CN116983245A - Application of aspergillus oryzae fermentation filtrate in promotion of collagen and/or elastin generation and/or antioxidation - Google Patents

Abstract

The invention provides an application of aspergillus oryzae fermented filtrate in promoting collagen and/or elastin generation and/or antioxidation. The invention also relates to application of the aspergillus oryzae fermented filtrate in preparing skin external preparations, medicines and health-care foods with the functions of promoting collagen and/or elastin generation and/or antioxidation.

Description

Application of aspergillus oryzae fermentation filtrate in promotion of collagen and/or elastin generation and/or antioxidation

Technical Field

The invention relates to the field of natural pharmaceutical chemistry, in particular to application of aspergillus oryzae fermentation filtrate in promoting collagen and/or elastin generation and/or antioxidation, and application of aspergillus oryzae fermentation filtrate in preparing skin external preparations, medicines and health-care foods with the effects of promoting collagen and/or elastin generation and/or antioxidation.

Background

The determinant of skin aging is the destruction of collagen in the dermis by the internal and external environment. Collagen in the skin accounts for 72%, dermis accounts for 80%, and the three-dimensional spiral structure of the collagen is like a dyke in a reservoir to lock deep water in the skin. The skin is supported in the form of springs in the skin, and the human body can produce many collagens at younger age, but their yield decreases with age. Elastin is the major component of elastane fiber. The skin has the ability of stretching and folding, is responsible for maintaining and supporting the elasticity of the skin, and the content of elastin in the skin is reduced along with the aging, so that the network support required for maintaining the compact skin is reduced, and the skin starts to relax, has fine wrinkles and other aging phenomena.

Fibroblasts (Fb) are the main cells constituting the dermis layer of the skin, and are the main cells of the dermis layer that produce collagen and other cellular interstitium, and their normal differentiation and proliferation maintain the normal structure and physiological functions of the skin. Fb has strong protein synthesis capability, can synthesize and secrete a large amount of matrix components such as elastin, collagen, glycosaminoglycan, glycoprotein and the like, further generates elastic fibers, collagen fibers and reticular fibers, and secretes various cell repair factors, so that the skin has strong renewal and self-repair capability, and substances with increased thickness and density of dermis of the skin have potential wrinkle stretching and skin elasticity and luster recovering effects.

Aspergillus oryzae (Aspergillus oryzae), a common species among filamentous fungi, belongs to the genus Aspergillus and, unlike Aspergillus flavus, does not secrete oncogenic aflatoxins, is a food-grade recognized and safe strain certified by the world health organization and the United states official, and its metabolites are also widely used in the fields of medicine, cosmetics, agriculture, etc.

Red rice, which is obtained by removing the husk from rice of Gramineae, is used as the original plant. Unlike ordinary rice, however, red rice has no early rice, only medium rice or one-season late rice, so that the yield is not as high as rice. Modern researches have shown that red rice has antioxidant and whitening activities. For example, in the literature, "research on nutritional quality evaluation of different rice varieties based on principal component analysis and cluster analysis", the rice varieties are classified into colored rice and ordinary rice, and research analysis is performed on the same, and it is first found that the colored rice has an overall stronger antioxidant activity than ordinary rice, and that procyanidin components are detected only in red rice. The literature shows to a great extent that the red rice is different from the common rice, but the varieties are different, the nutrition components are greatly different.

For example, chinese patent application CN 202010232010.3 relates to the use of red rice fermentation broth, reporting lactobacillus plantarum as a fermentation broth capable of acting on type IV collagen and VII collagen as anti-aging active ingredients in skin external preparations. For example, chinese patent application CN202010642140.4 relates to a red rice fermentation filtrate, a preparation method and application thereof in preparing cosmetics, discloses a fermentation process of red rice by using lactobacillus plantarum, and has the effects of moisturizing, whitening and anti-aging. For example, the literature "study of rice fermentation filtrate up-regulation of tight junction proteins and enhancement of skin barrier effects" reports that yeast fermented rice has skin barrier repair effects.

However, none of these documents reports that Aspergillus oryzae fermentation filtrate of red rice has a function of promoting collagen and/or elastin and/or antioxidation. According to the invention, through efficacy comparison of 6 bacteria fermentation filtrate of red rice, the aspergillus oryzae fermentation filtrate of red rice has excellent effect of promoting type I collagen, type III collagen and elastin. Meanwhile, according to the comparative research on the components of the red rice and the common rice fermented by the aspergillus oryzae, the content of the fermented components is different and cannot be equal although the rice is fermented.

Disclosure of Invention

In one aspect, the invention provides the use of Aspergillus oryzae fermented filtrate of red rice to promote collagen and/or elastin production and/or antioxidant.

In a preferred embodiment, the collagen is selected from the group consisting of: type I collagen, type III collagen, or a combination thereof. In a preferred embodiment, the volume concentration of the Aspergillus oryzae fermentation filtrate is at least 0.1%, more preferably at least 1%.

In another aspect, the invention relates to the use of Aspergillus oryzae fermented filtrate in the preparation of skin external preparations, medicines and health foods with collagen and/or elastin production promoting and/or antioxidant effects.

In a preferred embodiment, the content of the Aspergillus oryzae fermented filtrate in the skin external preparation, the medicine and the health food is 0.001-20 wt%, preferably 0.005-10 wt%, more preferably 0.01-5 wt%.

In a preferred embodiment, the skin external agent is selected from the group consisting of: face cream, milky lotion, jelly, pack, face toilet, pack, aerosol cleansing foam, spray, body wash, or facial cleanser.

In a preferred embodiment, the total sugar in the Aspergillus oryzae fermentation filtrate is greater than or equal to 9.9wt%, wherein the glucose content of the total sugar is greater than or equal to 40%.

In a preferred embodiment, the total protein in the Aspergillus oryzae fermentation filtrate is greater than or equal to 0.27wt%, wherein the peptide content in the total protein is greater than or equal to 80%.

Brief description of the drawings

Fig. 1 shows the results of Elastin experiments.

FIG. 2 shows the results of HAS1 experiments.

FIG. 3 shows the results of the Collagen I experiment.

FIG. 4 shows the results of the Collagen III experiment.

Fig. 5 shows ROS experimental results.

Detailed Description

The invention relates to the effect of aspergillus oryzae fermented filtrate in promoting collagen and/or elastin generation and/or antioxidation, and discovers that the aspergillus oryzae fermented filtrate can be used as an efficacy additive for external skin preparations, medicines and health-care foods for helping to realize the effects of maintaining skin compact, improving skin relaxation and/or wrinkles, resisting skin aging and the like.

In order to provide a more concise description, some quantitative representations presented herein are not modified by the term "about". It will be understood that each quantity given herein is intended to refer to an actual given value, whether or not the term "about" is explicitly used, and is also intended to refer to approximations of such given values, including approximations of such given values resulting from experimental and/or measurement conditions, as reasonably deduced by one of ordinary skill in the art.

To provide a more concise description, some quantitative expressions herein are recited as a range from about X to about Y. It should be understood that when a range is recited, the range is not limited to the recited upper and lower limits, but rather, includes the entire range of about X to about Y amounts or any amount therebetween.

The aspergillus oryzae fermented filtrate described herein may optionally be in the form of a finished package. In one embodiment, the package is a container such as a plastic, metal or glass tube or jar containing the fermentation filtrate of Aspergillus oryzae. The product may additionally have a package such as a plastic or cardboard box for storing the container. In one embodiment, the product comprises aspergillus oryzae fermented filtrate and has instructions directing the user to apply the aspergillus oryzae fermented filtrate to the skin to treat signs of skin aging as discussed below. Such instructions may be printed on the container, on the label insert, or on any other package.

As used herein, "topical application" means directly applying or spreading on the external skin, scalp or hair, for example, using the hand or an applicator such as a wipe, roller or sprayer.

As used herein, "cosmetically acceptable" means that the ingredients described by the term are suitable for use in contact with tissue (e.g., skin or hair) without undue toxicity, incompatibility, instability, irritation, allergic response, or the like.

The present invention has found that Aspergillus oryzae fermented filtrate is suitable for treating signs of skin aging. As used herein, "signs of skin aging" include the presence of fine lines and wrinkles, loss of elasticity, uneven skin tone, and scarring. In a particularly preferred embodiment, the sign of aging is the presence of fine lines and wrinkles and/or loss of elasticity.

As used herein, "treating signs of skin aging" refers to reducing, preventing, ameliorating, or eliminating the presence or signs of skin aging described above.

As used herein, "wrinkles" includes fine lines, fine wrinkles, or coarse wrinkles. Examples of wrinkles include, but are not limited to, fine lines around the eyes (e.g., "fish tail lines"), forehead and cheek wrinkles, eyebrow lines, and smiles around the mouth.

As used herein, "loss of elasticity" includes loss of elasticity or structural integrity of skin or tissue, including but not limited to sagging, laxity, and loose tissue. Loss of elasticity or tissue structural integrity may be caused by a variety of factors including, but not limited to, disease, aging, hormonal changes, mechanical trauma, environmental damage, or as a result of the application of a product such as a cosmetic or pharmaceutical to the tissue.

As used herein, "uneven skin tone" refers to skin conditions associated with diffuse or mottled pigmentation, which may be classified as hyperpigmentation, such as post-inflammatory hyperpigmentation.

As used herein, "scar" means a skin condition associated with redness or erythema.

As used herein, "cosmetic" refers to a cosmetic substance or article that retains, restores, imparts, stimulates or enhances the appearance of a body or appears to enhance a beauty or youthful appearance, particularly when it relates to the appearance of tissue or skin.

As used herein, "cosmetically effective amount" means an amount of physiologically active compound or composition sufficient to treat one or more signs of skin aging, but low enough to avoid serious side effects. The cosmetically effective amount of a compound or composition will vary depending on: the particular condition being treated, the age and physical condition of the end user, the severity of the condition being treated/prevented, the duration of the treatment, the nature of the other treatments, the particular compound or product/composition employed, the particular cosmetically acceptable carrier utilized, and the like.

Aspergillus oryzae fermented filtrate

Aspergillus oryzae (Aspergillus oryzae), a common species among filamentous fungi, belongs to the genus Aspergillus and, unlike Aspergillus flavus, does not secrete oncogenic aflatoxins, is a food-grade recognized and safe strain certified by the world health organization and the United states official, and its metabolites are also widely used in the fields of medicine, cosmetics, agriculture, etc.

The present invention is based on the following unexpected findings: the Aspergillus oryzae fermented filtrate has effects in promoting collagen and/or elastin production and/or antioxidant production of human fibroblast. Therefore, the aspergillus oryzae fermented filtrate can be used as an effective component to be added into skin care products to help improve skin relaxation, wrinkle problems and the like. The invention aims to provide the effects of promoting the generation of human collagen and/or elastin and/or antioxidation of the aspergillus oryzae fermented filtrate and the application thereof in cosmetics.

In some embodiments, the process for preparing the aspergillus oryzae fermented filtrate comprises the steps of:

(a) Providing red rice, crushing to obtain crushed rice, and adding water to cover the crushed rice;

(b) Adding amylase for liquefying;

(c) Adding saccharifying enzyme for saccharification;

(d) Adding Aspergillus oryzae for fermentation;

(e) Filtering to obtain Aspergillus oryzae fermented filtrate.

In a preferred embodiment, step (a) comprises a heating step, for example to a temperature of 40-60 ℃, preferably 50 ℃. In a preferred embodiment, the liquefaction time of step (b) is 1 hour. In a preferred embodiment, step (c) comprises saccharification by adding a saccharifying enzyme after the rice slurry has cooled for a period of 1 hour. In a preferred embodiment, the fermentation time of step (d) is from 36 to 168 hours. In a preferred embodiment, step (d) is followed by sterilization, centrifugation, filtration, activated carbon decolorization steps.

In applications that promote the production of collagen and/or elastin and/or antioxidant by fibroblasts, the volume concentration of the fermentation filtrate of Aspergillus oryzae is at least 0.1%, more preferably at least 1%.

In some embodiments, the volume concentration of the aspergillus oryzae fermentation filtrate is preferably less than 10%, more preferably less than 5%.

In some embodiments, the volume concentration of the Aspergillus oryzae fermentation filtrate is 1% -3%, preferably 1.25% -3%.

In a preferred embodiment, the total sugar in the Aspergillus oryzae fermentation filtrate is greater than or equal to 9.9wt%, wherein the glucose content of the total sugar is greater than or equal to 40%.

In a preferred embodiment, the total protein in the Aspergillus oryzae fermentation filtrate is greater than or equal to 0.27wt%, wherein the peptide content in the total protein is greater than or equal to 80%.

In a preferred embodiment, glutamic acid is more than or equal to 4.21mg/L and tyrosine is more than or equal to 1.30mg/L in the fermentation filtrate of the aspergillus oryzae. In a preferred embodiment, the total polyphenols in the fermentation filtrate of Aspergillus oryzae is not less than 0.01wt%. In a preferred embodiment, the peptide content of the Aspergillus oryzae fermentation filtrate is greater than or equal to 0.22wt%. In a preferred embodiment, the glucose in the Aspergillus oryzae fermentation filtrate is greater than or equal to 4.03wt%. In a preferred embodiment, the gamma-aminobutyric acid in the fermentation filtrate of the aspergillus oryzae is more than or equal to 13.21mg/L and VB ₃ ≥17.61mg/L。

External preparation for skin

In some embodiments, the aspergillus oryzae fermented filtrate can be used in the preparation of a skin external agent. The skin external preparation is preferably a cosmetic composition including, but not limited to, products in the form of face cream, milky lotion, jelly, lotion, essence, pack, eye cream, aerosol (cleansing foam), spray, body wash, facial cleanser, and the like.

The content of Aspergillus oryzae fermented filtrate in the skin external preparation is 0.001-20wt%, preferably 0.005-10wt%, more preferably 0.01-5wt%.

The external skin preparation is a general concept of all ingredients commonly used outside the skin, and may be, for example, a cosmetic composition. The cosmetic composition may be basic cosmetic, facial makeup cosmetic, body cosmetic, hair care cosmetic, etc., and its dosage form is not particularly limited and may be reasonably selected according to different purposes. The cosmetic composition also contains various cosmetically acceptable medium or matrix excipients depending on dosage form and purpose.

A skin external preparation comprising the fermentation filtrate of Aspergillus oryzae may be topically applied to human skin and/or hair. The skin external preparation may further comprise a cosmetically acceptable topical carrier, which may be about 50% to about 99.99% by weight of the skin external preparation (e.g., about 80% to about 99% by weight of the skin external preparation). In a preferred embodiment of the invention, the cosmetically acceptable topical carrier comprises water. The cosmetically acceptable topical carrier may include one or more materials selected from the group consisting of moisturizers, emollients, oils, humectants, and the like. In one embodiment, the cosmetically acceptable topical carrier includes a substrate such as a nonwoven or film material.

Skin external preparations may be formulated into a variety of product types including, but not limited to, lotions, creams, gels, sticks, sprays, ointments, cleansing liquid lotions and solid soaps, shampoos and hair conditioners, hair fixatives, pastes, foams, powders, mousses, shave creams, wipes, patches, hydrogels, film-forming products, masks and skin films, films and cosmetics such as foundations and mascaras. These product types may contain several types of cosmetically acceptable topical carriers including, but not limited to, solutions, suspensions, emulsions (e.g., microemulsions and nanoemulsions), gels, solids, and liposomes.

A skin external preparation containing Aspergillus oryzae fermented filtrate can be formulated into solution. The solution typically comprises an aqueous or organic solvent (e.g., about 50% to about 99.99% or about 90% to about 99% of a cosmetically acceptable aqueous or organic solvent). Examples of suitable organic solvents include propylene glycol, polyethylene glycol, polypropylene glycol, glycerol, 1,2, 4-butanetriol, sorbitol esters, 1,2, 6-hexanetriol, ethanol and mixtures thereof.

The skin external preparation may be formulated as a solution containing an emollient. Such skin external preparations preferably comprise from about 2% to about 50% of one or more emollients. As used herein, "emollient" refers to a substance used to prevent or reduce dryness, for example, by preventing the loss of skin moisture through the skin. Examples of emollients include, but are not limited to, vegetable oils, mineral oils, aliphatic esters, and the like.

Lotions can be prepared from such solutions. Lotions typically contain from about 1% to about 20% (e.g., from about 5% to about 10%) of one or more emollients and from about 50% to about 90% (e.g., from about 60% to about 80%) of moisture.

Another type of product that can be formulated from solutions is a cream. A cream typically contains from about 5% to about 50% (e.g., from about 10% to about 20%) of one or more emollients and from about 45% to about 85% (e.g., from about 50% to about 75%) of moisture.

While it is preferred that the skin external preparation comprising the fermentation filtrate of aspergillus oryzae comprises water, the skin external preparation may alternatively be anhydrous or an ointment which does not comprise water but rather is an organic and/or silicone solvent, grease, lipid and wax. Ointments may contain simple bases of animal or vegetable oils or semi-solid hydrocarbons. Ointments may contain from about 2% to about 10% of one or more emollients and from about 0.1% to about 2% of one or more thickeners.

The skin external preparation can be formulated as an emulsion. If the topical carrier is an emulsion, from about 1% to about 10% (e.g., from about 2% to about 5%) of the topical carrier contains one or more emulsifying agents. The emulsifier may be nonionic, anionic or cationic. Examples of suitable emulsifiers include those commonly identified as suitable emulsifiers in the personal care and cosmetic formulations arts.

Lotions and creams can be formulated as emulsions. Typically such lotions contain from 0.5% to about 5% of one or more emulsifying agents. Such creams typically contain from about 1% to about 20% (e.g., from about 5% to about 10%) of one or more emollients; about 20% to about 80% (e.g., 30% to about 70%) water; and from about 1% to about 10% (e.g., from about 2% to about 5%) of one or more emulsifiers.

Oil-in-water and water-in-oil single emulsion skin care formulations, such as lotions and creams, are well known in the cosmetic arts and can be used in the present invention. Multiple emulsion skin external preparations (e.g., water-in-oil-in-water and oil-in-water) are also useful in the present invention. Typically, such single-phase or multiple-phase emulsions contain moisture, emollients, and emulsifiers as their essential ingredients.

The skin external preparation comprising the fermentation filtrate of Aspergillus oryzae may also be formulated as a gel (e.g., an aqueous gel, an alcoholic gel, an alcohol/water gel, or an oily gel using a suitable gelling agent). Suitable gelling agents for aqueous and/or alcoholic gels include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethyl cellulose and hydroxypropyl cellulose). Suitable gellants for oils (e.g., mineral oils) include, but are not limited to, hydrogenated butene/ethylene/styrene copolymers and hydrogenated ethylene/propylene/styrene copolymers. Such gels typically contain between about 0.1% and 5% by weight of such gelling agents.

External preparations for skin containing the fermentation filtrate of Aspergillus oryzae may also be formulated as solid preparations (e.g., wax-based sticks, bar soaps, powders, or wipes containing powders).

In addition to the above components, the skin external preparations usable in the present invention may contain various other oil-soluble substances and/or water-soluble substances which are conventionally used in skin external preparations for use on the skin and hair at levels determined in the technical field thereof.

The skin external preparation of the present invention may contain additional components commonly found in skin care compositions, such as emollients, skin conditioning agents, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, etc., as long as they are physically and chemically compatible with the other components of the skin external preparation and do not affect the effect of the fermentation filtrate of aspergillus oryzae of the present invention.

In some embodiments of the skin external preparation of the present invention, one or more preservatives may be used. Suitable preservatives include p-hydroxyacetophenone, alkyl C1-C4 p-hydroxybenzoates and phenoxyethanol. The preservative is used in an amount of about 0.5 to about 2 wt%, preferably about 0.5 to 1 wt%, based on the total weight of the composition.

In one example of the skin external agent of the present invention, one or more antioxidants may be used. Suitable antioxidants include Butylated Hydroxytoluene (BHT), ascorbyl palmitate (BHA), butylated hydroxyanisole, phenyl-alpha-naphthylamine, hydroquinone, propyl gallate, nordihydroguaiaretic acid, vitamin E or derivatives of vitamin E, vitamin C and its derivatives, calcium pantothenate, green tea extracts and mixed polyphenols, and mixtures of the foregoing. The antioxidants are used in an amount ranging from about 0.02 to 0.5 weight percent, more preferably from about 0.002 to 0.1 weight percent, based on the total weight of the composition.

In one example of the skin external agent of the present invention, one or more emollients may be used which act as lubricants to reduce flaking and improve the appearance of the skin by their ability to remain on the skin surface or in the stratum corneum. Typical emollients include fatty esters, fatty alcohols, mineral oils, polyether siloxane copolymers, and the like. Examples of suitable emollients include, without limitation, polypropylene glycol ("PPG") -15 stearyl ether, PPG-10 cetyl ether, steareth-10, oleth-8, PPG-4 lauryl ether, vitamin E acetate, lanolin, cetyl alcohol, cetostearyl alcohol ethyl hexanoate, cetostearyl alcohol, glyceryl stearate, octyl hydroxystearate, dimethylpolysiloxane, and combinations thereof. Cetyl alcohol, cetostearyl alcohol ethyl hexanoate, cetostearyl alcohol, glycerol stearate, and combinations thereof are preferred. When used, the emollient is in an amount ranging from about 0.1 to about 30 weight percent, preferably from about 1 to about 30 weight percent, based on the total weight of the composition.

In one example of the skin external agent of the present invention, one or more moisturizers may be used. Humectants, also known as humectants, help to enhance the effectiveness of emollients, reduce flaking, stimulate removal of constituent scales and enhance skin feel. Polyols may be used as humectants including, but not limited to, glycerin, polyalkylene glycols, alkylene polyols and derivatives thereof, including butylene glycol, propylene glycol, dipropylene glycol, polyglycerol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1, 3-dibutylene glycol, 1,2, 6-hexanetriol, ethoxylated glycerin, propoxylated glycerin and combinations thereof. When used, the humectant is present in an amount of about 0.1 to about 20 weight percent, preferably about 1 to about 15 weight percent, based on the total weight of the composition.

In one example of the skin external agent of the present invention, one or more emulsifying agents may be used. The emulsifier may be used in an effective stabilizing amount. Preferably, the emulsifier is used in an amount of about 1.0 to about 10.0 wt%, more preferably about 3.0 to about 6.0 wt%, based on the total weight of the composition. Any emulsifier that is compatible with the components of the composition may be used. Suitable emulsifiers include stearic acid, cetyl alcohol, glyceryl stearate, lecithin, stearyl alcohol, steareth-2, steareth-20, acrylic/C10-30 alkanol acrylate cross-linked polymers, and combinations thereof.

In one example of the skin external agent of the present invention, one or more pH adjusting agents may be used. The pH adjuster useful in the skin external preparation of the present invention includes tromethamine. When used, the pH adjustor is used in an amount of about 0.1 to about 2 weight percent, preferably about 0.1 to about 1 weight percent, based on the total weight of the composition.

In one embodiment of the present invention, the skin external preparation comprises acrylic/C10-30 alkanol acrylate cross-linked polymer, glycerol, p-hydroxyacetophenone, glycerol stearate and lecithin, cetyl/stearyl alcohol, cetostearyl alcohol ethyl hexanoate, tromethamine or combinations thereof.

Additional cosmetic active agents

In some embodiments, the skin external preparation may further comprise additional cosmetic active agents. As used herein, a "cosmetically active agent" is a compound that has a cosmetic or therapeutic effect on skin or hair (e.g., a synthetic compound or a compound isolated from a natural source or natural extract), including but not limited to anti-acne agents, oil control agents, antimicrobial agents, anti-inflammatory agents, antifungal agents, antiparasitic agents, topical analgesics, sunscreens, photoprotective agents, antioxidants, keratolytic agents, surfactants, moisturizers, nutrients, vitamins, energy enhancers, antiperspirants, astringents, deodorants, solidifying agents, anti-sclerokeratotic agents, and agents for hair and/or skin conditioning.

In one embodiment, these cosmetically active agents are selected from (but are not limited to): hydroxy acids, benzoyl peroxide, D-panthenol, octyl methoxycinnamate, titanium dioxide, octyl salicylate, homosalate, avobenzone, carotenoids, radical scavengers, spin traps, amines, retinoids such as retinol and retinyl palmitate, ceramides, polyunsaturated fatty acids, essential fatty acids, enzymes, enzyme inhibitors, minerals, hormones such as estrogens, steroids such as hydrocortisone, 2-dimethylaminoethanol, copper salts such as copper chloride, copper-containing peptides such as Cu: gly-His-Lys, coenzyme Q10, peptides, amino acids such as proline, vitamins, lactobionic acid, acetyl-coa, niacin, riboflavin, thiamine, ribose, electron transfer substances such as NADH and FADH2, and other plant extracts such as aloe vera, feverfew, oatmeal, and derivatives and mixtures thereof. The cosmetically active agent is typically present in an amount of about 0.001% to about 20%, for example about 0.005% to about 10%, such as about 0.01% to about 5%, by weight of the skin external agent of the present invention.

Examples of vitamins include, but are not limited to, vitamin a, vitamin B (e.g., vitamin B3, vitamin B5, and vitamin B12), vitamin C, vitamin K, and different forms of vitamin E (e.g., alpha, beta, gamma, or delta tocopherol) or mixtures thereof, and derivatives thereof.

Examples of hydroxy acids include, but are not limited to, glycolic acid, lactic acid, malic acid, salicylic acid, citric acid, and tartaric acid.

Examples of antioxidants include, but are not limited to: water-soluble antioxidants such as mercapto compounds and their derivatives (e.g., sodium metabisulfite and N-acetyl-cysteine), lipoic acid and dihydrolipoic acid, resveratrol, lactoferrin and ascorbic acid derivatives (e.g., ascorbyl palmitate and ascorbyl polypeptide). Oil-soluble antioxidants suitable for use in the skin external preparations of the present invention include, but are not limited to, butylated hydroxytoluene, retinoids (e.g., retinol and retinyl palmitate), tocopherols (e.g., ethyl tocopheryl), tocotrienols, and ubiquinone. Natural extracts containing antioxidants suitable for use in the skin external preparations of the present invention include, but are not limited to: extracts containing flavonoids and isoflavones and their derivatives (e.g., genistein and diad zein), extracts containing resveratrol, etc. Examples of such natural extracts include grape seed, green tea, pine bark and propolis.

Application method

The skin external preparation of the present invention can be topically applied to mammalian skin in need of treatment for one or more signs of skin aging as described above. In one embodiment, the skin external agent may be applied to skin in need of treatment for fine lines and wrinkles and/or loss of elasticity. The skin external agent may be applied to the skin in need of such treatment according to a suitable treatment regimen, such as monthly, weekly, every other day, daily, twice daily, etc.

In certain embodiments, the skin external preparations of the present invention may also be used to treat other needs associated with the skin. For example, the skin external preparation of the present invention can be used for treating post-inflammatory hyperpigmentation, for reducing pore size, for reducing sebum production, and for alleviating scars. In certain other embodiments, the skin external agents of the present invention may be applied simultaneously with or within hours of a mechanical or physical exfoliating treatment (e.g., microdermabrasion treatment), or simultaneously with a chemical exfoliating agent or a keratolytic agent such as salicylic acid. In certain other embodiments, the skin external agents of the present invention may be applied to mucous membranes or other tissues such as vaginal tissue, oral tissue, or ocular tissue. In certain other embodiments, the skin external preparations of the present invention may be applied to mild wounds or post-operative sites to promote healing, to insect bites, to poison vine skin disorders or similar skin conditions, or are generally used to reduce itching.

Examples

The invention will be further illustrated by the following examples. It is noted herein that the examples are given solely for the purpose of illustration and are not to be construed as limitations on the scope of the invention, since many insubstantial modifications and variations will become apparent to those skilled in the art in light of the above teachings. The test methods in the following examples, in which specific conditions are not specified, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. All percentages and parts are by weight unless otherwise indicated.

Microscope olympus CKX53, camera Canon EOS 750D, magnification 200 times was used in the examples below. The red rice used in the following examples was purchased from Lijianning Yikang agricultural product development Co., ltd; the five-spice rice is a rice flower fragrance five-spice rice; aspergillus oryzae, saccharomyces cerevisiae, pichia guilliermondii, lactobacillus plantarum, bacillus subtilis, and Saccharomyces cerevisiae are all available from Jiangsu Hengshu group, inc.

Example 1: preparation of red rice combined yeast fermentation filtrate

Weighing 100g of red rice, and pulverizing (Dade machine DFY-600C) to obtain broken rice. The crushed rice was covered with 400g of water, heated to 50℃and, after stirring, 0.04g of amylase (Long Kete 240000 u/g) was added to liquefy for 1 hour. After the rice slurry had cooled, 0.03g of saccharifying enzyme (Long Kete 290000 u/g) was added to saccharify for 1 hour. And 5% of combined yeast (supplied by Jiangsu Hengshun group Co., ltd.) is added into the mash to ferment for 36-168 h. Sterilizing (steam sterilizing), centrifuging (Lu Xiangyi, DL-5M) at 3500rpm for 15min, and filtering with filter paper (Xinxing qualitative filter paper, 60 x 60 cm). Adding 1.5% active carbon powder (national medicine group chemical reagent Co., ltd.) into the filtrate, shaking the flask at 50deg.C and 200rpm for 1h (full temperature shaker, shanghai Zhi City ZWY-2102C), and filtering with filter paper. The filtrate was filtered through a 0.45 μm microfiltration membrane (Shanghai Xingjing sub purification materials factory). Obtaining red rice combined yeast fermentation filtrate for standby.

Example 2: preparation of red rice and Pichia pastoris fermentation filtrate

Weighing 100g of red rice, and pulverizing (Dade machine DFY-600C) to obtain broken rice. The crushed rice was covered with 400g of water, heated to 50℃and, after stirring, 0.04g of amylase (Long Kete 240000 u/g) was added to liquefy for 1 hour. After the rice slurry had cooled, 0.03g of saccharifying enzyme (Long Kete 290000 u/g) was added to saccharify for 1 hour. And 5% of Pichia guilliermondii (supplied by Jiangsu Hengshun group Co., ltd.) is inoculated into the mash for fermentation for 36-168 h. Sterilization (steam sterilization), centrifugation at 3500rpm for 15min, and filtration with filter paper. Adding 1.5% active carbon powder into the filtrate, shaking with 200rpm shaking table at 50deg.C for 1 hr (full temperature shaker, shanghai Zhi City ZWY-2102C), and filtering with filter paper. The filtrate was filtered through a 0.45 μm microfiltration membrane. Obtaining the fermentation filtrate of the red rice Pichia pastoris for standby.

Example 3: preparation of Aspergillus oryzae fermentation filtrate

Weighing 100g of red rice, and pulverizing (Dade machine DFY-600C) to obtain broken rice. The crushed rice was covered with 400g of water, heated to 50℃and, after stirring, 0.04g of amylase (Long Kete 240000 u/g) was added to liquefy for 1 hour. After the rice slurry had cooled, 0.03g of saccharifying enzyme (Long Kete 290000 u/g) was added to saccharify for 1 hour. 5% aspergillus oryzae (supplied by Jiangsu Hengshun group Co., ltd.) is added into the mash to ferment for 36-168 h. Sterilization (steam sterilization), centrifugation at 3500rpm for 15min, and filtration with filter paper. Adding 1.5% active carbon powder into the filtrate, shaking with 200rpm shaking table at 50deg.C for 1 hr (full temperature shaker, shanghai Zhi City ZWY-2102C), and filtering with filter paper. The filtrate was filtered through a 0.45 μm microfiltration membrane. Obtaining the Aspergillus oryzae fermented filtrate for standby.

Example 4: preparation of lactobacillus casei fermented filtrate of red rice

Weighing 100g of red rice, and pulverizing (Dade machine DFY-600C) to obtain broken rice. The crushed rice was covered with 400g of water, heated to 50℃and, after stirring, 0.04g of amylase (Long Kete 240000 u/g) was added to liquefy for 1 hour. After the rice slurry had cooled, 0.03g of saccharifying enzyme (Long Kete 290000 u/g) was added to saccharify for 1 hour. Inoculating 5% lactobacillus plantarum (supplied by Jiangsu Hengshun group Co., ltd.) into the mash, and fermenting for 36-168 h. Sterilization (steam sterilization), centrifugation at 3500rpm for 15min, and filtration with filter paper. Adding 1.5% active carbon powder into the filtrate, shaking with 200rpm shaking table at 50deg.C for 1 hr (full temperature shaker, shanghai Zhi City ZWY-2102C), and filtering with filter paper. The filtrate was filtered through a 0.45 μm microfiltration membrane. Obtaining lactobacillus plantarum fermentation filtrate for standby.

Example 5: preparation of bacillus subtilis fermented filtrate

Weighing 100g of red rice, and pulverizing (Dade machine DFY-600C) to obtain broken rice. The crushed rice was covered with 400g of water, heated to 50℃and, after stirring, 0.04g of amylase (Long Kete 240000 u/g) was added to liquefy for 1 hour. After the rice slurry had cooled, 0.03g of saccharifying enzyme (Long Kete 290000 u/g) was added to saccharify for 1 hour. 5% bacillus subtilis (supplied by Jiangsu Hengshun group Co., ltd.) is inoculated into the mash for fermentation for 36-168 h. Sterilization (steam sterilization), centrifugation at 3500rpm for 15min, and filtration with filter paper. Adding 1.5% active carbon powder into the filtrate, shaking with 200rpm shaking table at 50deg.C for 1 hr (full temperature shaker, shanghai Zhi City ZWY-2102C), and filtering with filter paper. The filtrate was filtered through a 0.45 μm microfiltration membrane. Obtaining the bacillus subtilis fermented filtrate for standby.

Example 6: preparation of red rice saccharomyces cerevisiae fermentation filtrate

Weighing 100g of red rice, and pulverizing (Dade machine DFY-600C) to obtain broken rice. The crushed rice was covered with 400g of water, heated to 50℃and, after stirring, 0.04g of amylase (Long Kete 240000 u/g) was added to liquefy for 1 hour. After the rice slurry had cooled, 0.03g of saccharifying enzyme (Long Kete 290000 u/g) was added to saccharify for 1 hour. 5% of Saccharomyces cerevisiae (supplied by Jiangsu Hengshun group Co., ltd.) is added into the mash to ferment for 36-168 h. Sterilization (steam sterilization), centrifugation at 3500rpm for 15min, and filtration with filter paper. Adding 1.5% active carbon powder into the filtrate, shaking with 200rpm shaking table at 50deg.C for 1 hr (full temperature shaker, shanghai Zhi City ZWY-2102C), and filtering with filter paper. The filtrate was filtered through a 0.45 μm microfiltration membrane. Obtaining the fermented filtrate of the red rice saccharomyces cerevisiae for standby.

Example 7: preparation of five-cereal aspergillus oryzae fermentation filtrate

100g of ordinary rice (commercially available) is weighed and crushed (Dade machine DFY-600C) to obtain crushed rice. The crushed rice was covered with 400g of water, heated to 50℃and, after stirring, 0.04g of amylase (Long Kete 240000 u/g) was added to liquefy for 1 hour. After the rice slurry had cooled, 0.03g of saccharifying enzyme (Long Kete 290000 u/g) was added to saccharify for 1 hour. And 5% of aspergillus oryzae is inoculated into the mash for fermentation for 36-168 hours. Sterilization (steam sterilization), centrifugation at 3500rpm for 15min, and filtration with filter paper. Adding 1.5% active carbon powder into the filtrate, shaking with 200rpm shaking table at 50deg.C for 1 hr (full temperature shaker, shanghai Zhi City ZWY-2102C), and filtering with filter paper. The filtrate was filtered through a 0.45 μm microfiltration membrane. Obtaining the Aspergillus oryzae fermented filtrate for standby.

Example 8: performance evaluation of red rice fermentation filtrate on collagen and elastin

1. Toxicity test experimental method

Cell inoculation: skin fibroblast (Fb 20081902, guangdong Boxi Biotechnology Co., ltd.) was cultured at a rate of 8X 10 ³ Seed Density of each well cells were seeded into 96 well plates, incubator (Thermo, 150I) (37 ℃, 5% CO ₂ ) Incubate overnight.

Test grouping: the test set was zeroed, solvent control, positive control and examples 1-6 ferments. Each set of examples set up 8 concentration gradients, with 3 duplicate wells per concentration gradient.

Preparing liquid: the solutions were prepared as follows.

TABLE 1

Administration: and when the cell plating rate in the 96-well plate reaches 40% -60%, the administration is carried out. 200. Mu.L of DMEM medium (Gibco) was added to each well of the solvent control group; 200. Mu.L of 10% DMSO (Sigma) in culture medium was added to each well of TGF-. Beta.1 group (Peprotech); example groups 200. Mu.L of the culture medium containing the examples at the corresponding concentrations was added per well; the zeroed group was inoculated without cells and only 200. Mu.L of cell culture medium was added. After the completion of the administration, the 96-well plate was placed in an incubator for culturing for 24 hours.

And (3) detection: after incubation of the cells for 24 hours, the supernatant was discarded, 0.5mg/mL MTT (Sigma) was added, incubated at 37℃for 4 hours in the absence of light, after the incubation was completed, 150. Mu.L of DMSO was added to each well, OD was read at 490nm and cell viability was calculated. Results are expressed as mean±sd.

The toxicity judging method comprises the following steps: the OD value of the sample group after MTT detection is compared with the OD value of the control group to judge, and after turning points are screened, about 2 concentrations are selected from the upper part and the lower part of the turning points to conduct morphological photographing so as to verify the MTT result.

Experimental results:

TABLE 2

More than 30% of lesions are defined as toxic according to ISO 10993-5 (in vitro cytotoxicity test) standard. Thus, 70% cell viability is internationally recognized as a non-toxic dose. From the above MTT results, it is believed that the example 1 fermentate did not exhibit significant cytotoxicity over a concentration range of 10% (v/v) based on fibroblasts; example 2 the fermentate showed no significant cytotoxicity in the 10% (v/v) concentration range based on fibroblasts; example 3 the fermentate was based on fibroblasts, showing no significant cytotoxicity in the concentration range of 3% (v/v); example 4 the fermentate showed no significant cytotoxicity in the 10% (v/v) concentration range based on fibroblasts; example 5 the fermentate showed no significant cytotoxicity in the 10% (v/v) concentration range based on fibroblasts; example 6 the fermentate showed no significant cytotoxicity in the 10% (v/v) concentration range based on fibroblasts.

2. Tightening anti-wrinkle efficacy test experiment method

The adopted kit comprises: elastin (Elastin), hyaluronan synthase 1 (HAS 1), collagen type I (Collagen I), collagen type III (Collagen III), RNAiso Plus (Ai Kerui organism), reverse transcription kit (Ai Kerui organism), fluorescent dye (Ai Kerui organism).

Cell inoculation: 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: the group was divided into a blank group, a TGF-. Beta.1 group (0.1. Mu.g/mL) and examples 1 to 6 (volume ratio: 3% and 1.25%).

Administration: and (3) when the cell plating rate in the 6-hole plate reaches 40% -60%, administration is carried out. 2mL of sample is added to each well, and 3 compound wells are arranged in each group. After the administration, the 6-well plate was placed in an incubator to be cultured for 24 hours.

And (3) sample collection: after incubation for 24h, the old solution was aspirated, washed twice with PBS (Soxhobao), 1mL RNAAiso Plus (Ai Kerui organisms) was added to each well, lysed cells were blown on, and samples were collected.

And (3) gene expression detection: RNA was extracted, reverse transcribed into cDNA using a reverse transcription kit (Ai Kerui organism), and then fluorescent quantitative PCR was performed (Ai Kerui organism), and the content was expressed as mean.+ -. SD. Comparisons between groups were performed using t-test statistical analysis. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences, significance being expressed in x.

Elastin (Elastin) experimental results:

TABLE 3 Table 3

Compared with the blank control group, the content of Elastin in the positive control group (TGF-beta 1) is up-regulated by 109%, which proves that the positive control of the test is effective. The Elastin content was up-regulated by 20% at 3% by volume and by 12% at 1.25% by volume in combination with yeast fermentation. The Elastin content was up-regulated by 9% when fermenting pichia pastoris in red rice at a volume concentration of 1.25%. The content of Elastin is up-regulated by 159% when the volume concentration is 3% and by 65% when the volume concentration is 1.25%. Lactobacillus plantarum fermentation was up-regulated by 100% at 3% by volume and 94% by volume at 1.25% by volume. Bacillus subtilis in red rice fermentation was up-regulated by 100% at 3% by volume and by 149% at 1.25% by volume. The Elastin content is up-regulated by 30% when the volume concentration is 3% and by 18% when the volume concentration is 1.25% when the red rice saccharomyces cerevisiae is fermented. The method shows that red rice combined with yeast, pichia guilliermondii fermentation, aspergillus oryzae fermentation, lactobacillus plantarum, bacillus subtilis and saccharomyces cerevisiae fermentation can well promote fibroblast to generate Elastin, so that a compacting effect is achieved. And aiming at the results, the results show that the fermented red rice of different bacteria has different expression effects on Elastin genes, and when the volume concentration is 3%, aspergillus oryzae is more than bacillus subtilis=lactobacillus plantarum is more than Saccharomyces cerevisiae, and pichia pastoris is also more than Saccharomyces cerevisiae.

Results of hyaluronate synthase 1 (HAS 1) experiments:

TABLE 4 Table 4

Compared with the blank control group, the HAS1 content of the positive control group (TGF-beta 1) is up-regulated by 26%, which proves that the positive control of the test is effective. The HAS1 gene expression levels of the fermentate groups of examples 1-6 were not significantly changed.

Experimental results of type I Collagen (Collagen I):

TABLE 5

The positive control (TGF-. Beta.1) had a Collagen I content up-regulated by 32% compared to the blank, indicating that the positive control was effective. In the fermented product groups of examples 1 to 6, only Aspergillus oryzae in red can well promote fibroblasts to generate Collagen I, so that the anti-wrinkle effect is achieved, and the content of the Collagen I is up-regulated by 17% when the volume concentration is 3%.

Experimental results of Collagen iii (Collagen iii):

TABLE 6

The positive control (TGF-. Beta.1) had a 62% up-regulation of the Collagen III content compared to the blank, indicating that the positive control was effective. The content of Collagen III was up-regulated by 34% at a volume concentration of 1.25% by fermentation of Pichia pastoris. Aspergillus oryzae fermentation was up-regulated by 47% at 3% volume concentration and by 15% at 1.25% volume concentration. Bacillus subtilis in red rice fermentation was up-regulated by 53% at 3% by volume and by 30% at 1.25% by volume. The method shows that the fermentation of the red rice Pichia pastoris, the fermentation of the Aspergillus oryzae and the fermentation of the bacillus subtilis can well promote fibroblasts to generate the Collagen III, and the anti-wrinkle effect is achieved. At a volume concentration of 3%, bacillus subtilis > aspergillus oryzae; pichia pastoris > Bacillus subtilis > Aspergillus oryzae at a volume concentration of 1.25%.

Example 9: evaluation of antioxidant Property of Red Rice fermentation filtrate

ROS kit: and (3) the green cloud is a green cloud.

Cell inoculation: 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: the control group was divided into a blank group, a negative control group, a VE group (0.5 mg/mL) and examples 1 to 6 (volume ratio: 3% and 1.25%).

Administration: and (3) when the cell plating rate in the 6-hole plate reaches 40% -60%, administration is carried out. 2mL of sample is added to each well, and 3 compound wells are arranged in each group. After the administration, the 6-well plate was placed in an incubator to be cultured for 24 hours.

UVA irradiation: according to the grouping, the groups with UVA (Philips) irradiation were subjected to 30J/cm ² Is placed in an incubator for continuous cultivation for 24 hours.

And (3) flow detection: cells were washed 3 times with PBS, 1ml of DCFH-DA probe (Biyun day) at a concentration of 10. Mu.M was added to each well, incubated in an incubator for 30min, DCFH-DA broth was discarded, the cells were washed 3 times with PBS, 0.25% pancreatin was digested, and after 1 washing of the cells with PBS, a certain amount of PBS was added, and the results were expressed as mean.+ -. SD. Comparisons between groups were performed using t-test statistical analysis. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences, with significance expressed as # compared to the blank group and significance expressed as x compared to the negative group.

Antioxidant (ROS) experimental results:

TABLE 7

Compared with the blank control group, the ROS content of the negative control group is obviously up-regulated by 47%, which proves that the test stimulation condition is effective. The ROS content of the positive control (VE) was down-regulated by 57%, indicating that the positive control was effective for this test. Pichia pastoris fermentation in red rice is reduced by 54% at 3% by volume and 50% at 1.25% by volume. Aspergillus oryzae fermentation was reduced by 63% at 3% by volume and by 61% at 1.25% by volume. Lactobacillus plantarum red ferments were down-regulated by 53% ROS at 3% volume concentration and by 44% at 1.25% volume concentration. The results show that the red rice Pichia pastoris fermentation, the Aspergillus oryzae fermentation and the Lactobacillus plantarum can well inhibit the expression of ROS in fibroblasts, and the antioxidation effect is achieved. And aiming at the results, the results show that the red rice fermented by different bacteria has different ROS expression effects, and when the volume concentration is 1.25% -3%, aspergillus oryzae is more than Pichia pastoris is more than Lactobacillus plantarum.

Example 10: component comparison research of Aspergillus oryzae fermentation filtrate

1. Determination of the free amino acid content (cf. GB/T30987-2020)

1.1 preparation of standard solution

0.01mg of each of L-glutamic acid (Shanghai An Spectrometry laboratory sciences Co., ltd.) and L-tyrosine (Shanghai An Spectrometry laboratory sciences Co., ltd.) was weighed, and dissolved in water to prepare a mixed solution, and the concentration of each amino acid was 1. Mu.g/mL for use.

1.2 sample pretreatment

Taking 5g of the fermentation filtrate of the example 1 and the fermentation filtrate of the example 7 in a 10mL volumetric flask, adding 0.02mol/L hydrochloric acid (national drug group) to fix the volume, carrying out ultrasonic treatment for 20min, centrifuging for 5min at 6000rpm, and taking 2.5mL of supernatant. 1.5mL of 0.02mol/L hydrochloric acid was added to the column. 100. Mu.L of the sample was dried to constant weight at 60℃in a 15mL centrifuge tube. Add 50 μl ethanol: phenyl isothiocyanate (CNW): water: triethylamine (national drug group) (7:1:1:1), ready to use and ready to prepare. Adding 0.45mL of mobile phase A, mixing, and filtering with 0.45 μm filter membrane for use.

1.3 content determination

Chromatographic column: c (C) ₁₈ SHISEIDO(4.6mm*250mm*5μm)

Sample injection amount: 10 mu L

Column temperature: 40 DEG C

Mobile phase: mobile phase a: anhydrous sodium acetate (national drug group) 0.1 mol/L: acetonitrile (Merck) (97:3), after mixing, adjust pH to 6.5; mobile phase B: acetonitrile: water (80:20)

Eluting: gradient elution according to the following Table

TABLE 8

Detection wavelength: 254nm

Amino acid content calculation formula:

wherein: w-the content of each amino acid (mg/kg) in the sample; c-concentration of target in sample (mg/L); c (C) ₀ -target concentration (mg/L) in the blank; v-volume (mL); n-dilution factor; m-sample sampling amount (g).

2. Protein content determination (cf. GB 5009.5-2016)

2.1 preparation of methyl Red ethanol solution and bromocresol Green ethanol solution

Weighing 0.1g of methyl red (national drug group), and diluting to 100mL with 95% ethanol for later use; 0.1g of bromocresol green was weighed and diluted to 100mL with 95% ethanol for further use.

2.2 automatic Kjeldahl method

10g of the fermentation filtrate of example 1 and the fermentation filtrate of example 7 were weighed into a digestion tube. 0.4g of copper sulfate (national drug group), 6g of potassium sulfate (national drug group) and 12mL of sulfuric acid (national drug group) are added into a digestion furnace to digest, and when the temperature of the digestion furnace reaches 420 ℃, the liquid in the digestion pipe is blue-green. After the mixture is clear and transparent, digestion is continued for 1 hour. Taking out, cooling, adding 20mL of water, distilling on an automatic Kjeldahl nitrogen determination instrument (Jinan Hecan instruments Co., ltd., K9840) for 7min, and adding 1-2 drops of methyl red ethanol solution into a receiving bottle: bromocresol green ethanol solution (1:5), ready-to-use and ready-to-use. Then, 10mL of a 20g/L boric acid (national reagent) solution was added thereto, and the distillate was taken up to 200mL. Titration was performed with 0.1mol/L hydrochloric acid at the end of light grey-red color, while reagent blank was made.

Protein content calculation formula:

wherein: protein content in W-sample (g/100 g); v (V) ₁ -the volume of hydrochloric acid solution consumed (mL) in the sample; v (V) ₂ -reagent blank consuming hydrochloric acid solution volume (mL); v (V) ₃ -aspirating a volume of digestive juice (mL); c-hydrochloric acid solution concentration (mol/L); m-sample sampling amount (g); f-nitrogen conversion to protein coefficient (meter conversion coefficient 5.95).

3. Determination of Total Polyphenol content (reference T/AHFIA 005-2018)

3.1 preparation of standard curve

Gallic acid (Shanghai source leaf biotechnology Co., ltd.) solution: transferring 1mL, 2mL, 3mL, 4mL and 5mL of 100mg/L gallic acid standard stock solution into a 100mL volumetric flask, and shaking uniformly with water until the volume reaches the scale for standby.

Gallic acid solution 1mL was taken in a 10mL tube. 5mL of 10% Fu Lin Fen (national drug Co., ltd.) was added, and the mixture was shaken and reacted for 5min. Adding 4mL of 75% sodium carbonate solution (national medicine group), adding water to a certain volume to scale, shaking uniformly, and standing for 60min. The absorbance was measured with a cuvette at 760nm wavelength using an ultraviolet spectrophotometer (Agilent 8453), and a standard curve was drawn.

3.2 sample measurement

10g of the fermentation filtrate of example 1 and the fermentation filtrate of example 7 are taken in a centrifuge tube, 5mL of 60% ethanol solution preheated at 70 ℃ is added, the mixture is stirred uniformly, and the mixture is put at 70 ℃ for leaching for 10min. Taking out, cooling to room temperature, centrifuging at 4000rpm for 10min, collecting supernatant, adding into 10mL volumetric flask, repeating the steps, mixing the extractive solutions, filtering with 0.45 μm filter membrane, and measuring total polyphenol content.

The total polyphenol content calculation formula:

wherein: w-total polyphenol content in sample (g/100 g); a-absorbance of the sample solution; a is that ₀ -blank control solution absorbance; v-volume (mL); n-dilution factor; k-gallic acid standard curve slope; m-sample sampling amount (g).

4. Determination of total sugar and monosaccharide content (cf. GB/T15672-2009)

4.1 determination of total sugar content

4.1.1 Standard Curve preparation

Glucose (Shanghai Source leaf Biotech Co.) solution: remove 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL of 100mg/L glucose standard stock solution in a 10mL stoppered tube, and volume to 1mL with water. 1mL of 5% phenol (national drug Co.) and 5mL of concentrated sulfuric acid (national drug Co.) were added, shaken well, and left to stand for 10min. The tube was then placed at 30℃for 20min. Absorbance was measured at 490nm using a cuvette and a standard curve was drawn.

4.1.2 sample measurement

10g of the sample was taken in a 250mL conical flask, 50mL of water and 15mL of concentrated hydrochloric acid (Lyyang Kande chemical Co., ltd.) were added, and the mixture was subjected to hydrolysis at 100℃for 3 hours. After cooling to room temperature, filtration was carried out, the volume was fixed with water to 250mL, and the total sugar content was measured as described above.

The total sugar content calculation formula:

wherein: w-total sugar content in sample (g/100 g); m is m ₁ -glucose mass (mg) in the sample; v (V) ₁ -total sample volume (mL); v (V) ₂ -solution volume at measurement (mL); m-sample sampling amount (g).

4.2 monosaccharide content determination

4.2.1 sample pretreatment

The fermentation filtrate of example 1 and the fermentation filtrate of example 7 were taken in 2g in a hydrolysis tube, 1mL of water and 1mL of 4mol/L of trifluoro acid (Guogui reagent) were added, and the mixture was hydrolyzed in a drying oven (Test 101-OAB) at 110℃for 5 hours. After removal, the mixture was cooled to room temperature, and 0.1mL of the hydrolysate was dried in a 4mL centrifuge tube at 60℃in a drying oven for 2 hours. 0.05mL of a 0.3mol/L sodium hydroxide solution (Guogui) and 0.05mL of a 1-phenyl-3-methyl-5-pyrazolone (Alasdine PMP) methanol solution were added and reacted at 70℃for 60 minutes. After removal, the mixture was cooled to room temperature, and 0.05mL of 0.3mol/L hydrochloric acid, 0.75mL of water and 1.5mL of chloroform (ACS) were added thereto, and the mixture was shaken, allowed to stand and separated into layers. The upper layer solution is taken and repeatedly filtered by a filter membrane with the thickness of 0.45 mu m for standby.

4.2.2 content determination

Chromatographic column: c (C) ₁₈ Agilent(4.6mm*250mm*5μm)

Sample injection amount: 10 mu L

Column temperature: 25 DEG C

Flow rate: 1.0mL/min

Mobile phase: 0.1mol/L potassium dihydrogen phosphate (pH adjusted to 6.8): acetonitrile (82:18)

Detection wavelength: 254nm

Glucose content calculation formula:

wherein: w-monosaccharide content in the sample (mg/kg); c-monosaccharide concentration in the sample (mg/L); c (C) ₀ -monosaccharide concentration (mg/L) in the blank solution; v-volume (mL); n-dilution factor; m-sample sampling amount (g).

5. Gamma-aminobutyric acid content determination (cf. QB/T4587-2013)

5.1 sample pretreatment

10g of the fermentation filtrate of example 1 and 10g of the fermentation filtrate of example 7 are taken, 25mL of 60% ethanol is added into the mixture for 3 hours in a water bath at 30 ℃, and the supernatant is collected and repeated once. The supernatants were combined and concentrated to an extract, and the extracts were fixed in a 5mL volumetric flask with 60% ethanol. Taking 120 mu L of sample, adding 600 mu L of phthalic aldehyde derivative solution (10 mg of phthalic aldehyde (CNW) dissolved in 0.5mL of methanol, adding 2mL of boric acid buffer solution (CNW) with the pH of 10.2 and 30 mu L of 2-mercaptoethanol (national medicine group) and uniformly mixing), and carrying out derivative for 10min for later use.

5.2 content determination

Chromatographic column: c (C) ₁₈ Agilent(4.6mm*250mm*5μm)

Sample injection amount: 10 mu L

Column temperature: 40 DEG C

Mobile phase: mobile phase a:25mmol/L sodium acetate (Guogui), pH was adjusted to 5.9 with 4% acetic acid; mobile phase B: acetonitrile

Eluting: gradient elution according to the following Table

TABLE 9

Detection wavelength: 332nm

Gamma-aminobutyric acid content calculation formula:

wherein: the content of gamma-aminobutyric acid (mg/kg) in the W-sample; c-concentration of target in sample (mg/L); c (C) ₀ -target concentration (mg/L) in the blank; v-volume (mL); n-dilution factor; m-sample sampling amount (g).

6.VB ₃ Content determination (reference GB 5009.89-2016)

6.1 sample pretreatment

Taking 2g of the fermentation filtrate of the example 1 and the fermentation filtrate of the example 7, putting the fermentation filtrate into a 50mL centrifuge tube, adding 25mL of water and 0.25g of amylase for enzymolysis for 30min, adjusting the pH to 1.7 and then adjusting the pH to 4.5. Ultrasonic extraction is carried out for 10min, and the volume is fixed to 50mL. Centrifuging, and filtering with 0.45 μm filter membrane.

6.2 content determination

Chromatographic column: c (C) ₁₈ SHISEIDO(4.6mm*250mm*5μm)

Sample injection amount: 10 mu L

Column temperature: 20 DEG C

Flow rate: 1.0mL/min

Mobile phase: dissolving 70mL of methanol, 20mL of isopropanol and 1g of sodium heptanesulfonate in 910mL of water, mixing, adjusting pH to 2.1 with perchloric acid, and filtering with 0.45 μm filter membrane

Detection wavelength: 261nm

VB ₃ The content calculation formula:

wherein: VB in W-sample ₃ Content (. Mu.g/kg); c-target concentration in sample (. Mu.g/L); c (C) ₀ Target concentration in blank control (μg/L); v-volume (mL); n-dilution factor; m-sample sampling amount (g).

7. Ceramide content determination

Experimental principle: the kit is used for measuring the level of the phytoceramide (Ceramide) in the specimen by using a double antibody sandwich method. Coating a microplate with a purified Ceramide antibody to prepare a solid-phase antibody, sequentially adding Ceramide into micropores coated with monoclonal antibody, combining with HRP-labeled Ceramide antibody to form an antibody-antigen-enzyme-labeled antibody complex, and thoroughly washing and then adding a substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme and to final yellow under the action of acid. The shade of the color and Ceramide in the sample were positively correlated. Absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader, and the concentration of Ceramide in the sample was calculated from a standard curve.

7.1 preparation of tissue homogenates

Tissue mass 0.2g was rinsed with PBS, dried, placed in a 5mL homogenization tube, and homogenized by adding 1.8mL of 0.05mol/L Tris-HCl to prepare a 10% homogenate. Centrifuging at 3000rpm for 15min, and collecting supernatant.

7.2 determination of ceramide content

Sample adding of standard substance: standard wells and sample wells were set, and 50 μl of different concentrations of standard was added to each standard well.

Sample adding: blank wells (blank control wells without sample and enzyme-labeled reagent, the rest steps are the same), and example wells are respectively provided. 40. Mu.L of sample diluent is added to the example wells of the enzyme-labeled coated plate, and 10. Mu.L of the fermentation filtrate of example 1 and the fermentation filtrate of example 7 are added respectively.

Adding enzyme: 100 mu L of enzyme-labeled reagent is added to each well, and blank wells are not added.

Incubation: sealing with sealing plate, and water-bathing at 37deg.C for 60min.

Preparing liquid: the 20-fold concentrated washing solution is diluted with water by 20-fold for later use.

Washing: carefully uncovering the sealing plate membrane, discarding the liquid, spin-drying, filling the washing liquid in each hole, standing for 30s, discarding, repeating for 5 times, and spin-drying.

Color development: 50 mu L of developer A solution and 50 mu L of developer B solution are added into each hole, and the mixture is uniformly mixed and developed for 15min at 37 ℃ in a dark place.

And (3) terminating: 50. Mu.L of stop solution was added to each well, at which time the solution turned blue to yellow.

And (3) measuring: the absorbance was measured at a wavelength of Kong Diaoling, 450nm, and the ceramide concentration in the test solution was calculated.

Ceramide content calculation formula:

wherein: ceramide content in W-sample (pg/mL); c-sample target concentration (ng/L); c (C) ₀ -target concentration (ng/L) in the blank; v-volume (mL); n-dilution factor; m-sample sampling amount (g).

Composition comparison study results:

table 10

Compared with the common Aspergillus oryzae fermentation filtrate, the red Aspergillus oryzae fermentation filtrate has the ceramide content lower than that of the common Aspergillus oryzae fermentation filtrate, and other detection results higher than that of the common Aspergillus oryzae fermentation filtrate. Wherein the glutamic acid content differs by at least a factor of 13; tyrosine content differs by 3.17 times; the total protein content differs by 18 times; the total sugar content differs by 4.5 times; glucose content differs by 33.58 times; the gamma-aminobutyric acid content differs by 66.05 times; VB (VB) ₃ The content is different by 117.4 times, thus, the fermentation of red aspergillus oryzae is considered to be almost not equivalent to the fermentation product of rice aspergillus oryzae.

The Aspergillus oryzae fermented filtrate can be used for preparing skin external preparation. The external preparation for skin is preferably a cosmetic composition such as a lotion, essence, cream, etc. The weight percentage of the external preparation for skin is 0.625% -20% (w/w). Preferably 1.25% -10% (w/w) by weight. More preferably 1.25% to 5% (w/w) by weight. Most preferably, the weight percentage is 1.25% -3% (w/w).

The following are examples of specific applications of Aspergillus oryzae fermented filtrate in skin external preparations, and formulations and preparation methods of these dosage forms. In the following tables, "-" indicates no addition.

Application example 1: preparation of face cream

Application example 2: preparation of the emulsion

Application example 13: preparation of jelly

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Application example 14: preparation of toning lotion

Application example 15: preparation of essence

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Application example 6: preparation of facial mask

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Application example 7: preparation of eye cream

Application example 8: preparation of aerosol (cleaning foam)

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Application example 9: preparation of the spray

Application example 10: preparation of bath lotion

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Application example 11: preparation of facial cleanser

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Claims (12)

1. The use of Aspergillus oryzae fermented filtrate for promoting collagen and/or elastin production and/or antioxidant is provided.

2. The use according to claim 1, wherein the collagen is selected from the group consisting of: type I collagen, type III collagen, or a combination thereof.

3. The use according to claim 1, wherein the volume concentration of the fermentation filtrate of aspergillus oryzae is at least 0.1%.

4. The use according to claim 3, wherein the volume concentration of the fermentation filtrate of aspergillus oryzae is at least 1%.

5. The application of Aspergillus oryzae fermented filtrate in preparing skin external preparation, medicine and health food with collagen and/or elastin generation promoting and/or antioxidant effects is provided.

6. The use according to claim 5, wherein the collagen is selected from the group consisting of: type I collagen, type III collagen, or a combination thereof.

7. The use according to claim 5, wherein the content of the fermentation filtrate of Aspergillus oryzae in the skin external preparation, the medicine and the health food is 0.001-20 wt%.

8. The use according to claim 7, wherein the content of the fermentation filtrate of aspergillus oryzae in the skin external preparation, the medicine and the health food is 0.005-10 wt%.

9. The use according to claim 7, wherein the content of the fermentation filtrate of aspergillus oryzae in the skin external preparation, the medicine and the health food is 0.01-5 wt%.

10. The use according to claim 5, wherein the external skin preparation is selected from the group consisting of: face cream, milky lotion, jelly, pack, face toilet, pack, aerosol cleansing foam, spray, body wash, or facial cleanser.

11. The use according to any one of claims 1 to 10, wherein the total sugar in the fermentation filtrate of aspergillus oryzae is greater than or equal to 9.9wt%, wherein the glucose content of the total sugar is greater than or equal to 40%.

12. The use according to any one of claims 1 to 10, wherein the total protein in the fermentation filtrate of aspergillus oryzae is not less than 0.27% by weight, wherein the peptide content of the total protein is not less than 80%.