CN114712297A - Herbal and pearl powder combined beautifying and skin brightening composition and preparation method thereof - Google Patents

Abstract

The application discloses a whitening and skin-brightening composition combining herbaceous plants and pearl powder and a preparation method thereof. The beauty composition containing one component is prepared by using pearl powder, ginseng root extract, aloe extract and nicotinamide as raw materials, and can enable skin to be glossy, clear and transparent, enable skin to be bright and shiny, enable skin to be glossy, clear and transparent and moisten and smooth skin; can also make the skin tender and bright, and has good market application prospect.

Description

Herbal and pearl powder combined beautifying and skin brightening composition and preparation method thereof

Technical Field

The application relates to the technical field of aloe, in particular to a herbal and pearl powder combined beauty and skin brightening composition and a preparation method thereof.

Background

Aloe has powerful vitality and remarkable injury repairing ability, and its magical effect is derived from various functional components contained in it. The aloe contains more than 160 chemical components, wherein 70 kinds of nutrients (polysaccharide, amino acid and the like), 20 kinds of organic acids, 20 kinds of minerals, 30 kinds of alkanes and 10 kinds of biological enzymes. In addition, the water content in the fresh aloe leaves is about 95 percent of the weight of the fresh leaves, is original natural biological water, is also called as slippery water, and has important effects on the aspects of beauty treatment, health care, medical treatment and the like. Although different origins, varieties and planting conditions may cause the content of some chemical components in aloe to be different, the types of the chemical components are approximately the same. Over 100 chemical compositions have been identified and used in research. The high content of aloe gel, aloe-emodin, aloe polysaccharide and barbaloin in various chemical components; in which, barbaloin is proved by modern pharmacology to specifically and obviously inhibit tyrosinase activity, has the effects of inhibiting the generation and deposition of skin melanin, and is a main component with whitening effect.

However, the existing aloe extracts have too low content of barbaloin, direct extraction of the barbaloin greatly promotes the production cost, and a single aloe extract has limited effects on whitening and brightening the skin.

Disclosure of Invention

In view of the above, the present application aims to provide a whitening and skin-brightening composition, which not only can increase the content of barbaloin in the composition, but also can better exert the whitening and skin-brightening effects through the combination of other herbs. The whitening and skin brightening composition can enable the skin to be glossy, clean and transparent, enables the skin to be bright and colorful, enables the skin to be glossy, clean and transparent, moistens and smoothens the skin, enables the skin to be tender and bright, and has good market application prospect.

In a first aspect, the embodiment of the application discloses a whitening and skin-brightening composition, which comprises 10-500 parts of the fermented extract, 10-200 parts of pearl powder and 10-100 parts of nicotinamide;

activating clostridium butyricum, inoculating the clostridium butyricum into a working seed culture solution, performing anaerobic culture to obtain a working seed solution, transferring the working seed solution into a fermentation culture medium, performing anaerobic culture to obtain a fermentation broth, and extracting the fermentation broth to obtain the fermentation extract;

the working seed culture solution comprises 1.25-8.5 g/L of aloe extract and 1.25-4.5 g/L of ginseng root extract, and the fermentation culture solution comprises 1.25-8.5 g/L of aloe extract, 1.25-4.5 g/L of ginseng root extract, 0.1-0.22 g/L of pearl powder and 0.02-0.12 g/L of nicotinamide.

In the embodiment of the application, the fermentation extract comprises 23.7-50.58 mg/g glycoprotein, 7.48-14.57 mg/g barbaloin, 41.49-58.19 mg/g soluble sugar, 7.72-10.98 mg/g ginsenoside Rg1, 5.46-6.95 mg/g ginsenoside Re, 3.39-5.41 mg/g ginsenoside Rb1, 4.06-5.05 mg/g ginsenoside Rb2, 2.8-3.46 ginsenoside Rc and 2.48-2.85 mg/g ginsenoside Rd.

In an embodiment of the present application, the whitening and skin-lightening composition further comprises at least one of a humectant, an emollient, a thickener, a preservative, and a fragrance; the addition amount of the humectant is 0.001-3 wt% based on the total mass of the composition; the addition amount of the thickening agent is 0.001-0.5%; the addition amount of the emollient is 0.001-0.5 wt%; the addition amount of the preservative is 0.001-1.5 wt%.

In embodiments herein, the humectant is selected from the group consisting of at least one of isopropyl alcohol, propylene glycol, butylene glycol, panthenol, glycerin, and glyceryl polyether-26; such emollients include, but are not limited to, olive oil, macadamia nut oil, sweet almond oil, grape seed oil, avocado oil, corn oil, sesame oil, soybean oil, peanut oil, meadowfoam seed oil, safflower seed oil, rosa canina oil, argan nut oil, jojoba seed oil, sunflower seed oil, vetiver oil, cantharidine, ethylhexyl palmitate, isopropyl myristate, hydrogenated polyisobutene, isohexadecane, isododecane, diethylhexyl carbonate, dioctyl carbonate, isopropyl lauroyl sarcosinate, isononyl isononanoate, hydrogenated polydecene, tris (ethylhexanoate), cetyl ethylhexanoate, bis-diethoxydiol cyclohexane 1,4 dicarboxylate, caprylic/capric glycerol triacetate, oleyl erucate, octyldodecanol phaseomannate, octyldodecanol, dimethicone, octylmethicone, polymethylsiloxane, hydrogenated polyethylene glycol, and mixtures thereof, At least one of cetyl dimethicone, cyclopentadimethicone, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, santalol, pelargonic alcohol, lauric acid, carnosic acid, palmitin acid, stearic acid, beeswax, candelilla wax, carnauba wax, lanolin, ozokerite, jojoba seed wax, paraffin, microcrystalline wax, hydrogenated rice bran wax, hydrogenated cocoglycerides, glycerol behenate/eicosanoate, carnosol phaseolalate, bis-diglycerol polyacyladipate-2, shea butter, and murumuru palm seed fat; the thickening agent comprises at least one of acrylic acid (ester) and derivatives thereof, xanthan gum, Arabic gum, polyethylene glycol-14M, polyethylene glycol-90M, succinoglycan, hydroxyethyl cellulose, hydroxypropyl cellulose and hydroxypropyl methyl cellulose; the antiseptic comprises at least one of methyl hydroxybenzoate, propyl hydroxybenzoate, phenoxyethanol, benzyl alcohol, phenethyl alcohol, bis (hydroxymethyl) imidazolidinyl, potassium sorbate, sodium benzoate, chlorphenesin, sodium dehydroacetate, phthalein fatty acid, 1, 2-hexanediol, 1, 2-pentanediol, p-hydroxyacetophenone, octaethylene glycol, caprylic glyceride, undecylenic acid glycerol acetate, sorbitan caprylate, and ethylhexyl glycerol.

In a second aspect, the present application discloses a preparation method of the whitening and skin-brightening composition of the first aspect, which includes the steps of mixing and homogenizing the combination according to the formula amount; wherein the preparation method of the fermentation extract comprises the following steps:

obtaining activated clostridium butyricum bacterial liquid;

preparing a primary seed solution, wherein the primary seed solution is obtained by coating the activated clostridium butyricum bacterial solution on a solid culture medium for anaerobic culture, and then transferring bacterial colonies on a flat plate to the primary seed culture solution for anaerobic culture;

preparing a working seed solution, wherein the working seed solution is obtained by inoculating the primary seed solution into a working seed culture solution for anaerobic culture; the working seed culture solution contains 1.25-8.5 g/L of aloe extract and 1.25-4.5 g/L of ginseng root extract;

preparing fermentation liquor, wherein the fermentation liquor is obtained by inoculating the working seed solution into a fermentation culture solution for anaerobic culture; the fermentation culture solution contains 1.25-8.5 g/L of aloe extract, 1.25-4.5 g/L of ginseng root extract, 0.1-0.22 g/L of pearl powder and 0.02-0.12 g/L of nicotinamide; and

a step of preparing the fermented extract.

In the examples of the present application, the working seed culture solution comprises 125-8.5 g/L aloe extract, 1.25-4.5 g/L ginseng root extract, 2-3 g/L yeast extract, 7-10 g/L beef extract, 6-10 g/L tryptone, 1-5 g/L glucose, 1-5 g/L NaCl, 1-3 g/L CH₃COONa·3H₂O and 0.1-0.5 g/L L-cysteine hydrochloride.

In the embodiment of the application, the fermentation culture solution contains 2-3 g/L of yeast extract, 7-10 g/L of beef extract, 6-10 g/L of peptone trypsin, 2-5 g/L of glucose, 1.25-8 g/L of aloe extract, 1.25-4.5 g/L of ginseng root extract, 1-5 g/L of NaCL and 1-3 g/L of CH₃COONa·3H₂O, 0.1-0.5 g/L L-cysteine hydrochloride, 0.1-0.22 g/L pearl powder and 0.02-0.12 g/L nicotinamide.

In the examples of the present application, the step of preparing the fermentation extract specifically comprises:

performing pulsed strong light irradiation treatment and/or ozone treatment on the fermentation liquor to inactivate; and

extracting the inactivated fermentation broth;

wherein, the conditions of the irradiation treatment of the pulsed strong light are as follows: adopting UV pulse strong light source to treat, setting irradiation distance of 5cm, fixed flash irradiation frequency of 5 times/s, single irradiation dose of 3.6J/cm² _,Irradiating for 3 times or more with single irradiation time of 40 s;

the ozone treatment condition is that the concentration of ozone in the fermentation liquor is 4mg/L, and the treatment time is 5 min.

In the embodiment of the present application, the step of extracting the inactivated fermentation broth specifically includes:

collecting fermentation liquor with inactivation rate of more than 99%, standing at 4 deg.C for 12 hr, centrifuging at 8000rpm for 10min, collecting precipitate, suspending in distilled water, salting out with 75% saturated ammonium sulfate solution, standing at 4 deg.C for 12 hr, centrifuging at 8000rpm for 10min at 4 deg.C, collecting precipitate, and freeze drying to obtain the fermented extract.

In the embodiment of the present application, the step of extracting the inactivated fermentation broth specifically includes:

collecting fermentation broth with inactivation rate over 99%, adding PEG6000 to make its concentration be 25 wt%, and adding Na₂SO₄Adding KCl to make it 6%, standing at 4 deg.C for 10h, centrifuging at 8000r/min for 10min to accelerate phase separation, collecting precipitate, and freeze drying to obtain the fermented extract.

Compared with the prior art, the application has at least the following beneficial effects:

the beauty composition containing the components is prepared by using the pearl powder, the ginseng root extract, the aloe extract and the nicotinamide as raw materials, and can enable the skin to be glossy, clear and transparent, enable the skin to be bright and shiny, enable the skin to be glossy, clear and transparent, and moisten and smooth skin; can also make skin tender and bright, and has good market application prospect.

Drawings

FIG. 1 is a typical HPLC chromatogram for detecting ginsenoside in fermented extract provided in the examples of this application.

FIG. 2 is a typical HPLC profile for detecting aloin in fermented extracts as provided in the examples of the present application.

FIG. 3 is a graph showing HE staining of skin tissue of rats in a typical normal group, which is provided in the animal experiment of the present application.

FIG. 4 is a graph showing HE staining of rat skin tissue in a typical model group provided in the animal experiments of the present application.

FIG. 5 is a graph of HE staining of rat skin tissue of a typical positive control group provided in the animal experiments of the present application.

FIG. 6 is a graph of HE staining of skin tissue of rats in a typical intervention group provided in the animal experiments of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. Reagents not individually specified in detail in this application are conventional and commercially available; methods not specifically described in detail are all routine experimental methods and are known from the prior art.

Preparation of aloe extract, ginseng root extract and fermented extract

Materials and methods

1. Preparation of aloe extract

Cleaning aloe leaf, peeling, homogenizing pulp for 5min, filtering to obtain filtrate, centrifuging at 8000rpm for 10min at low temperature, slowly adding 2 times volume of cold acetone solution at-20 deg.C into supernatant, mixing, sealing, treating in ice bath for 5 hr, centrifuging at 8000rpm, and collecting precipitate;

after heavy suspension precipitation is carried out by PBS (PBS) with pH value of 6.8, pectinase (CAS:9032-75-1, Shanghai Michelle chemical technology Co., Ltd.) is added according to the concentration of 15-50 mg/L, enzymolysis is carried out for 45-60 min under the condition of water bath at 45.0 ℃, then stewing is carried out for 10-15 min at 100-105 ℃, enzyme deactivation treatment is carried out, low-temperature centrifugation is carried out for 10min at 8000rpm, supernate is taken out, salting-out treatment is carried out by 75% saturated ammonium sulfate solution, standing is carried out for 12h at 4 ℃, centrifugation is carried out for 10min at 4 ℃ at 8000rpm, then precipitation is collected, and freeze drying is carried out, thus obtaining the aloe extract.

2. Preparation of ginseng root extract

Pulverizing Ginseng radix (Yilu Tang commercial Co., Ltd. of Jilin), sieving with 80 mesh sieve, mixing with 10 times of water, ultrasonic extracting in 40 deg.C water bath for 2 hr, centrifuging the extractive solution, filtering, mixing the residue with 10 times of water, heating in water bath to 100 deg.C, extracting for 1.5 hr, ultrafiltering and concentrating the extractive solution, and lyophilizing the concentrated solution to obtain Ginseng radix extract.

3. Fermentation of

The fermentation process of a specific example 1 was carried out as follows:

(1) bacterial strains

Clostridium butyricum, GIM1.676, beijing borrelim bathing biotechnology limited.

(2) Strain activation

Absorbing 1mL of clostridium butyricum strain preserved in a glycerol tube at the temperature of-80 ℃, inoculating the clostridium butyricum strain into an activated culture solution, and placing the clostridium butyricum strain in an anaerobic incubator for 24 hours at the temperature of 37 ℃. Wherein the formula of the activation culture solution comprises 10g/L of peptone, 10g/L of beef extract, 5g/L of glucose, 5g/L of sodium chloride, 1g/L of soluble starch and 0.5g/L of sodium acetate 3g/L, L-cysteine hydrochloride, the pH value is 7.0 +/-0.1, and the sterilization is carried out for 20min at 121 ℃.

(3) Preparation of Primary seed liquid

And (3) coating the activating solution cultured for 24 hours on a solid culture medium, carrying out anaerobic culture at 37 ℃ for 28 hours, transferring a larger bacterial colony again, inoculating the bacterial colony into a primary seed culture solution after obtaining a stable bacterial colony, and carrying out anaerobic culture at 37 ℃ for 24 hours to obtain the primary seed solution.

Wherein the solid medium comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose and 1g/L, NaCL 5g/L, CH soluble starch₃COONa·3H₂O3g/L, L-cysteine hydrochloride 0.5g/L, 0.5% methylene blue, agar 6g, adjusting pH to 7.1 + -0.1, and sterilizing at 121 deg.C for 20 min. The primary seed broth was formulated approximately the same as the solid medium, except that there was no methylene blue or agar.

(4) Preparation of working seed liquid

Inoculating the prepared primary seed solution into a 250mL culture bottle filled with 120mL of working seed culture solution according to the volume ratio of 3%, and culturing in an anaerobic incubator at 37 ℃ for 48h to obtain the working seed solution.

Wherein, the working seed culture solution comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose, 1.25g/L aloe extract, 1.25g/L ginseng root extract and 5g/L, CH NaCL₃COONa·3H₂O3g/L and L-cysteine hydrochloride 0.5g/L, adjusting pH to 7.1 +/-0.1, and sterilizing at 121 ℃ for 20min for later use. Wherein the aloe extract and the fermented extract are prepared by the above method.

(5) Preparation of fermentation broth

The method comprises the steps of filling 7L of fermentation medium into a 10L full-automatic stainless steel fermentation system (Jiangsu Beilang biological engineering equipment Co., Ltd.), cooling after sterilization, inoculating 210mL of working seed liquid into the fermentation medium, covering 2cm of paraffin oil into the inoculated culture liquid in an aseptic manner, continuously introducing nitrogen to maintain an anaerobic or low-oxygen state, controlling the temperature to be 37 ℃ and carrying out anaerobic culture for 30h at the pH value of 6.8-7.5, sampling and carrying out microscopic examination every 2h, observing the spore formation condition, and stopping fermentation if the spore formation rate is over 90%. And counting spores to determine the optimal anaerobic culture mode.

Wherein, the fermentation medium comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose, 1.25g/L aloe extract, 1.25g/L ginseng root extract and 5g/L, CH NaCL₃COONa·3H₂O3g/L, L-cysteine hydrochloride 0.5g/L, Margarita powder 0.22g/L, nicotinamide 0.12g/L, adjusting pH to 7.1 + -0.1, and sterilizing at 121 deg.C for 20 min. Wherein the pearl powder has a product number ZX-HZP-125, Xian ZXun Biotech Co. Nicotinamide, CAS, 98-92-0, Hangzhou platinum only Biotech, Inc.

The fermentation process of a specific example 2 is as follows: steps (1) to (3) and (5) are the same as in example 1; the seed culture medium used in step (4) comprises: 3g/L yeast extract, 7.5g/L beef extract, 8g/L tryptone, 3.5g/L glucose, 6.5g/L aloe extract and 2.5g/L, NaCL 5g/L, CH fermentation extract₃COONa·3H₂O3g/L and L-cysteine hydrochloride 0.5g/L, adjusting pH to 7.1 +/-0.1, and sterilizing at 121 ℃ for 20min for later use.

The fermentation process of a specific example 3 is as follows: steps (1) to (3) and (5) are the same as in example 1; the seed culture medium used in step (4) comprises: 2.2g/L yeast extract, 7.5g/L beef extract, 6.5g/L tryptone, 2.5g/L glucose, 8.5g/L aloe extract, 4.5g/L, NaCL 5g/L, CH fermented extract₃COONa·3H₂O3g/L and L-cysteine hydrochloride 0.5g/L, adjusting pH to 7.1 +/-0.1, and sterilizing at 121 ℃ for 20min for later use.

The fermentation process of a specific example 4 is as follows: steps (1) to (4) are the same as in example 1; the fermentation medium used in step (5) comprises: 3g/L yeast extract, 7.5g/L beef extract, 8g/L tryptone, 3.5g/L glucose, 6.5g/L aloe extract and 2.5g/L, NaCL 5g/L, CH fermentation extract₃COONa·3H₂O3g/L, L-cysteine hydrochloride 0.5g/L, pearl powder 0.22g/L, nicotinamide 0.12g/L, adjusting pH to 7.1 + -0.1, and sterilizing at 121 deg.C for 20 min.

The fermentation process of a specific example 5 was carried out as follows: the steps (1) to (4) are the same as in example 1; the fermentation medium used in step (5) comprises: 2.2g/L yeast extract, 7.5g/L beef extract, 6.5g/L tryptone, 2.5g/L glucose, 8.5g/L aloe extract and 4.5g/L, NaCL 5g/L, CH fermented extract₃COONa·3H₂O3g/L, L-cysteine hydrochloride 0.5g/L, Margarita powder 0.22g/L, nicotinamide 0.12g/L, adjusting pH to 7.1 + -0.1, and sterilizing at 121 deg.C for 20 min.

One specific fermentation implementation of comparative example 1 was: the steps (1) to (3) and (5) are the same as in example 1; the seed culture medium used in step (4) comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L, NaCL 5g/L, CH glucose₃COONa·3H₂O3g/L and L-cysteine hydrochloride 0.5g/L, adjusting pH to 7.1 +/-0.1, and sterilizing at 121 ℃ for 20min for later use.

One specific fermentation implementation of comparative example 2 was: steps (1) to (3) and (5) are the same as in example 1; the working seed culture solution used in the step (4) comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose and 1.25g/L, NaCL 5g/L, CH aloe extract₃COONa·3H₂O3g/L and L-cysteine hydrochloride 0.5g/L, adjusting pH to 7.1 +/-0.1, and sterilizing at 121 ℃ for 20min for later use.

One specific example of the fermentation process of comparative example 3 was carried out as follows: steps (1) to (3) and (5) are the same as in example 1; the working seed culture solution used in the step (4) comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose, 1.25g/L ginseng root extract and 5g/L, CH NaCL₃COONa·3H₂O3g/L and L-cysteine hydrochloride 0.5g/L, adjusting pH to 7.1 +/-0.1, and sterilizing at 121 ℃ for 20min for later use.

One specific example of the fermentation process of comparative example 4 was carried out as follows: steps (1) to (3) and (5) are the same as in example 1; the working seed culture solution used in the step (4) comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose, 0.5g/L aloe extract and 0.5g/L, NaCL 5g/L, CH fermented extract₃COONa·3H₂O3g/L and 0.5 g/LL-cysteine hydrochloride, adjusting pH to 7.1 +/-0.1, and sterilizing at 121 ℃ for 20min for later use.

One specific fermentation implementation of comparative example 5 was: steps (1) to (3) and (5) are the same as in example 1; the working seed culture solution used in the step (4) comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose, 10g/L aloe extract and 6g/L, NaCL 5g/L, CH fermented extract₃COONa·3H₂O3g/L and L-cysteine hydrochloride 0.5g/L, adjusting pH to 7.1 +/-0.1, and sterilizing at 121 ℃ for 20min for later use.

One specific fermentation implementation of comparative example 6 was: steps (1) to (4) are the same as in example 1; the fermentation culture solution used in the step (5) comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose, 1.25g/L aloe extract, 1.25g/L ginseng root extract and 5g/L, CH NaCL₃COONa·3H₂O3g/L and 0.5 g/LL-cysteine hydrochloride, adjusting pH to 7.1 +/-0.1, and sterilizing at 121 ℃ for 20min for later use.

One specific fermentation implementation of comparative example 7 was: the steps (1) to (4) are the same as in example 1; the fermentation culture solution used in the step (5) comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose, 0.5g/L aloe extract and 0.5g/L, NaCL 5g/L, CH fermented extract₃COONa·3H₂O3g/L, L-cysteine hydrochloride 0.5g/L, Margarita powder 0.22g/L, nicotinamide 0.12g/L, adjusting pH to 7.1 + -0.1, and sterilizing at 121 deg.C for 20 min.

One specific example of the fermentation process of comparative example 8 was carried out as follows: steps (1) to (4) are the same as in example 1; the fermentation culture solution used in the step (5) comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose, 10g/L aloe extract and 6g/L, NaCL 5g/L, CH fermented extract₃COONa·3H₂O3g/L, L-cysteine hydrochloride 0.5g/L, pearl powder 0.22g/L, nicotinamide 0.12g/L, adjusting pH to 7.1 + -0.1, and sterilizing at 121 deg.C for 20 min.

4. Preparation of extract of fermentation broth

The extraction of the fermentation broth of example 1 was carried out as follows:

(1) pulsed strong light irradiation treatment of fermentation liquor

The method adopts a blue spectrum Rick UV pulse hi-lite instrument, Shenzhen, blue spectrum Rick science and technology Limited company, and the flash illumination frequency is set to be 1 time/s at minimum; the energy of the ultraviolet region accounts for 21.1 percent of the total energy, and the single pulse dose at a position 13cm away from the center of the lamp tube is 150mJ/cm²(ii) a Ultraviolet peak value of 247 nm; irradiance of 615.0W/cm²(ii) a Pulse width 234 ms.

Setting irradiation distance of 5cm, fixed flash irradiation frequency of 5 times/s, single irradiation dose of 3.6J/cm, irradiation distance of 5cm, fixed flash irradiation frequency of 5 times/s, and single irradiation dose of 3.6J/cm in a plurality of sterile empty culture dishes containing 2.0mL of fermentation liquor² _,The single irradiation time was 40 s. The irradiation treatment was performed 3 times or more in total.

The influence of the compound on the bacterial and bud embracing inactivation rate takes a group which is not subjected to the pulsed strong light irradiation treatment as a control group. The experiment was set up in 3 replicates and the results are reported as mean values.

(2) Measurement of leakage amount of intracellular Material

Respectively representing the leakage relative content of nucleic acid and protein by using the ultraviolet absorbance at the wavelength of 260nm and 280nm, and using sterile physiological saline as a reference solution;

and simultaneously, counting the bacterial colonies of the inactivated fermentation liquor by adopting a plate bacterial colony counting method, and calculating the inactivation rate (the total number of the bacterial colonies of the control sample-the total number of the bacterial colonies of the irradiated sample)/the total number of the bacterial colonies of the control sample multiplied by 100 percent according to the following formula.

(4) Secondary inactivation treatment

In order to ensure that the inactivation rate of the fermentation liquor reaches more than 99 percent, in the embodiment of the actual inactivation treatment of the fermentation liquor, ozone is introduced into the fermentation liquor subjected to the irradiation treatment, the concentration of the ozone in the fermentation liquor is ensured to be 4mg/L, the treatment time is 5min, and after the treatment is finished, the fermentation liquor is subjected to ultrasonic treatment at 35 ℃ to fully volatilize the dissolved ozone.

(5) Extraction process

Detecting the inactivation rate of the fermentation liquor after irradiation treatment and/or ozone treatment, taking the fermentation liquor with the inactivation rate of more than 99%, standing at 4 ℃ for 12h, collecting the precipitate after centrifugation at 8000rpm for 10min, suspending in distilled water, performing salting-out treatment by using 75% saturated ammonium sulfate solution, standing at 4 ℃ for 12h, collecting the precipitate after centrifugation at 4 ℃ at 8000rpm for 10min, and freeze-drying to obtain the fermentation extract.

The fermentation liquid extraction processes of examples 2 to 5 and comparative examples 1 to 8 were the same as those of example 1.

The fermentation broth extraction procedure of example 6 was as follows:

the steps (1) to (4) are the same as those in example 1, and step (5) includes: detecting the inactivation rate of the fermentation liquid subjected to irradiation treatment and/or ozone treatment, collecting the fermentation liquid with inactivation rate of more than 99%, adding 25% (by mass) PEG6000 (Qianuo Biotechnology Co., Ltd., Hangzhou), and adding Na₂SO₄The mass fraction of (2) is 6%, and the mass fraction of the added KCl is 1%. Standing at 4 deg.C for 10h, centrifuging at 8000r/min for 10min to accelerate phase separation, collecting precipitate, and freeze drying to obtain the fermented extract.

5. Analysis of glycoproteins

The glycoproteins of the fermentation extracts prepared in the above examples 1 to 6 and comparative examples 1 to 8 were analyzed by the following methods:

test solution: dissolving the fermented extract in 5mM phosphate buffer (KH) with pH8.0₂PO₄-Na₂HPO₄) And 5% by mass as a chromatography sample.

First chromatography: loading a sample solution to be tested to a DEAE-cellulose52 chromatographic column (Qingdao Bishui Hantian biological limited, 2.5 x 45cm), standing for 15min to allow glycoprotein to be fully adsorbed, performing step gradient elution by using a phosphate buffer solution containing NaCl with the concentration of 0-0.5 mol/L (the concentration of NaCl is 0mol/L, 0.lmol/L, 0.2mol/L, 0.3mo1/L, 0.4mo1/L and 0.5mo1/L respectively), performing flow rate lml/min and elution volume of 2-3 BV for each concentration, collecting eluent at an elution peak at 280nm, collecting 6mL fractions in each tube, detecting the agglutination activity of each tube by using a V-type coagulation plate method, collecting and combining fractions with the agglutination activity, dialyzing distilled water to remove salts, and freeze-drying to obtain the crude glycoprotein extract.

And (3) second chromatography: dissolving glycoprotein crude extract lyophilized powder in distilled water, loading on gel Sephadex G75(2.4 × 45cm) column, eluting with 5mM phosphate buffer solution with pH8.0 under the conditions of flow rate of 5mL/h and 3mL per tube, collecting eluate at 280nm, measuring agglutination activity of each tube, collecting and combining components with agglutination activity, dialyzing, and freezing supernatant into dry powder to obtain glycoprotein lyophilized powder.

6. Detection of glycoprotein freeze-dried powder agglutination titer

Preparation of erythrocyte suspension: adding 3.8% sodium citrate into mouse plasma for anticoagulation, and centrifuging at 2000rpm for 10 min. Absorbing upper plasma and white blood cells, taking precipitated cells, treating the cells for 1h at 37 ℃ by using 0.1% trypsin, removing non-specifically adsorbed plasma globulin on the surfaces of red blood cells, washing the cells for 3-5 times by using normal saline, centrifuging the cells for 5min at 2000rpm each time, and finally preparing the red blood cell precipitate into 2% red blood cell suspension by using the normal saline.

Measurement of agglutination titer: on a v-type plate, 25. mu.L of a hemocyte buffer (0.075 mo1/L phosphate buffer pH7.2 containing 0.075mo1/L sodium chloride) was added to each well, and 25. mu.L of a sample (concentration 2.5mg/mL) was added to the 1 st well, followed by dilution in duplicate and mixing. 25 μ L of 2% fresh blood cell suspension was added to each well, shaken, and allowed to stand for 1 to 2 hours, and the results were observed, wherein the maximum sample dilution factor capable of agglutinating erythrocytes was defined as the agglutination titer (T), and the agglutination activity (HA) was defined as the reciprocal (mg/ml) of the minimum lectin concentration required for 50% agglutination of erythrocytes. Relative activity (rHA) refers to the agglutination activity per mg of lectin (HA/mg).

7. Analysis of glycoprotein content by SDS-polyacrylamide gel electrophoresis

The concentration of the separation gel is 7 percent, the pH value is 8.9, the concentration of the concentration gel is 2.5 percent, the pH value is 6.7, the electrophoresis is carried out on a vertical plate, and the voltage is 100-200V. The sample was dissolved in sample buffer (containing 1% SDS, 5% thioethanol, 10% glycerol, 0.2% bromophenol blue), heated at 100 ℃ for 5min, stained with 10% trichloroacetic acid-1% Coomassie Brilliant blue R250, and destained with 10% trichloroacetic acid. And after the electrophoresis is finished, scanning the electrophoresis spectrogram by using an Epalyzer 2 type full-automatic electrophoresis apparatus, and calculating the content of the glycoprotein in the corresponding sample according to the scanning result.

8. Detection of ginsenoside and barbaloin in fermented extract

Test solution: dissolving the fermented extract with 5% vanillin glacial acetic acid solution, adding 4 times volume of perchloric acid (needing to be stored in dark place), mixing, treating in water bath at 60 deg.C for 15min, cooling to room temperature, adding glacial acetic acid 5% of the volume of the above mixture, shaking, and filtering with 0.45 μm filter membrane to obtain test solution.

Standard solution:

ginsenoside Re (CAS:52286-59-6, purity: HPLC > -98%, Nanjing Dierg science and technology Limited), Rg1(CAS:22427-39-0, purity: HPLC > -98%, Nanjing origin Biotechnology Limited), ginsenoside Rbl (CAS:41753-43-9, purity:, Wuhantian Biotechnology Limited), ginsenoside Rb2(CAS:11021-13-9, purity: 99%, Kazakhstan chemical), ginsenoside Rc (CAS:11021-14-0 purity: 98%, Beijing Bailingwei science and technology Limited), and ginsenoside Rd (CAS: 52rd-93-8, purity: 98%, Beijing Bailingwei science and technology Limited), were each 1mg/mL of standard solutions made with methanol and filtered with 0.45 μm filter.

HPLC chromatographic conditions: an Agilent ZORBAX Eclipse XDB-C18 column (250 mm. times.4.6 mm, 5 μm); the mobile phase A is acetonitrile, the B is water, the gradient elution program is 0 → 5min, 5% A; 5 → 25min, 5% → 40% a; 25 → 35min, 40% A → 5% A; the detection wavelength is 203 nm; the column temperature is 30 ℃; the flow rate was 1 mL/min.

Standard curve: sucking each standard substance solution, diluting according to the proportion of 2X, 4X, 6X and 8X, respectively detecting according to the HPLC chromatographic conditions, making a standard curve according to the corresponding relation of the characteristic peak area and the concentration, detecting the test substance solution by the same detection method, calculating the corresponding ginsenoside concentration in each test substance according to the characteristic peak area, and calculating the ginsenoside content in the fermented extract. FIG. 1 shows a typical HPLC chromatogram for detecting ginsenoside in a sample (fermentation extract).

9. Detection of barbaloin content

Test solution: refer to the above steps.

Standard solution: precisely weighing aloin standard substance, and dissolving with methanol to obtain 1mg/mL aloin standard substance.

HPLC chromatographic conditions: a chromatographic column: an Agilent ZORBAX Eclipse XDB-C18 column (250 mm. times.4.6 mm, 5 μm); the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid solution with the mass fraction of 0.05 percent; gradient elution program (volume fraction) 0 → 5min, 20% a; 5 → 30min, 20% A → 90% A; 30 → 31min, 90% A → 20% A; 31 → 35min, 20% A; the flow rate is 1.0 mL/min; the detection wavelength is 300 nm; the column temperature is 25 ℃; the injection volume is 10 muL. FIG. 2 shows the HPLC chromatogram for detecting aloin in a typical sample (fermentation extract).

10. Data processing

All test data are expressed as mean and standard deviation, processed using SPSS13.0 software, and subject to multiple comparisons and marked for significant differences.

Second, result in

TABLE 1mg/g

TABLE 2 mg/g

Tables 1 and 2 list the fermented extracts provided in examples 1 to 6 and comparative examples 1 to 8, and the contents of relevant active ingredients in the ginseng root extract and aloe extract. As can be seen from table 1, the contents of glycoproteins, barbaloins and soluble sugars in the fermented extracts provided in examples 1 to 6 were significantly increased relative to the aloe extract and the fermented extracts provided in comparative examples 1 to 8, respectively. In conjunction with the foregoing description of the "fermentation" process, the seed culture medium used in "preparing the working seed solution" of examples 1-6, and the fermentation culture medium used in "preparing the fermentation broth" of examples 1-6 are different from those of comparative examples 1-6, so that the content of glycoprotein, aloin, and soluble sugar in the fermentation extracts provided in examples 1-6 is significantly increased.

As can be seen from table 2, the contents of ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd in the fermented extracts provided in examples 1 to 6 were all significantly increased compared to the aloe extract and the fermented extracts provided in comparative examples 1 to 8, respectively. In conjunction with the foregoing description of the "fermentation" process, the seed culture medium used in "preparation of working seed liquid" in examples 1 to 6 and the fermentation culture medium used in "preparation of fermentation liquid" in examples 1 to 6 are different from those in comparative examples 1 to 6, so that the contents of ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd in the fermented extract provided in examples 1 to 6 are significantly increased.

Fermentation extract Performance test

To further clarify the effect of the content of glycoprotein, barbaloin, soluble sugar, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd in the fermented extract provided in the embodiments of the present application being significantly higher than that of the aloe extract or the ginseng extract, the embodiments of the present application also performed the following performance tests:

1. DPPH radical scavenging test

The antioxidant activity of the fermentation extracts provided in examples 1 to 6 and comparative examples 1 to 8, respectively, was evaluated by constructing DPPH radical scavenging reaction systems, as shown in table 3. Test solution: after each fermentation extract was dissolved in a phosphate buffer solution having a pH of 6.8, the resulting solution was diluted to a concentration of 0.5 wt% (mass percentage concentration) corresponding to the test concentration. The DPPH free radical scavenging reaction system is characterized in that liquid is added into three groups of A, B and C according to the table 4, after the liquid is added, the reaction is carried out for 20min at constant temperature in water bath at 25 ℃, and the absorbance values of A, B and C are measured at 517 nm. The DPPH clearance is [ (B + C) -A ]/BX100%, where A, B, C is the absorbance at 517nm after the reaction in the corresponding reaction tube.

Table 3(mL)

Reagent	A	B	C
				Test solution	1.5	－	1.5
0.2mM DPPH solution	1.5	1.5	－
				Anhydrous ethanol	－	1.5	1.5

2. Inhibition of tyrosinase assay

A tyrosinase catalysis reaction system is constructed to evaluate the inhibition effect of the fermentation extracts respectively provided in examples 1-6 and comparative examples 1-8 on tyrosinase.

Test solution: after each fermentation extract was dissolved in a phosphate buffer solution having a pH of 6.8, the resulting solution was diluted to a concentration of 0.5 wt% (mass percentage concentration) corresponding to the test concentration.

The tyrosinase catalysis reaction system is divided into A, B, C groups and D groups according to the table 4 for adding liquid, after the liquid is added, the reaction is carried out for 20min under the constant temperature water bath at 37 ℃, and the absorbance values of A, B, C and D are measured at 475 nm. The tyrosinase inhibition rate was [ (A-B) - (C-D) ]/(A-B) × 100%, where A, B, C, D is the absorbance at 475nm after the reaction in the reaction tube.

TABLE 4(μ L)

Reagent	A	B	C	D
					Test solution	－	－	400	400
500U/mL tyrosinase solution	250	－	250	－
					0.5 wt% L-tyrosine	400	400	400	400
phosphate buffer pH6.8	750	1000	350	600

3. Hyaluronidase inhibition assay

An Elson-Morgan method reaction system for inhibiting hyaluronidase in vitro is constructed to evaluate the inhibition effect of the fermentation extracts provided in examples 1-6 and comparative examples 1-8 on hyaluronidase. Test solution: after each fermentation extract was dissolved in an acetic acid buffer solution having a pH of 5.6, the solution was diluted to a concentration of 0.5 wt% (mass percentage concentration) corresponding to the test concentration.

The reaction system of the Elson-Morgan method for inhibiting hyaluronidase in vitro is divided into A, B, C and D four groups according to the table 5 and is added with liquid according to the following steps: keeping the temperature of the calcium chloride solution and the hyaluronidase solution at 37 ℃ for 20 min; adding a test sample, and keeping the temperature at 37 ℃ for 20 min; adding sodium hyaluronate solution, keeping the temperature at 37 deg.C for 30min, and standing at room temperature for 5 min; adding 0.4mol/L sodium hydroxide solution and acetylacetone solution, heating in boiling water bath for 15min, and immediately cooling with ice water for 5 min; adding Ellisib reagent and absolute ethyl alcohol for dilution, standing for 20min for color development, and measuring the absorbance value of the reaction system at 555nm by using a spectrophotometer. The hyaluronidase inhibition rate is [ (a-B) - (C-D) ]/(a-B) × 100%, where A, B, C, D is the absorbance at 555nm after the reaction in the reaction tube is completed.

TABLE 5(mL)

Reagent	A	B	C	D
					Test solution	－	－	0.5	0.5
500U/mL hyaluronidase solution	0.5	－	0.5	－
					0.25mM calcium chloride solution	0.1	0.1	0.1	0.1
0.25mM sodium hyaluronate solution	0.5	0.5	0.5	0.5
					0.4mol/L sodium hydroxide solution	0.1	0.1	0.1	0.1
Acetylacetone solution	0.5	0.5	0.5	0.5
					Ellisib reagent	1.0	1.0	1.0	1.0
Anhydrous ethanol	3.0	3.0	3.0	3.0
					Acetic acid buffer at pH 5.6	0.5	1.0	－	0.5

4. Results

TABLE 6 mg/g

Table 6 lists the DPPH free radical scavenging rate, tyrosinase inhibition rate, and hyaluronidase inhibition rate for the fermented extracts, the aloe extracts, and the ginseng root extracts provided in each of examples 1-6 and comparative examples 1-8, respectively. As can be seen from table 6, the DPPH free radical scavenging rate, the tyrosinase inhibition rate and the hyaluronidase inhibition rate of the fermented extracts provided in examples 1 to 6 are significantly higher than those of the fermented extracts, the aloe extracts and the ginseng root extracts provided in comparative examples 1 to 8, which indicates that the fermented extracts provided in the examples of the present application have better antioxidant ability, and the stronger inhibition of the tyrosinase inhibition rate suggests that the fermented extracts may have a stronger effect of inhibiting melanin deposition in vivo, and have the potential of developing whitening or skin-brightening cosmetics; meanwhile, hyaluronidase is an enzyme capable of reducing the activity of hyaluronic acid in vivo and thus improving the liquid permeability of tissues, and the fermented extract provided in examples 1 to 6 having a higher hyaluronidase inhibition rate means that the content of hyaluronic acid in the fermented extract can be up-regulated in vivo or in skin tissues, the moisture retention and brightness of the skin tissues can be improved, the young state of the skin tissues can be maintained, and the skin tissues can be helped to resist inflammation.

Animal experiments

In order to further confirm the efficacy of the fermented extracts provided in embodiments 1 to 6 of the present application in whitening and brightening skin, the embodiments of the present application further perform related animal experiments, and the experimental methods are as follows:

materials and methods

1. Laboratory animal

Wistar rats, female, with a weight of about 300g and an age of 9-12 weeks, purchased from Jiangsu Wukong Biotechnology Limited, maintained in a constant temperature and humidity environment during the experiment, were fed normally.

2. Test article

As can be seen from the foregoing, the present application discloses a composition for whitening and brightening skin, comprising the fermented extract prepared by the method described in the above examples 1 to 6.

Wherein the fermentation extract comprises 23.7-50.58 mg/g glycoprotein, 7.48-14.57 mg/g barbaloin, 41.49-58.19 mg/g soluble sugar, 7.72-10.98 mg/g ginsenoside Rg1, 5.46-6.95 mg/g ginsenoside Re, 3.39-5.41 mg/g ginsenoside Rb1, 4.06-5.05 mg/g ginsenoside Rb2, 2.8-3.46 ginsenoside Rc and 2.48-2.85 mg/g ginsenoside Rd.

The fermentation extract is obtained by activating clostridium butyricum, inoculating the clostridium butyricum into a working seed culture solution for anaerobic culture to obtain a working seed solution, transferring the working seed solution into a fermentation culture medium for anaerobic culture to obtain fermentation liquor, and extracting the fermentation liquor. Wherein the working seed culture solution contains 1.25-8.5 g/L of aloe extract and 1.25-4.5 g/L of ginseng root extract, and the fermentation culture medium contains 1.25-8.5 g/L of aloe extract, 1.25-4.5 g/L of ginseng root extract, 0.22g/L of pearl powder and 0.12g/L of nicotinamide.

In a further embodiment, the whitening and skin-brightening composition further comprises pearl powder and niacinamide. Specifically, the whitening and skin-brightening composition comprises, by weight, 10-500 parts of the fermented extract, 10-200 parts of pearl powder and 10-100 parts of nicotinamide.

In order to further apply the whitening and skin-brightening composition to the field of cosmetics, the composition provided by the embodiment of the application further comprises at least one of a humectant, an emollient, a thickener, a preservative and a fragrance; the addition amount of the humectant is 0.001-3 wt% based on the total mass of the composition; the addition amount of the thickening agent is 0.001-0.5%; the addition amount of the emollient is 0.001-0.5 wt%; the addition amount of the preservative is 0.001-1.5 wt%.

Wherein the humectant is selected from at least one of isopropyl alcohol, propylene glycol, butylene glycol, panthenol, glycerin and glyceryl polyether-26.

Wherein the emollient includes, but is not limited to, olive oil, macadamia nut oil, sweet almond oil, grape seed oil, avocado oil, corn oil, sesame oil, soybean oil, peanut oil, meadowfoam seed oil, safflower seed oil, rosa canina oil, argan nut oil, jojoba seed oil, sunflower seed oil, vetiver oil, cantharidine, ethylhexyl palmitate, isopropyl myristate, hydrogenated polyisobutene, isohexadecane, isododecane, diethylhexyl carbonate, dioctyl carbonate, isopropyl lauroyl sarcosinate, isononyl isononanoate, hydrogenated polydecene, tris (ethylhexanoate), cetyl ethylhexanoate, bis-diethoxydiol cyclohexane 1,4 dicarboxylate, caprylic/capric glycerol triacetate, oleyl erucate, octyldodecanol carnosine, octyldodecanol, polydimethylsiloxane, canola oil, and mixtures thereof, Octyl methicone, cetyl dimethicone, cyclopentadimethicone, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, santalol, turtle liver alcohol, lauric acid, carnosic acid, palmityl acid, stearic acid, beeswax, candelilla wax, carnauba wax, lanolin, ozokerite, jojoba seed wax, paraffin wax, microcrystalline wax, hydrogenated rice bran wax, hydrogenated coconut oil glycerides, glycerol behenate/eicosanoate, carnosic alcohol phaseolalate, bis-diglycerol polyacyladipate-2, shea butter, and mugwort palm seed fat.

Wherein the thickening agent comprises at least one of acrylic acid (ester) and derivatives thereof, xanthan gum, Arabic gum, polyethylene glycol-14M, polyethylene glycol-90M, succinoglycan, hydroxyethyl cellulose, hydroxypropyl cellulose and hydroxypropyl methyl cellulose.

Wherein the preservative comprises at least one of methylparaben, propylparaben, phenoxyethanol, benzyl alcohol, phenethyl alcohol, bis (hydroxymethyl) imidazolidinyl, potassium sorbate, sodium benzoate, chlorphenesin, sodium dehydroacetate, caprylic phthalate fatty acid, 1, 2-hexanediol, 1, 2-pentanediol, p-hydroxyacetophenone, caprylic glycol, glyceryl caprylate, undecylenic acid glyceryl acetate, sorbitan caprylate, and ethylhexyl glycerin.

Wherein the cosmetic formulation of the whitening and skin-brightening composition is at least one selected from the group consisting of cream, essence, lotion and brightening lotion.

In order to further use the whitening and skin-brightening composition provided in the examples of the present application as a specific application, the formulation thereof is shown in table 7, and it is prepared, mixed, homogenized, etc. according to the conventional cosmetic preparation method to prepare the final formulation.

TABLE 7

2. Establishment of aging model

Wistar rats were injected with 1000mg/kg of D-galactose subcutaneously in the neck and back every day, and were used as aging model rats after 7 weeks of continuous injection. It was divided into model group, positive control group and intervention group. The positive group is coated with 1-2 g of vitamin E in a back injection area of a rat every day, the intervention group is coated with 1-2 g of the whitening and skin-brightening compositions provided in examples 1-6 and comparative examples 1-10 in the back injection area of the rat every day, and after the positive group and the intervention group are continuously coated for 3 months, the detection of relevant indexes is carried out. Normal Wistar rats that were not molded were additionally set up as a normal group.

3. Determination of Water content of rat skin

After the rats of the model group, the positive control group and the intervention group are subjected to the last administration and the rats of the normal group, after hairs in a back injection area of the rats are removed by using depilatory cream, skin tissues of the back are taken to be about 1.5 multiplied by 1.5cm, the skin tissues are rinsed by normal saline, subcutaneous fat and the like are removed, and the rats are wiped dry by filter paper and weighed as wet weight. And then drying the mixture in a drying oven at 80 ℃ for 12 hours, weighing the dry weight and recording the dry weight, and calculating the water content. The moisture content of the skin is (skin wet weight-skin dry weight)/skin wet weight × 100%.

4. Detection of related preparations in rat skin tissue

After the moisture content of the skin is measured, adding 9 times of normal saline with the temperature of 4 ℃ by weight into the skin tissue of the liver, homogenizing for 3-5 min, centrifuging for 15min at 3000rpm, taking the supernatant, storing at the temperature of 4 ℃ for detection, and detecting the content of the supernatant by using a Hydroxyproline (HYP), Hyaluronic Acid (HA), superoxide dismutase (SOD), Malondialdehyde (MDA) and Lipofuscin (LF) H kit. Hydroxyproline kit, 50T/48S, BOXBIO. Hyaluronic acid kit, 48T/96T, Zheke biology. Superoxide dismutase kit, 48T/96T, ER0332, Finey organism. Malondialdehyde kit, 48T/96T, LE-B0424, Lele organism. Lipofuscin kit, 96T, an Orchio organism.

5. Rat skin tissue observation

Taking the middle 0.5cm of the back of the rat²Fixing the large and small skins in 4% paraformaldehyde for 12-24h, dehydrating, transparentizing, waxing, embedding, slicing with paraffin, and sticking to a glass slide. Dewaxing by xylene, gradually rehydrating with ethanol to water washing, staining by hematoxylin, washing with tap water, differentiating by hydrochloric acid and ethanol, soaking in tap water, staining by HE, dehydrating by conventional method, sealing with transparent neutral gum, and observing skin tissue of rats of each dosage group under an optical microscope.

6. Content detection of melanin and heme in rat skin

The content of melanin and heme in the skin is determined by measuring the amount of reflection from the skin by light of a specific wavelength. The specific test method comprises the following steps: the melanin and hemoglobin content of skin was measured using melanin and hemoglobin test probe MX 18 of the Conemeter MPA 580 skin tester.

7. Evaluation of skin Brightness in rat

The skin brightness L value of each group of rats is detected according to the method of the cosmetic contact dermatitis diagnostic standard and the treatment principle.

8. Data processing

All test data are expressed as mean and standard deviation, processed using SPSS13.0 software, and subject to multiple comparisons and marked for significant differences.

Second, result in

The skin tissues of the mice of each group were sectioned and HE stained, and micrographs were taken, as shown in FIG. 3, in which the normal control group rats had intact epidermal tissue structure, clear cell stratification, thick dermis, and densely arranged wavy collagen fibers were visible therein. The skin cells of the rats in the aging model group are not clearly layered, the dermis layer is obviously thinned, the collagen fibers are reduced, and the arrangement is loose. The dermis of the positive control group rat is not much different from that of the model group, and the collagen fiber is more and the arrangement is more compact. Compared with the aging model group, the collagen fibers of the dermis layer of the rat in the intervention group are obviously increased, the arrangement is compact, and the thickness of the dermis is obviously increased and is even close to that of the normal control group.

TABLE 8

Table 8 shows the HYP, HA, SOD, MDA and LF content in the skin tissue of rats in each experimental group. As can be seen from Table 8, compared with the normal group, the HYP, HA and SOD contents of the rats in the model group are all obviously increased, and the MDA and LF contents are all obviously increased, which indicates that the construction of the Wistar rat model of aging is successful.

In the intervention group, after the whitening and skin-brightening composition provided in examples 1-6 is applied to the skin of an injection region of a model rat, the contents of HYP, HA and SOD in skin tissues are obviously increased compared with those of the model group, and the difference between the contents of HYP, HA and SOD in the skin tissues is not large compared with that in a normal group; the contents of MDA and LF in skin tissues are remarkably reduced to be the same as those of normal compared with a model group, so that the whitening and skin-brightening composition provided by the embodiment of the application HAs the effect of obviously regulating the contents of HYP, HA, SOD, MDA and LF in skin tissues of rats in aging models. HYP is related to synthesis of skin collagen, HA can improve skin moisture retention and keep metabolism of skin cells normal and young, SOD can improve oxidation resistance of skin tissues, MDA can reduce lipid peroxide accumulation of the skin tissues, and LF content represents the deposition condition of pigments in the skin tissues.

In the intervention group, after the whitening and skin-brightening composition provided in the comparative examples 1-10 is smeared on the skin of an injection region of a model rat, HYP, HA and SOD in skin tissues are raised relative to the model group, but the difference of the HYP, HA and SOD in the skin tissues is still significant compared with that of a normal group; the contents of MDA and LF in skin tissues are both reduced relative to the model group, but the differences from the normal skin tissues are still significant, so that the whitening and skin-brightening compositions provided in comparative examples 1-10 have poor effects on the efficacy of model rats.

TABLE 9

Table 9 shows the water content, MI index and EI index in the skin tissue of rats in each experimental group, and the rate of change in L value with respect to the normal group. As can be seen from Table 9, the water content and EI index of the skin tissue of the model group rat are significantly reduced, the MI index is significantly increased, and the L value is significantly reduced, which indicates that the skin of the model group rat has reduced water content, dry skin and serious melanin deposition, and the reduced heme index indicates that the skin has insufficient heme and insufficient blood gas, so that the skin brightness is insufficient, and the L value is significantly reduced.

In the intervention group, after the whitening and skin-brightening composition provided by the embodiments 1-6 is smeared on the skin of an injection region of a model rat, the water content, EI index and L value in the skin tissue are obviously increased relative to the model group, and the difference between the moisture content, EI index and L value is not large compared with that of a normal group; the MI indexes in the skin tissues are obviously reduced to be the same as those of the normal MI indexes in comparison with those of the model group, so that the whitening and skin-brightening composition provided by the embodiment of the application obviously reduces melanin deposition in the skin tissues of the model rat, improves the heme content of the skin, enables the skin to be ruddy, promotes the water content, and further promotes the skin whitening and brightness increase.

In the intervention group, the whitening and skin-brightening compositions provided in comparative examples 1 to 10 have poor effects on the water content, melanin deposition, heme improvement and brightness promotion effects of model rats.

In summary, the embodiments of the present application use clostridium butyricum to ferment the aloe extract and the ginseng root extract, so as to obtain a fermented extract, which contains high content of glycoprotein, barbaloin, soluble sugar and various ginsenosides, contains various active ingredients in the aloe extract and the ginseng root extract, and has a content significantly higher than that of the aloe extract and the ginseng root extract. Performance tests prove that the fermentation extract has better antioxidant capacity, and the stronger inhibition rate of tyrosinase suggests that the fermentation extract has stronger effect of inhibiting melanin deposition in vivo and has the potential of developing whitening or skin-brightening cosmetics; meanwhile, the fermented extract has higher hyaluronidase inhibition rate, which means that the content of hyaluronic acid in the fermented extract can be up-regulated in vivo or in skin tissues, the moisture retention and brightness of the skin tissues are improved, the young state of the skin tissues is kept, and the skin tissues can resist inflammation to provide help.

Animal experiments show that the whitening and skin-brightening composition prepared from the fermented extract can promote the synthesis of collagen of an aging model rat, reduce the accumulation of lipid peroxides of skin tissues, improve the oxidation resistance of the composition, reduce the accumulation of pigments in the composition and improve the skin brightness; not only can resist skin aging, but also can whiten and lighten the skin.

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.

Claims (10)

1. A whitening and skin-brightening composition comprises 10-500 parts of the fermented extract, 10-200 parts of pearl powder and 10-100 parts of nicotinamide;

activating clostridium butyricum, inoculating the clostridium butyricum into a working seed culture solution, performing anaerobic culture to obtain a working seed solution, transferring the working seed solution into a fermentation culture medium, performing anaerobic culture to obtain fermentation liquor, and extracting the fermentation liquor to obtain the fermentation extract;

the working seed culture solution comprises 1.25-8.5 g/L of aloe extract and 1.25-4.5 g/L of ginseng root extract, and the fermentation culture solution comprises 1.25-8.5 g/L of aloe extract, 1.25-4.5 g/L of ginseng root extract, 0.1-0.22 g/L of pearl powder and 0.02-0.12 g/L of nicotinamide.

2. The whitening and skin-lightening composition of claim 1, wherein the fermented extract comprises 23.7-50.58 mg/g glycoprotein, 7.48-14.57 mg/g barbaloin, 41.49-58.19 mg/g soluble sugar, 7.72-10.98 mg/g ginsenoside Rg1, 5.46-6.95 mg/g ginsenoside Re, 3.39-5.41 mg/g ginsenoside Rb1, 4.06-5.05 mg/g ginsenoside Rb2, 2.8-3.46 ginsenoside Rc and 2.48-2.85 mg/g ginsenoside Rd.

3. The whitening and skin-lightening composition according to claim 1 or 2, further comprising at least one of a moisturizing agent, an emollient, a thickening agent, a preservative, and a fragrance; the addition amount of the humectant is 0.001-3 wt% based on the total mass of the composition; the addition amount of the thickening agent is 0.001-0.5%; the addition amount of the emollient is 0.001-0.5 wt%; the addition amount of the preservative is 0.001-1.5 wt%.

4. The whitening and skin-lightening composition of claim 3, wherein the humectant is selected from the group consisting of at least one of isopropyl alcohol, propylene glycol, butylene glycol, panthenol, glycerin, and glyceryl polyether-26;

such emollients include, but are not limited to, olive oil, macadamia nut oil, sweet almond oil, grape seed oil, avocado oil, corn oil, sesame oil, soybean oil, peanut oil, meadowfoam seed oil, safflower seed oil, rosa canina oil, argan nut oil, jojoba seed oil, sunflower seed oil, vetiver oil, cantharidine, ethylhexyl palmitate, isopropyl myristate, hydrogenated polyisobutene, isohexadecane, isododecane, diethylhexyl carbonate, dioctyl carbonate, isopropyl lauroyl sarcosinate, isononyl isononanoate, hydrogenated polydecene, tris (ethylhexanoate), cetyl ethylhexanoate, bis-diethoxydiol cyclohexane 1,4 dicarboxylate, caprylic/capric glycerol triacetate, oleyl erucate, octyldodecanol phaseomannate, octyldodecanol, dimethicone, octylmethicone, polymethylsiloxane, hydrogenated polyethylene glycol, and mixtures thereof, At least one of cetyl dimethicone, cyclopentadimethicone, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, santalol, pelargonic alcohol, lauric acid, carnosic acid, palmitin acid, stearic acid, beeswax, candelilla wax, carnauba wax, lanolin, ozokerite, jojoba seed wax, paraffin, microcrystalline wax, hydrogenated rice bran wax, hydrogenated cocoglycerides, glycerol behenate/eicosanoate, carnosol phaseolalate, bis-diglycerol polyacyladipate-2, shea butter, and murumuru palm seed fat;

the thickening agent comprises at least one of acrylic acid (ester) and derivatives thereof, xanthan gum, Arabic gum, polyethylene glycol-14M, polyethylene glycol-90M, succinoglycan, hydroxyethyl cellulose, hydroxypropyl cellulose and hydroxypropyl methyl cellulose;

the antiseptic comprises at least one of methyl hydroxybenzoate, propyl hydroxybenzoate, phenoxyethanol, benzyl alcohol, phenethyl alcohol, bis (hydroxymethyl) imidazolidinyl, potassium sorbate, sodium benzoate, chlorphenesin, sodium dehydroacetate, phthalein fatty acid, 1, 2-hexanediol, 1, 2-pentanediol, p-hydroxyacetophenone, octaethylene glycol, caprylic glyceride, undecylenic acid glycerol acetate, sorbitan caprylate, and ethylhexyl glycerol.

5. The method for preparing the whitening and skin-brightening composition according to any one of claims 1 to 4, which comprises the steps of mixing and homogenizing the combination according to the formula amount; wherein the preparation method of the fermentation extract comprises the following steps:

obtaining activated clostridium butyricum bacterial liquid;

preparing a primary seed solution, wherein the primary seed solution is obtained by coating the activated clostridium butyricum bacterial liquid on a solid culture medium for anaerobic culture, and then transferring bacterial colonies on a flat plate to the primary seed culture solution for anaerobic culture;

preparing a working seed solution, wherein the working seed solution is obtained by inoculating the primary seed solution into a working seed culture solution for anaerobic culture; the working seed culture solution contains 1.25-8.5 g/L of aloe extract and 1.25-4.5 g/L of ginseng root extract;

preparing fermentation liquor, wherein the fermentation liquor is obtained by inoculating the working seed solution into a fermentation culture solution for anaerobic culture; the fermentation culture solution contains 1.25-8.5 g/L of aloe extract, 1.25-4.5 g/L of ginseng root extract, 0.1-0.22 g/L of pearl powder and 0.02-0.12 g/L of nicotinamide; and

a step of preparing the fermented extract.

6. The method of claim 5, wherein the working seed culture fluid comprises 1.25-8.5 g/L aloe extract, 1.25-4.5 g/L ginseng root extract, 2-3 g/L yeast extract, 7-10 g/L beef extract, 6-10 g/L tryptone, 1-5 g/L glucose, 1-5 g/L NaCl, 1-3 g/L CH₃COONa·3H₂O and 0.1-0.5 g/L L-cysteine hydrochloride.

7. The preparation method according to claim 5, wherein the fermentation culture solution comprises 2-3 g/L yeast extract, 7-10 g/L beef extract, 6-10 g/L peptone trypsin, 2-5 g/L glucose, 1.25-8 g/L aloe extract, 1.25-4.5 g/L ginseng root extract, 1-5 g/L NaCL, and 1-3 g/L CH₃COONa·3H₂O, 0.1-0.5 g/L L-cysteine hydrochloride, 0.1-0.22 g/L pearl powder and 0.02-0.12 g/L nicotinamide.

8. The method of claim 5, wherein the step of preparing the fermentation extract comprises:

a step of inactivating the fermentation liquor by performing pulsed strong light irradiation treatment and/or ozone treatment; and

extracting the inactivated fermentation broth;

wherein, the conditions of the irradiation treatment of the pulsed strong light are as follows: adopting UV pulse strong light source to treat, setting irradiation distance of 5cm, fixed flash irradiation frequency of 5 times/s, single irradiation dose of 3.6J/cm^2,Irradiating for 3 times or more with single irradiation time of 40 s;

the ozone treatment condition is that the concentration of ozone in the fermentation liquor is 4mg/L, and the treatment time is 5 min.

9. The method according to claim 8, wherein the step of extracting the inactivated fermentation broth comprises:

collecting fermentation liquor with inactivation rate of more than 99%, standing at 4 deg.C for 12 hr, centrifuging at 8000rpm for 10min, collecting precipitate, suspending in distilled water, salting out with 75% saturated ammonium sulfate solution, standing at 4 deg.C for 12 hr, centrifuging at 4 deg.C and 8000rpm for 10min, collecting precipitate, and freeze drying to obtain the fermented extract.

10. The method according to claim 8, wherein the step of extracting the inactivated fermentation broth comprises:

collecting fermentation broth with inactivation rate over 99%, adding PEG6000 to make its concentration be 25 wt%, and adding Na₂SO₄Adding KCl to make it 6%, standing at 4 deg.C for 10h, centrifuging at 8000r/min for 10min to accelerate phase separation, collecting precipitate, and freeze drying to obtain the fermented extract.

Images