diatomaceous earth in a chamber filter press to obtain a filtered mixture.

[0145] The filtered mixture was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to obtain a crude fish collagen peptide.

[0146] C: Preparation of collagen peptide of Example 8

[0147] Weigh 1 g of crude fish collagen peptide and dissolve it in water with the aforementioned carbohydrate composition to obtain an aqueous solution, then add 0.2 g of sodium chondroitin sulfate, stir at 70°C for 40 minutes, adjust the pH value of the aqueous solution to 8, and reduce the temperature of the aqueous solution to 4°C to obtain a precipitate. After freeze-drying the precipitate, obtain a glycosylated crude fish collagen peptide.

[0148] The crude glycosylated fish collagen peptide was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 15,000 Da, and then concentrated, sterilized, spray-dried and sieved to obtain the collagen peptide of Example 8.

[0149] Embodiment 9

[0150] The steps of Example 9 are the same as those of Example 8, except that, in Example 9, 0.75 g of the prepared acetylated custard apple polysaccharide and 2.5 g of osmanthus leaf polysaccharide were weighed and mixed with 1.25 g of trehalose to obtain the carbohydrate composition of Example 9.

[0151] Embodiment 10

[0152] The steps of Example 10 are the same as those of Example 1, except that, in Example 10, the crude glycosylated fish collagen peptide is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5000Da, and then concentrated, sterilized, spray-dried and sieved to obtain the collagen peptide of Example 10.

[0153] Comparative Example 1

[0154] The following steps are involved:

[0155] A: Preparation of fish collagen peptides:

[0156] Take cod bones, pick out impurities from the cod bones and clean the cod bones to obtain 150g of cod bones.

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

[0158] The temperature of the heated mixture is controlled to be 60°C and the pH value is 8. The first step of enzymolysis is performed using 0.4wt% papain from cod bones. The time for the first step of enzymolysis is 1.2h to obtain a product after the first step of enzymolysis. Then, the temperature of the product after the first step of enzymolysis is controlled to be 70°C and the pH value is 9. The second step of enzymolysis is performed using a composite protease comprising alkaline protease and trypsin (wherein the mass ratio of alkaline protease to trypsin is 2:1). The time for the second step of enzymolysis is 1.2h to obtain a mixture after enzymolysis.

[0159] The enzymatically hydrolyzed mixture was cooled to 45° C. and filtered using diatomaceous earth in a chamber filter press to obtain a filtered mixture.

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

[0161] Comparative Example 2

[0162] The steps of Comparative Example 2 are the same as those of Example 1, except that 4.5 g of acetylated custard apple polysaccharide is used in Comparative Example 2 to replace the carbohydrate composition of Example 1 (3 g of acetylated custard apple polysaccharide + 1.5 g of trehalose).

[0163] Comparative Example 3

[0164] The steps of Comparative Example 3 are the same as those of Example 1, except that Comparative Example 3 uses 4.5 g of trehalose to replace the carbohydrate composition (3 g of acetylated-modified annona polysaccharide+1.5 g of trehalose) of Example 1.

[0165] Comparative Example 4

[0166] The steps of Comparative Example 4 are the same as those of Example 1, except that Comparative Example 4 uses 4.5 g of arabinose to replace the carbohydrate composition of Example 1 (3 g of acetylated-modified annona polysaccharide+1.5 g of trehalose).

[0167] Moisture retention test:

[0168] Weigh the same mass of the collagen peptide samples prepared in Examples 1 to 10 and Comparative Examples 1 to 4, place them in a weighing bottle that has been dried to constant weight, add 3 times the mass of water, stir evenly, and weigh accurately. Place the weighing bottles open in a silica gel dryer with a relative humidity of 33%, keep the room temperature at 20°C, weigh each weighing bottle at 12h and 60h, and take two parallel experimental values. Calculate the moisture retention rate of the sample according to the following formula: Moisture retention rate (%) = W2 / W1×100; Where: W1 is the water mass before placement; W2 is the water mass after a certain period of placement.

[0169] Moisture absorption test:

[0170] Place a saturated NaCl solution and a saturated MgCl2 solution in a sealed desiccator to form an environment with a relative humidity of 33%, then place the desiccator in a constant temperature environment, accurately weigh a certain amount of collagen peptide samples prepared in Examples 1 to 10 and Comparative Examples 1 to 4 that have been dried to constant weight in weighing bottles that have been dried to constant weight, and place the weighing bottles open in desiccators with relative humidity of 73% and 33%, respectively, keep the room temperature at 25°C, weigh each weighing bottle at 12h and 60h, and take the average of two parallel experiments. Calculate the moisture absorption rate of the sample according to the following formula: Moisture absorption rate (%) = (W1-W0) / W0×100; Where: W0 is the mass of the dried sample; W1 is the mass of the sample after being placed for a certain period of time.

[0171] DPPH free radical scavenging activity assay:

[0172] Weigh the same mass of the collagen peptide samples prepared in Examples 1 to 10 and Comparative Examples 1 to 4, take 500 μL of a sample solution with a concentration of 30 mg / mL in a 96-well plate, add 100 μL of a DPPH ethanol solution with a concentration of 0.04 mg / mL, and react at room temperature in the dark for 30 minutes; the DPPH ethanol solution in the control group is replaced with 100 μL of anhydrous ethanol, the sample solution in the blank group is replaced with 100 μL of deionized water, and the sample solution in the positive control is replaced with 100 μL of ascorbic acid, and the rest is consistent with the preparation method of the detection solution. Measure the absorbance at 517 nm and perform three parallel experiments. Clearance rate (%) = [1 [A sample A control group] / A blank group] × 100%.

[0173] Determination of hydroxyl free radical scavenging ability:

[0174] Take 0.2 mL of FeSO4-EDTA mixed solution (10.0 mmol / L) and place it 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 samples of Examples 1 to 10 and Comparative Examples 1 to 4, dilute to 1.8 mL with phosphate buffer (pH = 7.4, 0.1 mol / L), and then add 0.2 mL H2O2 (10.0 mol / L), mix well and place in a 37°C constant temperature water bath to react for 1 hour, then add 1.0 mL of 2.8% (w / w) trichloroacetic acid (TCA) solution, centrifuge at 4000 r / min for 20 minutes, take 2.0 mL of supernatant and place in another test tube, add 1.0 mL of 1.0% (w / w) thiobarbituric acid (TBA) solution, mix well and place in a boiling water bath to react for 15 minutes, dilute 5 times after cooling, and measure the absorbance value at a wavelength of 532 nm. Clearance SA (%) = [(Ac-As) / (Ac-A0)] × 100; where: 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] Those skilled in the art will readily appreciate other embodiments of the present invention after considering the specification and practicing the disclosure disclosed herein. The present invention is intended to cover any variations, uses or adaptations of the present invention that follow the general principles of the present invention and include common knowledge or customary technical means in the art that are not disclosed by the present invention.

Claims

1. A method for preparing a 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 washing the fish tissue; 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, and standing for 1-2 hours after the reaction is completed 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; 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: 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.

2. The preparation method according to claim 1, characterized in that: The two-step enzymatic hydrolysis in step 3a includes: The temperature of the heated mixture is controlled to be 55° C.-65° C. and the pH value is 7-8, and papain is used to perform a first step of enzymolysis, and the time of the first step of enzymolysis is 1-1.5 hours to obtain a product after the first step of enzymolysis, and then the temperature of the product after the first step of enzymolysis is controlled to be 65° C.-75° C. and the pH value is 8-9, and a composite protease is used to perform a second step of enzymolysis, and the time of the second step of enzymolysis is 1-1.5 hours to obtain a mixture after enzymolysis; Wherein, the composite protease is at least two of alkaline protease, papain, trypsin and bromelain.

3. 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, and the solution is stirred at 65° C.-75° C. for 0.5-1 h, and the pH value of the aqueous solution is adjusted to 8, and the temperature of the aqueous solution is reduced to below 30° C. to obtain a precipitate, and the precipitate is freeze-dried to obtain the glycosylated crude fish collagen peptide.

4. The preparation method according to claim 1, characterized in that: The carbohydrate composition comprises plant polysaccharides and at least one disaccharide.

5. The preparation method according to claim 4, characterized in that: The disaccharide is maltose, lactose, sucrose, trehalose or cellobiose.

6. The preparation method according to claim 5, characterized in that: The disaccharide is trehalose.

7. The preparation method according to claim 4, characterized in that: The plant polysaccharide comprises annona polysaccharide.

8. The preparation method according to claim 7, characterized in that: The annona polysaccharide is prepared by the following steps: Step 1b: taking the pulp of a ripe custard apple fruit and preparing custard apple powder from the pulp; Step 2b: putting the sugar apple powder obtained in step 1b into a reactor, adding water into the reactor, and ultrasonically extracting for 0.5-1h under the conditions of ultrasonic temperature of 40°C-45°C and ultrasonic power of 300-400W to obtain a crude extract of sugar apple polysaccharide; Step 3b: decolorizing and deproteinizing the crude custard apple polysaccharide extract obtained in step 2b to obtain a custard apple polysaccharide extract; Step 4b: The annona polysaccharide extracts obtained in step 3b are combined and concentrated, and 95% ethanol is added until the concentration of ethanol 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.

9. The preparation method according to claim 7 or 8, characterized in that: The annona polysaccharide is also acetylated, and the acetylation step comprises: Step 5b: dissolving the annona polysaccharide obtained in step 4b in water, adjusting the pH value to 9-10, adding 5-15wt% acetic anhydride of the annona polysaccharide every 15 minutes, while controlling the pH value to remain unchanged, the number of times the acetic anhydride is added is 4-7 times, and after the acetic anhydride is added, stirring is continued for 0.5-1h, and then hydrochloric acid is added until the pH value of the solution is 6-7 to terminate the reaction, and then 95% ethanol is added until the concentration of ethanol in the final solution is 28%-32% to obtain a precipitate, and the precipitate is washed and dried to obtain acetylated annona polysaccharide.

10. The preparation method according to claim 1, characterized in that: An ultrafiltration membrane with a molecular weight cutoff of 3000Da was used for the first ultrafiltration, and an ultrafiltration membrane with a molecular weight cutoff of 15000Da was used for the second ultrafiltration.

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