Green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects, preparation method and application thereof

Abstract

The present invention discloses a green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects, and a preparation method and application thereof, which belong to the field of cosmetic technology. The preparation method of the green tea symbiotic fermentation liquid of the present invention comprises the following steps: first, green tea is made into green tea powder, and then the activated composite bacteria are connected to the fermentation matrix containing the green tea powder for fermentation, and the mixed solution obtained after fermentation is filtered, and the obtained filtrate is the green tea symbiotic fermentation liquid; wherein, the composite bacteria include saccharomyces cerevisiae, lactic acid bacteria and Brazilian Aspergillus, and the inoculation amount of the composite bacteria is 3-9%; the sign of the end of the fermentation is: the pH of the mixed solution drops to 3.0-4.5. The present invention uses saccharomyces cerevisiae, lactic acid bacteria and Brazilian Aspergillus to ferment green tea, and controls the fermentation parameters, and the obtained green tea symbiotic fermentation liquid has excellent antioxidant, oil-control and anti-wrinkle effects.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics, and in particular to a green tea symbiotic fermentation liquid having anti-oxidation, oil-control and anti-wrinkle effects, and a preparation method and application thereof. Background Art

[0002] Green tea, also known as "unfermented" tea, is made from tea tree shoots through processes such as withering, rolling and drying. Green tea contains a large amount of tea polyphenols, theanine, catechin, epicatechin and many other active ingredients that are beneficial to humans. Studies have shown that green tea has moisturizing, anti-oxidation and anti-aging effects. At present, common green tea extracts are obtained by aqueous solvent extraction (CN102984951A, CN101686701A). Some green tea extracts are prepared by bacterial fermentation (CN113908093A). In the prior art, green tea extracts are mostly prepared by water extraction. Although some green tea extracts are prepared by fermentation, the fermentation strains used are yeasts, and the fermented strains are single, and the prepared green tea extracts have poor skin care effects and cannot meet people's skin care needs. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle properties and a preparation method and application thereof.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] In a first aspect, a method for preparing a green tea symbiotic fermentation liquid having antioxidant, oil-control and anti-wrinkle effects is provided, comprising the following steps:

[0006] S1: preparing green tea powder from green tea;

[0007] S2: inoculating the activated composite bacteria into a fermentation matrix for fermentation to obtain a mixed solution;

[0008] S3: filtering the mixed solution obtained in step S2, and the obtained filtrate is the green tea symbiotic fermentation liquid;

[0009] The composite bacteria in step S2 include saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis;

[0010] The inoculation amount of the composite bacteria in step S2 is 3-9%; the fermentation matrix contains the green tea powder in step S1;

[0011] The sign of the end of fermentation in step S2 is that the pH of the mixed solution after fermentation drops to 3.0-4.5.

[0012] As a preferred embodiment of the method for preparing the green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects of the present invention, in the step S2, the inoculation ratio of brewer's yeast, lactic acid bacteria and brasiliensis is brewer's yeast: lactic acid bacteria: brasiliensis = 1-3:1-3:1-3.

[0013] As a preferred embodiment of the method for preparing the green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects of the present invention, in step S1, the particle size of the green tea powder is 60-100 meshes.

[0014] As a preferred embodiment of the method for preparing the green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects of the present invention, it can also include a step of activating the composite bacteria; the step of activating the composite bacteria includes preparing brewer's yeast seed liquid, lactic acid bacteria seed liquid and Brazilian Aspergillus seed liquid.

[0015] As a preferred embodiment of the method for preparing the antioxidant, oil-controlling and anti-wrinkle green tea symbiotic fermentation liquid of the present invention, the fermentation matrix further includes a carbon source, a nitrogen source and a balance of water.

[0016] As a preferred embodiment of the method for preparing the antioxidant, oil-controlling and anti-wrinkle green tea symbiotic fermentation liquid of the present invention, at least one of the following (a)-(h) is included:

[0017] (a) the amount of green tea added is 0.5-2.0% of the total mass of the fermentation substrate;

[0018] (b) the amount of the carbon source added is 0.2-2.0% of the total mass of the fermentation substrate;

[0019] (c) the nitrogen source is added in an amount of 0.1-1.0% of the total mass of the fermentation substrate;

[0020] (d) the carbon source is at least one of glucose, sucrose and fructose;

[0021] (e) the nitrogen source is at least one of sulfate, ammonium salt, peptone, distiller's grains, corn steep liquor, and yeast extract;

[0022] (f) the fermentation temperature is 28-38°C;

[0023] (g) the fermentation time is 40-100h;

[0024] (h) The fermentation speed is 100-300 rpm.

[0025] In a second aspect, a green tea symbiotic fermentation liquid having antioxidant, oil-control and anti-wrinkle effects is provided, wherein the green tea symbiotic fermentation liquid is prepared by the above-mentioned method for preparing the green tea symbiotic fermentation liquid having antioxidant, oil-control and anti-wrinkle effects.

[0026] In a third aspect, the invention provides application of the green tea symbiotic fermentation liquid having antioxidant, oil-control and anti-wrinkle effects in cosmetics.

[0027] In a fourth aspect, a cosmetic composition is provided, wherein the cosmetic composition contains the green tea symbiotic fermentation liquid having antioxidant, oil-control and anti-wrinkle effects.

[0028] As a preferred embodiment of the cosmetic composition of the present invention, the addition amount of the green tea symbiotic fermentation liquid having antioxidant, oil-control and anti-wrinkle effects is 0.1-50% of the total mass of the cosmetic composition.

[0029] In a fifth aspect, a method for improving skin condition for non-therapeutic purposes is provided, comprising topically applying a safe and effective amount of the green tea symbiotic fermentation liquid or the cosmetic composition having antioxidant, oil-control and anti-wrinkle effects to the skin of a person in need thereof.

[0030] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses brewer's yeast, lactic acid bacteria and Brazilian Aspergillus to ferment green tea, and the obtained green tea symbiotic fermentation liquid has excellent antioxidant, oil-control and anti-wrinkle effects. DETAILED DESCRIPTION

[0031] With reference to the detailed description of the following embodiments, it will be easier to understand the advantages and features of the present invention and the methods for achieving the advantages and features. However, the present invention can be implemented in various other forms and should not be construed as being limited to the embodiments set forth herein. The embodiments disclosed herein are provided so that the disclosure of the present invention will be thorough and complete, and the scope of the present invention will be fully conveyed to those skilled in the art.

[0032] The terms used in the specification are intended to describe certain embodiments only and should by no means limit the present invention.

[0033] Unless clearly used otherwise, expressions in the singular include the meaning of the plural form.

[0034] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as those commonly understood by ordinary technicians in the field to which the present invention belongs. Any term defined in a comprehensive dictionary should be interpreted as having the same meaning in the context of the relevant field, and, unless explicitly defined otherwise, should not be interpreted as having an idealistic or overly formalistic meaning.

[0035] Hereinafter, a cosmetic for skin anti-oxidation, oil control, anti-wrinkle, whitening and prevention of ultraviolet damage containing green tea symbiotic fermentation liquor as a cosmetic active ingredient according to the present invention will be described in detail.

[0036] The term "skin whitening" as used herein generally refers to a regimen for increasing the brightness of skin having reduced brightness due to excess pigments such as melanin or maintaining the brightness of skin at a certain level, and the brightness of the skin is increased by the above regimen.

[0037] In addition, the term "skin wrinkles" used herein refers to fine lines formed on the skin due to weakening of the skin, which may be caused by genetic factors, a decrease in collagen and elastin present in the dermis of the skin, the external environment, and the like.

[0038] In addition, the term "anti-wrinkle / anti-wrinkle" as used herein means to inhibit or suppress the generation of wrinkles on the skin or to reduce wrinkles that have already occurred.

[0039] The term "skin antioxidant" used in this article refers to the abbreviation of antioxidant free radicals. The human body is constantly exposed to the outside world, including breathing (oxidation reaction), external pollution, radiation exposure and other factors, which constantly produce free radicals in the human body. Antioxidants are any substances that can effectively inhibit the oxidation reaction of free radicals in low concentrations. Their mechanism of action can be directly acting on free radicals, or indirectly consuming substances that are prone to generating free radicals to prevent further reactions.

[0040] The term "skin oil control" as used herein refers to regulating the amount of oil secreted by the skin.

[0041] The term "UV damage" used in this article refers to the skin being exposed to ultraviolet rays, which causes massive necrosis of dermal cells and metabolic disorders, making it impossible for impurities in the cells to be metabolized away, causing the skin to develop sun spots, loss of elasticity, premature aging, sensitivity, wrinkles and other symptoms.

[0042] The term "preventing UV damage" as used herein refers to preventing or inhibiting the occurrence of various skin symptoms caused by skin exposure to UV rays.

[0043] As used herein, the term "safe and effective" amount means an amount of a compound or composition sufficient to induce a positive benefit as described herein, but low enough to avoid any serious side effects in the judgment of the skilled artisan.

[0044] The brewer's yeast used in the present invention is EBY100 yeast (bio-82069), the lactic acid bacteria are Lactococcus lactis subsp. lactis (bio-67315) and Lactobacillus plantarum (bio-60181), and the Aspergillus brasiliensis is Aspergillus brasiliensis (bio-74735). The above strains were purchased from Beijing Biobo Biotechnology Co., Ltd.

[0045] The present invention provides a method for preparing green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects, characterized in that it comprises the following steps:

[0046] S1: preparing green tea powder from green tea;

[0047] S2: inoculating the activated composite bacteria into a fermentation matrix for fermentation to obtain a mixed solution;

[0048] S3: filtering the mixed solution obtained in step S2, and the obtained filtrate is the green tea symbiotic fermentation liquid;

[0049] The composite bacteria in step S2 include Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis;

[0050] The inoculation amount of the composite bacteria in step S2 is 3-9%; the fermentation matrix contains the green tea powder in step S1;

[0051] The sign of the end of fermentation in step S2 is that the pH of the mixed solution drops to 3.0-4.5.

[0052] Preferably, the inoculation ratio of the Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis is Saccharomyces cerevisiae: lactic acid bacteria: Aspergillus brasiliensis = 1-3:1-3:1-3.

[0053] Green tea is one of the main types of tea in China. It is made from new leaves or buds of tea trees, which are not fermented but are processed through processes such as withering, shaping, and drying. It retains the natural substances of fresh leaves, including tea polyphenols, catechins, chlorophyll, caffeine, amino acids, vitamins and other nutrients.

[0054] The green tea of ​​the present invention may be naturally occurring (eg, "wild") or cultivated. Green tea may be purchased from commercial sources or from channels known to those skilled in the art.

[0055] For the use of industrial cosmetic applications, cultivation reduces the risk that the supply of green tea will become limited due to changes in the growing environmental conditions over time, and the use of cultivated green tea is preferred. Such cultivated green tea can control the growing environment and the resulting harvest can have a consistent quality, which is very desirable for industrial applications (such as cosmetics).

[0056] Green tea can be made by methods known to those skilled in the art. In an exemplary method, new leaves or buds of tea plants are subjected to processes such as withering, shaping, and drying to obtain green tea.

[0057] In the present invention, compared with the case where yeast, lactic acid bacteria or Aspergillus is used alone for fermentation, when Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis are used to symbiotically ferment green tea, Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis have a synergistic effect, so that the obtained green tea symbiotic fermentation liquid presents a more excellent skin care effect.

[0058] Specifically, the inoculation amount of the composite bacteria and the pH at the end of fermentation are key factors affecting the performance of the green tea symbiotic fermentation liquid. The present invention obtains a green tea symbiotic fermentation liquid with excellent performance by selecting the inoculation amount of the composite bacteria to be 3-9% and the pH at the end of fermentation to be 3.0-4.5. More preferably, the pH of the mixed solution at the end of fermentation is 4.0.

[0059] In addition, the ratio of the inoculation amounts of Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus braziliensis is also one of the main factors affecting the performance of the green tea symbiotic fermentation liquid. The present invention preferably has an inoculation amount ratio of Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus braziliensis of Saccharomyces cerevisiae: lactic acid bacteria: Aspergillus braziliensis = 1-3:1-3:1-3, and more preferably, an inoculation amount ratio of Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus braziliensis of Saccharomyces cerevisiae: lactic acid bacteria: Aspergillus braziliensis = 1:1:1; so as to obtain a green tea symbiotic fermentation liquid with better performance.

[0060] Preferably, the composite bacteria need to be activated before inoculation, and the activation can restore the activity of the strains and also provide a sufficient amount of strains for inoculation; wherein the step of activating the composite bacteria includes preparing brewer's yeast seed liquid, lactic acid bacteria seed liquid and Brazilian Aspergillus seed liquid.

[0061] Preferably, in step S1, the particle size of the green tea powder is 60-100 mesh.

[0062] The particle size of green tea powder should not be too large or too small. If the particle size of green tea powder is too large, it is not conducive to the dissolution of active substances in cells during the fermentation process; if the particle size of green tea powder is too small, due to electrostatic effect, the green tea powder cannot be evenly dispersed during the preparation of the fermentation matrix, which is not conducive to the subsequent fermentation.

[0063] Preferably, in step S2, the fermentation substrate further contains a carbon source, a nitrogen source and water; the specific preparation method of the fermentation substrate is: mixing the nitrogen source, the carbon source, water and the green tea powder obtained in step S1 to obtain the fermentation substrate.

[0064] In step S2, before the activated composite bacteria are inoculated into the fermentation matrix, the fermentation matrix needs to be sterilized at 121° C. and 0.1 MPa for 30 min.

[0065] In the present invention, the fermentation raw materials and the fermentation parameters jointly affect the progress of the fermentation. The present invention obtains a green tea symbiotic fermentation liquid with better performance and effect by optimizing various parameters. The preferred fermentation parameters are as follows:

[0066] (a) the amount of green tea added is 0.5-2.0% of the total mass of the fermentation substrate; more preferably, the amount of green tea added is 1.0% of the total mass of the fermentation substrate;

[0067] (b) the amount of the carbon source added is 0.2-2.0% of the total mass of the fermentation substrate; more preferably, the amount of the carbon source added is 0.5% of the total mass of the fermentation substrate;

[0068] (c) the amount of the nitrogen source added is 0.1-1.0% of the total mass of the fermentation substrate; more preferably, the amount of the nitrogen source added is 0.2% of the total mass of the fermentation substrate;

[0069] (d) the carbon source is at least one of glucose, sucrose and fructose; more preferably, the carbon source is glucose;

[0070] (e) the nitrogen source is at least one of sulfate, ammonium salt, peptone, distiller's grains, corn steep liquor, and yeast extract; more preferably, the nitrogen source is a compound of soy peptone and distiller's grains, and the mass ratio of soy peptone to distiller's grains is 2-5:1; more preferably, the mass ratio of soy peptone to distiller's grains is 3:1;

[0071] (f) the fermentation temperature is 28-38°C; more preferably, the fermentation temperature is 30°C;

[0072] (g) the fermentation time is 40-100h;

[0073] (h) The fermentation speed is 100-300 rpm; more preferably, the fermentation speed is 200 rpm.

[0074] The saccharomyces cerevisiae seed solution can be prepared by the following steps:

[0075] Use an inoculation loop to pick up a colony of Saccharomyces cerevisiae and place it in a liquid culture medium. Cultivate it at 25-30°C and 100-300 rpm for 24-72 hours to obtain a Saccharomyces cerevisiae seed solution. The cell concentration of the Saccharomyces cerevisiae seed solution is 1x10 7 -1x10 9 cfu / mL. Preferably, the cell concentration of the Saccharomyces cerevisiae seed solution is 2±1x10 8 cfu / mL;

[0076] The liquid culture medium for culturing the saccharomyces cerevisiae colony includes one of malt extract culture medium, YM culture medium and PDB culture medium. Preferably, it is YM culture medium.

[0077] The lactic acid bacteria seed liquid can be prepared by the following steps:

[0078] Use an inoculation loop to pick up lactic acid colonies and place them in liquid culture medium. Cultivate at 30-42°C and 100-300 rpm for 24-72 hours to obtain lactic acid bacteria seed solution. The bacterial concentration of the lactic acid bacteria seed solution is 1x10 7 -1x10 9 cfu / mL. Preferably, the culture is carried out at 37°C and 200 rpm for 48 hours; the bacterial concentration of the lactic acid bacteria seed liquid is 2±1x10 8 cfu / mL;

[0079] The liquid culture medium for culturing Lactococcus lactis subsp. lactis includes MRS liquid culture medium.

[0080] The steps for preparing the Brazilian Aspergillus seed solution are as follows:

[0081] Use an inoculation loop to pick up Aspergillus braziliensis colonies and place them in a liquid culture medium. Cultivate the culture medium at 28-35°C and 100-300 rpm for 72-120 hours to obtain Aspergillus braziliensis seed solution. The cell concentration of the Aspergillus braziliensis seed solution is 1x10 7 -1x10 9 cfu / mL. Preferably, the cell concentration of the Aspergillus brasiliensis seed solution is 2±1x10 8 cfu / mL;

[0082] Liquid culture media for culturing Aspergillus brasiliensis include potato complex medium.

[0083] The above-mentioned culture medium is a common culture medium for culturing bacteria in the prior art, and its preparation method is as follows:

[0084] Maltose medium: Add 4 times the amount of water to dry malt powder, keep it warm at 55-65℃ for 3-4 hours, check with iodine solution until it is completely saccharified, and obtain maltose. Dilute the maltose with water to a sugar content of 8-10 barin degrees, adjust the pH to 6.0-6.5, sterilize at 120℃ for 20 minutes, and store for later use.

[0085] YM medium: 3.0 g yeast extract, 3.0 g malt extract, 10.0 g glucose, 5.0 g peptone, 20.0 g agar (not included in liquid culture medium), distilled water to make up to 1.0 L, pH 6.2±0.2, sterilize at 120°C for 20 min and store for later use.

[0086] PDB culture medium: 200 g potatoes (peeled), 20 g sucrose, dilute to 1000 mL with water, sterilize at 120°C for 20 min and store for later use.

[0087] MRS liquid culture medium: Mix 10g of peptone, 5g of beef powder, 4g of yeast powder, 2g of glucose, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate, 0.2g of magnesium sulfate, and 0.05g of manganese sulfate, add distilled water to 1L, sterilize at 120℃ for 20min, and store for later use.

[0088] Potato comprehensive culture medium: Take 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate heptahydrate, 8mg of vitamin B1, 20g of glucose, and 20% potato juice to a total volume of 1000mL, sterilize at 120℃ for 20min, and store for later use; the preparation method of 20% potato juice: take 200g of peeled potatoes, cut into small pieces, add 1000mL of water and boil for 20min, filter out the potato pieces, and make up the filtrate to 1000mL.

[0089] In step S3, the mixed solution obtained in step S2 is sterilized to prevent further fermentation and improve the stability of the green tea symbiotic fermentation liquid; preferably, the sterilization conditions are as follows: at 85°C to 115°C, keep warm for 15 to 30 minutes; more preferably, the sterilization conditions are: at 90°C, keep warm for 20 minutes. After sterilization, the mixed solution obtained in step S2 is filtered using a diatomaceous earth filter or a microfiltration membrane. In this step, the mixed solution is filtered through 25μm, 2.5μm and 0.8μm polypropylene microfiltration membranes in sequence to remove residual green tea powder particles and fermentation organisms.

[0090] The present invention also provides green tea symbiotic fermentation liquid prepared by the above-mentioned preparation method of green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects. The obtained green tea symbiotic fermentation liquid has antioxidant, oil-control and anti-wrinkle effects.

[0091] In the present invention, only green tea symbiotic fermentation liquid is used to maximize the skin antioxidant, oil control, anti-wrinkle and efficacy. In addition, in order to enhance the skin antioxidant, oil control and anti-wrinkle effects by increasing the skin absorption rate of the active ingredient, as a cosmetic composition for skin antioxidant, oil control and anti-wrinkle, the cosmetic active ingredient of the present invention uses green tea symbiotic fermentation liquid, which is obtained by fermenting a fermentation matrix containing green tea with composite bacteria, thereby improving the skin care effect of the green tea symbiotic fermentation liquid.

[0092] The cosmetic composition for skin anti-oxidation, oil control and anti-wrinkle containing green tea symbiotic fermentation liquor as a cosmetic active ingredient according to the present invention shows excellent effects and excellent stability in the applied products.

[0093] According to a preferred embodiment of the present invention, the cosmetic composition comprises 0.1-50wt%, preferably 2-20wt%, more preferably 3-10wt% of green tea symbiotic fermentation liquid based on the total weight of the cosmetic composition, wherein the green tea symbiotic fermentation liquid is obtained by fermenting a fermentation matrix containing green tea with a composite bacteria.

[0094] In addition to using green tea symbiotic fermentation broth as an active ingredient, the ingredients contained in the cosmetic composition of the present invention also include ingredients commonly used in cosmetic compositions, such as conventional adjuvants, such as antioxidants, stabilizers, solubilizers, vitamins, colorants, pigments, and fragrances and carriers.

[0095] The cosmetic composition of the present invention can be prepared as any preparation conventionally prepared in the art, and can be formulated into, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, detergents containing surfactants, oils, powder foundations, emulsion foundations, wax foundations, sprays, etc., but the present invention is not limited thereto. More specifically, the cosmetic composition of the present invention can be prepared into the following preparations: moisturizing lotions, nourishing lotions, nourishing creams, massage creams, essences, facial masks, eye creams, cleansing creams, cleansing foams, cleansing waters, moisturizing creams, fermented creams, ampoules, shampoos, conditioners, sprays or powders.

[0096] When the preparation of the present invention is a paste, cream or gel, at least one selected from animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicon dioxide, talc and zinc oxide can be used as a carrier component.

[0097] When the preparation of the present invention is a powder or spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component. In particular, when the preparation of the present invention is a spray, a propellant (such as chlorofluorocarbons, propane / butane or dimethyl ether) can be additionally included.

[0098] When the preparation of the present invention is a solution or emulsion, a solvent, a solubilizer or an emulsifier may be used as a carrier component. For example, at least one selected from the group consisting of water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, butylene glycol, 1,3-butyl glycol oil, polyoxyethylene hydrogenated castor oil, glycerin, glycerol, fatty esters, phenoxyethanol, triethanolamine, polyethylene glycol, beeswax, polysorbate 60, sorbitan sesquioleate, paraffin, sorbitan stearate, glyceryl monostearate (lipophilic), stearic acid, glyceryl stearate / PEG-400 stearate, carboxy polymer, sitosterol, polyglyceryl 2-oleate, ceramide, cholesterol, steareth-4, dicetyl phosphate, macadamia oil, carboxyvinyl polymer, xanthan gum, and fatty acid ester of sorbitan can be used.

[0099] When the preparation of the present invention is a suspension, at least one selected from the following can be used as a carrier component: a liquid diluent such as water, ethanol, glycerol, butylene glycol or propylene glycol; a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, hydroxyethylcellulose, sodium hyaluronate, phenoxyethanol, aluminum metahydroxide, bentonite, stearic acid, cetyl alcohol, glyceryl monostearate, polyoxyethylene sorbitan monostearate, sorbitan sesquioleate, glyceryl monostearate / glyceryl stearate / polyoxyethylene stearate, wax, paraffin, squalane, caprylic / capric triglyceride, carboxyvinyl polymer, triethanolamine, agar and tragacanth gum.

[0100] When the preparation of the present invention is a surfactant-containing cleanser, at least one selected from the following can be used as a carrier component: fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamido betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives and ethoxylated glyceryl fatty acid esters.

[0101] The cosmetic compositions described herein are particularly useful as part of a cosmetic treatment method for treating skin having signs of aging or UV damage, wherein such compositions may be applied to skin in need of such treatment for a period of time necessary to improve the condition of such skin. Although such duration will vary depending on the potency of such cosmetic composition, it is preferred to apply the cosmetic composition to the skin at least once a day.

[0102] The present invention can be further demonstrated by way of the following examples, which should not be considered as limiting. Unless otherwise stated, the ratios given in any example herein are expressed as percentages by weight. Temperature is degrees Celsius, and pressure is atmospheric pressure.

[0103] In the following examples, the seed solution was prepared by the following method:

[0104] Saccharomyces cerevisiae seed solution: Use an inoculation loop to pick up a colony of Saccharomyces cerevisiae and place it in YM medium. Culture it at 28°C and 200 rpm for 48 h to obtain Saccharomyces cerevisiae seed solution. The cell concentration of the Saccharomyces cerevisiae seed solution is 2±1x10 8 cfu / mL;

[0105] Lactic acid bacteria seed solution: Use an inoculation loop to pick up Lactococcus lactis subsp. lactis or Lactobacillus plantarum colonies and place them in MRS liquid culture medium. Culture them at 37°C and 200 rpm for 48 h to obtain Lactococcus lactis subsp. lactis seed solution or Lactobacillus plantarum seed solution. The cell concentration of Lactococcus lactis subsp. lactis or Lactobacillus plantarum seed solution is 2±1x10 8 cfu / mL;

[0106] Aspergillus braziliensis seed solution: Use an inoculation loop to pick up Aspergillus braziliensis colonies and place them in potato comprehensive culture medium. Culture them at 28°C and 200 rpm for 96 hours to obtain Aspergillus braziliensis seed solution. The cell concentration of Aspergillus braziliensis seed solution is 2±1x10 8 cfu / mL;

[0107] Fission yeast seed solution: Use an inoculation loop to pick up a fission yeast colony and place it in YM medium. Incubate at 28°C and 200 rpm for 48 h to obtain a fission yeast seed solution. The cell concentration of the fission yeast seed solution is 2±1x10 8 cfu / mL.

[0108] Wherein, in the embodiment of the present invention, the composite bacteria used for fermentation are saccharomyces cerevisiae, lactic acid bacteria and brazilian aspergillus. The saccharomyces cerevisiae in the embodiment of the present invention is EBY100 yeast (bio-82069), the lactic acid bacteria are Lactococcus lactis subsp. lactis (bio-67315) and Lactobacillus plantarum (bio-60181), and the brazilian aspergillus is brazilian aspergillus (bio-74735), and the above strains are purchased from Beijing Biobowei Biotechnology Co., Ltd.

[0109] It should be noted that the brewer's yeast, lactic acid bacteria and Aspergillus brasiliensis purchased from commercial channels can be used to prepare the green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects of the present invention, and are not limited to the above-mentioned EBY100 yeast (bio-82069), Lactococcus lactis subsp. lactis (bio-67315), Lactobacillus plantarum (bio-60181) and Aspergillus brasiliensis (bio-74735).

[0110] Example 1

[0111] Example 1 The preparation method of green tea symbiotic fermentation liquid with antioxidant, oil control and anti-wrinkle effects is as follows:

[0112] S1: Select dried green tea leaves, grind them into powder using a grinding machine, pass them through an 80-mesh sieve, remove impurities on the upper layer, and take the tea powder on the lower layer as green tea powder;

[0113] S2: Dissolve 1% green tea powder, 0.5% glucose, 0.15% soy peptone and 0.05% distiller's grains in the remaining water, mix well, seal the fermentation tank, heat to 121°C, keep for 30 minutes for sterilization, and cool to room temperature to obtain a fermentation matrix; wherein the above percentages are expressed as percentages based on the total weight of the fermentation matrix;

[0114] Add yeast seed liquid, Lactococcus lactis subsp. lactis seed liquid and Aspergillus braziliensis seed liquid to the fermentation matrix, stir and ferment at 30°C, the speed of the stirring paddle is 200rpm; fermentation is completed when the pH value of the fermentation liquid reaches 4.0; wherein the inoculation amount of the yeast seed liquid, the Lactococcus lactis subsp. lactis seed liquid and the Aspergillus braziliensis seed liquid are 2% respectively;

[0115] S3: sterilize the microorganisms in the fermentation liquid obtained in step S2 by pasteurization, the sterilization temperature is 90°C, and the sterilization time is 20 minutes; after sterilization, the obtained fermentation liquid is filtered through 25μm, 2.5μm and 0.8μm polypropylene microfiltration membrane columns in sequence to remove impurities and obtain green tea symbiotic fermentation liquid.

[0116] The preparation method of the green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects in Examples 2-7 and Comparative Examples 1-10 is the same as that in Example 1, and the only difference is the amount of nitrogen source added in step S2, the inoculation amount of the composite bacteria in step S2 or the pH when the fermentation is completed in step S2; the differences between Examples 2-7 and Comparative Examples 1-10 are shown in Table 1.

[0117] Table 1

[0118]

[0119]

[0120]

[0121] Example 8

[0122] Example 8 The preparation method of green tea symbiotic fermentation liquid with antioxidant, oil control and anti-wrinkle effects is as follows:

[0123] S1: Select dried green tea leaves, grind them into powder using a grinding machine, pass them through an 80-mesh sieve, remove impurities on the upper layer, and take the tea powder on the lower layer as green tea powder;

[0124] S2: Dissolve 0.5% green tea powder, 2% fructose, 0.667% soy peptone and 0.333% ammonium sulfate in the remaining water, mix well, seal the fermentation tank, heat to 121°C, keep for 30 minutes for sterilization, cool to room temperature, and obtain a fermentation matrix; wherein the above percentages are expressed as percentages based on the total weight of the fermentation matrix;

[0125] Adding saccharomyces cerevisiae seed liquid, Lactococcus lactis seed liquid and Aspergillus braziliensis seed liquid to a fermentation matrix, stirring and fermenting at 30°C, with a stirring paddle speed of 300 rpm; fermentation is completed when the pH value reaches 3.8; wherein the inoculation amounts of EBY100 yeast seed liquid, Lactococcus lactis seed liquid and Aspergillus braziliensis seed liquid are 3% respectively;

[0126] S3: The microorganisms in the fermentation liquid obtained in step S2 are sterilized by pasteurization, the sterilization temperature is 85°C, and the sterilization time is 30 minutes; after the sterilization, the obtained fermentation liquid is filtered through 25μm, 2.5μm and 0.8μm polypropylene microfiltration membrane columns in sequence to remove impurities and obtain green tea symbiotic fermentation liquid.

[0127] Example 9

[0128] Example 9 The preparation method of green tea symbiotic fermentation liquid with antioxidant, oil control and anti-wrinkle effects is as follows:

[0129] S1: Select dried green tea leaves, grind them into powder using a grinding machine, pass them through an 80-mesh sieve, remove impurities on the upper layer, and take the tea powder on the lower layer as green tea powder;

[0130] S2: Dissolve 2% green tea powder, 0.2% sucrose, 0.8333% yeast extract and 0.1667% distiller's grains in the remaining water, mix well, seal the fermentation tank, heat to 121°C, keep for 30 minutes for sterilization, cool to room temperature, and obtain a fermentation matrix; wherein the above percentages are expressed as percentages based on the total weight of the fermentation matrix;

[0131] Adding saccharomyces cerevisiae seed liquid, Lactococcus lactis seed liquid and Aspergillus braziliensis seed liquid to a fermentation matrix, stirring and fermenting at 30° C., with a stirring paddle speed of 100 rpm; fermenting to a pH value of 4.2 to complete the fermentation; wherein the inoculation amounts of saccharomyces cerevisiae seed liquid, Lactococcus lactis seed liquid and Aspergillus braziliensis seed liquid are 1% respectively;

[0132] S3: The microorganisms in the fermentation liquid obtained in step S2 are sterilized by pasteurization at a temperature of 115°C for 15 minutes. After sterilization, the fermentation liquid is filtered through 25 μm, 2.5 μm and 0.8 μm polypropylene microfiltration membrane columns in sequence to remove impurities and obtain green tea symbiotic fermentation liquid.

[0133] Example 10

[0134] The antioxidant properties of the green tea symbiotic fermentation liquids obtained in Examples 1-9 and Comparative Examples 1-10 were tested;

[0135] 1. DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) method

[0136] 1.1DPPH concentration 0.2mM (prepared with anhydrous ethanol): weigh 0.007875g DPPH powder and dissolve it in 100mL anhydrous ethanol to prepare 0.2mM DPPH.

[0137] 1.2 Sample preparation: The green tea symbiotic fermentation liquid obtained in the above Examples 1-9 and Comparative Examples 1-10 was diluted with deionized water to aqueous solutions with mass fractions of 0.005% and 0.02%, respectively, and placed in a brown bottle for later use.

[0138] 1.3 Test steps: Add samples in sequence according to Table 2, mix the reaction solutions in each test tube, react at room temperature for 30 minutes in the dark, detect the absorbance of each group at 517nm, and record the data.

[0139] Table 2 Sample addition requirements

[0140]

[0141] 1.4 Data Analysis:

[0142] The clearance rate was calculated as follows:

[0143] Where: T—absorbance of sample tube, i.e. absorbance of solution after sample reacts with DPPH;

[0144] T0—sample background OB value, i.e., the absorbance value of the sample solution;

[0145] C—DPPH tube absorbance, that is, the absorbance of DPPH solution without adding sample;

[0146] C0—Blank tube absorbance value, that is, the solvent absorbance value.

[0147] 2. O2 ·- (Superoxide Anion Radical) Pyrogallol Method

[0148] Reference method: Cheng Chao, Xue Feng, Li Wei, Wang Xingping. Effects of three treatment methods on the ability of phycobiliprotein of Pueraria lobata to scavenge superoxide anion radicals[J]. Food Science, 2014, 35(13): 26-31.

[0149] 2.1 Reagent preparation

[0150] (1) Preparation of 0.1mol / L Tris-HCl solution: Accurately weigh 2.4228g of Tris particles, add 100mL of deionized water, stir and mix to dissolve to obtain 0.2mol / L Tris solution. Take 1.67mL of concentrated hydrochloric acid and add 98.33mL of deionized water to dilute to obtain 0.2mol / L HCl solution. Take 100mL of Tris solution and 44.76mL of 0.2mol / L HCl solution, mix, adjust pH to 8.2, and add deionized water to make up to 200ml.

[0151] (2) Preparation of 10 mmol / L HCl solution: Take 0.83 mL of concentrated hydrochloric acid and add deionized water to make up to 100 mL.

[0152] (3) Preparation of 45 mmol / L pyrogallol solution: Accurately weigh 0.2837 g of pyrogallol powder and add 50 ml of 10 mmol / L HCl solution to dissolve and mix.

[0153] (4) Sample preparation: The above Examples 1-9 and Comparative Examples 1-10 were diluted with deionized water to aqueous solutions with a mass fraction of 0.1% and a mass fraction of 0.3%, respectively, and placed in a brown bottle for later use.

[0154] 2.2 Test steps: Add samples in sequence according to Table 3. Mix the reaction solutions in each test tube, incubate at 25°C for 20 minutes in the dark, transfer to a 4 cm cuvette, and then add pyrogallol solution that has been preheated at 25°C for 20 minutes to start the reaction. Detect the absorbance at 420 nm, record the absorbance every 30 seconds for 3 minutes, calculate the slope, save and record.

[0155] Table 3 Sample addition requirements

[0156]

[0157]

[0158] 2.3 The calculation formula for the clearance rate of superoxide anion free radicals is:

[0159]

[0160] Where, T is the absorbance of the sample tube, i.e. the slope of the solution after the sample reacts with pyrogallol;

[0161] T0—sample background slope;

[0162] C—absorbance of blank tube, i.e. slope of pyrogallol solution when no sample is added;

[0163] C0—Solvent background slope.

[0164] 3. Cell experiment method

[0165] Experimental principle: Free radical theory is an important branch of skin aging, and anti-free radical substances can delay skin aging. Aerobic cells produce a series of reactive oxygen species (ROS) during metabolism, including: O2 ·- , H2O2 and HO2 · , ·OH, etc., which affect the normal functions and physiological activities of skin cells. DCF-DA (2',7'-dichlorofluorescein diacetate) itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by the esterase in the cell to generate DCF. However, DCF cannot penetrate the cell membrane, making it easy for the probe to be loaded into the cell. The reactive oxygen in the cell can oxidize the non-fluorescent DCF to generate fluorescent DCF. By detecting the fluorescence of DCF, the level of reactive oxygen in the cell can be known.

[0166] Instrument used: Multifunctional microplate reader (BioTek, Cytation 1)

[0167] Main reagents: DMEM culture medium (Gibco), fetal bovine serum (Gibco), DCF-DA (Sigma), DMSO (Solybdo), 1.5 mM H2O2 aqueous solution.

[0168] Cell line: human fibroblasts (HFF-1 cells)

[0169] Culture medium: DMEM medium containing 10% fetal bovine serum.

[0170] Culture conditions: 37°C, 5% CO2, saturated humidity.

[0171] Sample: 0.5% by mass of green tea symbiotic fermentation broth of Example 1-9 and Comparative Example 1-10, the above culture broth was used as solvent for preparation.

[0172] Human fibroblasts were seeded into 96-well plates at a density of 5000 / well, and 0.1 mL of culture medium was added, and the plates were incubated at 37°C in a CO2 incubator overnight. Then reagents were added to each well; sample group: 0.05 mL of H2O2 solution + 0.05 mL of each green tea symbiotic fermentation liquid; blank control group: 0.1 mL of culture medium; modeling group: 0.05 mL of mM H2O2 solution + 0.05 mL of culture medium, incubated at 37°C in a CO2 incubator for 24 h;

[0173] After incubation for 24 h, the cells were washed twice with PBS, 50 μM DCFH–DA was added to each well and incubated for 30 min, and the cells were washed twice with PBS. The cells in the sample group and the modeling group were treated with 0.1 mL of 1.5 mM H2O2 solution for 1 h, and the blank group was supplemented with 0.1 mL of complete culture medium, and then the cells were washed twice with PBS.

[0174] Oxidized dichlorofluorescein (DCF) was photographed in each group of samples using an excitation wavelength of 485 nm and an emission wavelength of 528 nm, and the fluorescence density of the pictures was analyzed using Image-Pro (IPP) software. The lower the fluorescence density, the better the antioxidant effect of the green tea symbiotic fermentation liquid.

[0175] The test results are shown in Table 4.

[0176] Table 4

[0177]

[0178]

[0179] The test results show that Examples 1-9 of the present invention have good scavenging effects on DPPH and O2 ·- Under the same conditions, Examples 1-9 removed DPPH and O2 ·- The effect is better than that of comparative examples 1-10. ·-The effect is better than that of comparative examples 1-7, indicating that the composite fermentation of Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis has better effect, and the obtained fermentation liquid has better antioxidant effect. ·- The effect of Example 1, 2, 4, and 5 is better than that of Example 8, and the fluorescence density of Examples 1, 2, 4, and 5 is lower than that of Example 8, indicating that during fermentation, the inoculation amount of Saccharomyces cerevisiae, lactic acid bacteria, and Aspergillus brasiliensis should be controlled at 1-3:1-3:1-3. If the ratio exceeds this, the effect of composite fermentation is poor, which affects the antioxidant effect of green tea symbiotic fermentation liquid. ·- The effect of is better than that of comparative examples 9 and 10, and the fluorescence density of 1, 6, and 7 is lower than that of comparative examples 9 and 10, indicating that the pH value of the fermentation completion index has a great influence on the antioxidant effect of the final product. When the pH value at the completion of fermentation is high, it means that the green tea has not been fully fermented, and there are fewer active substances in the fermentation liquid, resulting in poor antioxidant effect; when the pH value at the completion of fermentation is low, it means that the green tea is over-fermented, and the bacteria in the fermentation liquid are prone to cell autolysis, which consumes the active substances produced in the fermentation liquid.

[0180] From the results of comparative examples 1 and 3, it can be seen that the ratio of nitrogen source addition affects the fermentation effect of green tea, and further affects the antioxidant effect of the green tea symbiotic fermentation liquid. In the present invention, the nitrogen source is preferably a compound of soy peptone and distiller's grains. Most preferably, the mass ratio of soy peptone to distiller's grains is 3:1.

[0181] Embodiment 11

[0182] The oil control performance of the green tea symbiotic fermentation liquid obtained in Examples 1-9 and Comparative Examples 1-10 was tested; the specific method is as follows:

[0183] Sample treatment: Prepare a sample mask, use deionized water to prepare the green tea symbiotic fermentation liquid of Examples 1 to 9 and Comparative Examples 1 to 10 into an aqueous solution with a mass concentration of 1%, take 2 mL of the aqueous solution and add it to a non-woven fabric with an area of ​​about 4 cm*7 cm, so that the non-woven fabric is evenly wetted to obtain a sample to be tested. For the blank control, deionized water is used instead of the green tea composite fermentation liquid.

[0184] Test instrument: Skin oil tester (Sebumeter SM815).

[0185] Method of use: 190 volunteers aged 18 to 45 with vigorous skin oil secretion were selected and randomly divided into 19 groups (10 people in each group, using the same sample). The samples and blank control areas were randomly distributed in the left or right forehead area of ​​the volunteers according to the test area. No samples were used in the blank control test area. After cleansing, the volunteers took each mask to be tested and applied it to the corresponding test area for 5 minutes and then removed it. Wait for it to be naturally absorbed. The test area should not be touched within the next 3 hours. Immediately after cleansing, at the 0.5 hour, the 1 hour, and the 3 hour of use, the skin oil content of each test area was tested, and the data were recorded as the initial value, 1h, and 3h. Each sample was tested 5 times and the average value was taken. The larger the skin oil content value, the more oil the skin has. The greater the rate of change of skin oil content, the higher the rate of skin oil secretion. Conversely, the lower the rate of skin oil secretion, the better the oil control effect.

[0186] Among them, the skin oil content change rate (%) = (skin oil content at the nth time point after use - skin oil content detected before use) / skin oil content detected before use × 100%; the test results are shown in Table 5.

[0187] Table 5

[0188]

[0189]

[0190] The above results show that in the blank control group, skin oil not only did not decrease, but increased. Under the same conditions, the change rate of skin oil in Examples 1-9 is greater than that in Comparative Examples 1-10, indicating that the oil control effect of Examples 1-9 is better than that of Comparative Examples 1-10. Among them, the oil control effect of Examples 1 and 2 is better than that of Comparative Examples 1-7, indicating that the effect of composite fermentation using Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis is better. The oil control effect of Examples 1, 2, 4 and 5 is better than that of Comparative Example 8, indicating that during fermentation, the inoculation amount of Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis should be controlled at 1-3:1-3:1-3. Exceeding this ratio, the effect of composite fermentation is poor, which affects the oil control effect of the green tea symbiotic fermentation liquid. The oil control effects of Examples 1, 6 and 7 are better than those of Comparative Examples 9 and 10, indicating that the pH value, an indicator of fermentation completion, has a great influence on the oil control effect of the final product. A higher pH value at the completion of fermentation indicates that the green tea has not been fully fermented, and there are fewer active substances in the fermentation liquid, resulting in a poor oil control effect. A lower pH value at the completion of fermentation indicates that the green tea is over-fermented, and the bacteria in the fermentation liquid are prone to cell autolysis, which consumes the active substances produced in the fermentation liquid.

[0191] From the results of comparative examples 1 and 3, it can be seen that the ratio of nitrogen source addition affects the fermentation effect of green tea, and further affects the oil control effect of the green tea symbiotic fermentation liquid. In the present invention, the nitrogen source is preferably a compound of soy peptone and distiller's grains. Most preferably, the mass ratio of soy peptone to distiller's grains is 3:1.

[0192] Example 13

[0193] The anti-wrinkle performance of the green tea symbiotic fermentation liquid obtained in Examples 1-9 and Comparative Examples 1-10 was tested; the specific method is as follows:

[0194] 200 volunteers with facial wrinkles (30-50 years old) were selected as anti-wrinkle efficacy testers and randomly divided into 20 groups (10 people in each group, using the same sample, one of which used an essence matrix). The green tea symbiotic fermentation liquid of Examples 1-9 and Comparative Examples 1-10 was prepared into an essence and an essence matrix for comparison. 1-2 mL of the essence of Examples 1-9 and Comparative Examples 1-10 was applied to the face and tested in Week 0, Week 2, and Week 4. The facial conditions of the volunteers were tested by VISIA-CR before use, after 2 weeks of use, and after 4 weeks of use. The wrinkle area ratio of the facial condition was analyzed by the skin comprehensive analysis software Image-Pro Plus (MediaCybernetics, USA). The anti-wrinkle effect of the green tea symbiotic fermentation liquid of Examples 1-9 and Comparative Examples 1-10 was evaluated by the change rate of the wrinkle area ratio. Among them, the skin wrinkle area ratio change rate (%) = (skin wrinkle area ratio after the nth week of use-skin wrinkle area ratio before use) / skin wrinkle area ratio before use * 100%.

[0195] The formula of the essence is shown in Table 6 below; the test results are shown in Table 7.

[0196] The steps for preparing the serum are as follows:

[0197] (1) Heat the ingredients of group A to 70°C, stir evenly, add the pre-mixed group B, raise the temperature to 80°C, stir until completely dissolved, and keep warm for later use;

[0198] (2) Heat the ingredients in group C to 80°C and keep warm for later use;

[0199] (3) Homogenize at 8000 rpm, add the mixture in step (1) to group C, and homogenize until it is completely emulsified;

[0200] (4) Cooling to 40°C, adding the ingredients of group D and group E in sequence, stirring evenly, and obtaining an essence (in the matrix essence, the green tea symbiotic fermentation liquid of component D is replaced by water of equal mass).

[0201] Table 6

[0202]

[0203]

[0204] Table 7

[0205]

[0206] The above results show that the matrix essence still has no anti-wrinkle effect after 2 or 4 weeks of use. Under the same conditions, the anti-wrinkle effect of Examples 1-9 is better than that of Comparative Examples 1-10. Among them, the anti-wrinkle effect of Examples 1 and 2 is better than that of Comparative Examples 1-7, indicating that the effect of composite fermentation using Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis is better. The anti-wrinkle effect of Examples 1, 2, 4 and 5 is better than that of Comparative Example 8, indicating that during fermentation, the inoculation amount of Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis should be controlled at 1-3:1-3:1-3. Exceeding this ratio, the effect of composite fermentation is poor, which affects the anti-wrinkle effect of the green tea symbiotic fermentation liquid. The anti-wrinkle effects of Examples 1, 6 and 7 are better than those of Comparative Examples 9 and 10, indicating that the pH value, an indicator of fermentation completion, has a great influence on the anti-wrinkle effect of the final product. A higher pH value at the completion of fermentation indicates that the green tea has not been fully fermented, and there are fewer active substances in the fermentation liquid, resulting in a poor anti-wrinkle effect. A lower pH value at the completion of fermentation indicates that the green tea is over-fermented, and the bacteria in the fermentation liquid are prone to cell autolysis, which consumes the active substances produced in the fermentation liquid.

[0207] From the results of comparative examples 1 and 3, it can be seen that the ratio of nitrogen source added affects the fermentation effect of green tea, and further affects the anti-wrinkle effect of the green tea symbiotic fermentation liquid. In the present invention, the nitrogen source is preferably a compound of soy peptone and distiller's grains. Most preferably, the mass ratio of soy peptone to distiller's grains is 3:1.

[0208] Embodiment 14

[0209] The whitening properties of the green tea symbiotic fermentation liquids obtained in Examples 1-9 and Comparative Examples 1-10 were tested.

[0210] Experimental principle: Tyrosinase is the rate-limiting enzyme in the melanin synthesis pathway, which mainly affects the production of melanin by affecting the conversion of tyrosine to DOPA and the oxidation of DOPA to DOPAQUINONE. The principle is that tyrosine or DOPA is converted into DOPAQUINONE under the action of tyrosinase. This reaction is a color reaction. The inhibition rate of tyrosinase activity of different samples is determined by colorimetry, thereby determining the whitening effect of different samples.

[0211] Instrument used: UV spectrophotometer (HITACHI, U2910)

[0212] Main reagents: L-dopa, tyrosinase, disodium hydrogen phosphate, dipotassium hydrogen phosphate, kojic acid.

[0213] Reagent preparation: The green tea symbiotic fermentation broth of Examples 1-9 and Comparative Examples 1-10 were respectively prepared into 0.4 mg / mL, 2 mg / mL, 10 mg / mL, and 50 mg / mL aqueous solutions; 1 mg / mL kojic acid aqueous solution; PBS buffer;

[0214] Tyrosinase solution: Prepare a tyrosinase solution with an active concentration of 100 U / mL using PBS buffer at pH 6.8.

[0215] DOPA solution: Prepare a DOPA solution with a mass concentration of 1 mg / mL using PBS buffer and keep it away from light.

[0216] Add each reagent in sequence according to Table 8, then incubate in a 37°C water bath for 10 min, add 2 mL of DOPA solution, react at 37°C for 5 min, and measure the absorbance at 475 nm.

[0217] Data analysis: Tyrosinase inhibition rate (%) = [1-(OD C -OD D ) / (OD A -OD B )]×100%; where OD A is the absorbance value of the enzyme solution control group; OD B is the absorbance value of the enzyme solution control blank group; OD C is the absorbance value of the sample group; OD D is the absorbance value of the blank control of the sample group.

[0218] IC 50 Calculation: (i.e., the concentration at which the inhibition rate reaches 50%): GraphPadPrism was used to fit and calculate IC 50 IC 50 The lower the value, the stronger the ability of the corresponding sample to inhibit tyrosinase is, and the better the whitening effect of the sample is. The test results are shown in Table 9.

[0219] Table 8

[0220]

[0221] Table 9 IC of inhibition rate of each sample on tyrosinase activity 50 value

[0222]

[0223]

[0224] The above results show that the anti-wrinkle effect of Examples 1-9 is better than that of Comparative Examples 1-10. Among them, the whitening effect of Examples 1 and 2 is better than that of Comparative Examples 1-7, indicating that the effect of composite fermentation using Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus braziliensis is better. The whitening effect of Examples 1, 2, 4 and 5 is better than that of Comparative Example 8, indicating that during fermentation, the inoculation amount of Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus braziliensis should be controlled at 1-3:1-3:1-3. Exceeding this ratio, the effect of composite fermentation is poor, affecting the whitening effect of the green tea symbiotic fermentation liquid. The whitening effect of Examples 1, 6 and 7 is better than that of Comparative Examples 9 and 10, indicating that the pH value of the fermentation completion index has a great influence on the whitening effect of the final product. The pH at the time of fermentation completion is high, indicating that the green tea has not been fully fermented, and the active substances in the fermentation liquid are less, resulting in poor whitening effect; when the pH at the time of fermentation completion is low, it means that the green tea is over-fermented, and the strains in the fermentation liquid are prone to cell autolysis, consuming the active substances produced in the fermentation liquid.

[0225] From the results of comparative examples 1 and 3, it can be seen that the ratio of nitrogen source addition affects the fermentation effect of green tea, and further affects the whitening effect of the green tea symbiotic fermentation liquid. In the present invention, the nitrogen source is preferably a compound of soy peptone and distiller's grains. Most preferably, the mass ratio of soy peptone to distiller's grains is 3:1.

[0226] Embodiment 15

[0227] The UV damage prevention performance of the green tea symbiotic fermentation liquid obtained in Examples 1-9 and Comparative Examples 1-10 was tested; the specific method is as follows:

[0228] The green tea symbiotic fermentation liquids of Examples 1 to 9 and Comparative Examples 1 to 10 were respectively formulated into aqueous solutions with a mass concentration of 10% as samples. 190 volunteers were selected (one sample was used for every 10 people), and the inner side of the volunteer's forearm was used as the test area (the left arm or the right arm was randomly selected, and the other arm was used as a blank control without any sample). The staff applied the sample on the 1st to 3rd day of the test, 2 times / day, with an interval of 8 hours between the application of the sample. On the 4th day, the SPF In Vivo Analyzer 6500 cosmetic sun protection index test system was used to model the ultraviolet irradiation of the sample application position and the blank control area. On the 5th day, the modeling area was tested for skin melanin and hemoglobin, and the change rates of skin hemoglobin and skin melanin in the sample group and the blank control group were calculated to evaluate the ability of the sample group to prevent ultraviolet damage.

[0229] Skin hemoglobin change rate (%) = (skin hemoglobin value after irradiation - skin hemoglobin value before irradiation) / skin hemoglobin value before irradiation * 100%

[0230] Skin melanin change rate (%) = (skin melanin value after irradiation - skin melanin value before irradiation) / skin melanin value before irradiation * 100%

[0231] After UV damage, the values ​​of skin hemoglobin and skin melanin become higher, and the greater the corresponding change rate, the weaker the skin's ability to prevent UV damage. The test results are shown in Table 10.

[0232] Table 10

[0233]

[0234]

[0235] The test results show that compared with the blank control group, the green tea symbiotic fermentation liquids of Examples 1-9 of the present invention and Comparative Examples 1-10 all have the effect of preventing ultraviolet damage. Among them, the effect of the green tea symbiotic fermentation liquids of Examples 1-9 of the present invention in preventing ultraviolet damage is better than that of the green tea symbiotic fermentation liquids of Comparative Examples 1-10.

[0236] The effect of preventing ultraviolet damage in Examples 1 and 2 is better than that in Comparative Examples 1-7, indicating that the effect of composite fermentation using Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus braziliensis is better. The effect of preventing ultraviolet damage in Examples 1, 2, 4 and 5 is better than that in Comparative Example 8, indicating that during fermentation, the inoculation amount of Saccharomyces cerevisiae, lactic acid bacteria and Aspergillus braziliensis should be controlled at 1-3:1-3:1-3. Exceeding this ratio, the effect of composite fermentation is poor, affecting the effect of preventing ultraviolet damage in the symbiotic fermentation liquid of green tea. The effect of preventing ultraviolet damage in Examples 1, 6 and 7 is better than that in Comparative Examples 9 and 10, indicating that the pH value of the fermentation completion index has a great influence on the effect of preventing ultraviolet damage in the final product. When the pH is high when the fermentation is completed, it means that the green tea has not been fully fermented, and there are fewer active substances in the fermentation liquid, resulting in poor effect of preventing ultraviolet damage; when the pH is low when the fermentation is completed, it means that the green tea is over-fermented, and the strains in the fermentation liquid are prone to cell autolysis, consuming the active substances produced in the fermentation liquid.

[0237] From the results of comparative examples 1 and 3, it can be seen that the ratio of nitrogen source added affects the fermentation effect of green tea, and further affects the effect of the green tea symbiotic fermentation liquid in preventing ultraviolet damage. In the present invention, the nitrogen source is preferably a compound of soy peptone and distiller's grains. Most preferably, the mass ratio of soy peptone to distiller's grains is 3:1.

[0238] Embodiment 15

[0239] The irritation of the green tea symbiotic fermentation liquid obtained in Examples 1-9 and Comparative Examples 1-10 was tested.

[0240] The eye irritation test of the present invention refers to "SN / T2329-2009 Chicken Embryo Chorioallantoic Membrane Test for Eye Irritation and Corrosion of Cosmetics".

[0241] The chicken embryo chorioallantoic membrane experiment was used to test the ability of the nanoemulsion to cause toxic changes in the chicken embryo chorioallantoic membrane, thereby describing the potential eye irritation factors and processes of the nanoemulsion. The chorioallantoic membrane (CAM) is a respiratory membrane that surrounds the chicken embryo. Since the surface of the chicken embryo chorioallantoic membrane is rich in blood vessels and can be regarded as a complete organism, this experiment uses the characteristics of the complete, clear and transparent vascular system of the hatched chicken embryo chorioallantoic membrane. A certain amount of nanoemulsion is contacted with the chicken embryo chorioallantoic membrane, and the toxic effect indicators of the chorioallantoic membrane (such as changes in bleeding, coagulation and vascular lysis) are observed after a specified time, and a score (IS) is given, as shown in Table 11, and the mathematical mean is calculated to evaluate the eye irritation of the nanoemulsion.

[0242] The reaction time method was used to calculate the stimulus score (IS), and the calculation formula was as follows:

[0243]

[0244] Where:

[0245] sec H (hemorrhage time) - the average time to the onset of bleeding observed on the CAM membrane, in seconds;

[0246] sec L (vessellysis time) - the average time to the onset of vascular lysis observed on the CAM membrane, in seconds;

[0247] sec C (coagulation time) - the average time in seconds for the onset of coagulation observed on the CAM membrane.

[0248] Table 11 Evaluation of stimulation scoring method results

[0249] Stimulus score Irritation classification IS<1 Non-irritating 1≤IS<5 Mild irritation 5≤IS<9 Moderate irritation IS ≥ 10 Strong irritant / corrosive

[0250] The specific experimental methods are as follows:

[0251] The test subjects were 9-day-old chicken embryos.

[0252] The samples were respectively the green tea symbiotic fermentation broth (20 wt % aqueous solution) of Examples 1-9 and Comparative Examples 1-10, the negative control group was normal saline, and the positive control group was 1 % sodium dodecyl sulfate solution (SDS solution).

[0253] Preparation of chicken embryos: Place the purchased 0-day-old eggs in an incubator for 9 days (the temperature of the incubator is 37.5°C and the relative humidity is 55% to 70%). Select chicken embryos with well-developed blood vessels, mark the position of the air chamber on the eggshell surface, and allocate the eggs required for the samples to be tested (6 chicken embryos for one sample, 1 chicken embryo for the negative control, and 1 chicken embryo for the positive control).

[0254] Conduct the test: Use a tool to remove the marked eggshell. Use a pipette to drip 0.9% sodium chloride solution (normal saline) to moisten the egg membrane, and pour out the sodium chloride solution. Carefully use tweezers to remove the inner membrane, ensuring that the blood vessels are not damaged. Observe that there is no obvious bleeding or turbidity in the chicken embryo, and it can be judged that it can be used for the experiment. Take 300 μL of the test substance and cover it on the chorioallantoic membrane. After 3 minutes of action, rinse the test substance on the membrane with normal saline for 30 seconds. Immediately observe the reaction of the chorioallantoic membrane, take a photo to record the state of the chicken embryo, and end the observation. The results are shown in Table 12 below.

[0255] Table 12

[0256] sample IS Irritation classification Example 1 0.1 Non-irritating Example 2 0.2 Non-irritating Example 3 0.2 Non-irritating Example 4 0.4 Non-irritating Example 5 0.3 Non-irritating Example 6 0.3 Non-irritating Example 7 0.2 Non-irritating Example 8 0.4 Non-irritating Example 9 0.5 Non-irritating Comparative Example 1 0.7 Non-irritating Comparative Example 2 0.8 Non-irritating Comparative Example 3 0.7 Non-irritating Comparative Example 4 0.6 Non-irritating Comparative Example 5 0.7 Non-irritating Comparative Example 6 0.8 Non-irritating Comparative Example 7 0.7 Non-irritating Comparative Example 8 0.8 Non-irritating Comparative Example 9 0.8 Non-irritating Comparative Example 10 0.7 Non-irritating

[0257] The test results in Table 12 show that the green tea symbiotic fermentation liquid of the embodiment of the present invention and the comparative example are non-irritating to the eyes.

[0258] Finally, it should be noted that the above embodiments are used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects, characterized in that: The following steps are involved: S1: preparing green tea powder from green tea; S2: inoculating the activated composite bacteria into a fermentation matrix for fermentation to obtain a mixed solution; S3: filtering the mixed solution obtained in step S2, and the obtained filtrate is the green tea symbiotic fermentation liquid; The composite bacteria in step S2 include saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis; The inoculation amount of the composite bacteria in step S2 is 3-9%; the fermentation matrix contains the green tea powder of step S1; the fermentation matrix also includes a carbon source, a nitrogen source and residual water; the addition amount of the green tea is 0.5-2.0% of the total mass of the fermentation matrix; the addition amount of the nitrogen source is 0.1-1.0% of the total mass of the fermentation matrix; the nitrogen source is at least one of sulfate, ammonium salt, peptone, distiller's grains, corn steep liquor and yeast extract; The sign of the end of fermentation in step S2 is: the pH of the mixed solution drops to 3.0-4.5; In the step S2, the ratio of the inoculation amount of saccharomyces cerevisiae, lactic acid bacteria and Aspergillus brasiliensis is saccharomyces cerevisiae: lactic acid bacteria: Aspergillus brasiliensis = 1-3:1-3:1-3.

2. The preparation method according to claim 1, characterized in that In the step S1, the particle size of the green tea powder is 60-100 mesh.

3. The preparation method according to claim 1, characterized in that: At least one of the following (a)-(e): (a) the amount of the carbon source added is 0.2-2.0% of the total mass of the fermentation substrate; (b) the carbon source is at least one of glucose, sucrose and fructose; (c) the fermentation temperature is 28-38°C; (d) the fermentation time is 40-100h; (e) The fermentation speed is 100-300 rpm.

4. A green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects, characterized in that: The green tea symbiotic fermentation liquid is prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the green tea symbiotic fermentation liquid having antioxidant, oil-control and anti-wrinkle effects as claimed in claim 4 in cosmetics.

6. A cosmetic composition, characterized in that The cosmetic composition contains the green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects as claimed in claim 4.

7. The cosmetic composition according to claim 6, characterized in that The added amount of the green tea symbiotic fermentation liquid with anti-oxidation, oil-control and anti-wrinkle effects is 0.1-50% of the total mass of the cosmetic composition.

8. A method for improving skin condition for non-therapeutic purposes, characterized in that: The method comprises topically applying a safe and effective amount of the green tea symbiotic fermentation liquid with antioxidant, oil-control and anti-wrinkle effects according to claim 4 or the cosmetic composition according to any one of claims 6-7 to the skin of a person in need thereof.

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