CN117530911B

CN117530911B - Snow lotus fermentation product, preparation method and application thereof, and cosmetics

Info

Publication number: CN117530911B
Application number: CN202410028018.6A
Authority: CN
Inventors: 杨银; 谢敏婷; 杨小鲁
Original assignee: Guangzhou Huamiao Biotechnology Research Institute Co ltd
Current assignee: Guangzhou Huamiao Biotechnology Research Institute Co ltd
Priority date: 2024-01-09
Filing date: 2024-01-09
Publication date: 2024-05-07
Anticipated expiration: 2044-01-09
Also published as: CN117530911A

Abstract

The invention belongs to the field of new daily chemical materials, and discloses a preparation method of a snowflake fermentation product, which comprises the steps of inoculating zymophyte into a snowflake fermentation culture solution for aerobic fermentation treatment to obtain the snowflake fermentation product; the snow lotus fermentation culture solution contains carbon source, tremella extract, yeast extract and snow lotus powder; the fermentation bacteria are saccharomycetes and/or compound bacteria. The method adopts tremella extract, yeast extract and snow lotus powder for mixed fermentation, and the obtained fermentation product has the functions of tightening, relieving and anti-wrinkle skin care, and simultaneously has good safety, mildness and no irritation. Meanwhile, the invention also provides a snow lotus herb fermentation product, application and cosmetics thereof.

Description

Snow lotus fermentation product, preparation method and application thereof, and cosmetics

Technical Field

The invention relates to the field of new daily chemical materials, in particular to a snow lotus fermentation product, a preparation method, application and cosmetics thereof.

Background

With the progress of times and scientific technology, the cosmetic industry has undergone countless changes and innovations, and the demand of consumers for green, safe and functional cosmetics is increasing, so that the core competitiveness of cosmetic products is gradually focused on safety and efficacy. The biological fermentation technology is used as an emerging biological technology, uses certain functions unique to microorganisms, adopts modern biological engineering technology means, and is used for producing products on a large scale or directly applying the microorganisms to product terminals, so that the biological fermentation technology is gradually applied to the field of cosmetics, and the safety and the efficacy of the cosmetics are greatly improved. The biological fermentation technology is applied to the field of cosmetics, and has the advantages that on the one hand, microorganisms contain certain enzymes, and substances such as cellulose, hemicellulose and the like in cell walls and cell interstitials of plant raw materials can be degraded, so that plant cells are broken, cell gaps are increased, the mass transfer resistance of active ingredients in the extraction medium from the cells is reduced, and the extraction rate of the active ingredients is greatly improved. On the other hand, the biological fermentation process can enrich the active ingredients of the raw materials, and generate new effects. Compared with traditional physical, chemical and other extraction means, the biological fermentation technology can convert and modify the effective components to a greater extent by purposefully selecting strains, controlling fermentation conditions, regulating metabolic pathways and explaining the efficacy mechanism of fermentation products, thereby improving and optimizing the properties of the original efficacy components; the mild conditions of microbial transformation can also minimize the damage to the active ingredients during fermentation, thus promoting the efficacy of the cosmetic. In addition, natural plant resources are rich in nutrition components, but the components are complex, and the problems of excessive heavy metal, pesticide residues and the like exist, so that the application and development of the natural plant resources are limited. The microbial fermentation technology is adopted to carry out biological conversion, so that the toxicity of the original toxic substances can be reduced, and the use safety is ensured. Saussurea involucrata is a plant of saussurea of Compositae, and is also called herba Saussureae Involueratae, and flos Clerodendri Bungei. The characteristic of the saussurea involucrata adapting to the alpine environment is that the saussurea involucrata is formed under the conditions of severe cold and drought of the alpine for a long time, and the special survival habit and the growth environment bring about wide pharmacological action and precious practical value of the saussurea involucrata.

The application of herba Saussureae Involueratae in cosmetics is mainly divided into two types, one is the use of extract, and the other is the fermentation of extract.

Specifically, the following documents show:

Prior art 1: CN110680775A discloses a whitening, moisturizing and moisturizing essence liquid, which comprises the following components in parts by weight:

100-150 parts of damascus roseus flower water, 5-10 parts of ophiopogon root extract, 0.1-2 parts of yeast extract, 1-5 parts of lysate of fermentation products of two-split yeast, 1-5 parts of ganoderma lucidum extract, 3-10 parts of snow lotus herb extract, 1-5 parts of tremella fruit body extract, 5-10 parts of glabrous greenbrier rhizome extract and 0.5-1.5 parts of preservative.

The technology utilizes the snow lotus herb extract to be added into cosmetics for utilization, and has the effects of whitening, moisturizing and supplementing water.

Prior art 2: CN111728910a discloses a fermented extract of blueberry and snow lotus, a preparation method and application thereof. The fermented extract of the blueberries and the snow lotus flowers is obtained by fermenting the blueberries and the snow lotus flowers together by using saccharomyces cerevisiae.

The technology utilizes the snow lotus herb to ferment, and the obtained ferment extract is utilized, and has the effects of moisturizing and resisting oxidation.

Prior art 3: CN111249218B discloses a snow lotus fermentation stock solution, a preparation method and application thereof, wherein the method comprises the steps of mixing snow lotus powder, zymophyte bacterial liquid, glucose and water to obtain an initial system, wherein the zymophyte bacterial liquid is mixed bacterial liquid of lactobacillus plantarum and streptococcus thermophilus, regulating pH to 4.5-6.5 for fermentation, fermenting at 35-43 ℃ for 12-60 hours to obtain fermentation liquid, sterilizing the fermentation liquid, centrifuging, and collecting supernatant to obtain the snow lotus fermentation stock solution.

The technology utilizes the snow lotus herb to ferment, and the obtained ferment extract is utilized, and has the functions of antioxidation and whitening.

The technical problem to be solved by the scheme is as follows: how to enrich the efficacy of the snow lotus herb in cosmetics and improve the corresponding efficacy effect.

Disclosure of Invention

The invention aims to provide a preparation method of a snow lotus fermentation product, which adopts tremella extract, yeast extract and snow lotus powder for mixed fermentation, and the obtained fermentation product has the functions of tightening, relieving and anti-wrinkle, and simultaneously has good safety, mildness and no irritation.

Meanwhile, the invention also provides a snow lotus herb fermentation product, application and cosmetics thereof.

In order to achieve the above purpose, the present invention provides the following technical solutions: a preparation method of herba Saussureae Involueratae fermentation product comprises inoculating fermentation bacteria into herba Saussureae Involueratae fermentation broth, and performing aerobic fermentation to obtain herba Saussureae Involueratae fermentation product;

The snow lotus fermentation culture solution contains carbon source, tremella extract, yeast extract and snow lotus powder;

the fermentation bacteria are saccharomycetes and/or compound bacteria;

The compound bacteria comprise streptococcus thermophilus, lactobacillus acidophilus, lactobacillus reuteri, lactobacillus bulgaricus, bifidobacterium longum, bifidobacterium breve, bifidobacterium lactis, bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium infantis;

The concentration of the snow lotus powder in the snow lotus fermentation culture solution is 0.1-10wt%;

The concentration of the tremella extract in the snow lotus herb fermentation culture solution is 0.1-5 wt%;

the concentration of the yeast extract in the snow lotus herb fermentation culture solution is 0.1-5 wt%;

the concentration of the carbon source in the snow lotus herb fermentation culture solution is 0.1-10wt%;

the addition amount of the fermentation bacteria is 0.1-1 g/kg of the snow lotus herb fermentation culture solution.

The core of the invention is to ferment the culture medium containing tremella extract, yeast extract and snow lotus herb powder by utilizing saccharomycetes.

Specifically, the main component of the tremella extract is tremella polysaccharide, which can play a role in conditioning and moisturizing skin when acting on cosmetics alone, and when the tremella extract exists as a component of fermentation culture solution, the tremella extract can promote the activity of saccharomycetes, and the tremella extract is reported in related documents (research on the influence of tremella polysaccharide on lactobacillus acidophilus L101 fermentation growth, food research and development, volume 29, 11 th of 2008); during the research process of the project, we find that tremella polysaccharide has positive influence on the activity of saccharomycetes as well; meanwhile, the fermented product contains a large amount of active tremella polysaccharide, which can play a very good role in moisturizing, whitening and compacting;

the yeast extract is a yellow powdery or pasty product prepared by extracting soluble components (proteins, amino acids, peptides, nucleotides and the like) in yeast cells, wherein the soluble components serve as active ingredients for improving the activity of yeast on one hand, and the active ingredients contained in a fermentation product can play a good role in tightening, relieving and resisting wrinkles on the other hand.

The snow lotus flower is a common Chinese medicine, has the effects of being sweet and warm, slightly bitter, returning liver and kidney meridian, dispelling wind and dampness, strengthening tendons and bones, tonifying kidney yang, regulating menstruation and stopping bleeding. The saussurea involucrata pollen contains rich polyphenol compounds, flavonoid compounds, vitamin C, arbutin and proteins, natural plant polysaccharide, organic acid, lipid and the like, and can release active ingredients into fermentation liquor under the action of zymophyte, and meanwhile, the proteins and the like are decomposed to obtain various functional polypeptides, amino acids and the like.

The obtained saussurea involucrata fermentation product has excellent effects in tightening, relieving, resisting wrinkles and the like.

In the preparation method of the snowflake fermentation product, the carbon source is one or more of sucrose, glucose and molasses.

In the preparation method of the snowflake fermentation product, in the snowflake fermentation culture solution, the concentration of the tremella extract is 0.1-1 wt%, the concentration of the snowflake powder is 0.1-5 wt%, the concentration of the yeast extract is 0.1-1 wt%, and the concentration of the carbon source is 1-3 wt%.

In the preparation method of the snowflake fermentation product, the concentration of the tremella extract in the snowflake fermentation culture solution is 0.5wt%, the concentration of the snowflake powder is 1.25-5 wt%, the concentration of the yeast extract is 0.5wt%, the concentration of the carbon source is 2wt%, and the balance is water.

In the preparation method of the snowflake fermentation product, the fermentation bacteria are saccharomyces cerevisiae;

Or the fermentation bacteria are Saccharomyces cerevisiae and composite bacteria;

or, the zymophyte is a compound bacterium;

The composite bacteria are composite bacteria of a yogurt starter provided by Angel Yeast Co., ltd and having the name of bifidobacterium passii 10 bacteria.

In the preparation method of the snowflake fermentation product, the fermentation bacteria are added into the snowflake fermentation culture solution in the form of viable bacteria powder.

In the preparation method of the snowflake fermentation product, the method specifically comprises the following steps:

s1, mixing a carbon source, snow lotus herb powder, tremella extract, yeast extract and water to obtain snow lotus herb fermentation culture solution, and regulating the pH value of the solution;

S2, sterilizing the snow lotus fermentation culture solution;

S3, adding zymophyte into the cooled sterilized snow lotus fermentation culture solution, wherein the dissolved oxygen content value is 10% -50%, correlating the stirring rotation speed with the dissolved oxygen content, and automatically compensating and regulating the rotation speed;

S4, after fermentation culture is finished, sterilizing a fermentation culture solution;

s5, treating the snow lotus fermentation culture solution by using a centrifuge, and collecting a culture supernatant after centrifugation;

s6, clarifying and filtering the culture supernatant to obtain the saussurea involucrata fermentation product.

In the preparation method of the snowflake fermentation product, if the zymophyte only contains saccharomycetes, the fermentation temperature is 26-29 ℃ and the fermentation time is 6-72 h;

If the zymophyte is composite bacteria and saccharomycetes, firstly adding the saccharomycetes into the snow lotus fermentation culture solution, wherein the fermentation temperature is 26-29 ℃, and the fermentation time is 6-72 h; then adding the composite bacteria into the snow lotus fermentation culture solution, and increasing the fermentation temperature to 36-38 ℃ and the fermentation time to 6-72 h;

in S1, the pH is 5.5-7.5.

Meanwhile, the invention also discloses a snow lotus herb fermentation product which is prepared by adopting any one of the methods.

Meanwhile, the invention also provides application of the saussurea involucrate fermentation product in preparing daily chemicals.

Preferably, the daily chemical product is in the form of emulsion, cream, essential oil or essence with water as a continuous phase.

Finally, the invention also provides a cosmetic containing the snow lotus herb fermentation product.

Preferably, the cosmetic contains 0.01 to 10wt% of the snow lotus fermentation product.

Compared with the prior art, the invention has the beneficial effects that:

(1) In the aspect of fermentation process, different microorganism strains are selected, namely, composite bacteria such as saccharomyces cerevisiae of fungus type, bacterial microorganism bifidobacterium and the like and a combined bacterial agent containing bacterial agents such as the fungus saccharomycetes and the bifidobacterium and the like are adopted to ferment the snow lotus herb culture solution, the high-density culture of the microorganisms is realized by utilizing a fermentation tank, the efficient extraction of effective active substances of the snow lotus herb is facilitated, and meanwhile, various small molecular proteins, organic acids, lipids and the like in the culture solution are transformed in different forms by utilizing a unique metabolic enzyme system of the microorganisms, so that various beneficial novel active substances are generated.

The method utilizes the form of microbial fermentation to extract plants, so that on one hand, the content and activity of functional substances of the plants are extracted and maintained to a high degree, and on the other hand, microorganisms can generate various microbial active ingredients in the metabolic process of the plants, and simultaneously, the microorganisms are transformed to generate new functional ingredients and degrade phytotoxicity. The invention provides a preparation method of a saussurea involucrata fermentation product of different types of microorganisms, and the prepared saussurea involucrata fermentation product has the functions of tightening, relieving and resisting wrinkles, and has good safety, mildness and no irritation.

(2) In terms of raw material selection, tremella extract and yeast extract are selected to assist the fermentation preparation of snow lotus herb powder, so that the product with more abundant efficacy and stronger efficacy capability is obtained, in particular,

The tremella extract has main components of tremella polysaccharide, can play a role in conditioning and moisturizing skin when acting on cosmetics alone, and can promote the activity of saccharomycetes when being used as a component of fermentation culture solution, and is reported in related documents (research on influence of tremella polysaccharide on lactobacillus acidophilus L101 fermentation growth, food research and development, volume 29, 11 th of 2008); during the research process of the project, we find that tremella polysaccharide has positive influence on the activity of saccharomycetes as well; meanwhile, the fermented product contains a large amount of active tremella polysaccharide, which can play a very good role in moisturizing, whitening and compacting;

Through the optimization, the obtained saussurea involucrata fermentation product has excellent effects in aspects of compactness (elastase inhibition rate test), relaxation (hyaluronidase inhibition rate test), wrinkle resistance (DPPH free radical clearance rate test) and the like.

Drawings

FIG. 1 is a graph (0 min) showing the results of chick embryo chorioallantoic membrane test of the formulation of example 3 of the present invention;

FIG. 2 is a graph (5 min) showing the results of chick embryo chorioallantoic membrane test of the formulation of example 3 of the present invention;

FIG. 3 is a graph (0 min) showing the results of chick embryo chorioallantoic membrane assay for the formulation of example 4 of the present invention;

FIG. 4 is a graph (5 min) showing the results of chick embryo chorioallantoic membrane assay for the formulation of example 4 of the present invention;

FIG. 5 is a graph (0 min) showing the results of chick embryo chorioallantoic membrane assay for the formulation of example 5 of the present invention;

FIG. 6 is a graph (5 min) showing the results of chick embryo chorioallantoic membrane assay for the formulation of example 5 of the present invention;

FIG. 7 is a graph (0 min) showing the results of chick embryo chorioallantoic membrane assay using physiological saline;

FIG. 8 is a graph showing the results of chick embryo chorioallantoic membrane assay (5 min) using physiological saline;

FIG. 9 is a graph (0 min) showing the results of chick embryo chorioallantoic membrane assay using sodium hydroxide solution;

FIG. 10 is a graph showing the results of the chick embryo chorioallantoic membrane assay (5 min) using sodium hydroxide solution.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Raw materials and reagents

Tremella extract: henan Dragon bioengineering Co., ltd, polysaccharide content 50%,500 g/bag;

yeast extract: 500 g/bottle Guangzhou Ding national biotechnology Co., ltd., brand OXOID, model LP0021B, source: an inlet;

Snow lotus flower powder: 500 g/bag of Huifeng national medicine Limited, anhui, production lot number 230601, production date 20230615, and execution standard "Anhui province Chinese herbal pieces-processing Specification" 2019 edition; drug production license: anhui 20190401;

saccharomyces cerevisiae: the collection number of Guangdong microorganism strain collection center is CGMCC 2.1364; freeze-dried powder/1 branch;

Composite bacteria: angel Yeast Co., ltd 8 g/bag; brand: baker stream/drill; series: 10 bacteria; the production place: suzhou city of Jiangsu province in China; name: bifidobacterium bailii 10-strain yoghurt starter; production license number: SC10632050900407.

Example 1 preparation of Single fermentation inoculant Yeast snow lotus herb fermentation product

The saussurea involucrata fermentation culture solution is prepared by mixing and preparing the saussurea involucrata fermentation culture solution according to the proportion of 20 g of glucose, 5g of tremella extract, 5g of yeast extract and 12.5 g of saussurea involucrata powder per 1 kg, and the pH value of the solution is regulated to 6.0. After the preparation, the culture solution is sterilized at the temperature of 121 ℃ for 20min under high temperature and high pressure. And cooling the culture solution, and adding a saccharomyces cerevisiae microbial inoculum into the sterilized snow lotus fermentation culture solution, wherein the ratio of the saccharomyces cerevisiae microbial inoculum to the sterilized snow lotus fermentation culture solution is 0.5 gram per 1 kilogram of culture solution. Setting the constant-temperature culture temperature of the fermentation tank at 28 ℃, wherein the dissolved oxygen content value is 30%, correlating the stirring rotating speed with the dissolved oxygen content, automatically compensating and adjusting the rotating speed, and fermenting and culturing for 24 hours. After the fermentation culture is finished, the culture solution is sterilized at the temperature of 121 ℃ for 20min under high temperature and high pressure. After the sterilization, the culture broth of the snow lotus fermentation was treated with a centrifuge at 12000rpm, and the culture supernatant after centrifugation was collected. Clarifying the culture supernatant to obtain the saussurea involucrata fermentation product.

EXAMPLE 2 preparation of Single fermentation inoculant Yeast snow lotus herb fermentation product

The saussurea involucrata fermentation culture solution is prepared by mixing and preparing the saussurea involucrata fermentation culture solution according to the proportion of 20 g of glucose, 5 g of tremella extract, 5 g of yeast extract and 25 g of saussurea involucrata powder per 1 kg, and the pH value of the solution is regulated to 6.0. After the preparation, the culture solution is sterilized at the temperature of 121 ℃ for 20min under high temperature and high pressure. And cooling the culture solution, and adding a saccharomyces cerevisiae microbial inoculum into the sterilized snow lotus fermentation culture solution, wherein the ratio of the saccharomyces cerevisiae microbial inoculum to the sterilized snow lotus fermentation culture solution is 0.5 gram per 1 kilogram of culture solution. Setting the constant-temperature culture temperature of the fermentation tank at 28 ℃, wherein the dissolved oxygen content value is 30%, correlating the stirring rotating speed with the dissolved oxygen content, automatically compensating and adjusting the rotating speed, and fermenting and culturing for 24 hours. After the fermentation culture is finished, the culture solution is sterilized at the temperature of 121 ℃ for 20min under high temperature and high pressure. After the sterilization, the culture broth of the snow lotus fermentation was treated with a centrifuge at 12000rpm, and the culture supernatant after centrifugation was collected. Clarifying the culture supernatant to obtain the saussurea involucrata fermentation product.

Example 3 preparation of Single fermentation inoculant Yeast snow lotus herb fermentation product

The saussurea involucrata fermentation culture solution is prepared by mixing and preparing the saussurea involucrata fermentation culture solution according to the proportion of 20 g of glucose, 5 g of tremella extract, 5 g of yeast extract and 50 g of saussurea involucrata powder per 1 kg, and the pH value of the solution is regulated to 6.0. After the preparation, the culture solution is sterilized at the temperature of 121 ℃ for 20min under high temperature and high pressure. And cooling the culture solution, and adding a saccharomyces cerevisiae microbial inoculum into the sterilized snow lotus fermentation culture solution, wherein the ratio of the saccharomyces cerevisiae microbial inoculum to the sterilized snow lotus fermentation culture solution is 0.5 gram per 1 kilogram of culture solution. Setting the constant-temperature culture temperature of the fermentation tank at 28 ℃, wherein the dissolved oxygen content value is 30%, correlating the stirring rotating speed with the dissolved oxygen content, automatically compensating and adjusting the rotating speed, and fermenting and culturing for 24 hours. After the fermentation culture is finished, the culture solution is sterilized at the temperature of 121 ℃ for 20min under high temperature and high pressure. After the sterilization, the culture broth of the snow lotus fermentation was treated with a centrifuge at 12000rpm, and the culture supernatant after centrifugation was collected. Clarifying the culture supernatant to obtain the saussurea involucrata fermentation product.

Example 4 preparation of a fermentation product of Compound bacteria snow lotus

The saussurea involucrata fermentation culture solution is prepared by mixing and preparing the saussurea involucrata fermentation culture solution according to the proportion of 20 g of glucose, 5g of tremella extract, 5g of yeast extract and 50 g of saussurea involucrata powder per 1 kg, and the pH value of the solution is regulated to 6.0. After the preparation, the culture solution is sterilized at the temperature of 121 ℃ for 20min under high temperature and high pressure. And (3) adding a composite bacterial fermentation inoculant into the sterilized snow lotus fermentation culture solution after the culture solution is cooled, wherein the ratio of the composite bacterial fermentation inoculant to the sterilized snow lotus fermentation culture solution is 0.5 gram per 1 kilogram of culture solution. Setting the constant temperature culture temperature of the fermentation tank at 37 ℃, wherein the value of the dissolved oxygen content is 30%, correlating the stirring rotating speed with the dissolved oxygen content, automatically compensating and adjusting the rotating speed, and fermenting and culturing for 24 hours. After the fermentation culture is finished, the culture solution is sterilized at the temperature of 121 ℃ for 20min under high temperature and high pressure. After the sterilization, the culture broth of the snow lotus fermentation was treated with a centrifuge at 12000rpm, and the culture supernatant after centrifugation was collected. Clarifying the culture supernatant to obtain the saussurea involucrata fermentation product.

Example 5 preparation of a fermentation product of snow lotus with a Complex bacterium and Yeast Combined fermentation inoculant

The saussurea involucrata fermentation culture solution is prepared by mixing and preparing the saussurea involucrata fermentation culture solution according to the proportion of 20 g of glucose, 5g of tremella extract, 5g of yeast extract and 50 g of saussurea involucrata powder per 1 kg, and the pH value of the solution is regulated to 6.0. After the preparation, the culture solution is sterilized at the temperature of 121 ℃ for 20min under high temperature and high pressure. And cooling the culture solution, and adding a saccharomyces cerevisiae microbial inoculum into the sterilized snow lotus fermentation culture solution, wherein the ratio of the saccharomyces cerevisiae microbial inoculum to the sterilized snow lotus fermentation culture solution is 0.5 gram per 1 kilogram of culture solution. Setting the constant-temperature culture temperature of the fermentation tank at 28 ℃, wherein the dissolved oxygen content value is 30%, correlating the stirring rotating speed with the dissolved oxygen content, automatically compensating and adjusting the rotating speed, and fermenting and culturing for 24 hours. After 24h, the composite bacterial fermenting agent is added in a proportion of 0.5 g per 1 kg of culture solution. Setting the constant temperature culture temperature of the fermentation tank at 37 ℃, wherein the value of the dissolved oxygen content is 30%, correlating the stirring rotating speed with the dissolved oxygen content, automatically compensating and adjusting the rotating speed, and fermenting and culturing for 24 hours. After the fermentation culture is finished, the culture solution is sterilized at the temperature of 121 ℃ for 20min under high temperature and high pressure. After the sterilization, the culture broth of the snow lotus fermentation was treated with a centrifuge at 12000rpm, and the culture supernatant after centrifugation was collected. Clarifying the culture supernatant to obtain the saussurea involucrata fermentation product.

Example 6

The procedure was as in example 5, except that 10g of glucose, 10g of tremella extract, 10g of yeast extract and 10g of snow lotus powder were mixed and formulated in a ratio of 10g of glucose per 1 kg to obtain snow lotus fermentation broth.

Example 7

The procedure was as in example 5, except that 30 g of glucose, 1 g of tremella extract, 1 g of yeast extract and 1 g of snow lotus powder were mixed and formulated in a ratio of 30 g of glucose per 1 kg to obtain snow lotus fermentation broth.

Comparative example 1

The procedure was as in example 5, except that the fermentation broth of saussurea involucrata was prepared by mixing and formulating the mixture at a ratio of 20 g glucose, 10 g yeast extract and 50 g saussurea involucrata powder per 1 kg.

Comparative example 2

The procedure of example 5 was repeated except that the culture broth was prepared by mixing 20 g of glucose, 5g of ganoderan extract, 5g of yeast extract and 50 g of snow lotus powder per 1 kg of the culture broth.

Comparative example 3

The procedure of example 5 was repeated except that the culture broth was prepared by mixing 20 g of glucose, 5g of algal polysaccharide extract, 5g of yeast extract and 50 g of snow lotus powder per 1 kg of the culture broth.

Comparative example 4

The procedure is substantially as in example 5, except that the fermentation broth is a combination of bacteria and monascus, and that the first-step saccharomyces cerevisiae is replaced by monascus in equal proportions.

Comparative example 5

Preparing a mixed solution, wherein the mixed solution contains 5g of tremella extract, 5g of yeast extract, 50 g of snow lotus herb extract (SAUSSUREA INVOLUCRATA EXTRACT, supplier: huifeng national medicine Co., ltd.) and 1kg of deionized water; stirring and dispersing, fully dissolving and soaking the raw materials for 24 hours, and then filtering to obtain the essence.

Test example 1 efficacy test of snow lotus fermentation product

The testing method comprises the following steps:

1.1 elastase inhibition test

Control and test sample treatment:

Sample: the snow lotus fermentation products of examples 1 to 7, and the snow lotus fermentation products of comparative examples 1 to 4, and the essence of comparative example 5.

Positive control (EGCG): 0.1% epigallocatechin gallate (EGCG, 98%) in water.

Negative control: pure water.

Test procedure

Setting up a sample group, a sample background group, a solvent group and a solvent background group, setting up 3 groups in parallel, respectively adding different reagent solutions into a 96-well plate, gently shaking, incubating at 25 ℃ for 15min, placing the sample groups in an enzyme-labeled instrument, and measuring the absorbance at 410 nm.

Calculation formula

；

Wherein: a-is the absorbance of the reaction solution without the sample;

b-is the absorbance of the reaction solution without sample and enzyme;

C-absorbance of a reaction solution containing the sample and the enzyme;

D-is the absorbance of the reaction solution containing the sample and containing no enzyme.

Data analysis

The statistical analysis software is SPSS, and the comparison among the elastase inhibition rates of the test sample, the positive control and the negative control adopts independent sample t test. The above statistical analysis was a two-tailed test with a significance level of a=0.05. P is more than or equal to 0.05, which indicates that no significant difference exists between the two groups; p < 0.05, indicating a significant difference between the two groups.

1.2 Hyaluronidase inhibition test

Control and test sample treatment

Sample: examples 1 to 7, and comparative examples 1 to 4, and comparative example 5.

Positive control: 3% dipotassium glycyrrhizinate aqueous solution (dipotassium glycyrrhizinate, purity is more than or equal to 98%).

Negative control: pure water.

Test procedure

Setting up a sample group, a sample background group, a solvent group and a solvent background group, setting up 3 parallel groups each, adding different reagent solutions into the four groups respectively, shaking uniformly, standing at room temperature for 30min for color development, and measuring the absorbance value at the wavelength of 528nm by using an ultraviolet spectrophotometer.

Calculation formula

；

Wherein: a-is the absorbance of the reaction solution without the sample;

b-is the absorbance of the reaction solution without sample and enzyme;

C-absorbance of a reaction solution containing the sample and the enzyme;

Data analysis

The statistical analysis software is SPSS, and independent sample t test is adopted for comparison among the hyaluronidase inhibition rates of the test sample, the positive control substance and the negative control substance. The above statistical analysis was a two-tailed test with a significance level of a=0.05. P is more than or equal to 0.05, which indicates that no significant difference exists between the two groups; p < 0.05, indicating a significant difference between the two groups.

1.3 DPPH radical scavenging test

Control and test sample treatment:

Positive control: 0.1% vitamin E (purity not less than 95%) in ethanol (95%).

Negative control: pure water.

Test procedure

Setting up a sample tube, a sample background tube, a DPPH tube and a solvent background tube, setting up 3 parallel tubes in each group, adding different reagent solutions into the four groups respectively, slightly and uniformly shaking, and standing for 5min at room temperature. Each set of reaction solutions was transferred to a 1cm cuvette and absorbance was measured at 517 nm.

Calculation formula

；

Wherein:

t-is the absorbance of the sample tube, i.e. the absorbance of the solution after the reaction of the sample and DPPH;

T0-is the sample background absorbance;

C is the light absorption value of the DPPH tube for 3 times, namely the light absorption value of the DPPH solution when no sample is added;

C0-is the background absorbance of the solution.

Data analysis

Test results

The results of the% elastase inhibition test are shown in table 1:

TABLE 1 results of elastase inhibition rate

Project	Elastase inhibition%	P value
			Example 1	41.322±0.716	＜0.05
Example 2	43.388±2.581	＜0.05
			Example 3	45.868±0.716	＜0.05
Example 4	46.694±5.010	＜0.05
			Example 5	45.041±5.726	＜0.05
Example 6	20.909±2.727	＜0.05
			Example 7	19.091±1.575	＜0.05
Comparative example 1	8.182±2.727	＜0.05
			Comparative example 2	30.928±1.786	＜0.05
Comparative example 3	21.649±3.571	＜0.05
			Comparative example 4	23.711±3.093	＜0.05
Comparative example 5	6.186±3.093	＜0.05
			Negative control	-6.098±1.829	/
Positive control	87.805±2.794	＜0.05

As can be seen from Table 1, the saussurea involucrata fermentation products obtained in examples 1-5 have a relatively obvious inhibition effect on elastase, and the detection data shows that the saussurea involucrata fermentation products obtained by adopting different strains have a relatively excellent relieving effect by adopting a fermentation mode of saussurea involucrata, namely, using a single microbial agent, namely, saccharomyces cerevisiae, a composite microbial agent, and the combined microbial agent, wherein the Saccharomyces cerevisiae, the composite microbial agent and the like.

As can be seen from comparative examples 1 to 3, the tremella extract is a key component in the present invention, and its elastase inhibitory effect is remarkably reduced when it is replaced with other components having similar functional polysaccharides.

The results of the% hyaluronidase inhibition test are shown in table 2:

TABLE 2 hyaluronidase inhibition results

Project	Hyaluronidase inhibition%	P value
			Example 1	25.615±0.355	＜0.05
Example 2	39.754±0.939	＜0.05
			Example 3	41.393±1.280	＜0.05
Example 4	35.246±0.939	＜0.05
			Example 5	30.123±1.775	＜0.05
Example 6	22.417±0.338	＜0.05
			Example 7	35.283±1.217	＜0.05
Comparative example 1	43.080±0.585	＜0.05
			Comparative example 2	21.942±0.360	＜0.05
Comparative example 3	29.844±1.589	＜0.05
			Comparative example 4	31.175±1.362	＜0.05
Comparative example 5	40.048±0.719	＜0.05
			Negative control	-2.667±2.0367	/
Positive control	57.33±0.7689	＜0.05

As can be seen from Table 2, the snow lotus fermentation products obtained in examples 1-7 have a relatively obvious inhibition effect on hyaluronidase, and the detection data show that the snow lotus fermentation products obtained by fermentation have a relatively excellent tightening effect by adopting different strains to ferment snow lotus, namely using a single microbial agent, namely Saccharomyces cerevisiae, composite bacteria, a combined microbial agent comprising Saccharomyces cerevisiae, composite bacteria and the like.

It should be noted that: during the hyaluronidase assay, some data results which are more difficult to understand were presented, and the inhibition effect of comparative example 5 was superior to that of example 5, which was not expected during the experimental run. Or it is hypothesized that fermentation does not favor an increase in hyaluronidase inhibition.

At the same time, the inhibition effect of example 7 is superior to that of examples 5 and 6, which is also difficult to understand, and a reasonable mechanism explanation cannot be given for the result temporarily through the discussion of the meeting of the detection personnel.

It can be seen from examples 5 and comparative examples 1 to 3 that the tremella extract does not take absolute advantage of the inhibition of hyaluronidase.

The% DPPH radical scavenging test results are shown in table 3:

TABLE 3 DPPH radical scavenging results

Project	DPPH radical scavenging%	P value
			Example 1	46.247±0.951	＜0.05
Example 2	41.614±0.686	＜0.05
			Example 3	49.718±1.238	＜0.05
Example 4	56.250±1.145	＜0.05
			Example 5	47.582±1.011	＜0.05
Example 6	16.028±2.830	＜0.05
			Example 7	16.695±2.806	＜0.05
Comparative example 1	2.064±0.865	＜0.05
			Comparative example 2	26.108±1.156	＜0.05
Comparative example 3	18.059±1.654	＜0.05
			Comparative example 4	19.235±2.386	＜0.05
Comparative example 5	1.938±0.199	＜0.05
			Negative control	-2.721±0.012	/
Positive control	85.355±1.287	＜0.05

As shown in Table 3, the saussurea involucrata fermentation products obtained in examples 1-5 have obvious effect of scavenging DPPH free radicals, and the detection data shows that the saussurea involucrata fermentation products obtained by adopting different strains have excellent anti-wrinkle effects by adopting a single microbial agent of Saccharomyces cerevisiae, composite bacteria and a combined microbial agent of Saccharomyces cerevisiae, composite bacteria and the like.

Summarizing: the following conclusions can be drawn from tables 1 to 3:

1. The examples are generally superior to the comparative examples in terms of DPPH radical scavenging rate, elastase inhibition rate;

2. the individual test groups had some uncertainty in terms of hyaluronidase inhibition, but it is clear that fermentation with a single strain would be superior to that with a complex strain.

3. The formulations represented by examples 3-5 were chosen to be the most preferred of the present invention based on a comprehensive advantage judgment.

4. Based on comprehensive advantage judgment, the composite bacterial fermentation inoculant is adopted for fermentation due to secondary fermentation and single saccharomyces cerevisiae fermentation.

5. Based on comprehensive advantage judgment, the tremella extract is an important and indispensable means in the formula.

6. Based on the results in table 2, it is suggested that, in the practical application formulation, additional addition of tremella extract, yeast extract, and snow lotus extract on the basis of the fermentation product may be one direction for optimizing various performances. In subsequent experiments, research and discussion of this aspect will be conducted; 10 to 50% by weight of the essence shown in comparative example 5 was added on the basis of examples 1 to 5, in particular on the basis of example 4.

Test example 2 safety test of snow lotus fermentation product

1. Test method

Chick embryo chorioallantoic membrane assay

Control and test sample treatment

Sample: examples 3 to 5 snow lotus fermentation products.

Negative control: physiological saline.

Positive control: 0.1mol/L NaOH solution.

Test procedure

6 Chick embryos are selected for each group in the test, the condition of chorioallantoic membranes is recorded by using photographing equipment, and a polytetrafluoroethylene resin ring is placed on the chorioallantoic membranes of the chick embryos and photographed and recorded. And adding a sample to be tested into the polytetrafluoroethylene resin ring, recording the time of adding the sample, covering the air chamber with a wetted preservative film, transferring the chick embryo into a constant temperature and humidity box for culture, and observing the degree of each toxic effect change.

Calculation results:

Performing a test by adopting an end point evaluation method, calculating end point Evaluation (ES), and reserving two positions after decimal points; score per chick embryo = sum of extent of bleeding, coagulation and vascular thawing observed per chick embryo; mean of the mathematical sums obtained for ES-6 chick embryos. The ES average score is calculated according to the following formula:

；

Result determination criteria:

ES is less than or equal to 4, and has no irritation; ES is more than 4 and less than or equal to 12, and has light irritation; ES is more than 12 and less than 16, and has moderate irritation; ES is more than or equal to 16, and has strong irritation/corrosiveness.

2. Test results

The results of the chick embryo chorioallantoic membrane test are shown in Table 4:

TABLE 4 results of chick embryo chorioallantoic membrane test

Project	ES score	Results
			Example 3	3.67	No irritation
Example 4	4.00	No irritation
			Example 5	3.67	No irritation
Positive control	18.00	Strong irritation

Examples 1 to 3 all use a single microbial inoculum of Saccharomyces cerevisiae and the same fermentation method, except that the amount of snow lotus powder added in example 3 is larger, and the efficacy test data in examples 1 to 3 show that the efficacy data in example 3 are superior to those in examples 1 to 2, so that only the snow lotus fermentation products in examples 3 to 5 are subjected to safety test.

As can be seen from Table 4 and accompanying figures 1-10, the snow lotus fermentation products obtained in examples 3-5 have no irritation to chick embryo chorioallantoic membrane, so that the invention can be demonstrated to adopt different strains to ferment snow lotus, namely, single bacteria such as Saccharomyces cerevisiae, composite bacteria such as Lactobacillus bulgaricus and bifidobacterium are used, and the combined bacteria such as Saccharomyces cerevisiae and composite bacteria are used, so that the snow lotus fermentation products obtained by fermentation have good safety.

Application example

A galactose yeast snow lotus essence consists of A phase, B phase, C phase and D phase:

The phase A comprises the following components in percentage by mass: 3g of 1, 3-butanediol, 5g of glycerol, 0.2g Carbopol 941 g of disodium EDTA, 0.02g of hyaluronic acid;

the phase B comprises the following components in percentage by mass: 0.05g of octanoyl hydroxyvaleric acid, 0.05g of ethylhexyl glycerol, 0.6g of 1, 2-hexanediol, 1g of 1, 2-pentanediol;

the phase C comprises the following components in percentage by mass: 0.2g arginine;

The phase D comprises the following components in percentage by mass: 5g of snow lotus herb fermentation stock solution.

A galactose yeast snow lotus essence is prepared by adding phase A into an emulsifying pot, heating to 85deg.C, stirring, cooling to 45deg.C, sequentially adding B, C, D phases, and stirring at 30r/min for 10 min.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims

1. A preparation method of a snowflake fermentation product is characterized in that zymophyte is inoculated into a snowflake fermentation culture solution for aerobic fermentation treatment, and the snowflake fermentation product is obtained through sterilization, separation and collection of supernatant; the dissolved oxygen content value of the snow lotus fermentation culture solution is 10% -50%, the pH value is 5.5-7.5, the fermentation temperature of the aerobic fermentation is 37 ℃, and the fermentation time is 24 hours;

The fermentation bacteria are composite bacteria; the product name of the composite bacteria is the composite bacteria of the bifidobacterium passii 10-strain type yoghurt starter;

In the snow lotus fermentation culture solution, the concentration of the tremella extract is 0.1-1 wt%, the concentration of snow lotus powder is 0.1-5 wt%, the concentration of the yeast extract is 0.1-1 wt%, and the concentration of the carbon source is 1-3 wt%;

the addition amount of the zymophyte is 0.1-1 g/kg of snow lotus fermentation culture solution;

The fermentation bacteria are added into the snow lotus fermentation culture solution in the form of viable bacteria powder.

2. The method for producing a fermentation product of saussurea involucrate according to claim 1, wherein the carbon source is one or more of sucrose, glucose, and molasses.

3. The method for preparing a snow lotus fermentation product according to claim 1 or 2, characterized in that it comprises the following steps:

S2, sterilizing the snow lotus fermentation culture solution;

S3, adding zymophyte into the cooled sterilized snow lotus fermentation culture solution, correlating the stirring rotation speed with the dissolved oxygen content, and automatically compensating and regulating the rotation speed;

S5, treating the snow lotus fermentation culture solution subjected to fermentation culture and sterilization by using a centrifuge, and collecting a culture supernatant after centrifugation;

4. A snow lotus fermentation product, characterized in that it is prepared by the method according to any one of claims 1 to 3.

5. Use of the snow lotus fermentation product according to claim 4 for preparing daily chemicals.

6. The use according to claim 5, wherein the daily chemical product is in the form of an emulsion, cream, essential oil or water-based continuous phase concentrate.

7. A cosmetic comprising the fermentation product of saussurea involucrate according to claim 4.

8. The cosmetic according to claim 7, wherein the product of fermentation of saussurea involucrata is contained in an amount of 0.01 to 10 wt%.