CN114712297B

CN114712297B - Herbal and pearl powder combined beautifying and skin brightening composition and preparation method thereof

Info

Publication number: CN114712297B
Application number: CN202210505536.3A
Authority: CN
Inventors: 贺英
Original assignee: Guangdong Hanrun Biotechnology Co ltd
Current assignee: Guangdong Hanrun Biotechnology Co ltd
Priority date: 2022-05-10
Filing date: 2022-05-10
Publication date: 2022-10-14
Anticipated expiration: 2042-05-10
Also published as: CN114712297A

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 colorful, enable 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.

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 a powerful vitality and a surprising ability to repair damage, the magical efficacy of which derives from the various functional ingredients it contains. The aloe contains more than 160 chemical components, wherein 70 nutrients (polysaccharides, amino acids and the like), 20 organic acids, 20 minerals, 30 alkanes and 10 biological enzymes. In addition, the water content in the fresh aloe leaves accounts for about 95 percent of the weight of the fresh leaves, is original natural biological water, is also called 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. More than 100 chemical compositions have been identified and used in research. Among the various chemical components, the higher content is aloe vera gel, aloe-emodin, aloe polysaccharides and barbaloin; in the composition, modern pharmacology proves that the barbaloin has obvious effects of inhibiting tyrosinase activity, inhibiting skin melanin generation and deposition, 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 invention aims to provide a skin whitening and brightening composition, which not only can increase the content of barbaloin in the composition, but also can better exert the skin whitening and 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 fermentation 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 solution, performing anaerobic culture to obtain a fermentation broth, and extracting the fermentation broth to obtain the fermentation extract;

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 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.

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 to 3 weight percent based on the total mass of the composition; the addition amount of the thickening agent is 0.001 to 0.5 percent; the addition amount of the emollient is 0.001 to 0.5 weight percent; 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; <xnotran> , , , , , , , , , , , , , , , , , , , , , , , , , , , ( ) , , - 1,4 , / , , , , , , , , , , , , , , , , , , , , , , , , , , , / , , - -2, ; </xnotran> 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 preservative comprises at least one of methylparaben, propylparaben, phenoxyethanol, benzyl alcohol, phenethyl alcohol, bis (hydroxymethyl) imidazolidinyl vein, potassium sorbate, sodium benzoate, chlorphenesin, sodium dehydroacetate, caprylyl caproic acid, 1, 2-hexanediol, 1, 2-pentanediol, p-hydroxyacetophenone, caprylic glycol, caprylic glyceride, glyceryl undecylenate, sorbitan caprylate and ethylhexyl glycerin.

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 to 8.5g/L of aloe extract, 1.25 to 4.5g/L of ginseng root extract, 0.1 to 0.22g/L of pearl powder and 0.02 to 0.12g/L of nicotinamide; and

a step of preparing the fermented extract.

In the embodiment of the application, the working seed culture solution contains 1.25-8.5 g/L of aloe extract, 1.25-4.5 g/L of ginseng root extract, 2-3 g/L of yeast extract, 7-10 g/L of beef extract, 6-10 g/L of tryptone, 1-5 g/L of glucose, 1-5 g/L of NaCl and 1-3 g/L of CH ₃ COONa·3H ₂ O and 0.1-0.5 g/L L-cysteine hydrochloride.

In the embodiment of the application, the fermentation culture solution comprises 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 CH ₃ COONa·3H ₂ O, 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:

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 for treatment, 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 ^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 5min.

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

collecting fermentation liquor with inactivation rate over 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.

collecting fermentation broth with inactivation rate over 99%, adding PEG6000 to make its concentration be 25wt%, and adding Na ₂ SO ₄ Making it to be 6%, adding KCl to make its concentration to be 1%, standing at 4 deg.C for 1 hr, 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 the skin tender and bright, and has good market application prospect.

Drawings

FIG. 1 is a typical HPLC chromatogram for the detection of ginsenosides in fermented extracts as provided in the examples of the present 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 of HE staining of skin tissue of rats in a typical normal group provided in the animal experiments 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 showing HE staining of rat skin tissue in a typical positive control group provided in the animal experiment of the present application.

FIG. 6 is a graph showing 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

1. 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, slowly adding 2 times volume of cold acetone solution of-20 deg.C into supernatant, mixing, sealing, placing in ice bath for 5h, centrifuging at 8000rpm, and collecting precipitate;

after the heavy suspension precipitation is carried out by PBS solution with pH =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 the mixture is cooked for 10-15 min at 100-105 ℃, enzyme deactivation treatment is carried out, low-temperature centrifugation is carried out for 10min at 8000rpm, supernatant is taken, salting-out treatment is carried out by 75% saturated ammonium sulfate solution, the mixture is placed for 12h at 4 ℃, centrifugation is carried out for 10min at 4 ℃ and 8000rpm, then the precipitate is collected and freeze-dried, and the aloe extract is obtained.

2. Preparation of ginseng root extract

Pulverizing Ginseng radix (Yilu Tang commercial and trade Co., ltd., 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, 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 bath biotechnology limited.

(2) Bacterial 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, 3g/L of sodium acetate and 0.5g/L of 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 activation solution cultured for 24 hours on a solid culture solution, carrying out anaerobic culture at 37 ℃ for 28 hours, transferring a larger bacterial colony again to obtain a stable bacterial colony, inoculating the bacterial colony into a primary seed culture solution, and carrying out anaerobic culture at 37 ℃ for 24 hours to obtain a primary seed solution.

Wherein, the solid culture solution comprises: 3g/L of yeast extract, 10g/L of beef extract, 10g/L of tryptone, 5g/L of glucose, 1g/L of soluble starch, 5g/L of NaCl and CH ₃ COONa·3H ₂ O3 g/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 20min. The primary seed culture solution and the solid culture solution have the same formula, and are differentOnly in the absence of methylene blue and 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 of yeast extract, 10g/L of beef extract, 10g/L of tryptone, 5g/L of glucose, 1.25g/L of aloe extract, 1.25g/L of ginseng root extract, 5g/L of NaCl and CH ₃ COONa·3H ₂ O3 g/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 culture solution into a 10L full-automatic stainless steel fermentation system (Jiangsu Beilang biological engineering equipment Co., ltd.), cooling after sterilization, inoculating 210mL of working seed solution into the fermentation culture solution, covering 2cm of paraffin oil in the inoculated culture solution 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 microscopic examination every 2h, observing the spore formation condition, and stopping fermentation if the spore formation rate is over 90%. And counting spores and determining the optimal anaerobic culture mode.

Wherein, the fermentation 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, 5g/L NaCl, CH ₃ COONa·3H ₂ O3 g/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 20min. 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 solution used in the step (4) comprises: yeast extract 3g/L, beef extract 7.5g/L, pancreatic egg8g/L of white peptone, 3.5g/L of glucose, 6.5g/L of aloe extract, 2.5g/L of ginseng root extract, 5g/L of NaCl, and CH ₃ COONa·3H ₂ O3 g/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 solution used in the 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 ginseng root extract, 5g/L NaCl, CH ₃ COONa·3H ₂ O3 g/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 culture solution used in the step (5) comprises: 3g/L yeast extract, 7.5g/L beef extract, 8g/L tryptone, 3.5g/L glucose, 6.5g/L aloe extract, 2.5g/L ginseng root extract, 5g/L NaCl, CH ₃ COONa·3H ₂ O3 g/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 20min.

The fermentation process of a specific example 5 is as follows: the steps (1) to (4) are the same as in example 1; the fermentation culture solution used in the 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, 4.5g/L ginseng root extract, 5g/L NaCl, CH ₃ COONa·3H ₂ O3 g/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 20min.

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 solution used in the step (4) comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose, 5g/L NaCl, CH ₃ COONa·3H ₂ O3 g/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 2 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 aloe extract, 5g/L NaCl, CH ₃ COONa·3H ₂ O3 g/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 3 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, 1.25g/L ginseng root extract, 5g/L NaCl, CH ₃ COONa·3H ₂ O3 g/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, 0.5g/L ginseng root extract, 5g/L NaCl, CH ₃ COONa·3H ₂ O3 g/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 example of the fermentation process of comparative example 5 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, 10g/L aloe extract, 6g/L ginseng root extract, 5g/L NaCl, CH ₃ COONa·3H ₂ O3 g/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: the steps (1) to (4) are the same as in example 1; the fermentation culture solution used in step (5) comprises: 3g/L of yeast extract, 10g/L of beef extract, 10g/L of tryptone, 5g/L of glucose, 1.25g/L of aloe extract, 1.25g/L of ginseng root extract, 5g/L of NaCl and CH ₃ COONa·3H ₂ O3 g/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 step (5) comprises: 3g/L of yeast extract, 10g/L of beef extract, 10g/L of tryptone, 5g/L of glucose, 0.5g/L of aloe extract, 0.5g/L of ginseng root extract, 5g/L of NaCl, and CH ₃ COONa·3H ₂ O3 g/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 20min.

One specific example of the fermentation process of comparative example 8 was carried out as follows: the steps (1) to (4) are the same as in example 1; the fermentation culture solution used in step (5) comprises: 3g/L yeast extract, 10g/L beef extract, 10g/L tryptone, 5g/L glucose, 10g/L aloe extract, 6g/L ginseng root extract, 5g/L NaCl, CH ₃ COONa·3H ₂ O3 g/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 20min.

4. Preparation of fermentation broth extract

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/cm ² (ii) a Ultraviolet peak value is 247nm; irradiance of 615.0W/cm ² (ii) a Pulse width 234ms.

Taking a plurality of sterile empty culture dishes containing 2.0mL of fermentation liquor, setting the irradiation distance to be 5cm, the fixed flash irradiation frequency to be 5 times/s and the single irradiation dose to be 3.6J/cm ² The single irradiation time was 40s. The irradiation treatment was performed 3 times or more in total.

The control group is the group which is not subjected to intensive pulse light irradiation treatment. 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 bacterial colonies of the control sample-the total bacterial colonies of the irradiated sample)/the total bacterial colonies of the control sample multiplied by 100% according to the following formula.

(4) Inactivating again

In order to ensure that the inactivation rate of the fermentation liquor reaches more than 99 percent, in the practical embodiment of inactivating 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, centrifuging at 8000rpm for 10min, collecting the precipitate, suspending in distilled water, salting out with 75% saturated ammonium sulfate solution, standing at 4 ℃ for 12h, centrifuging at 4 ℃ at 8000rpm for 10min, collecting the precipitate, 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 in 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 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 (Kyoho Biotechnology Co., ltd.), and adding Na ₂ SO ₄ The mass fraction of (2) is 6%, and the mass fraction of the added KCl is 1%. Standing at 4 deg.C for 10h, and 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 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) pH8.0 ₂ PO ₄ -Na ₂ HPO ₄ ) And the mass concentration is 5 percent so as to be used as a chromatography test sample.

First chromatography: loading the sample solution onto DEAE-cellulose52 chromatographic column (2.5 × 45cm, qingdao Bishui Hantian biological Co., ltd.), standing for 15min to allow glycoprotein to be fully adsorbed, performing stepwise gradient elution with phosphate buffer solution containing 0-0.5 mol/L NaCl (the NaCl concentrations are 0mol/L, 0.l mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L and 0.5 mol/L), flowing speed lml/min, the elution volume of each concentration is 2-3 BV, collecting eluent at 280nm elution peak, collecting 6mL fractions in each tube, detecting the agglutination activity of each tube by V-type blood coagulation plate method, collecting and combining fractions with agglutination activity, dialyzing distilled water to remove salt, and freeze-drying to obtain crude glycoprotein extract.

And (3) second chromatography: dissolving glycoprotein crude extract freeze-dried powder in distilled water, loading on a gel SephadexG75 (2.4X 45 cm) column, eluting with 5mM phosphate buffer solution with the pH of 8.0 under the conditions of flow rate of 5mL/h and 3mL per tube, collecting eluent at an elution peak at 280nm, measuring the agglutination activity of each tube, collecting and combining components with the agglutination activity, and freezing supernatant into dry powder after dialysis, namely the glycoprotein freeze-dried 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 10min. Absorbing the upper plasma and white blood cells, taking the precipitated cells, treating the cells for 1h at 37 ℃ by using 0.1% trypsin, removing the plasma globulin non-specifically adsorbed on the surfaces of the red blood cells, washing the cells for 3 to 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 mol/L phosphate buffer pH7.2 containing 0.075mol/L sodium chloride) was added to each well, and 25. Mu.L of a sample (concentration 2.5 mg/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, and after shaking, the mixture was left 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 lowest lectin concentration required for producing 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 separation gel is 7 percent, the pH value is 8.9, the concentration of concentration gel is 2.5 percent, the pH value is 6.7, 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 an electrophoresis spectrogram by using an Epalyzer2 type full-automatic electrophoresis apparatus, and calculating the content of the glycoprotein in the corresponding sample according to a scanning result.

8. Detection of ginsenosides and barbaloin in fermented extracts

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 medicine science and technology Limited), rg1 (CAS: 22427-39-0, purity: HPLC > =98%, nanjing origin plant biotechnology Limited), ginsenoside Rbl (CAS: 41753-43-9, purity: =98%, wuhan Tian plant biotechnology Limited), ginsenoside Rb2 (CAS: 11021-13-9, purity: 99%, kazakh chemistry), ginsenoside Rc (CAS: 11021-14-0 purity: 98%, beijing Bailingwei science and technology Limited), and ginsenoside Rd (CAS: 525293-8, purity: 705%, beijing Bailingwei science and technology Limited), were prepared into standard solutions of 1mg/mL with methanol, respectively, and filtered with 0.45 μm filter membrane.

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

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 procedure (volume fraction) 0 → 5min,20% A;5 → 30min, 20%; 30 → 31min,90% > -A → 20% >; 31 → 35min, 20% A; the flow rate is 1.0mL/min; the detection wavelength is 300nm; 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 multiple comparisons and marked for significant differences.

2. As a result, the

TABLE 1mg/g

Detailed description of the preferred embodiments	Glycoprotein content	Aloin (Bar)	Soluble sugar content
				Example 1	29.82±6.12bc	8.75±1.27c	46.76±5.27b
Example 2	31.54±5.25b	8.92±1.01bc	46.93±4.52b
				Example 3	32.02±4.72b	9.21±1.34c	47.03±6.11b
Example 4	35.15±3.16b	11.21±1.68b	49.51±4.64a
				Example 5	36.22±4.35b	11.69±1.34b	50.36±5.36a
Example 6	43.31±7.27a	13.14±1.43a	52.02±6.17a
				Comparative example 1	15.78±3.24d	4.84±1.35ef	37.04±4.22c
Comparative example 2	15.63±2.95d	4.91±1.18e	36.96±3.17c
				Comparative example 3	15.48±4.06d	4.63±1.24f	37.13±5.16c
Comparative example 4	16.21±3.78d	5.21±0.87e	37.84±3.23c
				Comparative example 5	16.36±4.02d	5.43±0.96e	37.91±3.52c
Comparative example 6	16.43±4.05d	5.62±0.56d	37.83±4.15c
				Comparative example 7	16.36±6.21d	5.78±0.72d	37.57±5.23c
Comparative example 8	16.52±7.07d	5.67±0.61d	37.62±3.11c
				Aloe extract	15.35±2.68	4.71±1.84f	36.82±5.76c

TABLE 2mg/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 the aloe extract. As can be seen from table 1, the content of glycoprotein, barbaloin and soluble sugar 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 solution used in "preparation of working seed solution" of examples 1-6, and the fermentation culture solution used in "preparation of fermentation broth" of examples 1-6 were 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 was 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 solution used in "preparation of working seed solution" of examples 1 to 6 and the fermentation culture solution used in "preparation of fermentation broth" of examples 1 to 6 were different from those of 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 extracts provided in examples 1 to 6 were 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 further perform 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 0.5wt% (mass percentage concentration) corresponding to the test concentration. The DPPH free radical scavenging reaction system is divided into three groups A, B and C according to the table 3, liquid is added, after the sample addition is finished, the reaction is carried out for 20min under the constant temperature of 25 ℃ water bath, and the light absorption value of each group A, B and C is measured at 517 nm. DPPH clearance = [ (B + C) -A ]/B x 100%, wherein A, B and C are absorbance values at 517nm after the reaction of the corresponding reaction tube is finished.

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

Tyrosinase catalytic reaction systems were constructed to evaluate the inhibitory effects of the fermentation extracts provided in examples 1 to 6 and comparative examples 1 to 8, respectively, 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 0.5wt% (mass percentage concentration) corresponding to the test concentration.

The tyrosinase catalysis reaction system is divided into four groups A, B, C and D according to the table 4 for adding liquid, after the sample adding is finished, the constant temperature reaction is carried out for 20min in water bath at 37 ℃, and the light absorption values of A, B, C and D are measured at 475 nm. Tyrosinase inhibition = [ (A-B) - (C-D) ]/(A-B) × 100%, where A, B, C, and D are absorbance values at 475nm after the reaction in the corresponding reaction tubes.

TABLE 4 (μ L)

Reagent	A	B	C	D
					Test solution	－	－	400	400
500U/mL tyrosinase solution	250	－	250	－
					0.5wt.% of L-tyrosine	400	400	400	400
phosphate buffer pH =6.8	750	1000	350	600

3. Hyaluronidase inhibition assay

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

The reaction system of the Elson-Morgan method for inhibiting hyaluronidase in vitro is divided into four groups A, B, C and D according to the table 5 to be added with liquid according to the following steps: keeping the temperature of the calcium chloride solution and the hyaluronidase solution at 37 ℃ for 20min; adding the sample to be tested, and keeping the temperature at 37 ℃ for 20min; adding sodium hyaluronate solution, keeping the temperature at 37 deg.C for 30min, and standing at room temperature for 5min; adding 0.4mol/L sodium hydroxide solution and acetylacetone solution, heating in boiling water bath for 15min, and immediately cooling with ice water for 5min; adding an 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. Hyaluronidase inhibition = [ (A-B) - (C-D) ]/(A-B). Times.100%, wherein A, B, C, D are absorbance values at 555nm after the reaction in the corresponding reaction tube is finished.

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 agents	1.0	1.0	1.0	1.0
Anhydrous ethanol	3.0	3.0	3.0	3.0
					Acetic acid buffer pH =5.6	0.5	1.0	－	0.5

4. Results

TABLE 6mg/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 to 6 and comparative examples 1 to 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 capacity, and the stronger inhibition of the tyrosinase inhibition rate suggests that the fermented extracts may have stronger in vivo melanin deposition inhibition effect, and have the potential of developing whitening or skin-brightening cosmetics; meanwhile, since hyaluronidase is an enzyme that can reduce the activity of hyaluronic acid in vivo and thus improve the fluid permeability in tissues, the higher hyaluronidase inhibition ratio of the fermented extracts provided in examples 1 to 6 means that the content of hyaluronic acid in vivo or in skin tissues can be up-regulated, the moisture retention and brightness of skin tissues can be improved, the youthful state of 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 examples 1 to 6 of the present application in whitening and brightening skin, the examples of the present application further performed related animal experiments, and the experimental methods were as follows:

1. materials and methods

1. Laboratory animal

Wistar rats, female, weighing about 300g, 9-12 weeks old, purchased from Jiangsu Wukong Biotechnology Limited, and kept the animal environment at constant temperature and humidity during the experiment, were fed normally.

2. Test article

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

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, performing anaerobic culture to obtain a working seed solution, transferring the working seed solution into the fermentation culture solution, performing anaerobic culture to obtain fermentation liquor, and extracting the fermentation liquor. Wherein, the working seed culture solution contains 1.25 to 8.5g/L of aloe extract and 1.25 to 4.5g/L of ginseng root extract, and the fermentation culture solution contains 1.25 to 8.5g/L of aloe extract, 1.25 to 4.5g/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 also 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 to 3 weight percent 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 content of the first and second substances, 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 oil, jojoba oil, sunflower seed oil, oil of mauritika palm, squalane, ethylhexyl palmitate, isopropyl myristate, hydrogenated polyisobutene, isohexadecane, isododecane, diethylhexyl carbonate, dioctyl carbonate, isopropyl lauroylsarcosine, isononyl isononanoate, hydrogenated polydecene, tris (ethylhexanoate), cetyl ethylhexanoate, bis-diethoxydiol cyclohexane 1, 4-dicarboxylate, caprylic/capric triglyceride oleyl erucate, octyldodecanol carnosine, octyldodecanol, dimethicone, octylmethicone, cetyl dimethicone, cyclopentadimethicone, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, behenyl alcohol, batyl alcohol, lauric acid, carnosine, palmitic 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, carnosine and phaseolus vulgaris litchi, bis-diglycerol polyacyladipate-2, shea butter, and bruised palm kernel 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 caproic acid, 1, 2-hexanediol, 1, 2-pentanediol, p-hydroxyacetophenone, caprylic glycol, glyceryl caprylate, glyceryl undecylenate, 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 on the 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 on the back injection area of the rat every day, and after the compositions 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 in the model group, the positive control group and the intervention group are subjected to the last administration and the rats in the normal group, after hair in a back injection area of the rats is removed by using depilatory cream, skin tissues at the back part are taken for about 1.5 multiplied by 1.5cm, the skin tissues at the back part 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. Moisture content of skin = (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 ℃ into the liver skin tissue, 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 in the supernatant by using Hydroxyproline (HYP), hyaluronic Acid (HA), superoxide dismutase (SOD), malondialdehyde (MDA) and Lipofuscin (LF) H kits. Hydroxyproline kit, 50T/48S, BOXBIO. Hyaluronic acid kit, 48T/96T, zheke biology. Superoxide dismutase kit, 48T/96T, ER0332, feien 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 rat ² Fixing 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.

2. Results

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 and arranged compactly, 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 significantly reduced, while the MDA and LF contents are all significantly 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 to 6 is applied to the skin of the injection region of a model rat, the contents of HYP, HA and SOD in the skin tissue are obviously increased compared with the model group, and the difference between the contents of HYP, HA and SOD in the skin tissue is not large; 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 compositions provided in the comparative examples 1 to 10 are applied to the skin of an injection area of a model rat, HYP, HA and SOD in skin tissues are obviously different from those of a normal group although the HYP, HA and SOD are increased relative to those of the model group; the contents of MDA and LF in the skin tissues were both reduced compared to the model group, but still were significantly different from normal, thereby demonstrating that the whitening and skin-lightening compositions provided in comparative examples 1 to 10 had 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 skin whitening and skin brightening composition provided in examples 1 to 6 is applied to the skin of an injection area of a model rat, the water content, EI index and L value in the skin tissue are all obviously increased relative to the model group, and the difference between the water 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 had poor effects on the water content, melanin deposition, heme improvement and brightness enhancement efficacy 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 oxidation resistance, and the stronger inhibition rate of tyrosinase suggests that the fermentation extract may have stronger action of inhibiting melanin deposition in vivo, and has the potential of developing cosmetics with whitening or skin brightening effects; 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 provide help for resisting inflammation.

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

1. A preparation method of a whitening and skin-brightening composition is characterized in that the whitening and skin-brightening composition comprises 10 to 500 parts of fermentation extract, 10 to 200 parts of pearl powder and 10 to 100 parts of nicotinamide; the whitening and skin-brightening composition further comprises 2.5wt% of glycerin, 0.5wt% of lanolin and 0.5wt% of acacia gum based on the total mass of the composition; wherein the fermented extract comprises 23.7 to 50.58mg/g glycoprotein, 7.48 to 14.57mg/g barbaloin, 41.49 to 58.19mg/g soluble sugar, 7.72 to 10.98mg/g ginsenoside Rg1, 5.46 to 6.95mg/g ginsenoside Re, 3.39 to 5.41mg/g ginsenoside Rb1, 4.06 to 5.05mg/g ginsenoside Rb2, 2.8 to 3.46 ginsenoside Rc and 2.48 to 2.85mg/g ginsenoside Rd;

the preparation method comprises the steps of mixing and homogenizing the components 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 comprises 1.25-8.5g/L of aloe extract, 1.25-4.5g/L of ginseng root extract, 2-3g/L of yeast extract, 7-10g/L of beef extract, and,6 to 10g/L tryptone, 1 to 5g/L glucose, 1 to 5g/L NaCl and 1 to 3g/L CH ₃ COONa·3H ₂ O and 0.1 to 0.5g/L L-cysteine hydrochloride;

preparing a fermentation liquor, wherein the fermentation liquor is obtained by inoculating the working seed liquid into a fermentation culture solution for anaerobic culture; the fermentation culture solution comprises 2 to 3g/L yeast extract, 7 to 10g/L beef extract, 6 to 10g/L peptone trypsin, 2 to 5g/L glucose, 1.25 to 8g/L aloe extract, 1.25 to 4.5g/L ginseng root extract, 1 to 5g/L NaCl, and 1 to 3g/L CH ₃ COONa·3H ₂ O, 0.1 to 0.5g/L L-cysteine hydrochloride, 0.1 to 0.22g/L pearl powder and 0.02 to 0.12g/L nicotinamide; and

a step of preparing the fermented extract.

2. The method of claim 1, wherein the step of preparing the fermentation extract comprises:

performing pulsed strong light irradiation 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: the UV pulse strong light source is adopted for processing, the irradiation distance is 5cm, the fixed flash irradiation frequency is 5 times/s, the single irradiation dose is 3.6J/cm, the single irradiation time is 40s, and the irradiation is 3 times or more.

3. The method according to claim 2, wherein the step of extracting the inactivated fermentation broth comprises:

4. The method according to claim 2, 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 25wt%, and adding Na ₂ SO ₄ Making the concentration of the extract to be 6%, adding KCl to make the concentration of the extract to be 1%, standing at 4 ℃ for 10h, centrifuging at 8000r/min for 10min to accelerate phase separation, collecting precipitate, and freeze drying to obtain the fermented extract.