CN115161207A

CN115161207A - Preparation method of galactose yeast solution, galactose yeast powder and application of galactose yeast powder in skin brightening and wrinkle diminishing

Info

Publication number: CN115161207A
Application number: CN202211020457.XA
Authority: CN
Inventors: 王晓娟; 储秀秀; 朱源源
Original assignee: Ningbo Gelukang Biotechnology Co ltd
Current assignee: Ningbo Gelukang Biotechnology Co ltd
Priority date: 2022-08-24
Filing date: 2022-08-24
Publication date: 2022-10-11

Abstract

The invention discloses a preparation method of galactose yeast liquid, galactose yeast powder and application of the galactose yeast powder in skin brightening and wrinkle diminishing. The galactose yeast solution takes the eupatorium adenophorum primary sugar and the galactose as nutrients, galactose yeast is adopted for fermentation, the galactose yeast solution is obtained by processing fermentation liquor, saccharide components in the galactose yeast solution are discovered, wherein oligosaccharide and oligosaccharide from eupatorium adenophorum are contained, and the formed emulsion or paste can be found to play a role in obviously improving the moisture content, elasticity and wrinkles of the skin of a subject, and is suitable to be used as a cosmetic raw material for brightening the skin, preserving the moisture and reducing the wrinkles.

Description

Preparation method of galactose yeast solution, galactose yeast powder and application of galactose yeast powder in skin brightening and wrinkle reducing

Technical Field

The invention relates to galactose yeast and the technical field of fermentation thereof, in particular to a preparation method of galactose yeast liquid, galactose yeast powder and application of the galactose yeast powder in skin brightening and wrinkle diminishing.

Background

The galactose yeast-like bacteria fermentation product filtrate is named as PITERA in English, and has the main functions of whitening and removing freckles, a skin conditioner, an antioxidant and a humectant in cosmetics and skin care products, has the risk coefficients of safety, can be used at ease, generally has no influence on pregnant women, and has no pox-causing property. The galactose yeast-like bacteria fermentation product filtrate is a natural yeast extract, can generate small molecules which comprise vitamin B group, mineral substances, amino acids and the like and are beneficial to skin care, and is high-quality yeast essence only used for skin care. Is often used in certain high-grade skin care products to help improve the natural physiological function of the skin. In actual use, the skin care product can balance grease and adjust the overall state of the skin, and the most obvious point is that the skin is transparent.

Disclosure of Invention

The invention aims to provide a preparation method of galactose yeast liquid, galactose yeast powder and application of galactose yeast liquid in brightening skin and reducing wrinkles.

In a first aspect, the present invention provides a preparation method of galactose yeast solution, wherein the preparation method comprises:

activating galactose yeasts by adopting an activation culture medium to obtain activation solution of the galactose yeasts;

inoculating the activation solution into a seed culture medium to obtain a seed solution;

inoculating the seed liquid into a fermentation culture medium to obtain a fermentation liquid; and

processing the fermentation liquor to obtain the galactose yeast solution;

wherein the activation medium, the seed medium, and the fermentation medium each comprise a eupatorium adenophorum initial sugar.

Further, the preparation method of the lycopus esculentus primary sugar comprises the following steps:

sieving dried herba Lycopi with 50 mesh sieve, adding 10 times of 95% ethanol water solution, heating and refluxing at 90 deg.C for 3 times, each time for 3 hr, filtering, and volatilizing ethanol from residue;

adding 10 times weight of deionized water into the residue, extracting at 90 deg.C for 3 times (each for 1.5 hr), filtering, and mixing filtrates. The filtrate was again concentrated to about 1/4 volume;

removing protein by sevag method, repeatedly treating for 4 times, and collecting the upper layer solution. The complete removal of the protein is verified by adopting a Coomassie brilliant blue method;

adding the extracted upper layer solution into 80% ethanol solution 4 times of the weight of the upper layer solution, fully stirring, standing for 24h, and taking precipitate;

washing the extracted precipitate with anhydrous alcohol, acetone and petroleum ether, and drying to obtain the final product.

Further, the activation culture medium comprises 3.0-15.0 g/L of hydrolyzed casein, 15.0-25.0 g/L of sucrose, 6.0-15.0 g/L of galactose, 0.5-1.0 g/L of arabinose concentration, 0.1g/L of eupatorium japonicum initial sugar, and NaNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.25。

Further, the seed culture medium comprises 2.0-15.0 g/L of hydrolyzed casein, 15.0-20.0 g/L of sucrose, 8.0-15.0 g/L of galactose, 0.5-1.5 g/L of arabinose concentration, 0.8-1.5 g/L of eupatorium japonicum initial sugar, and NaNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.3。

Further, the fermentation medium comprises 8.0-10.0 g/L of hydrolyzed casein, 8.0-15.0 g/L of sucrose, 20.0g/L of galactose, 2.5g/L of arabinose, 6.5-8.5 g/L of eupatorium japonicum primary sugar, naNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.3。

Further, the step of processing the fermentation liquor to obtain the galactose yeast solution comprises the following steps:

collecting the fermentation liquor, centrifuging at 12000rpm, collecting supernatant, sterilizing with 0.25um filter membrane, filtering, adding 10 times of 95% ethanol water solution, heating and refluxing at 90 deg.C for 3 times, each time for 3 hr, filtering, and volatilizing ethanol from residue;

adding 10 times weight of deionized water into residue, extracting at 90 deg.C for 3 times (each for 1.5 hr), filtering, mixing filtrates, and concentrating to about 1/4 volume;

removing protein by utilizing a sevag method, repeatedly treating for 4 times, taking the upper layer solution, and verifying complete protein removal by adopting a Coomassie brilliant blue method;

adding the upper layer solution into 80% ethanol solution 4 times the weight of the upper layer solution, stirring, standing for 24 hr, and collecting precipitate. The extracted precipitate is washed with absolute ethyl alcohol, acetone and petroleum ether in sequence.

In a second aspect, the invention provides galactose yeast powder prepared by the preparation method of the first aspect, which comprises eupatorium adenophorum polysaccharide with weight-average molecular weight of 12588-16344 and polydispersity index of 1.069-1.152; and galacto-oligosaccharides with a weight-average molecular mass of 2168-2792 and a polydispersity index of 1.234-4.523; the monosaccharide composition of the eupatorium adenophorum polysaccharide comprises glucose, galactose, xylxylogalacturonic acid and rhamnose.

Further, the weight percentage of the eupatorium adenophorum polysaccharide in the eupatorium adenophorum polysaccharide is 43.8-46.9%, and the weight percentage of the galacto-oligosaccharide in the eupatorium adenophorum polysaccharide is 32.7-34.4%.

In a third aspect, the invention provides a cream, which comprises 6-9 wt% of sweet almond oil, 5.5-10 wt% of avocado oil, 20-40 wt% of white oil, 1.5wt% of polyvinyl stearyl alcohol ether, 0.25-0.55 wt% of sodium polyacrylate, 0.15-0.30 wt% of hydrogenated polyporalene, 0.05-0.15 wt% of lauryl alcohol ether-5, 0.01-0.05 wt% of 1, 3-dihydroxy-5, 5-dimethylhydantoin, 0.05-0.5 wt% of hydroxyethyl cellulose, 0.02-0.1 wt% of potassium trisorbate and the balance of aqueous solution containing galactose yeast powder.

In a fourth aspect, the invention provides application of the galactose yeast powder prepared by the preparation method in preparation of cosmetics for brightening skin and reducing wrinkles.

Compared with the prior art, the technical scheme provided by the invention at least has the following technical effects:

the galactose yeast solution is fermented by galactose yeast with eupatorium adenophorum polysaccharide and galactose as nutrients, the galactose yeast is treated to obtain the galactose yeast solution, saccharide components in the galactose yeast solution are found to contain oligolactose and oligosaccharide from eupatorium adenophorum, and the formed emulsion or paste can obviously improve the moisture content, elasticity and wrinkles of the skin of a subject, and is suitable to be used as a cosmetic raw material for brightening the skin, preserving the moisture and reducing the wrinkles.

According to the composition analysis research of the galactose yeast solution provided by the invention, as the galactose yeast solution mainly contains eupatorium adenophorum polysaccharide and galacto-oligosaccharide, and the structure of the galacto-oligosaccharide is a 12-sugar structure, the galactose yeast solution has the effects of oxidation resistance, tyrosinase activity inhibition, skin protection from sunlight radiation, collagen decomposition and hardening reduction, and fibroblast proliferation and metabolism stimulation, so that the collagen of a real skin layer can be generated to play the roles of filling the skin and relieving wrinkles, and therefore, under the long-term use condition, the galactose yeast solution has a remarkable effect of reducing the wrinkle area of the skin, can permeate the skin, improve the skin moisture and protect the skin barrier, and the moisture effect is favorable for improving the skin wrinkles from depth, and has the effects of brightening the skin, tightening and weakening the textures.

Drawings

FIG. 1 is a graph showing the results of skin moisture content after short-term treatment of galactose yeast solutions provided in examples 1 to 3 according to the present invention and comparative examples 1 to 8, respectively.

FIG. 2 is a graph showing the results of skin moisture contents after short-term treatment of ointments provided in examples 1 to 3 of the present invention and comparative examples 1 to 8, respectively.

FIG. 3 is a graph showing the results of skin moisture contents after long-term treatment of galactose yeast solutions provided in examples 1 to 3 of the present invention and comparative examples 1 to 8, respectively.

FIG. 4 is a graph showing the results of skin moisture contents after long-term treatment of the ointments provided in examples 1 to 3 of the present invention and comparative examples 1 to 8, respectively.

FIG. 5 is a graph showing the results of the skin elasticity parameter R2 after the short-term treatment of galactose yeast solutions provided in examples 1 to 3 according to the present invention and comparative examples 1 to 8, respectively.

FIG. 6 is a graph showing the results of the skin elasticity parameter R2 after the short-term treatment of the ointments provided in examples 1 to 3 of the present invention and comparative examples 1 to 8, respectively.

FIG. 7 is a graph showing the results of the skin elasticity parameter R2 after the long-term treatment of the galactose yeast solutions provided in examples 1 to 3 according to the present invention and comparative examples 1 to 8, respectively.

FIG. 8 is a graph showing the results of the skin elasticity parameter R2 after long-term treatment of the ointments provided in examples 1 to 3 of the present invention and comparative examples 1 to 8, respectively.

FIG. 9 is a graph showing the results of the total wrinkle area after the galactose yeast solution according to examples 1 to 3 of the present invention and comparative examples 1 to 8, respectively, is treated for a long period of time.

FIG. 10 is a graph showing the results of the total wrinkle area of the skin after the long-term treatment with the ointments provided in examples 1 to 3 according to the present invention and comparative examples 1 to 8, respectively.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention 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 invention and are not intended to limit the invention. Reagents which are not detailed and are not separately described in the invention are conventional reagents and can be obtained from commercial sources; methods not specifically described in detail are all routine experimental methods and are known from the prior art.

It should be noted that the terms "first", "second", and the like in the description and claims of the present invention and in the drawings are used for distinguishing similar objects, and do not necessarily have to be used for describing a specific order or sequence or have a substantial limitation on technical features thereafter. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in other sequences than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

In the embodiment of the invention, eupatorium adenophorum spreng polysaccharide and galactose are used as nutrients, yeast is adopted for fermentation, fermentation liquor is extracted, the extracting solution is separated, saccharide components in the extracting solution are found, wherein the saccharide components comprise oligolactose and oligosaccharide from eupatorium adenophorum spreng, and the formed emulsion or paste can be found to play a role in obviously improving the moisture content, elasticity and wrinkles of the skin of a subject, and is suitable to be used as a cosmetic raw material for brightening the skin, preserving the moisture and reducing the wrinkles. For this reason, the present invention has carried out the following tests and examples.

1. Fermentation strain

Galactose yeast (Galactomyces geotrichum, KTAPG711.67, institute of microbiology, china academy of sciences).

2. Herba lycopi primary sugar

Sieving dried herba Lycopi medicinal powder with 50 mesh sieve, collecting 100g, placing in round-bottom flask, adding 10 times of 95% ethanol water solution, heating and refluxing at 90 deg.C for 3 times, each time for 3 hr, filtering, and volatilizing ethanol from residue. Adding 10 times weight of deionized water into the residue, extracting at 90 deg.C for 3 times (each for 1.5 hr), filtering, and mixing filtrates. The filtrate was again concentrated to about 1/4 of the volume. Removing protein by sevag method, repeatedly treating for 4 times, and collecting the upper layer solution. Complete removal of the protein was verified by Coomassie Brilliant blue. Adding the upper layer solution into 80% ethanol solution 4 times the weight of the upper layer solution, stirring, standing for 24 hr, and collecting precipitate. Washing the extracted precipitate with anhydrous alcohol, acetone and petroleum ether, and drying to obtain the initial eupatorium adenophorum sugar. The total sugar content of the eupatorium adenophorum spreng initial sugar is 86.3 percent (weight percentage) by adopting a phenol-sulfuric acid method for detection.

3. Culture medium

Example 1:

the activation medium comprises hydrolyzed casein 10.0g/L, sucrose 20.0g/L, galactose 13.0g/L, arabinose concentration 1.0g/L, herba Lycopi initial sugar 0.1g/L, naNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.25。

The seed culture medium contains hydrolyzed casein 10.0g/L, sucrose 20.0g/L, galactose 13.0g/L, arabinose concentration 0.5g/L, eupatorium japonicum initial sugar 1.5g/L, and NaNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.3。

The fermentation medium comprises hydrolyzed casein 8.0g/L, sucrose 8.0g/L, galactose 20.0g/L, arabinose 2.5g/L, eupatorium japonicum initial sugar 6.5g/L, and NaNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.3。

Example 2:

the activation medium comprises hydrolyzed casein 3.0g/L, sucrose 25.0g/L, galactose 6.0g/L, arabinose concentration 0.5g/L, herba Lycopi initial sugar 0.1g/L, naNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.25。

The seed culture medium contains hydrolyzed casein 15.0g/L, sucrose 15.0g/L, galactose 15.0g/L, arabinose concentration 0.5g/L, herba Lycopi initial sugar 0.8g/L, and NaNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.3。

The fermentation medium comprises hydrolyzed casein 10.0g/L, sucrose 10.0g/L, galactose 20.0g/L, arabinose 2.5g/L, eupatorium japonicum initial sugar 6.5g/L, and NaNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.3。

Example 3:

the activation medium comprises hydrolyzed casein 15.0g/L, sucrose 15.0g/L, galactose 15.0g/L, arabinose concentration 0.5g/L, eupatorium japonicum initial sugar 0.8g/L, naNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.25。

The seed culture medium contains hydrolyzed casein 2.0g/L, sucrose 20.0g/L, galactose 8.0g/L, arabinose concentration 1.5g/L, herba Lycopi initial sugar 1.0g/L, and NaNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.3。

The fermentation medium comprises 8.0g/L of hydrolyzed casein, 15.0g/L of sucrose, 20.0g/L of galactose, 2.5g/L of arabinose, 8.5g/L of eupatorium japonicum primary sugar, and NaNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.3。

Comparative example 1:

the activation culture medium comprises yeast extract 10.0g/L, sucrose 20.0g/L, galactose 15.0g/L, and NaNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L, pH6.25. The seed medium and the fermentation medium used therein were the same as in example 1.

Comparative example 2

The activation culture medium comprises yeast extract 10.0g/L, sucrose 20.0g/L, galactose 13.0g/L, arabinose concentration 1.0g/L, eupatorium japonicum initial sugar 1.5g/L, naNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L, pH6.25. The seed medium and the fermentation medium used therein were the same as in example 1.

Comparative example 3

The activation medium comprises hydrolyzed casein 10.0g/L, sucrose 20.0g/L, galactose 13.0g/L, herba LycopiInitial sugar 1.5g/L, naNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L, pH6.25. The seed medium and the fermentation medium used therein were the same as in example 1.

Comparative example 4

The activation medium comprises hydrolyzed casein 10.0g/L, sucrose 20.0g/L, galactose 13.0g/L, arabinose concentration 1.0g/L, naNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L, pH6.25. The seed medium and the fermentation medium used therein were the same as in example 1.

Comparative example 5:

the activation medium, seed medium and fermentation medium used therein were the same as in comparative example 1.

Comparative example 6:

the activation medium, seed medium and fermentation medium used therein were the same as in comparative example 2.

Comparative example 7:

the activation medium, seed medium and fermentation medium used therein were the same as in comparative example 3.

Comparative example 8:

the activation medium, seed medium and fermentation medium used therein were the same as in comparative example 4.

4. Activation of bacterial strains

Thawing freeze-dried tube of galactose yeast rapidly, inoculating into liquid activation culture medium, and culturing at 28-30 deg.C for 3 days to obtain activation solution. The liquid activation medium contains hydrolyzed casein (CAS: 65072-00-6, purity: 99%) 10.0g/L, sucrose 20.0g/L, galactose 13.0g/L, arabinose concentration 1.0g/L, eupatorium sugar 1.5g/L, naNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.25。

5. Preparation of seed liquid

Inoculating the activated solution into a shake flask of the seed culture solution according to the inoculation amount of 1.0 percent (volume ratio), at the temperature of 30 ℃, the rotating speed of 160r/min, the liquid loading amount of 100mL/500mL triangular flask, and culturing for 24h to obtain the seed solution.

6. Preparation of fermentation broth

Inoculating the seed solution into the fermentation culture solution according to the inoculation amount of 1.5% (volume ratio), and culturing at 30 ℃ and the rotation speed of 160r/min for 72h to obtain the fermentation liquid.

7. Fermentation broth treatment

Centrifuging at 12000rpm, collecting supernatant, sterilizing with 0.25um filter membrane, filtering, adding 10 times of 95% ethanol water solution, heating and refluxing at 90 deg.C for 3 times, each time for 3 hr, filtering, and volatilizing ethanol from residue. Adding 10 times weight of deionized water into the residue, extracting at 90 deg.C for 3 times (each for 1.5 hr), filtering, and mixing filtrates. The filtrate was again concentrated to about 1/4 of the volume. Removing protein by sevag method, repeatedly treating for 4 times, and collecting upper layer solution. Complete removal of the protein was verified by Coomassie Brilliant blue. Adding the above extracted upper layer solution into 4 times weight of 80% ethanol solution, stirring, standing for 24 hr, and collecting precipitate. Washing the extracted precipitate with anhydrous alcohol, acetone and petroleum ether, drying to obtain galactose yeast powder.

8. Ingredient analysis of galactose yeast powder

The molecular weight, monosaccharide composition and content of the eupatorium adenophorum polysaccharide in galactose yeast fermentation liquor are detected by adopting a method disclosed in 'eupatorium adenophorum seed polysaccharide separation and purification, structure characterization and antioxidant activity [ J ] food science, 2021, vol.42, no. 19'.

A liquid chromatography double-column method and a high performance ion exchange chromatography are adopted to determine the content of galactooligosaccharides, and the comparison [ J ] Chinese food additive, 2022 and No. 2 discloses a method for determining the content of the galactooligosaccharides in the fermentation liquor of the galactooligosaccharides.

9. Analysis of eupatorium japonicum polysaccharide in galactose yeast powder

The molecular mass is an important structural index of the polysaccharide, and the properties of the polysaccharide, such as physicochemical property, biological activity and the like, are influenced. The weight average molecular mass (mw), polydispersity index (mw/mn), and percentage content of eupatorium adenophorum polysaccharide in the galactose yeast powder were determined by HPGPC methods in examples 1-3 and comparative examples 1-8, respectively, and the results are shown in table 1.

TABLE 1

In table 1, "-" indicates no detection. As can be seen from Table 1, no eupatorium adenophorum polysaccharide was detected in the galactose yeast powders provided in comparative examples 1, 4, 5 and 8. While the weight average molecular weight of the eupatorium adenophorum polysaccharide provided by the galactose yeast powder provided by the examples 1-3 and the comparative examples 2, 3, 6 and 7 is between 12588 and 16344, and the eupatorium adenophorum polysaccharide in the galactose yeast powder provided by the examples 1-3 has lower polydispersity index and higher percentage content.

TABLE 2 molar ratio of monosaccharide composition of eupatorium adenophorum polysaccharide in galacto-fermentation broth

The analysis result of monosaccharide composition molar ratio of herba Lycopi polysaccharide in galacto-fermented powder is shown in Table 2, "-" indicates no detection. As can be seen from Table 2, the monosaccharide composition molar ratios of the eupatorium adenophorum polysaccharides provided in examples 1-3, respectively, are approximately equivalent and comprise GalA (galacturonic acid) Rha (rhamnose), while the eupatorium adenophorum polysaccharides provided in comparative examples 2, 3, 6 and 7, respectively, do not comprise GalA and Rha.

In addition, gel chromatography is adopted to find that the galactose yeast powder provided by the examples 1 to 3 and the comparative examples 2, 3, 6 and 7 of the invention contains the eupatorium adenophorum polysaccharide with the molecular weight of 12588 to 16344, the weight-average molecular weight of 2168 to 2792 and the polydispersity index of 1.234 to 4.523. The results are shown in Table 3. As shown in Table 3, the weight average molecular weight of the galactooligosaccharide in the galactose yeast powder provided in examples 1 to 3 was 2168 to 2235, and the polydispersity index was 1.096 to 1.118, and it was concluded that the galactooligosaccharide had a 12-sugar structure. The weight average molecular weight of the galacto-oligosaccharide in the galacto-yeast powder provided in the comparative examples 2 and 3 is 2357-2368, and the polydispersity index is 1.964-2.455, and the galacto-oligosaccharide is deduced to be 13 sugar structures. The weight average molecular weight of the galactooligosaccharide in the galactose yeast powder provided by the comparative examples 6 and 7 is 2684-2792, the polydispersity index is 2.268-2.354, and the galactooligosaccharide is deduced to be 15 sugar structure. Also, comparative examples 2, 3, 6 and 7 provided galactose yeast powder having a significantly lower content of galactooligosaccharides than examples 1 to 3.

TABLE 3 molecular weight, dispersion coefficient and content of galactooligosaccharides in galactose fermentation broth

10. Antioxidant capacity of galactose yeast solution

The galactose yeast powders provided in the above examples 1 to 3 and comparative examples 1 to 8 were respectively prepared into galactose yeast solutions of different concentrations of 0.5mg/mL, 1mg/mL, 2mg/mL, 5mg/mL, 10mg/mL, 20mg/mL and 50mg/mL with deionized water as test sample solutions. Adding 5.7mL of 50mmol/L Tris-HCl buffer solution with pH of 8.2 into a 10mL EP tube, adding 0.2mL of test solution, uniformly mixing, keeping the temperature at 25 ℃ for 10min, adding 0.1mL of 10mmol/L pyrogallol solution preheated at 25 ℃, keeping the total volume at 6mL, rapidly shaking, and recording the absorbance increase (AS) of the solution at the wavelength of 320nm for 1min by using an ultraviolet-visible spectrophotometer. The increase in absorbance per minute in the linear range was calculated. The reagent is taken as above, the sample solution is replaced by equal volume of water, and the increase value (Ao) of absorbance within 1min at the wavelength of 320nm is also measured. And calculating the superoxide anion free radical clearance according to a formula of the superoxide anion free radical clearance (%) = (Ao-AS)/Ao x 100%, drawing an inhibition curve of the test solution with different concentrations, and estimating the concentration (IC 50-O2) when the superoxide anion free radical clearance of the test solution reaches 50% according to the drawn curve.

The same test solutions as above were prepared and the tests were divided into T1, T2, T3 and T4 groups. In the T1 group, 3mL of PBS solution and 1mL of 1mM L-tyrosine solution were reacted AT 37 ℃ for 10min, and the absorbance AT1 value was measured AT 475 nm. In the T2 group, 1mL of tyrosinase solution (3130U/mg) and 2mL of PBS solution, as well as 1mL of 1mM L-tyrosinase solution, were reacted AT 37 ℃ for 10min, and the absorbance AT2 value was measured AT 475 nm. And in the T3 group, 2mL of LPBS solution, 1mL of test solution and 1mL of 1mM L-tyrosine solution are taken to react for 10min AT 37 ℃, and the absorbance AT3 value is measured AT 475 nm. And in the T4 group, 1mL of tyrosinase solution (3130U/mg), 1mL of PBS solution, 1mL of 1mM L-tyrosine solution and 1mL of test solution are uniformly mixed, reacted AT 37 ℃ for 10min, and the absorbance AT4 value of the sample is measured AT 475 nm. According to the formula, the tyrosinase activity inhibition rate = [1- (AT 4-AT 3)/(AT 2-AT 1) ] × 100%, inhibition curves of the test solution with different concentrations are drawn, and the concentration (IC 50-TYR) AT which the tyrosinase activity inhibition rate of the test solution reaches 50% is estimated according to the drawn curves.

TABLE 4 (mg/mL)

As shown in Table 4, the IC50-O2 and IC50-TYR values of the galactose yeast solutions provided in examples 1 to 3 were significantly lower than those of comparative examples 2, 3, 6 and 7, while those of comparative examples 1, 4 and 5 were not detected to have antioxidant properties and to inhibit tyrosinase activity.

11. Evaluation of efficacy of galactose yeast solution and cream

The above tests revealed that the galactose yeast solutions provided in examples 1 to 3 can be used as raw materials for cosmetics for whitening skin and lightening wrinkles. The specific test is as follows:

(1) Test article

Galactose yeast solutions provided in examples 1 to 3 and comparative examples 1 to 8 were used as test samples. In addition, a cream was prepared from the above raw material according to the formulation shown in the following table, and used as a test sample.

To this end, the embodiment of the present invention further discloses a cream, which comprises sweet almond oil 6-9 wt%, avocado oil 5.5-10 wt%, white oil 20-40 wt%, polyvinyl stearyl alcohol ether 1.5wt%, sodium polyacrylate 0.25-0.55 wt%, hydrogenated polyprenol 0.15-0.30 wt%, lauryl alcohol ether-5.05-0.15 wt%,1, 3-dihydroxy-5, 5-dimethyl hydantoin 0.01-0.05 wt%, hydroxyethyl cellulose 0.05-0.5 wt%, potassium trisorbate 0.02-0.1 wt%, and the balance of galactose yeast solution containing galactooligosaccharide with a concentration of not less than 5.0mg/mL and eupatorium polypeptide 5.0 mg/mL.

The preparation method of the cream comprises the steps of fully mixing sweet almond oil, avocado oil and white oil according to the formula, heating the mixture and galactose yeast solution containing galacto-oligosaccharide and eupatorium polypeptide with the concentration of not less than 5.0mg/mL to 90 ℃, homogenizing at 10000r/min for 2min, simultaneously adding polyvinyl stearate, sodium polyacrylate, hydrogenated polyporalene, lauryl alcohol ether-5 and 1, 3-dihydroxy-5, 5-dimethyl hydantoin in the homogenizing process, adding hydroxyethyl cellulose after the temperature is reduced to 50 ℃, stirring to room temperature, adding potassium sorbate, stirring uniformly, and cooling to obtain the cream.

(2) Subject requirements and test conditions

Healthy subjects between 20 and 60 years of age are selected according to the voluntary principle, and have no history of skin diseases and cosmetic allergy. The subjects were required to clean their face before the test, to be stable for at least 30min under the test conditions before the test, and to have no need to drink, eat, move vigorously during the test, and to use other products of the same type during the test. The test environment is that the temperature is 20-22 ℃, the Relative Humidity (RH) is 50%, and all the testees voluntarily cooperate with the experiment.

(3) The tested sample is used

And in the short-term test group, each test subject uses a different test article on the left arm and the right arm respectively, the moisture content, the elasticity and the wrinkles of the test articles used for 0h, 1h, 2h, 4h and 6h are measured respectively, and finally the data are counted according to different test articles. Test Point labeling, at each subjectThe inner sides of the left forearm and the right forearm are marked with a test area (4 cm multiplied by 4 cm) by a mould, holes are dug on the mould, and the sides of the holes are marked with test point positions of each test probe. The sample concentration is 2.0 + -0.1 mg/cm ² The dosage of the composition is uniformly coated on a test area, and 8 testees are randomly selected from each test sample.

Long-term test group, one test article was administered to each subject, and the moisture content, elasticity and wrinkles of the test articles were measured at 0d, 20d and 40 d. Each subject was applied to the face of the patient in the morning and evening, about 0.5g of the test article was applied each time, and the test article was applied to the face of the patient uniformly. The test site location was marked with a locating film and 8 subjects were randomly selected for each test article.

(4) Determination of skin moisture content

In the short-term test group, the moisture tester CM825 was used to measure the 0 th, 1h, 2h, 4h and 6h of the test article applied to the inner marker point of the forearm of the subject, and as shown in FIGS. 1 and 2, the galactose yeast solutions or ointments provided in examples 1-3 in the short term significantly increased the moisture content of the skin, while the galactose yeast solutions or ointments provided in comparative examples 2, 3, 6 and 7 showed good moisture retention performance in the short term, but did not significantly increase the moisture content in the later period of 6h, while the galactose yeast solutions provided in comparative examples 1, 4, 5 and 8 did not have the effect of significantly increasing the moisture content of the skin.

In the long-term test group, the moisture content change at 0d, 20d and 40d was used at the mark point of the face, and the results are shown in fig. 3 and 4. As can be seen from FIGS. 3 and 4, the ointments provided in examples 1 to 3 significantly increase the moisture content of the skin after long-term use, while the ointments provided in comparative examples 1 to 8 do not have the effect of significantly increasing the moisture content of the skin after long-term use.

Therefore, the galactose yeast solution or the ointment prepared on the basis of the galactose yeast solution provided by the embodiment of the invention has better effects of increasing the skin moisture content and reducing the skin moisture loss, which is related to the galactooligosaccharide structure contained in the galactose yeast solution and the component and content of eupatorium adenophorum polysaccharide.

(5) Skin elasticity measurement

Referring to "Rodrigues L, eemco. Eemco side to the in vivo performance of the Skin. Part 2".

The influence of the test article on the skin elasticity in the short and long term was evaluated by measuring the change in the skin elasticity of the test article at the medial forearm mark point (short-term test group) of the subject at 0h, 1h, 2h, 4h and 6h and the skin elasticity of the test article at the facial mark point (long-term test group) before and after 20d and 40d, respectively, by the skin elasticity tester MPA580, and the result was expressed by the elasticity parameter R2 without unit. The skin elasticity changes of the test article at 0h, 1h, 2h, 4h and 6h of the forearm medial marker (short-term test group) measured by the skin elasticity tester MPA580 are shown in fig. 5 and 6, and it can be seen from fig. 5 and 6 that the short-term skin elasticity is significantly increased after the application of the galactose yeast solution or ointment provided in examples 1 to 3, while the skin elasticity is slightly increased or unchanged by the application of the galactose yeast solution or ointment provided in comparative examples 1 to 8.

The results of the long-term skin elasticity changes at the facial marker points (long-term test group) using the test articles on day 0, 20d and 40d are shown in fig. 7 and 8. As can be seen from FIGS. 7 and 8, the galactose yeast solution provided in examples 1 to 3 was used to improve skin elasticity, but it was not as effective as the ointment prepared from galactose yeast solution, thus demonstrating that the effect of galactose yeast in ointment preparation on skin elasticity was enhanced. Comparative examples 1 to 8, however, provide either a galacto-yeast solution or an ointment thereof, which has a limited effect of improving skin elasticity during short-term or long-term use.

(6) Skin wrinkle determination

The principle of the wrinkle analysis system Visioline VL650 is that shadows are formed at positions with wrinkles on a silica gel copy model of the skin by virtue of oblique illumination, a high-resolution camera is arranged above a host platform, and the change of the area, the length and the wrinkle depth value of the shadow parts is analyzed by special software to reflect the wrinkle change. When the wrinkle model is copied, the subject lies down, the left side of the head faces upwards, the eyes are closed, and the positioning glue is stuck to the mark points. Pouring a certain amount of silica gel into a plastic box, adding a proper amount of catalyst coagulant, quickly and uniformly stirring, uniformly coating the silica gel on the area defined by the positioning gel, and removing the silica gel after 10min and pasting the silica gel on the positioning paper board special for the Visioline VL650 wrinkle analysis system. Fine line changes in the test samples at the site of the subject's facial markers (long term test group) were analyzed using the Visoline VL650 wrinkle analysis system with 20d, 40d consecutive skin gel replicates prior to use. Placing the prepared silica gel replica on a Visoline VL650 platform, adjusting the focus and light to be vertical to wrinkle lines, and analyzing the total wrinkle area (mm) after photographing ² )。

The total wrinkle area change at the site of the subject's facial marker (long term test group) using the test article's 0 th, 20 th and 40 th d silicone rubber replicas was analyzed using the Visioline VL650 wrinkle analysis system is shown in fig. 9 and 10. As can be seen from FIGS. 9 and 10, the ointments provided in examples 1 to 3 all had a good effect of improving the total wrinkle area of the skin.

The effects of free radical oxidation and light radiation are important reasons for skin wrinkles, and by combining the above component analysis research on the galactose yeast solution provided by the embodiment, the galactose yeast solution mainly contains eupatorium adenophorum polysaccharide and galacto-oligosaccharide, and the galacto-oligosaccharide has a 12-saccharide structure, so that the galactose yeast solution has oxidation resistance and tyrosinase activity inhibition, can protect the skin from being irradiated by sunlight, reduce the decomposition and hardening of collagen, and stimulate the proliferation and metabolism of fibroblasts, thereby being beneficial to the generation of collagen in a real skin layer to play a role in filling the skin and relieving wrinkles, so that under the long-term use condition, the skin wrinkle area is remarkably reduced, the skin can be permeated in long-term use, the skin moisture and the skin barrier are improved, and the moisture effects are beneficial to improving skin wrinkles from the depth, and the effects of brightening the skin color, tightening and lightening the textures are achieved.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention 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 invention are included in the scope of the present invention.

Claims

1. A preparation method of galactose yeast solution is characterized by comprising the following steps:

activating galactose yeasts by adopting an activation medium to obtain an activation solution of the galactose yeasts;

processing the fermentation liquor to obtain the galactose yeast solution;

2. The method according to claim 1, wherein the method for preparing the eupatorium adenophorum spreng sugar comprises:

removing protein by sevag method, repeatedly treating for 4 times, and collecting upper layer solution. The complete removal of the protein is verified by adopting a Coomassie brilliant blue method;

3. The preparation method of claim 2, wherein the activation medium comprises hydrolyzed casein 3.0-15.0 g/L, sucrose 15.0-25.0 g/L, galactose 6.0-15.0 g/L, arabinose concentration 0.5-1.0 g/L, eupatorium japonicum initial sugar 0.1g/L, naNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.25。

4. The method according to claim 3, wherein the seed medium comprises hydrolyzed casein 2.0-15.0 g/L, sucrose 15.0-20.0 g/L, galactose 8.0-15.0 g/L, arabinose concentration 0.5-1.5 g/L, eupatorium japonicum initial sugar 0.8-1.5 g/L, naNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl 0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.3。

5. The method of claim 3, wherein the fermentation medium comprises hydrolyzed casein 8.0-10.0 g/L, sucrose 8.0-15.0 g/L, galactose 20.0g/L, arabinose 2.5g/L, eupatorium japonicum sugar 6.5-8.5 g/L, naNO ₃ 3.0g/L、MgSO ₄ ·7H ₂ O 0.5g/L、KCl0.5g/L、FeSO ₄ ·4H ₂ O0.01g/L and K ₂ HPO ₄ 1.0g/L，pH6.3。

6. The method according to claim 1, wherein the step of treating the fermentation broth to obtain the galactose yeast solution comprises:

collecting the fermentation liquor, centrifuging at 12000rpm to obtain supernatant, filtering with 0.25um filter membrane for sterilization, adding 10 times of 95% ethanol water solution, heating and refluxing at 90 deg.C for 3 times, each time for 3 hr, filtering, and evaporating ethanol from residue;

adding 10 times of deionized water into the residue, extracting at 90 deg.C for 3 times (each for 1.5 hr), filtering, mixing filtrates, and concentrating the filtrate to about 1/4 volume;

7. Galactose yeast powder prepared by the preparation method of any one of claims 1 to 6, characterized by comprising eupatorium japonicum polysaccharide with weight-average molecular weight of 12588 to 16344 and polydispersity index of 1.069 to 1.152; and galacto-oligosaccharides with a weight-average molecular mass of 2168-2792 and a polydispersity index of 1.234-4.523;

the monosaccharide composition of the eupatorium adenophorum polysaccharide comprises glucose, galactose, xylxylogalacturonic acid and rhamnose.

8. The galactose yeast powder according to claim 7, wherein the weight percentage of the eupatorium adenophorum polysaccharide in the eupatorium adenophorum polysaccharide is 43.8-46.9%, and the weight percentage of the galacto-oligosaccharide in the eupatorium adenophorum polysaccharide is 32.7-34.4%.

9. A cream is characterized by comprising 6-9 wt% of sweet almond oil, 5.5-10 wt% of avocado oil, 20-40 wt% of white oil, 1.5wt% of polyvinyl stearate alcohol ether, 0.25-0.55 wt% of sodium polyacrylate, 0.15-0.30 wt% of hydrogenated polyporalene, 0.05-0.15 wt% of lauryl alcohol ether-5, 3-dihydroxy-5, 5-dimethylhydantoin 0.01-0.05 wt%, 0.05-0.5 wt% of hydroxyethyl cellulose, 0.02-0.1 wt% of potassium trisorbate and the balance of aqueous solution containing galactose yeast powder.

10. Use of galactose yeast powder prepared by the preparation method according to any one of claims 1 to 6 for preparing a cosmetic for skin lightening and wrinkle reduction.