CN117045547A - Preparation method and application of antioxidant compact supermolecule composite tea fermentation extract - Google Patents
Preparation method and application of antioxidant compact supermolecule composite tea fermentation extract Download PDFInfo
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- CN117045547A CN117045547A CN202211599480.9A CN202211599480A CN117045547A CN 117045547 A CN117045547 A CN 117045547A CN 202211599480 A CN202211599480 A CN 202211599480A CN 117045547 A CN117045547 A CN 117045547A
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- fermentation
- tea
- supermolecule
- antioxidant
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Classifications
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- 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
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
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- 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
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- A61K8/602—Glycosides, e.g. rutin
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- A—HUMAN NECESSITIES
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- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- A—HUMAN NECESSITIES
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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- A—HUMAN NECESSITIES
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- 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
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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Abstract
The application relates to the technical field of cosmetics, and discloses a preparation method and application of an antioxidant compact supermolecule compound tea fermentation extract, wherein tea leaves are taken as raw materials, crushed and sieved to obtain tea powder; adding pure water into tea powder, dispersing uniformly, transferring into a fermentation tank, heating for sterilization, and adding supermolecule composition (betaine glycerol glucoside) and fermentation broth (saccharomycete/acetobacter xylinum) for fermentation to obtain fermentation broth; the application applies the tea fermentation extract to cosmetics, has the effects of antioxidation, moisture preservation, wrinkle resistance and tightening, and can effectively solve the increasingly serious skin problem of human beings.
Description
Technical Field
The application relates to the technical field of cosmetic raw materials, in particular to a preparation method and application of an antioxidant compact supermolecule compound tea fermentation extract.
Background
The tea leaves can be classified into green tea, oolong tea, yellow tea, black tea, white tea, black tea, and tea leaves are rich in various active ingredients such as tea polyphenols, catechin, caffeine, theanine, organic acid, tea polysaccharide, tea saponin, vitamins, amino acids, mineral elements, etc. The tea has the functions of resisting oxidation, resisting radiation, resisting inflammation, resisting allergy and the like, and mainly plays the roles of nourishing skin and delaying skin aging through the functions of resisting wrinkle, moisturizing, whitening, removing freckles and the like in the aspect of skin.
Skin is the most direct organ in contact with the outside, and its barrier effect is critical to skin health; research shows that the tea has the functions of DPPH, OH and O 2– The active oxygen free radical has better scavenging effect, which shows that the active oxygen free radical has protection and repair effects on free radical oxidative damage, and has certain anti-damage and repair effects on skin; aquaporin 3 (AQP 3) is a transport protein involved in the transmembrane transport of water, glycerol and urea, etc., through neutral small molecules on cell membranes. Aquaporin 3 (AQP 3) is involved not only in skin hydration, skin barrier function and wound healing, maintaining the normal morphology and function of the skin, but also in keratinocyte proliferation and migration. Aquaporin 3 (AQP 3) is expressed in epidermis and basal layer, is a natural moisturizing repair factor, maintains the interstitial and intracellular hydration level of skin cells, and plays a key role in skin moisture, moisturizing and repairing。
Many ingredients beneficial to skin exist in tea, but the traditional method for extracting the effective ingredients in the tea is concentrated, and the problems of poor stability, deep color, undefined efficacy mechanism and the like exist. The application adopts supermolecule composite fermentation to prepare fermentation extract, combines the separation and purification technologies of multistage filter membranes and the like, and thoroughly solves the application problem of tea extract in cosmetics. At present, the application of the tea extract prepared by fermentation in cosmetics is less, most of fermentation conditions are relatively original in the aspect of the fermentation and purification process of the raw materials of the cosmetics, the strain activity and activity stability are poor, the fermentation time is long, pollution is easy, the variability is large, and the like.
Supermolecules are generally defined as complex, organized aggregates formed by two or more molecules held together by intermolecular interactions (e.g., intermolecular hydrogen bonds, van der Waals forces, hydrophilic and hydrophobic interactions, pi-pi interactions, etc.), and maintaining a degree of integrity that allows for well-defined microscopic and macroscopic properties. The supermolecule has complete structure and special performance, and the specific combination is applied to fermentation technology to promote the stability of the product and protect the activity of fermentation strain.
Chinese patent CN 106119172A is a preparation method of a direct-throwing type black tea fungus starter, which uses tea extract as a main freeze-drying protective agent, respectively prepares saccharomycetes, acetobacter pasteurii and Acetobacter xylinum into freeze-drying fungus powder, and then prepares the direct-throwing type black tea fungus starter by mixing according to a certain proportion. The ferment prepared by the preparation method of the application has stable quality and the viable count (1-2) multiplied by 10 11 cfu/g, long shelf life, and can be stored for two years at-18 ℃, and the activity of the thalli is maintained to be more than 85%; compared with the traditional manual workshop type black tea fungus fermentation, the direct-feeding black tea fungus starter can be directly added into tea soup to prepare black tea fungus fermentation liquor by fermentation, and can be applied to the production of large-scale industrialized black tea fungus beverages; and with skim milkCompared with the direct-vat starter taking the powder as the main freeze-drying protective agent, the direct-vat starter of the application has no milk flavor, and the flavor of tea is not affected by the fermentation of the black tea fungus by using the direct-vat starter of the application, and the flavor of the direct-vat starter of the black tea fungus is consistent with that of the traditional black tea fungus fermentation liquid. Chinese patent CN110150420A is a preparation method of Kang Pucha rich in antioxidant, mixing water, sucrose and black tea leaves (125-130 wt.% (12.5-13 wt.% (1-1.5 wt.%) and boiling to obtain a base liquid; inoculating the base solution with a concentration of 10 5 -10 7 CFU/mL Acetobacter pasteurium seed solution, 10 6 ～10 8 CFU/mL Acetobacter xylinum seed solution and 10 5 ～10 7 Seed liquid formed by mixing CFU/mL Bayer joint yeast according to the volume ratio of 1:1:1 is obtained to obtain liquid to be fermented: the volume ratio of the mixed seed liquid to the base liquid is as follows: (3-3.5) (97-97.5); and (3) placing the liquid to be fermented in a container with the open end covered with gauze, and standing and fermenting for 10 days at the temperature of 25-30 ℃ under the humidity of 30-40% to obtain the Kangpu tea.
The preparation method is mainly applied to the food direction, cosmetics are different from foods, main active substances are absorbed through skin, and tea fermentation products have the problems of dark color, large taste and the like. However, no report has been made in the prior art.
Disclosure of Invention
The application aims to provide a preparation method and application of an antioxidant compact supermolecule composite tea fermentation extract, and aims to solve the problems of strain activity, color, taste and stability of fermentation liquor in the tea fermentation process and promote the application and industrialization of the tea fermentation extract in cosmetics.
In order to achieve the aim, the preparation method and the application of the antioxidant compact supermolecule composite tea fermentation extract are characterized by comprising the following steps:
s1: preparing fermentation base liquid: pulverizing folium Camelliae sinensis with low temperature pulverizer, sieving (200 mesh) to obtain tea powder, adding pure water according to solid-to-liquid ratio (1:20-300), dispersing uniformly, and sterilizing to obtain fermentation matrix;
s2: preparation of a supramolecular composition: weighing betaine and glyceroglycosides respectively according to the weight ratio of 1 (0.1-5), and grinding at low speed for 20-100 minutes by using a colloid mill to prepare the supermolecule composition.
S3: preparing a composite seed bacterial liquid: inoculating saccharomycetes and acetobacter xylinum into an MRS liquid culture medium, and performing shake culture to obtain compound seed bacterial liquid;
s4: adding the supermolecule composition prepared in the step S2 and the composite seed bacterial liquid prepared in the step S3 into fermentation base liquid for fermentation, wherein the fermentation temperature is 25-36 ℃, the fermentation time is 2-5 days, and the fermentation liquid is obtained, the addition of the supermolecule composition is 0.1-5% of the volume of the fermentation base liquid, and the addition of the composite seed bacterial liquid is 1-5% of the volume of the fermentation base liquid.
S5: sterilizing the fermentation liquor obtained in the step S4 for 20min at 121 ℃, cooling to normal temperature, adding 0.1-5.0% of active carbon, stirring and adsorbing, and then centrifuging by a butterfly type centrifuge to obtain a centrifugate, and filtering the centrifugate by a ceramic membrane with the aperture of 20-300 nm to obtain a ceramic membrane permeate; filtering the ceramic membrane permeate by using an ultrafiltration membrane to remove macromolecular substances to obtain ultrafiltration membrane permeate, further filtering and purifying the ultrafiltration membrane permeate by using a sodium filter membrane to remove ionic components, collecting nanofiltration membrane filtrate, adding 0.01-1.0% of p-hydroxyacetophenone and polyalcohol (0.01-1.0% of dipropylene glycol or 0.01-1.0% of 1, 2-hexanediol or 0.01-1.0% of octanediol or 0.01-1.0% of isopentane diol or a combination of two or more of the above) into the filtrate, and finally obtaining the tea fermentation extract.
Preferably, the solid-to-liquid ratio of the tea powder to the water in the step S1 is 1:50-150;
preferably, in the step S2, the weight ratio of betaine to glyceroglycoside is 1 (2-5);
preferably, the adding amount of the supermolecule composition in the step S4 is 1-2% of the volume of the fermentation base solution, and the inoculating amount of the composite seed bacterial solution is 3-5% of the volume of the fermentation base solution;
preferably, in the step S5, the ceramic membrane is a ceramic membrane with the aperture of 20-100 nm, the ultrafiltration membrane is a membrane with the molecular weight cut-off of 1000-5000Da, and the sodium filter membrane is a membrane with the molecular weight cut-off of 150-500Da.
The application of the antioxidant compact supermolecule composite tea fermentation extract in cosmetics can promote fermentation bacteria propagation, shorten fermentation time, ensure fermentation quality, effectively solve the problems of heavy taste, dark color and instability of tea fermentation liquor, and has the advantages of low cytotoxicity of the obtained raw materials and excellent antioxidant, moisturizing, anti-wrinkle and compact effects.
The application has the beneficial effects that: the technical scheme of the application skillfully provides the application of the supermolecule combined betaine-glycerol glucoside in the black tea fermentation process, and has the following specific advantages:
1. the characteristics of betaine are reserved in the supermolecule composition betaine and glycerol glucoside, so that the supermolecule composition has a buffer effect on the pH value of fermentation liquor, can stabilize the pH value of the fermentation liquor, and can slowly produce glucose under the combined action of saccharomycetes, so that a carbon source is provided for acetobacter xylinum to promote the mass growth and propagation of acetobacter xylinum, the supermolecule composition is favorable for shortening the fermentation time, keeping the activity of fermentation strains and stabilizing the quality of fermentation products;
2. aiming at the problems of dark color and heavy smell of fermentation liquor, activated carbon and a multi-stage filter membrane are adopted for separation and purification, so as to achieve decolorization and deodorization, enrich active ingredients and remove impurity ingredients, and obtain a fermentation extract which can be widely applied to cosmetics;
3. the tea fermentation liquor is obtained by adding activated carbon into the fermentation liquor for adsorption and centrifugation after sterilization, and filtering, purifying and separating the centrifugate by a ceramic membrane, an ultrafiltration membrane and a nanofiltration membrane, and the p-hydroxyacetophenone and the polyalcohol are added into the tea fermentation liquor to obtain the raw material of cosmetics.
Drawings
FIG. 1 is a flow chart of a method of preparation according to an embodiment of the present application;
FIG. 2 is a graph showing OD values of fermentation tubes according to an embodiment of the present application;
FIG. 3 is a graph showing the rise in moisture content of the stratum corneum of the skin according to an embodiment of the present application;
FIG. 4 is a graph showing the AQP3 protein content of human keratinocytes according to an embodiment of the present application.
Detailed Description
The present application will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
As shown in fig. 1 to 4, the preparation method and application of the antioxidant compact supermolecule composite tea fermentation extract of the application are as follows:
example 1
Taking 500g of tea leaves, crushing and sieving by a low-temperature crusher to obtain tea powder, adding 25000g of pure water, uniformly dispersing, and sterilizing to obtain a fermentation substrate; according to the weight ratio of 1: weighing betaine and glyceroglycosides, and grinding at low speed for 50 minutes by using a colloid mill to obtain a supermolecule composition; inoculating saccharomycetes and acetobacter xylinum into an MRS liquid culture medium, and performing shake culture to obtain compound seed bacterial liquid; adding 1% of the supermolecule composition and 5% of the compound seed bacterial liquid into the fermentation base liquid for fermentation at the fermentation temperature of 35 ℃ for 3 days to obtain fermentation liquid; sterilizing the fermentation liquor at 121 ℃ for 20min, cooling to normal temperature, adding 0.5% active carbon, stirring and adsorbing, centrifuging by a butterfly centrifuge to obtain a centrifugate, and filtering the centrifugate by a ceramic membrane with the aperture of 200nm to obtain a ceramic membrane permeate; filtering the ceramic membrane permeate with ultrafiltration membrane (membrane cut-off molecular weight 5000 Da) to remove macromolecular substances to obtain ultrafiltration membrane permeate; the ultrafiltration membrane permeate is further filtered and purified by a sodium filter membrane (300 Da) to remove ionic components, nanofiltration membrane filtrate is collected, and 0.5% of p-hydroxyacetophenone and 0.1% of isopentyl glycol are added into the nanofiltration membrane filtrate to finally obtain the tea fermentation extract.
Example 2
Taking 1000g of tea leaves, crushing and sieving the tea leaves by a low-temperature crusher to obtain tea powder, adding 100000g of pure water, uniformly dispersing, and sterilizing to obtain a fermentation substrate; according to the weight ratio of 1: weighing betaine and glyceroglycosides, and grinding at low speed for 80 minutes by using a colloid mill to obtain a supermolecule composition; inoculating saccharomycetes and acetobacter xylinum into an MRS liquid culture medium, and performing shake culture to obtain compound seed bacterial liquid; adding 5% of the supermolecule composition and 3% of the compound seed bacterial liquid into the fermentation base liquid for fermentation at the fermentation temperature of 30 ℃ for 5 days to obtain fermentation liquid; sterilizing the fermentation liquor at 121 ℃ for 20min, cooling to normal temperature, adding 5% active carbon, stirring and adsorbing, centrifuging by a butterfly centrifuge to obtain a centrifugate, and filtering the centrifugate by a ceramic membrane with the aperture of 50nm to obtain a ceramic membrane permeate; filtering the ceramic membrane permeate with ultrafiltration membrane (membrane cut-off molecular weight 1000 Da) to remove macromolecular substances to obtain ultrafiltration membrane permeate; the ultrafiltration membrane permeate is further filtered and purified by a sodium filter membrane (150 Da) to remove ionic components, the nanofiltration membrane permeate is collected, and 0.5% of p-hydroxyacetophenone, 0.1% of isopentyl glycol, 0.4% of dipropylene glycol, 0.1% of 1, 2-hexanediol and 0.1% of octanediol are added into the nanofiltration membrane permeate to finally obtain the tea fermentation extract.
Comparative example 1
Preparation method example 2, except that no supramolecular composition was added to the fermentation broth;
comparative example 2
The fermentation process and example 1, taking 500g of black tea, crushing and sieving by a low-temperature crusher to obtain tea powder, adding 25000g of pure water, uniformly dispersing, and sterilizing to obtain a fermentation substrate; according to the weight ratio of 1: weighing betaine and glyceroglycosides, and grinding at low speed for 50 minutes by using a colloid mill to obtain a supermolecule composition; inoculating saccharomycetes and acetobacter xylinum into an MRS liquid culture medium, and performing shake culture to obtain compound seed bacterial liquid; adding 1% of the supermolecule composition and 5% of the compound seed bacterial liquid into the fermentation base liquid for fermentation at the fermentation temperature of 35 ℃ for 3 days to obtain fermentation liquid; sterilizing the fermentation liquor at 121 ℃ for 20min, cooling to normal temperature, adding 0.5% of active carbon, stirring and adsorbing, centrifuging by a butterfly centrifuge to obtain a centrifugate, and adding 0.5% of p-hydroxyacetophenone and 0.1% of isopentyl glycol into the centrifugate to finally obtain the tea fermentation extract.
1. Bacterial activity test and stability test
1. Fermentation bacteria vitality test
The MTT method for measuring the bacterial activity can distinguish dead and alive bacteria, and is simple, quick and good in repeatability, and suitable for testing the bacterial activity in fermentation liquor.
As shown in FIG. 2, the fermentation broth in the fermentation vessel in various time periods was taken, centrifuged at 10000rp, the supernatant was removed, the pellet was washed with PBS, 40. Mu.l of MTT of 0.5mg/ml was added after centrifugation, incubated at 30℃for 4 hours, and OD was measured at 570nm by a microplate reader 570 Values. The OD value increases with the number of living bacteria, and the absorbance value is positively correlated with the number of bacteria within a certain range.
From the aspect of bacterial activity, the supermolecule compositions are added in the embodiment 1, the embodiment 2 and the comparative embodiment 2, and the added supermolecule composition group has a particularly good effect on the stability of bacterial activity, and the bacterial activity is obviously higher than that of the comparative embodiment 1 (without the supermolecule composition), so that the supermolecule composition has the effect of maintaining or improving the bacterial activity and is a key substance for guaranteeing the fermentation quality.
2. Tea fermentation extract stability test
(1) Heat resistance test: the sample parts are placed in an electric heating constant temperature incubator, the temperature is regulated to 40+/-1 ℃, the samples are taken out and placed to room temperature in 0,1,7, 15, 30, 45 and 60 days respectively, whether abnormal phenomena such as color change, layering, precipitation, taste change and the like exist or not is observed, meanwhile, the pH value of the sample is measured by a pH meter, and the heat resistance of the product is judged, wherein the heat resistance is shown in Table 1.
TABLE 1 Heat stability test
(2) Cold resistance test: the sample parts are placed in a constant temperature refrigerator, the temperature is regulated to be minus 15+/-1 ℃, the samples are taken out and placed to room temperature in 0,1,7, 15, 30, 45 and 60 days respectively, whether abnormal phenomena such as color change, layering, precipitation, taste change and the like exist or not is observed, meanwhile, the pH value of the sample is measured by a pH meter, and the cold resistance of the product is judged, wherein the cold resistance is shown in Table 2.
Table 2 cold resistance stability test
(3) Strong light irradiation test: the sample parts are placed in an illumination box with fluorescent lamps, the illuminance is 4500 lx+/-500 lx, the sample parts are taken out and placed to room temperature in 0,1,7, 15, 30, 45 and 60 days respectively, whether abnormal phenomena such as color change, layering, precipitation, taste change and the like exist or not is observed, meanwhile, the pH value of the sample is measured by a pH meter, and the light resistance of the product is judged, wherein the light resistance is shown in Table 3.
TABLE 3 light stability test
The stability test results show that the appearance smell of the tea fermentation extract prepared by combining supermolecule composite fermentation with a multi-stage filter membrane in examples 1 and 2 under the conditions of high temperature, low temperature and strong light has no change and stable pH, the method adopted by the scheme of the application can solve the defects of color change, smell change, precipitation and the like of the traditional fermentation mode, and the raw materials obtained by the method have wide application prospects in cosmetics.
2. Analysis of antioxidant and moisturizing effects of tea fermentation extract
1. In vitro antioxidant Activity test and assay
(1) DPPH radical scavenging: the sample tube (T), the sample background (T0), the DPPH tube (C) and the solvent background (C0), and 3 parallel tubes are required to be arranged for each sample tube (T) with tested concentration. 1ml of the same concentration of sample solution was added to each of the sample tube (T) and the sample background (T0). 2ml of the mixture was filled in a tube and mixed well. 1ml of DPPH ethanol solution was added to the sample tube (T) and the DPPH tube (C), and the mixture was gently shaken and allowed to stand at room temperature for 5 minutes. Each reaction solution was transferred to a 1cm cuvette, and absorbance was measured at 517 nm. The half clearance concentration IC50 was calculated.
(2)ABTS + Radical scavenging: and setting proper mass concentration gradient according to the characteristics of the sample and the recommended addition amount, and respectively preparing sample liquid to be tested by taking PBS buffer liquid as a solvent. The sample tube (As), the sample background (Ab) and the sample blank tube (A0) are established, 3 parallel tubes are required to be established for each sample tube (As) of each tested concentration of each sample, and meanwhile, the sample blank tube (A0) is also 3 parallel tubes. 0.2ml of the same concentration of sample solution was added to each of the sample tube (As) and the sample background (Ab), and 0.2ml of PBS buffer was added to the sample blank tube (A0). To each of As and A0, 0.8ml of ABTS+ working solution was added, and to Ab, 0.8ml of PBS buffer was added. The reaction was carried out in the dark for 6min. Each reaction tube solution was transferred to a 1cm cuvette and absorbance was measured at 734 nm.
The half clearance concentration IC50 was calculated.
(3) Reduction capability test: setting up a sample tube (A) 1 ) Sample background tube (A) 2 ) Sample blank tube (A) 3 ) And solvent background tube (A) 4 ) The sample tube (A1) of each tested concentration of each sample needs to be provided with 3 parallel tubes. In the sample tube (A) 1 ) To the mixture, 50. Mu.L of a sample solution, 250. Mu.L of a PBS buffer having pH of 6.5 and 250. Mu.L of a 0.1% potassium ferricyanide solution were added, the mixture was uniformly mixed, the mixture was incubated in a water bath at 50℃for 20 minutes, then rapidly cooled to room temperature, 250. Mu.L of a 10% trichloroacetic acid solution, which may be centrifuged, was added, 1mL of ultrapure water and 400. Mu.L of a 0.1% ferric chloride solution were further added, the mixture was uniformly mixed, and the absorbance was measured at a wavelength of 700nm after standing at room temperature for 10 minutes. Wherein the sample background tube (A) 2 ) Ultrapure water was used instead of potassium ferricyanide, trichloroacetic acid and ferric chloride, and a sample blank tube (A 3 ) Replace sample with PBS buffer, solvent background tube (a 4 ) After the sample background tube (A) 2 ) Based on (2), the sample is replaced by PBS buffer. The relative reduction rate is calculated by taking the ferrous ion reduction amount of VC as 100% reduction：
Table 4 antioxidant test results
The results are shown in Table 4, and the antioxidant test results show that the raw materials obtained in the examples are DPPH and ABTS + IC with reducing ability 50 The values are much lower than the comparative examples, indicating that the examples have superior antioxidant effects to the comparative examples.
2. Human body moisturizing effect test
The apparent age of the skin has a close relationship with the moisture content, and sufficient moisture is one of the important factors for maintaining skin health and good vision. The moisture content of the stratum corneum is closely related to the skin surface softness, smoothness and flexibility, the complete barrier protection function of the epidermis and the maintenance of the basic structure and function of the skin, and the experiment uses the moisture content of the stratum corneum as an index to evaluate the efficacy of the product. The experiment adopts a conductivity method to measure the moisture content of the skin cuticle, the dry cuticle has weaker conductivity, the conductivity of the skin can be increased when the moisture content is increased, and the conductivity and the moisture content are positively correlated.
Test instrument: skin stratum corneum hydration measuring instrument, efficacy evaluation laboratory (constant temperature: 25 ℃, constant humidity: 40%); subject standard: a total of 15 persons, aged 20-50 years, were tested for each sample, and during the test period, oral or topical administration was prohibited for claiming anti-wrinkle, skin tightening or other formulations similarly claiming. The subject is prohibited from receiving cosmetic surgery or other cosmetic means that may affect the anti-wrinkle, moisturizing efficacy test. The subject should be protected from long-term exposure to light by taking indoor activity as a major factor. The testing method comprises the following steps: selecting the same tester's armMeasuring skin, taking 2cm of each adjacent part 2 The moisture content of the horny layer was measured, and then an equal amount of test sample was applied to each of them, and the moisture content of the horny layer of the skin was measured for various periods of time, and the moisture rise value of the horny layer was calculated, as shown in FIG. 3.
From the viewpoint of the moisture rising value of the horny layer, example 1 and example 2 each had a good moisturizing effect, example 2 was slightly superior to example 1, and the comparative example had substantially no moisturizing effect.
3. Cytotoxicity and mechanism of action analysis
1. Reagents, cells, etc
Human skin fibroblasts (bosch); PBS (Solarbio); cell culture medium MEM (containing 4.5g/L D-Glucose, BI) +15% fetal bovine serum (BI) +1% cocktail antibiotic (100 x, beyotime); cell culture broth (low sugar DMEM) (4.5 g/LD-Glucose, vivaCell) +10% fetal bovine serum (BI) +1% cocktail antibiotic (100 x, beyotime); 0.25% Trypsin (Gibco); thiazole blue (MTT) solution (MACKLIN); DMSO solution (Coolaber); 96-well cell culture plate (Nest); 12 well cell culture plate (Nest); 6 well cell culture plate (Nest); cell culture flask (CORNING); trypsin/EDTA solution (basal media); lipopolysaccharide LPS (guangzhou degree); WY14643 (Beyotime); TRIzol reagent (Beyotime); isopropyl alcohol (Macklin); chloroform (Macklin); ethanol (Macklin); TRIzol reagent (Biotek). IL-1. Alpha. ELISA detection kit (Multisciences); IL-8ELISA detection kit (Multisciences); human type I procollagen ELISA kit.
2. Cytotoxicity test procedure
Human skin fibroblasts at 1.0X10 4 The cells were seeded at a density of 100 μl/well into 96-well plates and incubated for 24h. After 24h of cell adherence, the culture medium was discarded, 100 μl of the sample solution to be tested was added to each well, and a blank group and a negative group were set. After incubation of the cells for 24h, 10. Mu.L of MTT solution was added to each well, and the cells were cultured in an incubator for 4h. The culture broth was discarded, 150. Mu.L of DMSO was added to each well, and after shaking for 10min, the absorbance at 490nm (OD 490 nm) was compared with a control sample not treated with the test substance and the results were calculated.
Wherein:
relative survival% -relative OD 490 nm ，％
Test OD 490 nm Average OD of test substance 490 nm
Neg OD 490 nm Average OD of negative control 490 nm
TABLE 5 cytotoxicity test results
The results are shown in Table 5, and the cytotoxicity test results show that the examples of the present application have excellent cell compatibility with both human keratinocytes and human skin fibroblasts, and the cytotoxicity is far lower than that of the comparative examples.
3. Human type I procollagen content assay
Cell plating: at 5X 10 3 Cell/well density cells were seeded into 96-well plates overnight for adherence. Preparing liquid: test object working solutions were prepared according to the test protocol as follows.
Adding the medicine: according to the test scheme of the table, when the cell fusion rate in the 96-well plate reaches 40% -60%, grouping dosing is carried out, 200 mu L of sample is added to each well, and each group of 3 compound wells is acted for 72 hours. Cell supernatants were collected and assayed for human type I procollagen content using ELISA kits.
TABLE 6 detection results of human type I procollagen content based on human skin fibroblast model
Table 6 shows that the fermentation liquor obtained by the method has obvious effect of improving the content of human skin fibroblast protein, and the rising rates are 62.88% and 89.46%, respectively, which shows that the fermentation extract obtained by the method has the capability of promoting the synthesis of human collagen, and has the anti-wrinkle and tightening effects at the concentration.
4. Assay for determining AQP3 protein content in human keratinocytes
Aquaporin 3 (AQP 3) is the most abundantly expressed aquaporin subtype of AQPs family in human skin, mainly expressed in basal layer, acantha cell layer, granulosa layer of human epidermis, gradually disappearing to stratum corneum; the high expression of AQP3 in the basal membrane band can promote the transportation of water, glycerol and urea; in addition, AQP3 plays an important role in stratum corneum glycerol transport, and AQP3 transports endogenous glycerol and triglycerides in sebaceous glands into epidermis to directly or indirectly influence skin moisturizing effect, so that the effect of a sample to be tested can be evaluated by detecting the content of AQP3 protein.
Reagents and cells: human keratinocytes (purchased for Yu Punuo race); cell culture involves reagents and consumables: 1 x PBS (Solarbio); cell culture medium MEM (1 x, 4.5g/L D-Glucose, BI) +15% fetal bovine serum (BI) +1% cocktail antibiotic (100 x, beyotide); 0.25% Trypsin (1 x, gibco); thiazole blue (MTT) solution (MACKLIN); DMSO solution (Coolaber); 6 well cell culture plate (Nest); cell culture flask (CORNING); caCl (CaCl) 2 (Aladin); paraformaldehyde (Aladin).
Cell culture: according to 2X 10 5 Cell/well seeding Density cells were seeded into 6-well plates, incubator (37 ℃, 5% CO) 2 ) Incubate overnight. Preparing liquid: and preparing the working fluid of the test object according to the test group.
Table 7 test grouping table
Administration of drugs: according to the test group, see Table 7, when the cell plating rate in the 6-well plate reaches 40% -60%, group administration is carried out, the administration amount of each well is 2mL, 3 compound wells are arranged in each group, and the incubator (37 ℃, 5% CO) 2 ) And incubated for 24h.
AQP3 detection: after fixation with 4% paraformaldehyde for 30min, immunofluorescence detection of aquaporin 3 (AQP 3) was performed, and the pictures were taken and analyzed under a microscope, as shown in fig. 4 and table 8.
TABLE 8 AQP3 protein content in human keratinocytes
The results in table 8 show that the fermented extract obtained by the method can obviously increase the content of aquaporin 3 (AQP 3) in human keratinocytes, the increase rates are 49.00% and 63.00% respectively, and the fermented extract obtained by the method has the effect of up-regulating the expression of aquaporin 3 (AQP 3) genes and has the moisturizing effect under the concentration.
The application has the beneficial effects that: the technical scheme of the application skillfully provides the application of the supermolecule combined betaine glycerol glucoside in the tea fermentation process, and has the following specific advantages:
1. the characteristics of betaine are reserved in the supermolecule composition betaine and glycerol glucoside, so that the supermolecule composition has a buffer effect on the pH value of fermentation liquor, can stabilize the pH value of the fermentation liquor, and can slowly produce glucose under the combined action of saccharomycetes, so that a carbon source is provided for acetobacter xylinum to promote the mass growth and propagation of acetobacter xylinum, the supermolecule composition is favorable for shortening the fermentation time, keeping the activity of fermentation strains and stabilizing the quality of fermentation products;
2. aiming at the problems of dark color and heavy smell of fermentation liquor, activated carbon and a multi-stage filter membrane are adopted for separation and purification, so as to achieve decolorization and deodorization, enrich active ingredients and remove impurity ingredients, and obtain a fermentation extract which can be widely applied to cosmetics;
the tea fermentation liquor is obtained by adding activated carbon into the fermentation liquor for adsorption and centrifugation after sterilization, and filtering, purifying and separating the centrifugate by a ceramic membrane, an ultrafiltration membrane and a nanofiltration membrane, and the p-hydroxyacetophenone and the polyalcohol are added into the tea fermentation liquor to obtain the raw material of cosmetics.
In conclusion, the method can promote the reproduction of zymophyte, shorten the fermentation time, ensure the fermentation quality, effectively solve the problems of heavy taste, dark color, instability and the like of the black tea fermentation liquid, has low cytotoxicity of the raw materials, has excellent effects of resisting oxidation, moisturizing, resisting wrinkles and compacting, and can be widely applied to cosmetics.
The foregoing description is only illustrative of the present application and is not intended to limit the scope of the application, and all equivalent modifications made by the teachings of the present application, or direct or indirect application in the relevant art, are intended to be included within the scope of the present application.
Claims (6)
1. The preparation method of the antioxidant compact supermolecule composite tea fermentation extract is characterized by comprising the following steps of:
s1: preparing fermentation base liquid: pulverizing folium Camelliae sinensis with low temperature pulverizer, sieving (200 mesh) to obtain tea powder, adding pure water according to solid-to-liquid ratio (1:20-300), dispersing uniformly, and sterilizing to obtain fermentation matrix;
s2: preparation of a supramolecular composition: weighing betaine and glyceroglycosides respectively according to the weight ratio of 1 (0.1-5), and grinding at low speed for 20-100 minutes by using a colloid mill to prepare the supermolecule composition.
S3: preparing a composite seed bacterial liquid: inoculating saccharomycetes and acetobacter xylinum into an MRS liquid culture medium, and performing shake culture to obtain compound seed bacterial liquid;
s4: adding the supermolecule composition prepared in the step S2 and the composite seed bacterial liquid prepared in the step S3 into fermentation base liquid for fermentation, wherein the fermentation temperature is 25-36 ℃, the fermentation time is 2-5 days, and the fermentation liquid is obtained, the addition of the supermolecule composition is 0.1-5% of the volume of the fermentation base liquid, and the addition of the composite seed bacterial liquid is 1-5% of the volume of the fermentation base liquid.
S5: sterilizing the fermentation liquor obtained in the step S4 for 20min at 121 ℃, cooling to normal temperature, adding 0.1-5.0% of active carbon, stirring and adsorbing, and then centrifuging by a butterfly type centrifuge to obtain a centrifugate, and filtering the centrifugate by a ceramic membrane with the aperture of 20-300 nm to obtain a ceramic membrane permeate; filtering the ceramic membrane permeate by using an ultrafiltration membrane to remove macromolecular substances to obtain ultrafiltration membrane permeate, further filtering and purifying the ultrafiltration membrane permeate by using a sodium filter membrane to remove ionic components, collecting nanofiltration membrane filtrate, adding 0.01-1.0% of p-hydroxyacetophenone and polyalcohol (0.01-1.0% of dipropylene glycol or 0.01-1.0% of 1, 2-hexanediol or 0.01-1.0% of octanediol or 0.01-1.0% of isopentane diol or a combination of two or more of the above) into the filtrate, and finally obtaining the tea fermentation extract.
2. The method for preparing an antioxidant compact supermolecule composite tea fermentation extract according to claim 1, wherein the solid-to-liquid ratio of tea powder to water in the step S1 is 1:50-150.
3. The method for preparing the antioxidant compact supermolecule composite tea fermentation extract according to claim 1, wherein the weight ratio of betaine to glyceroglycosides in the step S2 is 1 (2-5).
4. The method for preparing an antioxidant compact supermolecule composite tea fermentation extract according to claim 1, wherein the adding amount of the supermolecule composition in the step S4 is 1-2% of the volume of fermentation base solution, and the inoculating amount of the composite seed bacterial solution is 3-5% of the volume of fermentation base solution.
5. The method for preparing the antioxidant compact supermolecular composite tea fermentation extract according to claim 1, wherein in the step S5, a ceramic membrane with the pore diameter of 20-100 nm is selected, an ultrafiltration membrane with the membrane retention molecular weight of 1000-5000Da is selected, and a sodium filter membrane with the membrane retention molecular weight of 150-500Da is selected.
6. The application of the antioxidant compact supermolecule composite tea fermentation extract in cosmetics can promote fermentation bacteria propagation, shorten fermentation time, ensure fermentation quality, effectively solve the problems of heavy taste, dark color and instability of tea fermentation liquor, and has the advantages of low cytotoxicity of the obtained raw materials and excellent antioxidant, moisturizing, anti-wrinkle and compact effects.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117598940A (en) * | 2023-11-16 | 2024-02-27 | 广州中妆美业化妆品有限公司 | Tea tender protein composition with antioxidant, anti-wrinkle and ultraviolet injury repairing effects and application thereof |
| CN117598964A (en) * | 2023-11-16 | 2024-02-27 | 广州中妆美业化妆品有限公司 | Tea and caviar composition with repairing, antioxidant, anti-wrinkle and tightening effects and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117598940A (en) * | 2023-11-16 | 2024-02-27 | 广州中妆美业化妆品有限公司 | Tea tender protein composition with antioxidant, anti-wrinkle and ultraviolet injury repairing effects and application thereof |
| CN117598964A (en) * | 2023-11-16 | 2024-02-27 | 广州中妆美业化妆品有限公司 | Tea and caviar composition with repairing, antioxidant, anti-wrinkle and tightening effects and application thereof |
| CN117598964B (en) * | 2023-11-16 | 2024-04-30 | 广州中妆美业化妆品有限公司 | Tea and caviar composition with repairing, antioxidant, anti-wrinkle and tightening effects and application thereof |
| CN117598940B (en) * | 2023-11-16 | 2024-06-21 | 广州中妆美业化妆品有限公司 | Tea tender protein composition with antioxidant, anti-wrinkle and ultraviolet injury repairing effects and application thereof |
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