CN106399437B - Preparation method of small water turtle polypeptide and small water turtle polypeptide mask - Google Patents
Preparation method of small water turtle polypeptide and small water turtle polypeptide mask Download PDFInfo
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- CN106399437B CN106399437B CN201610808564.7A CN201610808564A CN106399437B CN 106399437 B CN106399437 B CN 106399437B CN 201610808564 A CN201610808564 A CN 201610808564A CN 106399437 B CN106399437 B CN 106399437B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
Abstract
The invention discloses a preparation method of a small water turtle polypeptide, which comprises the following steps: taking the minced meat of the stone small water turtle, and mixing the materials according to the proportion of 1: 5-1: adding ultrapure water according to the solid-liquid ratio of 10 to obtain meat paste water; adding neutral protease into the meat paste water, and carrying out enzymolysis for 8-12 h to obtain an enzymolysis liquid; carrying out enzyme deactivation operation on the enzymolysis liquid to obtain a small water turtle polypeptide solution; centrifuging the small water turtle polypeptide solution, removing the precipitate, and freeze-drying to obtain the small water turtle polypeptide. The average molecular weight range of the extracted small water turtle polypeptide is 2000-3000, the small water turtle polypeptide belongs to small molecular bioactive polypeptide, and the small water turtle polypeptide has an antioxidation effect and can resist oxygen free radicals. The invention also discloses a small water turtle polypeptide mask.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of a small water turtle polypeptide and a small water turtle polypeptide mask.
Background
The stone small water turtle, named Huanghoushui turtle, is mainly distributed in provinces such as Guangdong, Hainan, Guangxi, Fujian, Hunan, etc. of China. The Chinemys reevesii is reptile of the genus Tortoise of the family Testudinidae. According to the present scientific research, the body of the small water turtle not only contains rich protein, but also contains a plurality of vitamins, ossein, peptides, unsaturated lipids, a plurality of enzymes, cutin, fatty acid, blood sugar and a plurality of nutrient elements such as calcium, potassium, phosphorus and the like which are easy to be absorbed by the human body, thereby having extremely high economic value and medicinal value,
the small water turtle polypeptide has wide application in food industry, medicine industry, daily chemical industry, bioengineering and other aspects, and the latest research shows that the small water turtle polypeptide belongs to a bioactive polypeptide, has the characteristics of low toxicity, antioxidation and the like, and is very favorable for product development as a main functional raw material of the facial mask.
Bioactive polypeptide refers to amino acid polymer with certain physiological function for life activity of living body, and can be called biological peptide, and its distributed molecular weight range is very large, it can be dipeptide or macromolecular polypeptide formed from several hundred amino acids connected by peptide bond. The bioactive polypeptide has the characteristics of good water solubility and physicochemical properties, low viscosity, low foaming rate and low gelling property, so the bioactive polypeptide has higher application value.
Since the industry of large-scale extraction and separation technology is mature, the natural ingredients slowly found in skin care products on the market are from land to sea, and from plants to animals, and all the natural ingredients are fully available. Some people, even in places where they are rarely found, try to find special materials and create skin care curiosity, including rainforests. Naturally, most of the ingredients are gimmicks, most of the base materials are still ingredients in the mineral oil era, and only some natural ingredients are occasionally added, so that the problems of corrosion prevention and the like are still difficult to overcome due to the mixing of the ingredients, and therefore, the skin care effect is poor, the toxicity is high, and the health of consumers is hurt.
Disclosure of Invention
The invention aims to provide a preparation method of a small water turtle polypeptide and a small water turtle polypeptide mask.
The technical scheme adopted by the invention is that the preparation method of the small water turtle polypeptide comprises the following steps: taking the minced meat of the stone small water turtle, and mixing the minced meat of the stone small water turtle with the solid-liquid ratio of 1: 5-1: 10, adding ultrapure water into the minced meat of the stone coin turtle to obtain minced meat water; adding neutral protease into the meat paste water for enzymolysis, adjusting the pH of the system in the enzymolysis process, and performing enzymolysis for 8-12 h to obtain an enzymolysis liquid; carrying out enzyme deactivation operation on the enzymatic hydrolysate to obtain a small water turtle polypeptide solution; and centrifuging the small water turtle polypeptide solution, removing precipitates, reserving filtrate, and freeze-drying to obtain the small water turtle polypeptide.
Further, the temperature of the ultrapure water is 40-50 ℃, and the pH of the ultrapure water is 7-8.
Further, NaOH is used for adjusting the pH value of the reaction system in the enzymolysis process.
Further, the enzyme deactivation operation is: and (3) putting the enzymolysis liquid into an environment with the temperature of 85 ℃ to inactivate enzyme for 10 min.
Further, the enzyme deactivation operation is to deactivate the enzyme for 10min by putting the enzymolysis liquid in the environment of 85 ℃.
Further, in the operation of centrifuging the small water turtle polypeptide solution, the rotation speed of the centrifugation is 8000 r/min.
The invention also provides a small water turtle polypeptide mask, which comprises the following components in part by weight: the feed comprises the components of small water turtle polypeptide, deionized water, 1, 3-butanediol, sodium hyaluronate, glyceryl polyether-26, giant kelp extract, hydroxyethyl cellulose, glycerol, polysorbate-80, dipotassium glycyrrhizinate, xanthan gum, hydroxyethyl acrylate and methyl hydroxybenzoate.
Further, the small water turtle polypeptide is prepared in any one of the above manners.
Further, the formula comprises the following components in percentage by mass: 0.05-0.2 part of small water turtle polypeptide, 60-90 parts of deionized water, 1-4 parts of 1, 3-butanediol, 0.1-0.3 part of sodium hyaluronate, 1-5 parts of glyceryl polyether-26, 0.5-2 parts of giant kelp extract, 0.05-0.2 part of hydroxyethyl cellulose, 5-20 parts of glycerol, 0.05-0.2 part of polysorbate-80, 0.1-1 part of dipotassium glycyrrhizinate, 0.1-0.3 part of xanthan gum, 0.05-0.2 part of methylparaben and 2-5 parts of compound amino acid.
The average molecular weight range of the extracted cyclemys trifasciata polypeptide is 2000-3000, the cyclemys trifasciata polypeptide belongs to micromolecular bioactive polypeptide, the cyclemys trifasciata polypeptide has an antioxidation effect, can resist Reactive Oxygen Species (ROS), can protect cells from oxidative damage caused by excessive reactive oxygen species, and can enable facial skin to absorb the antioxidation components of the cyclemys trifasciata polypeptide by applying the cyclemys trifasciata polypeptide to the mask, so that the generation of free radicals is reduced, the skin care effect is remarkable, and the generated toxicity is low.
Drawings
FIG. 1 is a data chart of PSC experiment results of in vitro antioxidant hydrogen peroxide radical scavenging ability of a small water turtle polypeptide;
FIG. 2 is a data diagram of ORAC experimental results of in vitro antioxidant oxygen radical absorption capacity of the small water turtle polypeptide;
FIG. 3 is a graph of the toxicity of the small water turtle polypeptide against H2pG2 cells;
FIG. 4 is a graph of data showing in vivo anti-CAA results of using PBS to wash Chinemys reevesii polypeptides;
figure 5 is a graph of data on in vivo anti-CAA results for scarab polypeptides without PBS wash.
Detailed Description
The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. It is within the scope of the present invention to modify or replace methods, steps or conditions of the present invention without departing from the spirit and substance of the present invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The technical scheme adopted by the invention is that the preparation method of the small water turtle polypeptide comprises the following steps: taking the minced meat of the stone small water turtle, and mixing the minced meat of the stone small water turtle with the solid-liquid ratio of 1: 5-1: 10 adding ultrapure water into the minced meat of the stone coin turtle to obtain minced meat water; adding neutral protease into the meat paste water for enzymolysis, adjusting the pH of the system in the enzymolysis process, and performing enzymolysis for 8-12 h to obtain an enzymolysis liquid; carrying out enzyme deactivation operation on the enzymolysis liquid to obtain a small water turtle polypeptide solution; centrifuging the small water turtle polypeptide solution, removing the precipitate, reserving the filtrate, and freeze-drying to obtain the small water turtle polypeptide.
The temperature of the ultrapure water is 40-50 ℃, and the pH of the ultrapure water is 7-8.
NaOH is used for adjusting the pH value of the reaction system in the enzymolysis process.
The enzyme deactivation operation comprises the following steps: and (3) putting the enzymolysis liquid into an environment with the temperature of 85 ℃ to inactivate enzyme for 10 min.
In the operation of centrifuging the small water turtle polypeptide solution, the centrifugation rotating speed is 8000 r/min.
The small water turtle polypeptide extracted by the method has the average molecular weight range of 2000-3000, has an antioxidant function, and can resist Reactive Oxygen Species (ROS), wherein the ROS is a byproduct in a biological aerobic metabolic process and comprises oxygen ions, peroxides, oxygen-containing free radicals and the like. These particles are quite small and are very active due to the presence of unpaired free electrons. Excessive levels of reactive oxygen species can cause damage to cells and genetic structures. Active oxygen, which is an oxygen-containing, chemically active molecule. Including oxygen ions (oxygen ions) and hydrogen peroxide (peroxide). Has strong chemical reactivity because of the existence of unpaired electrons outside the nucleus. ROS are byproducts of normal oxygen metabolism and play a major role in cell signaling and maintaining homeostasis. However, the amount of ROS increases dramatically over time and under the influence of the external environment (e.g., exposure to Ultraviolet (UV) or heat sources, etc.), and the cause of such changes may be due to significant damage to the cell structure. This phenomenon is called oxidative stress. ROS can also be generated by external factors such as ionizing radiation. Antioxidant action is the process of counteracting collective oxidation. The antioxidant substances of the human body are synthesized by themselves and are also supplied by food. Enzymatic and non-enzymatic antioxidants play a crucial role in protecting peroxidative damage due to exercise. The supplement of antioxidant substances is beneficial to the exercise body to reduce the generation of free radicals or accelerate the elimination of the free radicals so as to resist the side effects of the free radicals, thereby being beneficial to the health of the general people and possibly delaying the occurrence of exercise-induced fatigue and aging. The oxidation is the biggest threat of skin aging, and free radicals of the skin can be flooded by sunshine, pressure, environmental pollution and the like, so that the oxidation phenomena of dark complexion, water shortage and the like are generated. The antioxidant skin care product can prevent skin relaxation and wrinkle, improve skin pigmentation caused by ultraviolet, and remove senile plaque and other pigmented spots. We live in oxygen every day, without leaving it. However, excessive oxygen is inhaled and contacts with excessive oxygen, which can accelerate skin aging and damage cell tissues in the body. Therefore, some antioxidant skin care products are used at present, contain rich moisturizing essence, and can remold the natural repair function of the skin and save the pressed skin in the daytime.
The invention also provides a small water turtle polypeptide mask, which comprises the following components in part by weight: the feed comprises the components of small water turtle polypeptide, deionized water, 1, 3-butanediol, sodium hyaluronate, glyceryl polyether-26, giant kelp extract, hydroxyethyl cellulose, glycerol, polysorbate-80, dipotassium glycyrrhizinate, xanthan gum, methylparaben and compound amino acid. The stone small water turtle polypeptide mask takes stone small water turtle polypeptide as a main functional raw material, and all the components are prepared into the mask according to a conventional method.
The specific formula comprises the following components in percentage by mass: 0.05-0.2 part of small water turtle polypeptide, 60-90 parts of deionized water, 1-4 parts of 1, 3-butanediol, 0.1-0.3 part of sodium hyaluronate, 1-5 parts of glyceryl polyether-26, 0.5-2 parts of giant kelp extract, 0.05-0.2 part of hydroxyethyl cellulose, 5-20 parts of glycerol, 0.05-0.2 part of polysorbate-80, 0.1-1 part of dipotassium glycyrrhizinate, 0.1-0.3 part of xanthan gum, 0.05-0.2 part of methylparaben and 2-5 parts of compound amino acid.
1, 3-butanediol is one of polyhydric alcohols, is often used as a humectant and a solvent in cosmetics, and has small water-holding proportion and certain bacteriostatic action in the aspect of moisture preservation because butanediol is a small-molecular moisture-preserving component.
SODIUM HYALURONATE (SODIUM HYALURONATE) is extracted from rooster comb, or is prepared by fermenting lactococcus lactis, and is white or white-like granule or powder, and has no odor, nitrogen content of 2.8-4.0% and glucuronic acid content of 37.0-51.0% when dried. Is widely used in the field of cosmetics and has the function of moisturizing.
The glyceryl polyether-26 is obtained by polymerizing glycerin and ethylene oxide, belongs to nonionic surfactant-polyhydric alcohol polyoxyethylene ether, is used as a moisturizing agent and a spreading agent of cosmetics, and belongs to one of international cosmetic raw materials.
The giant kelp extract is extracted from giant kelp of Phaeophyceae and Laminariales, and contains components for whitening and removing blackhead. Is a used cosmetic raw material published by the No. 11 document of the State food and drug administration.
Hydroxyethyl cellulose (HEC) is a white or light yellow, tasteless and nontoxic fibrous or powdery solid, is prepared by etherification of alkali cellulose and ethylene oxide (or chloroethanol), and belongs to nonionic soluble cellulose ethers. HEC is widely used in cosmetics due to its good properties of thickening, suspending, dispersing, emulsifying, binding, film forming, moisture protecting, and protective colloid providing.
Polysorbate-80 as non-ionic surfactant, medicinal supplementary material, solubilizer, emulsifier, etc.
Dipotassium glycyrrhizinate is white or quasi-white fine powder, has the purity of 98 percent, has special sweet taste, good water solubility and pure mouthfeel. The dipotassium glycyrrhizinate has multiple effects of bacteriostasis, inflammation diminishing, detoxification, anti-allergy, deodorization and the like. Has wide application in the industries of medicine, cosmetics, daily chemicals, food and the like.
Xanthan gum is an extracellular microbial multi-pool produced by intoxication of saccharides by xanthomonas sp. Due to the special structure and colloid characteristics of macromolecules, the modified starch has multiple functions and can be used as an emulsifier, a stabilizer, a gel thickener, a sizing agent, a film forming agent and the like.
Methylparaben, also known as methylparaben. Methylparaben. Methylparaben, white crystalline powder or colorless crystals, readily soluble in alcohol, ether and acetone, very slightly soluble in water, boiling point 270-. Is mainly used as a sterilization preservative for organic synthesis, foods, cosmetics and medicines.
The compound amino acid is: contains various amino acids such as glycine, alanine, leucine, isoleucine, valine, cystine, cysteine, methionine, threonine, serine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, glutamic acid, aspartic acid, etc., and can adjust the proportion of each component or delete one or more of the components according to the requirement.
In order to increase the viscosity of the mask, 0.01-6 parts of hydroxyethyl acrylate can be added appropriately; in order to increase the fragrance of the mask, a proper amount of essence can be added, and the mass ratio range is 0.01-0.2.
Example 1:
taking 10g of the meat paste of the stone small water turtle, adding 50ml of ultrapure water with the temperature of 40 ℃ and the pH value of 7, adding neutral protease, adjusting the pH value of a reaction system by NaOH in the enzymolysis process, carrying out hydrolysis for 8h, then carrying out enzyme deactivation for 10min in the environment of 85 ℃, then carrying out centrifugation for 30min at the rotation speed of 8000r/min, then removing the precipitate, and carrying out freeze drying to obtain the stone small water turtle polypeptide.
Example 2
Taking 10g of the meat paste of the small water turtle, adding 100ml of ultrapure water with the temperature of 50 ℃ and the pH value of 8, adding neutral protease, adjusting the pH value of a reaction system by NaOH in the enzymolysis process, carrying out hydrolysis for 10h, then carrying out enzyme deactivation for 10min at the temperature of 85 ℃, carrying out centrifugation for 30min at the rotating speed of 8000r/min, removing the precipitate, and carrying out freeze drying to obtain the small water turtle polypeptide.
Example 3
Taking 10g of the meat mash of the small water turtle, adding 80ml of ultrapure water with the temperature of 45 ℃ and the pH value of 7.5, adding neutral protease, adjusting the pH value of a reaction system by NaOH in the enzymolysis process, hydrolyzing for 9h, then inactivating the enzyme for 10min in an environment of 85 ℃, centrifuging for 30min at the rotating speed of 8000r/min, removing the precipitate, and freeze-drying to obtain the small water turtle polypeptide.
Example 4
Research on the antioxidant effect of the small water turtle polypeptide.
The obtained small water turtle polypeptide is prepared into 1mg/mL aqueous solution by pure water, and is stored at-20 ℃ for standby.
The small water turtle polypeptide was diluted to different concentrations (starting concentration 1 g/ml).
The small water turtle polypeptide is diluted to different concentrations (the initial concentration is 4.8mg/ml), and the in vitro antioxidant capacity is measured by adopting a method of hydrogen peroxide radical scavenging capacity PSC.
The method for measuring the hydrogen peroxide radical scavenging ability of the small water turtle polypeptide is appropriately improved on the basis of the scheme proposed by Adom et al (Wang, H.; Cao, G.H.; Prior, R.L., Oxygen radial absorbing 395 capacity of the anticancer chemins, journal of Agricultural and Food Chemistry 1997,45, 304-. The experimental reaction system adopts 75mmol/L KH except for illustration2PO4-K2HPO4(pH 7.4). The DCFH-DA, ascorbic acid and ABAP solutions used in the experiment are prepared in situ. The small water turtle polypeptide sample is diluted by the phosphate buffer solution and then measured by using a 96-well plate, and 100 mu L of small water turtle polypeptide diluent and 100 mu L of DCFH-DA are added in each well, and 50 mu L of 200mmol/L ABAP is added. DCFH-DA working solutionPrepared before the measurement by adding 1340. mu.L of 2.48mM DCFH-DA to 160. mu.L of 1mM KOH, mixing well, reacting in the dark for 5min, and then diluting to 12mL with 75mM phosphate buffer. The reaction process is carried out in a fluorescence microplate reader preheated to 37 ℃, the excitation wavelength of the microplate reader is set to be 485nm, the absorption wavelength is set to be 538nm, and the reaction is completed within 40 min.
The antioxidant capacity calculation equation is as follows:
PSC unit＝1-(SA/CA)
in the equation, CA is the fluorescence accumulation of the blank, and SA is the fluorescence accumulation of the sample or the standard. The antioxidant value of the samples is expressed as PSC values in units of micromoles equivalent to ascorbic acid equivalents per gram of dry weight of the sample (μmol vit.c. equi./g, DW).
The results of the in vitro antioxidant capacity measurement by the method of hydrogen peroxide radical scavenging PSC are shown in fig. 1.
As can be seen from FIG. 1, the polypeptide of Chinemys reevesii has a significant effect on the scavenging ability of hydrogen peroxide free radicals.
Example 5
The scarab polypeptide was diluted to different concentrations (starting concentration 1000 μ g/mL) and the in vitro antioxidant capacity was measured using oxygen radical absorbance capacity ORAC. The samples were measured by the modified ORAC method (Adom, K.K.; Liu, R.H., Rapid radiological screening capacity (PSC) assay for assaying the bottom of a hydraulic and lipophilic analytical method. journal of agricultural and food chemistry 2005,53, 6572-80). The reaction system adopts 75mmol/L KH2PO4-K2HPO4(pH 7.4) buffer solution, and the standard products used in the experiment, namely Trolox, ABAP and fluoroscein (fluorescein substrate), are prepared. The samples were prepared in advance with buffer to the appropriate concentration, 20. mu.L of sample or Trolox standard (concentration gradient between 6.25 and 50. mu.M) was added to each respective well, 200. mu.L of 0.96. mu.M fluorescein was added to each well using a multi-track pipette after incubation for 10min at 37 ℃ in a fluorescence microplate reader, 119mM of ABAP 20. mu.L was rapidly added to each well except for the fluorescence control well after incubation for 20min under the same conditions, and the reaction was monitored in the fluorescence microplate reader. The parameters set by the microplate reader are as follows: 37 ℃; excitation wavelength is 485nm, and measuring wavelength is 520 nm; every 5min, 35 times in total. All samples were run in triplicate. The ORAC values are expressed in micromoles of Trolox equivalents per gram of sample (dry weight) (μmol Trolox equiv./g, DW), and the results of the in vitro antioxidant oxygen radical absorption ORAC test of the scarab polypeptide are shown in fig. 2.
As can be seen from FIG. 2, Control is the result of ORAC (oxygen radical absorption coefficient) of the in vitro antioxidant oxygen radical absorption capacity of the small water turtle polypeptide, and the graph shows that the small water turtle polypeptide has good absorption capacity and long duration for the in vitro antioxidant oxygen radical absorption capacity.
Example 6
Toxicity of the Chinemys reevesii polypeptide was tested using a methylene blue staining method (Yang, J.; Liu R.H., synthetic Effect of applied extracts and Quercetin 3-beta-D-glucose Combination on anti-proliferative activity in MCF-7 Human Breast Cancer Cells in vitro. journal of agricultural and Food Chemistry 2009,57, 8581) to test the toxicity of the Chinemys reevesii polypeptide. Laying HepG2 cells in logarithmic growth phase on a 96-well plate at a density of 4 x 104, placing 100 mu L of each well, correspondingly adding the diluted cyclemys trifasciata polypeptide with a series of concentrations into each well after 24 hours of adherence, adding 50 mu L of methylene blue into each well, incubating for 1 hour according to a method of a literature after 24 hours of drug treatment, washing with water, adding an Elution solution to dissolve, detecting an absorption value under a wavelength of 570nm by using an enzyme labeling instrument, and detecting the toxicity of the cyclemys trifasciata polypeptide. The toxicity results of the scarab polypeptide on HepG2 cells are shown in fig. 3.
As can be seen from FIG. 3, the small water turtle polypeptide having a concentration of less than 1mg/ml is not toxic to HepG2 cells, and a high concentration of more than 1mg/ml has a toxic effect on HepG2 cells.
Example 7
Research on in vivo antioxidant CAA of polypeptide of small water turtle
The absorption level antioxidant effect was examined by the method of Wolfe et al (Wolfe, K.L.; Liu, R.H., Cellular Antioxidant Activity (CAA) assay for assaying antioxidants, foods, and dietary supplements, journal of agricultural aggregation and food chemistry 2007,55, 8896-. Laying HepG2 cells in logarithmic growth phase on a 96-well plate at a density of 4 x 104, wherein each well is 100 mu L, correspondingly adding a series of diluted cyclemys trifasciata polypeptides containing DCFH-DA dye into each well after 24 hours of adherence, placing the wells in an incubator at 37 ℃ for incubation for 1 hour, removing the culture medium, washing the wells with PBS or without PBS according to the experimental method, rapidly adding 100 mu L of 600mM ABAP into each well, removing blank wells, and monitoring the reaction in a fluorescence microplate reader. The parameters set by the microplate reader are as follows: 37 ℃; excitation wavelength is 485nm, and measuring wavelength is 520 nm; every 5min for 1 h. The cell level antioxidant effect was calculated according to the following formula.
CAA unit＝100-(∫SA/∫CA)×100
And ^ SA represents the corresponding fluorescence value of the sample, and ^ CA represents the corresponding fluorescence value of the blank.
The experimental results are shown in fig. 4 and 5, where fig. 4 shows the results of washing with PBS and fig. 5 shows the results of washing without PBS.
As can be seen from FIGS. 4 and 5, the polypeptide of Chinemys reevesii has a certain effect of resisting oxidation CAA in vivo, and the effect after PBS cleaning is better than that without PBS cleaning.
Example 8
The stone small water turtle polypeptide mask comprises the following components in percentage by mass: 0.05 part of small water turtle polypeptide, 1 part of 1, 3-butanediol, 0.1 part of sodium hyaluronate, 1 part of glyceryl polyether-26, 0.5 part of giant kelp extract, 0.05 part of hydroxyethyl cellulose, 5 parts of glycerin, 0.05 part of polysorbate-80, 0.1 part of dipotassium glycyrrhizinate, 0.1 part of xanthan gum, 0.05 part of methyl hydroxybenzoate, 2 parts of compound amino acid, 1 part of hydroxyethyl acrylate and 90 parts of deionized water.
Example 9
The stone small water turtle polypeptide mask comprises the following components in percentage by mass: 0.2 part of small water turtle polypeptide, 4 parts of 1, 3-butanediol, 0.3 part of sodium hyaluronate, 5 parts of glyceryl polyether-26, 2 parts of giant kelp extract, 0.2 part of hydroxyethyl cellulose, 20 parts of glycerin, 0.2 part of polysorbate-80, 1 part of dipotassium glycyrrhizinate, 0.3 part of xanthan gum, 0.2 part of methylparaben, 5 parts of compound amino acid, 0.1 part of essence and 60 parts of deionized water of 1.5 parts of hydroxyethyl acrylate.
Example 10
The stone small water turtle polypeptide mask comprises the following components in percentage by mass: 0.1 part of small water turtle polypeptide, 60-90 parts of deionized water, 3 parts of 1, 3-butanediol, 0.2 part of sodium hyaluronate, 3 parts of glyceryl polyether-26, 1 part of giant kelp extract, 0.1 part of hydroxyethyl cellulose, 10 parts of glycerin, 0.1 part of polysorbate-80, 0.5 part of dipotassium glycyrrhizinate, 0.2 part of xanthan gum, 0.1 part of methylparaben, 4 parts of compound amino acid, 0.2 part of essence and 77.5 parts of deionized water.
Claims (5)
1. The preparation method of the small water turtle polypeptide is characterized by comprising the following steps:
taking the minced meat of the stone small water turtle, and mixing the minced meat of the stone small water turtle with the solid-liquid ratio of 1: 5-1: 10, adding ultrapure water into the minced meat of the golden turtle to obtain minced meat water, wherein the temperature of the ultrapure water is 40-50 ℃, and the pH value of the ultrapure water is 7-8;
adding neutral protease into the meat paste water for enzymolysis, adjusting the pH of the system in the enzymolysis process, and performing enzymolysis for 8-12 h to obtain an enzymolysis liquid;
carrying out enzyme deactivation operation on the enzymatic hydrolysate to obtain a small water turtle polypeptide solution;
centrifuging the small water turtle polypeptide solution, removing precipitates, keeping filtrate, and freeze-drying to obtain the small water turtle polypeptide, wherein in the centrifuging operation of the small water turtle polypeptide solution, the rotating speed of the centrifugation is 8000r/min, the time of the centrifugation is 30min, and the average molecular mass of the small water turtle polypeptide is 2000-3000.
2. The method for preparing the polypeptide of Chinemys reevesii as claimed in claim 1, wherein NaOH is used to adjust the pH value of the reaction system during the enzymolysis.
3. The method for preparing the small water turtle polypeptide according to claim 1, wherein the enzyme deactivation operation is: and (3) putting the enzymolysis liquid into an environment of 85 ℃ to inactivate enzyme for 10 min.
4. The stone small water turtle polypeptide mask is characterized by comprising the following components in percentage by weight: the small water turtle polypeptide, deionized water, 1, 3-butanediol, sodium hyaluronate, glyceryl polyether-26, giant kelp extract, hydroxyethyl cellulose, glycerol, polysorbate-80, dipotassium glycyrrhizinate, xanthan gum, hydroxyethyl acrylate and methyl hydroxybenzoate, wherein the small water turtle polypeptide is prepared by the preparation method of the small water turtle polypeptide according to any one of claims 1-3.
5. The Chinemys reevesii polypeptide mask as claimed in claim 4, wherein the formula comprises the following components in percentage by mass:
0.05-0.2 part of small water turtle polypeptide, 60-90 parts of deionized water, 1-4 parts of 1, 3-butanediol, 0.1-0.3 part of sodium hyaluronate, 1-5 parts of glyceryl polyether-26, 0.5-2 parts of giant kelp extract, 0.05-0.2 part of hydroxyethyl cellulose, 5-20 parts of glycerol, 0.05-0.2 part of polysorbate-80, 0.1-1 part of dipotassium glycyrrhizinate, 0.1-0.3 part of xanthan gum, 0.05-0.2 part of methylparaben and 2-5 parts of compound amino acid.
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| CN201610808564.7A CN106399437B (en) | 2016-09-07 | 2016-09-07 | Preparation method of small water turtle polypeptide and small water turtle polypeptide mask |
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| CN201610808564.7A CN106399437B (en) | 2016-09-07 | 2016-09-07 | Preparation method of small water turtle polypeptide and small water turtle polypeptide mask |
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| CN107184948B (en) * | 2017-05-02 | 2021-02-05 | 深圳凯联龟业有限公司 | Application of small water turtle polypeptide mixture, fatty liver prevention and treatment health product or fatty liver prevention and treatment drug |
| CN108066186A (en) * | 2017-11-24 | 2018-05-25 | 广州赛莱拉干细胞科技股份有限公司 | Face cream and face cream applicator system based on light black huge marine algae extract |
| CN109467589B (en) * | 2018-10-31 | 2021-02-05 | 深圳凯联健康生物科技有限公司 | Active peptide, recombinant vector, recombinant cell, antioxidant composition, and preparation method and application thereof |
| CN109364238A (en) * | 2018-12-03 | 2019-02-22 | 佛山市肽硒其生物科技有限公司 | A kind of tortoise peptide combinations and its product |
| CN111662360B (en) * | 2020-05-12 | 2021-08-31 | 华中农业大学 | Turtle small peptide and preparation method and application thereof |
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