CN117904247A

CN117904247A - Lactobacillus soybean milk fermentation product with tightening and repairing effects, and preparation method and application thereof

Info

Publication number: CN117904247A
Application number: CN202311770657.1A
Authority: CN
Inventors: 刘婉; 覃贻佳; 周广祥
Original assignee: Guangzhou Xinghui Biotechnology Co ltd
Current assignee: Guangzhou Xinghui Biotechnology Co ltd
Priority date: 2023-12-20
Filing date: 2023-12-20
Publication date: 2024-04-19
Anticipated expiration: 2043-12-20
Also published as: CN117904247B

Abstract

The application discloses a lactobacillus soybean milk fermentation product with tightening and repairing effects, and a preparation method and application thereof. The preparation method of the soybean milk fermentation product comprises the following steps: (1) Uniformly mixing the soybean milk powder and water, and sterilizing to obtain a fermentation raw material; (2) Inoculating lactobacillus seed solution and schizophyllum strain seed solution into fermentation raw materials for fermentation to obtain a crude fermentation product; (3) Sterilizing and filtering the crude fermentation product to obtain the lactobacillus soybean milk fermentation product with the tightening and repairing effects. The application utilizes lactobacillus and schizophyllum commune to carry out mixed fermentation on soybean milk, the lactobacillus and the schizophyllum commune decompose macromolecular proteins into micromolecular peptides, and schizophyllum commune and a plurality of other skin care components are generated in the fermentation process, and the final product has good effects of moisturizing, tightening, repairing and reddening, and is suitable for being applied to preparing cosmetics.

Description

Lactobacillus soybean milk fermentation product with tightening and repairing effects, and preparation method and application thereof

Technical Field

The application belongs to the technical field of biological fermentation, and particularly relates to a lactobacillus soybean milk fermentation product with a tightening and repairing effect, and a preparation method and application thereof.

Background

Soybean is one of the important grain crops in china and has been cultivated for five thousand years. The soybean has rich nutrients, vegetable protein content over 35%, and the protein content is 3-8 times of that of potato and grain crops. In addition to proteins, soybeans contain nutrients necessary for the human body such as fat, calcium, phosphorus, iron, and vitamins.

People have a long history of eating and using soybeans. In daily life, people prepare soybean into various bean products such as soybean milk, bean curd, dried beancurd sticks and the like for eating. Part of people loving beauty also use the bean dregs remained after the preparation of the soybean milk to directly apply the face so as to achieve the effect of skin care. In modern cosmetics, bean products are also added as active ingredients to skin care products. The patent application with the application number of CN200710303768.6 discloses a facial mask, which is prepared by taking soybean protein as a main active ingredient and matching with water extracts of pearl powder and ginseng. The patent application with the application number of CN202310199077.5 discloses a high-efficiency ginseng soybean facial mask, which contains various active ingredients such as soybean extract, ginseng total saponin, deoxyarbutin, gingerol and the like, and can play a role in skin care.

The soybean milk contains abundant vegetable proteins, but the soybean proteins are macromolecular proteins, and when the soybean proteins or the soybean extracts are directly smeared on the face, the protein molecules cannot be directly absorbed by the skin, so that the skin care function of cosmetics taking the soybean extracts or the soybean proteins as active ingredients is poor, and the skin care effect of the soybean cannot be well exerted.

Disclosure of Invention

In order to solve the problems, the application provides a lactobacillus soybean milk fermentation product with a tightening and repairing effect, and a preparation method and application thereof.

In order to solve the technical problems, the technical scheme provided by the application is as follows:

in a first aspect, the present application provides a method for preparing a lactobacillus soymilk fermentation product with a tightening and repairing effect, comprising:

(1) Uniformly mixing the soybean milk powder and water, and sterilizing to obtain a fermentation raw material;

(2) Inoculating lactobacillus seed solution and schizophyllum strain seed solution to the fermentation raw material in the step (1) for fermentation to obtain a crude fermentation product;

(3) And (3) sterilizing and filtering the crude fermentation product in the step (2) to obtain the lactobacillus soybean milk fermentation product with the tightening and repairing effects.

The application adds most lactobacillus and a small amount of schizophyllum into the fermentation raw material containing soybean milk powder for fermentation. The soybean milk powder is dissolved in water to obtain soybean milk, and contains nutrients such as protein, vitamins, fat, etc. Lactobacillus and schizophyllum are used for completing growth and metabolism by utilizing nutrient substances in soybean milk. The large molecular proteins in the soybean milk are decomposed into small molecular peptides and other active substances by various enzymes in lactobacillus and schizophyllum commune; schizophyllum commune also produces schizophyllum commune during growth and metabolism. In the process of mixed fermentation, substances generated by lactobacillus and schizophyllum commune can be used by another strain, the growth and metabolism of the other strain are promoted, and the final fermentation product has high active substance content, so that the effects of moisturizing, tightening, repairing and reddening are better realized.

In some embodiments, the fermentation feedstock in step (1) comprises 3-8% soy milk powder and 92-97% water by mass percent.

In some embodiments, the soy milk powder in step (1) is a soy milk powder.

The applicant finds that the addition of a certain amount of soybean milk powder to the fermentation raw material can promote the growth and metabolism of lactobacillus and schizophyllum commune, so that the final product has better moisturizing, tightening, repairing and reddening effects. When the addition amount of the soybean milk powder is less than 3%, initial nutrients in the fermentation raw materials are less, the growth of lactobacillus and schizophyllum commune is slower, active substances generated in the fermentation process are less, and the moisturizing, tightening, repairing and reddening effects of the final product are poor. When the added amount of the soybean milk powder is more than 8%, the nutrition substances in the fermentation raw materials are excessive, the lactobacillus and the schizophyllum commune grow in a large amount initially, the skin care active substances are not metabolized, and the lactobacillus and the schizophyllum commune consume the skin care active substances generated by metabolism in order to continue to maintain growth when entering the later period of fermentation, so that the skin care effect of the final product is poor, and therefore, the added amount of the soybean milk powder is controlled to be 3-8% in the application.

In some embodiments, the sterilization in step (1) is to keep the mixed soybean milk powder and water at 115-130 ℃ for 15-30min.

In some embodiments, the total inoculum size of the lactobacillus seed liquid and the schizophyllum commune seed liquid in step (2) is 1-4% of the volume of the fermentation feedstock.

In some embodiments, the volume ratio of the lactobacillus seed solution to the schizophyllum commune seed solution in step (2) is (3-5): 1.

The applicant finds that the final product has good moisturizing, tightening, repairing and reddening effects through mixed fermentation of lactobacillus and schizophyllum commune to soybean milk through a large number of experiments. The skin care effect of the finally obtained fermentation product is poor by fermenting only lactobacillus or only schizophyllum commune. Further, the applicant found that the addition ratio of lactobacillus seed solution and schizophyllum commune was controlled during fermentation, and the moisturizing, tightening, repairing and reddening effects of the final fermentation product were different. If the lactobacillus seed solution is too much, schizophyllum commune is liable to grow in the fermentation process, and finally becomes only lactobacillus for fermentation. If the adding ratio of schizophyllum commune is too large, the fermentation raw materials become sticky gradually in the fermentation process, and the tightening, repairing and reddening effects of the final product are poor.

In some embodiments, the lactobacillus in step (2) is at least one of lactobacillus plantarum, lactobacillus brevis, lactobacillus rhamnosus.

In some embodiments, the method for preparing the lactobacillus seed solution in step (2) comprises:

A1, picking lactobacillus colony to activate in a seed culture medium A to obtain lactobacillus suspension;

A2, inoculating the lactobacillus suspension to the seed culture medium A for expansion culture to obtain the lactobacillus seed solution.

In some embodiments, the temperature of the activation in step A1 is 33-40 ℃ and the time of the activation is 20-30 hours.

In some embodiments, the lactobacillus suspension in step A2 is added in an amount of 0.5-3% of the volume of the seed medium a in step A2.

In some embodiments, the temperature of the expansion culture in step A2 is 33-40 ℃, the time of the expansion culture is 40-55h, and the rotation speed of the fermentation shake flask during the expansion culture is 100-180rpm.

In some embodiments, the seed medium a in step A1 or A2 is MRS medium.

In some embodiments, the method for preparing schizophyllum commune seed solution in step (2) comprises:

B1, picking schizophyllum commune colony into a seed culture medium B for activation to obtain schizophyllum commune suspension;

And B2, inoculating the schizophyllum commune suspension to the seed culture medium B for expansion culture to obtain the schizophyllum commune seed solution.

In some embodiments, the temperature of the activation in step B1 is 25-33 ℃ and the time of the activation is 72-144 hours.

In some embodiments, the schizophyllum commune suspension in step B2 is added in an amount of 0.5-3% by volume of the seed medium B in step B2.

In some embodiments, the temperature of the expansion culture in step B2 is 25-33 ℃, the time of the expansion culture is 72-144 hours, and the rotation speed of the fermentation shake flask during the expansion culture is 100-180rpm.

In some embodiments, the seed medium B in step B1 or B2 is at least one of potato dextrose aqueous medium, potato wheat bran medium.

In some embodiments, the temperature of the fermentation in step (2) is 32-37 ℃, the sterile air ventilation during the fermentation is 0.3-0.8vvm, and the fermentation time is 60-80 hours.

In some embodiments, the sterilization in step (3) is incubating the crude fermentation product at 80-95 ℃ for 15-30min.

In some embodiments, the filtration in step (3) is a diatom filter filtration.

In a second aspect, the application provides a lactobacillus soymilk fermentation product with tightening and repairing effects, which is prepared by adopting the method.

The lactobacillus soybean milk fermentation product prepared by the application has good effects of moisturizing, tightening, repairing and improving redness.

In a third aspect, the present application provides the use of the lactobacillus soymilk fermentation product with tightening and repairing effects in the preparation of cosmetics.

In some embodiments, the lactobacillus soymilk fermentation product with the tightening and repairing effects is added into the cosmetic in an amount of 0.1-20% by mass percent.

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

(1) The application inoculates most lactobacillus and a small amount of schizophyllum commune into the fermentation raw material containing soybean milk powder for fermentation, the lactobacillus and the schizophyllum commune utilize the nutrient substances of the soybean milk powder for growth and metabolism to generate various active substances including schizophyllum commune, and meanwhile, the schizophyllum commune breaks down macromolecular plant proteins in the soybean milk into micromolecular peptides in the growth process, and the micromolecular peptides are easy to permeate into the skin, so that the skin care effect is better exerted.

(2) The soybean milk fermentation product prepared by the application contains schizophyllan, small molecular peptide and other active substances generated in the metabolic process of lactobacillus and schizophyllan, and can well play the effects of moisturizing, tightening, repairing, reddening and relieving.

(3) The strain used in the application is the practical lactobacillus and schizophyllum, the fermentation raw material is soybean milk powder, and the soybean milk powder is a common food raw material, and the fermentation product is green, healthy, safe and non-irritating, and is suitable for being applied to cosmetics.

Detailed Description

The present application will be described in further detail with reference to examples, but embodiments of the present application are not limited thereto. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The reagents used in the examples are commercially available as usual unless otherwise specified.

All percentages mentioned in the present application are mass percentages unless otherwise indicated.

The soybean milk powder (soybean milk powder) and the black soybean milk powder used in the application are common soybean milk powder and black soybean milk powder in the market.

The schizophyllum commune model used in the application is GIM5.44, which is sourced from the collection of patent strains of the institute of microbiology, guangdong province.

The lactobacillus plantarum used in the application is BNCC187903 in model number, the lactobacillus brevis used in the application is BNCC337373 in model number, and the saccharomyces cerevisiae used in the application is BNCC337309 in model number, and the strains are all from Beijing Beidou Nakau biological technology institute.

The lactobacillus rhamnosus used in the application is CICC 20257 and is sourced from Beijing Yuwei technology Co.

The potato dextrose water culture medium, potato wheat bran culture medium and MRS culture medium used in the application can be obtained commercially, and can also be prepared by the following formula:

Potato dextrose water medium: 200g/L potato soaked powder, 20g/L glucose and the balance of water.

Potato wheat bran medium: 200g/L potato, 100g/L wheat bran, 20g/L glucose, 2g/L monopotassium phosphate, 5g/L magnesium sulfate and the balance water.

MRS medium: 10g/L peptone, 8g/L beef extract powder, 4g/L yeast powder, 20g/L glucose, 0.2g/L magnesium sulfate, 5g/L sodium acetate, 2g/L diammonium citrate, 2g/L dipotassium hydrogen phosphate, 0.04g/L manganese sulfate, 1g/L Tween 80 and the balance water.

The preparation method of the culture medium comprises the following steps: mixing the raw materials in the formula with water, sterilizing at 115-130deg.C for 20-30min, and storing.

The preparation method of the mung bean powder in the comparative example comprises the following steps: pulverizing dried semen Phaseoli Radiati in a pulverizer, sieving, and collecting lower layer powder.

The preparation method of the red bean powder in the comparative example comprises the following steps: pulverizing dried semen Ormosiae Hosiei in a pulverizer, sieving, and collecting lower layer powder.

Example 1

The preparation method of the lactobacillus soybean milk fermentation product with the tightening and repairing effects comprises the following steps:

(1) Uniformly mixing 5% of soybean milk powder and 95% of deionized water, and preserving heat at 121 ℃ for 20min for sterilization to obtain fermentation raw materials.

(2) Inoculating lactobacillus plantarum seed liquid and schizophyllum strain seed liquid (the volume ratio of the lactobacillus plantarum seed liquid to the schizophyllum strain seed liquid is 4:1, namely, the lactobacillus plantarum seed liquid volume fraction is 2.0 percent, and the schizophyllum strain seed liquid volume fraction is 0.5%) with the total volume fraction of 2.5 percent into the fermentation raw material in the step (1), and fermenting for 70 hours at 35 ℃ under the condition that the sterile air ventilation is 0.5vvm, so as to obtain a crude fermentation product.

(3) And (3) preserving the temperature of the crude fermentation product at 90 ℃ for 20min, cooling, and filtering the sterilized crude fermentation product through a diatom filter to obtain the lactobacillus soybean milk fermentation product with the tightening and repairing effects.

The preparation method of the lactobacillus plantarum seed solution in the embodiment 1 comprises the following steps:

a1, picking lactobacillus plantarum colonies into an MRS culture medium, and activating at 37 ℃ for 24 hours to obtain lactobacillus plantarum suspension.

A2, adding the lactobacillus plantarum suspension obtained in the step A1 into an MRS culture medium in an amount of 1.5% by volume, and performing expansion culture for 48 hours at 37 ℃ at the rotating speed of a fermentation shake flask of 150rpm to obtain lactobacillus plantarum seed liquid.

The preparation method of the schizophyllum commune seed solution in the embodiment 1 comprises the following steps:

B1, picking schizophyllum commune colony into potato dextrose water culture medium, and activating for 96 hours at 28 ℃ to obtain schizophyllum commune suspension.

B2, adding the schizophyllum commune suspension in the step B1 into a potato dextrose water culture medium in an amount of 1.5 percent by volume, and performing expansion culture for 96 hours at 28 ℃ at the rotating speed of a fermentation shake flask of 150rpm to obtain schizophyllum commune seed liquid.

Examples 2 to 11

Examples 2 to 11 the preparation method of lactobacillus soymilk fermentation product having firmness and maintenance efficacy is different from example 1 as shown in table 1, and the remaining parameters and steps are the same as example 1.

TABLE 1

The preparation methods of the Lactobacillus brevis seed solution and the Lactobacillus rhamnosus seed solution in examples 6 and 7 are the same as that of the Lactobacillus plantarum seed solution in example 1, except that Lactobacillus plantarum colonies in example 1 are replaced with Lactobacillus brevis or Lactobacillus rhamnosus colonies.

Example 12

(1) Uniformly mixing 6% of soybean milk powder and 94% of deionized water, and preserving the temperature at 115 ℃ for 30min for sterilization to obtain a fermentation raw material.

(2) Inoculating lactobacillus plantarum seed liquid and schizophyllum strain seed liquid with the total volume fraction of 4% (the volume ratio of the lactobacillus plantarum seed liquid to the schizophyllum strain seed liquid is 3:1, namely the lactobacillus plantarum seed liquid is 3% and the schizophyllum strain seed liquid is 1%) into the fermentation raw material in the step (1), and fermenting for 80 hours at 34 ℃ under the condition that the sterile air ventilation is 0.4vvm, so as to obtain a crude fermentation product.

(3) And (3) preserving the temperature of the crude fermentation product at 80 ℃ for 30min, cooling, and filtering the sterilized crude fermentation product through a diatom filter to obtain the lactobacillus soybean milk fermentation product with the tightening and repairing effects.

The preparation method of the lactobacillus plantarum seed solution of the embodiment 12 comprises the following steps:

a1, picking lactobacillus plantarum colonies into an MRS culture medium, and activating for 20 hours at 33 ℃ to obtain lactobacillus plantarum suspension.

A2, adding the lactobacillus plantarum suspension obtained in the step A1 into an MRS culture medium in an amount of 3% by volume, and performing expansion culture for 40 hours at 33 ℃ at the rotating speed of a fermentation shake flask of 180rpm to obtain lactobacillus plantarum seed liquid.

The preparation method of the schizophyllum commune seed solution of the embodiment 12 comprises the following steps:

b1, picking schizophyllum commune colony into a potato wheat bran culture medium, and activating at 33 ℃ for 144 hours to obtain schizophyllum commune suspension.

B2, adding the bacterial suspension in the step B1 into potato dextrose water culture medium in an amount of 0.5 percent by volume, and performing expansion culture for 144 hours at 33 ℃ at the rotating speed of a fermentation shake flask of 180rpm to obtain schizophyllum commune seed liquid.

Example 13

(1) Mixing 7% of soybean milk powder and 93% of deionized water, and sterilizing at 130 ℃ for 20min to obtain fermentation raw material.

(2) Inoculating lactobacillus rhamnosus seed liquid with the total volume fraction of 1% and schizophyllum commune seed liquid (the volume ratio of lactobacillus rhamnosus seed liquid to schizophyllum commune seed liquid is 3:1, namely lactobacillus plantarum seed liquid with the volume fraction of 0.75% and schizophyllum commune seed liquid with the volume fraction of 0.25%) into the fermentation raw material in the step (1), and fermenting for 60 hours at 36 ℃ under the condition that the sterile air ventilation is 0.7vvm to obtain a crude fermentation product.

(3) And (3) preserving the temperature of the crude fermentation product at 95 ℃ for 15min, cooling, and filtering the sterilized crude fermentation product through a diatom filter to obtain the lactobacillus soybean milk fermentation product with the tightening and repairing effects.

The preparation method of the lactobacillus rhamnosus seed solution of the embodiment 13 comprises the following steps:

a1, picking lactobacillus rhamnosus colonies into an MRS culture medium, and activating for 28 hours at 40 ℃ to obtain lactobacillus rhamnosus suspension.

A2, adding the lactobacillus rhamnosus suspension obtained in the step A1 into an MRS culture medium in an amount of 0.5% by volume, and performing amplification culture for 55 hours at 40 ℃ at a rotating speed of a fermentation shake flask of 100rpm to obtain lactobacillus rhamnosus seed liquid.

The preparation method of the schizophyllum commune seed solution of the embodiment 13 comprises the following steps:

b1, picking schizophyllum commune colony into potato dextrose water culture medium, and activating for 72 hours at 25 ℃ to obtain schizophyllum commune suspension.

And B2, adding the bacterial suspension obtained in the step B1 into potato dextrose water culture medium in an amount of 3% by volume, and performing expansion culture for 72 hours at 25 ℃ at a rotating speed of a fermentation shake flask of 100rpm to obtain schizophyllum commune seed liquid.

Comparative example 1

Comparative example 1 differs from example 1 in that the lactobacillus plantarum seed solution and the schizophyllum commune seed solution having a total volume fraction of 2.5% in example 1 were changed to only the lactobacillus plantarum seed solution having a volume fraction of 2.5% in comparative example 1, and the remaining steps and parameters were the same as in example 1.

The preparation method of the lactobacillus plantarum seed solution is the same as that of the example 1.

Comparative example 2

Comparative example 2 is different from example 1 in that the lactobacillus plantarum seed solution and the schizophyllum commune seed solution having a total volume fraction of 2.5% in example 1 are changed to only the schizophyllum commune seed solution having a volume fraction of 2.5% in comparative example 2, and the remaining steps and parameters are the same as in example 1.

The preparation method of the schizophyllum commune seed solution is the same as that of example 1.

Comparative example 3

Comparative example 3 differs from example 1 in that comparative example 3 was carried out by changing the lactobacillus plantarum seed solution having a volume fraction of 2% and the schizophyllum commune seed solution having a volume fraction of 0.5% in example 1 to the lactobacillus plantarum seed solution having a volume fraction of 1.25% and the schizophyllum commune seed solution having a volume fraction of 1.25%, and the remaining steps and parameters were the same as in example 1.

Wherein, the preparation method of lactobacillus plantarum seed solution and schizophyllum commune seed solution is the same as that of example 1.

Comparative example 4

Comparative example 4 differs from example 1 in that comparative example 4 was prepared by changing the volume fraction of 0.5% schizophyllum commune seed liquor from example 1 to 0.5% Saccharomyces cerevisiae seed liquor, and the remaining steps and parameters were the same as in example 1.

The preparation method of the saccharomyces cerevisiae seed liquid comprises the following steps:

And C1, selecting saccharomyces cerevisiae colonies into a potato dextrose water culture medium, and activating for 96 hours at 30 ℃ to obtain saccharomyces cerevisiae suspension.

And C2, adding the bacterial suspension in the step C1 into a potato dextrose water culture medium in an amount of 1.5 percent by volume, and performing expansion culture for 96 hours at the temperature of 30 ℃ and the rotating speed of a fermentation shake flask of 150rpm to obtain a saccharomyces cerevisiae seed liquid.

Comparative example 5

Comparative example 5 differs from example 1 in that comparative example 5 was changed to 5% soybean milk powder of example 1, and the remaining steps and parameters were the same as example 1.

Comparative example 6

Comparative example 6 differs from example 1 in that comparative example 6 changed the 5% soybean milk powder of example 1 to 5% mung bean powder, and the remaining steps and parameters were the same as example 1.

Comparative example 7

Comparative example 7 is different from example 1 in that comparative example 7 changes the 5% soybean milk powder of example 1 to 5% red bean powder, and the remaining steps and parameters are the same as example 1.

Comparative example 8

Comparative example 8 differs from example 1 in that comparative example 8 changed the fermentation temperature in step (2) of example 1 from 35 deg.c to 40 deg.c, and the remaining steps and parameters were the same as example 1.

Comparative example 9

Comparative example 9 differs from example 1 in that comparative example 9 changed the amount of introduction of sterile air in step (2) of example 1 from 0.5vvm to 1.0vvm, and the remaining steps and parameters were the same as example 1.

Application examples 1 to 22

Application examples 1-22 the final fermentation products of examples 1-13, comparative examples 1-9 were prepared into skin care lotions.

The formula of the skin care essence comprises a component A and a component B. Wherein,

The component A comprises the following components in percentage by mass: the total mass fraction of the skin care essence is 100 percent of deionized water, 5 percent of butanediol, 0.05 percent of sodium hyaluronate, 0.15 percent of allantoin, 0.08 percent of carbomer and 0.55 percent of p-hydroxyacetophenone.

The component B comprises the following components in percentage by mass: 0.08% triethanolamine, 0.3% phenoxyethanol and 3% final fermentation product of examples 1-13 or comparative examples 1-9 (only one example or comparative example final fermentation product was added per skin care concentrate).

The preparation method of the skin care essence comprises the following steps: sequentially adding the materials of the component A into a container, heating to 80 ℃, and uniformly mixing; and (3) sequentially adding the materials of the component B when the temperature is reduced to 45 ℃, uniformly mixing, and cooling to room temperature to obtain the skin care essence, wherein the relations of the application examples, the examples and the comparative examples are shown in table 2.

TABLE 2

Efficacy test example 1: long-acting moisture-keeping and tightening performance

The test basis is as follows: QB/T4256-2011, cosmetic moisturizing efficacy evaluation guidelines; T/TDCA 003-2021 cosmetic tightening efficacy test method.

The test indexes are as follows: moisture content of skin stratum corneum; after the sample was used, the higher the moisture content of the skin stratum corneum indicated the better moisturizing effect of the test sample. After use of the sample, the higher the skin elasticity R2, the better the tightening effect of the test sample (the closer the value of R2 to 1, the better).

Test instrument: skin moisture test probe Corneometer CM 825, courage & khazaka, germany; skin elasticity test probe, cutometer, MPA580, courage & Khazaka, germany.

Test sample: skin care essence, blank essence of application examples 1-22 (formulation and preparation method are the same as application examples 1-22 except that the final fermentation product of example or comparative example is replaced with deionized water of the same quality).

The testing steps are as follows: 230 volunteers with dry facial skin and skin cuticle moisture content below 20c.u were selected (randomly divided into 23 groups of 10 people each, each 10 people using one serum). Volunteers all signed informed consent and had no history of cosmetic allergies, no factors that could affect the test results, such as physical high sensitivity. Each design was random, controlled, and the evaluator blinded tested the efficacy of the sample. The skin horny layer moisture content and skin elasticity R2 of the face were tested after the volunteers were rested in a room with a relative humidity of 40% -60% for 20min at 20-22 ℃ before using the samples. The volunteers then self-used the test samples as required, taking the appropriate amount of samples each time, 1 time each morning and evening for a total of 2 weeks. The procedure was repeated after 2 weeks to test the moisture content of the facial skin horny layer, the rate of change of the moisture content of the facial skin horny layer and the rate of change of the skin elasticity R2 of the volunteers before and after use were calculated, and the test results were averaged. The greater the rate of change of the moisture content of the skin horny layer or the rate of change of the skin elasticity R2, the better the moisturizing effect and the tightening effect of the test sample are shown.

The rate of change of the moisture content of the skin horny layer and the rate of change of the skin elasticity R2 are calculated according to the formula:

Skin stratum corneum moisture content change rate (%) = (T _2w-T_0w)/T_0w ×100%

Rate of change (%) = (T _2w-T_0w)/T_0w ×100% of skin elasticity R2)

T _2w refers to the skin stratum corneum moisture content/skin elasticity R2 after 2 weeks of use; t _0w refers to the moisture content of the stratum corneum/skin elasticity R2 before use.

The test results are shown in table 3 (for convenience of distinction, the skin care serum of each application example is shown with the corresponding example or comparative example).

TABLE 3 Table 3

Compared with the blank essence, the skin care essence prepared by the embodiment and the comparative example has larger change rate of moisture content of skin cuticle and larger skin elasticity R2 after 2 weeks, which indicates that the soybean milk fermentation products of the embodiment and the comparative example have long-acting moisturizing effect and tightening effect. Wherein, the change rate of the examples 1-13 is larger than that of the comparative examples 1-9, which shows that the long-acting moisturizing effect and the tightening effect of the examples 1-13 are better than those of the comparative examples 1-9. The long-acting moisturizing effect and the tightening effect of the embodiment 1 are superior to those of the comparative examples 1, 2 and 4, which show that the long-acting moisturizing and tightening effects of the final fermentation product obtained by fermenting the soybean milk by adopting lactobacillus and schizophyllum commune are better, and the long-acting moisturizing and tightening effects of the final product are poorer by selecting one of the bacteria for fermentation or changing the schizophyllum commune into saccharomyces cerevisiae. The long-acting moisturizing effect and the tightening effect of the example 1 are superior to those of the comparative examples 5-7, which shows that the type of the fermentation raw materials has an influence on the long-acting moisturizing and tightening effects of the final product, and the long-acting moisturizing and tightening effects of the final product are poor by selecting black soybean milk, green bean powder or red bean powder to form the fermentation raw materials and fermenting with lactobacillus and schizophyllum commune. The long-acting moisturizing effect and the tightening effect of example 1 are superior to those of examples 2 and 3, and it is shown that the adding amount of the soybean milk powder affects the long-acting moisturizing effect of the final fermentation product, and the more the adding amount of the soybean milk powder is, the better the moisturizing and tightening effects are, but after the adding amount of the soybean milk powder exceeds 5%, although the moisturizing and tightening effects of the final fermentation product can be increased by further adding the soybean milk powder, the increase of the moisturizing and tightening effects is not obvious, and in order to control the production cost, the adding amount of the soybean milk powder is optimally 5%. The long-acting moisturizing effect and the tightening effect of the example 1 are superior to those of the examples 4 and 5 and the comparative example 2, and the fact that the inoculation proportion of lactobacillus and schizophyllum commune affects the growth and metabolism of strains in fermentation raw materials, further affects the long-acting moisturizing and tightening effect of the final product, and in the initial state of fermentation, small proportion of schizophyllum commune is inoculated into fermentation broth, so that the final fermentation product is beneficial to producing more skin care active substances, and when the inoculation proportion of schizophyllum commune is excessive, the initial bacterial load of the schizophyllum commune is excessive, the growth of lactobacillus is affected, and the moisturizing active substances and the tightening active substances of the final product are less, so that the moisturizing and tightening effects are poor. The moisturizing and tightening effects of examples 1, 8 and 9 are superior to those of comparative example 8, and the fact that the temperature has great influence on the whole fermentation process shows that the optimal fermentation temperatures of lactobacillus and schizophyllum commune are greatly different. The moisturizing and tightening effects of examples 1, 10 and 11 are superior to those of comparative example 9, which shows that the ventilation rate has a great influence on the whole fermentation process, and the ventilation rate of the sterile air in the application is controlled to be 0.3-0.8vvm, and the optimal ventilation rate is 0.5vvm, so that the obtained soybean milk fermentation product has the best long-acting moisturizing and tightening effects.

Efficacy test example 2: repair and redness performance

The test indexes are as follows: skin moisture loss TEWL value, skin redness (i.e., skin heme value). The skin moisture loss TEWL value can reflect the barrier function of the skin, and the larger the skin moisture loss TEWL value is, the more skin moisture loss in unit time is represented, and the worse the skin barrier function is; the skin heme value reflects the skin heme content of the test part, the greater the skin heme value, the more severe the redness of the skin.

Test instrument: skin moisture loss test probe TEWAMETER TM, courage & Khazaka, germany; skin heme test probe Mexameter MX, courage & Khazaka, germany.

Test sample: the same efficacy test example 1.

The testing steps are as follows: 230 volunteers (divided into 23 groups, 10 people each, one essence for each 10 people) with no damage, no spots and obvious pigment areas on the inner sides of the arms and no physical high sensitivity are selected, 1.5X1.5 cm skin areas on the inner sides of the arms of the volunteers are subjected to spot pasting by adopting SDS solution with the mass fraction of 1%, 0.08mL of SDS solution with the mass fraction of 1% is titrated by each spot tester, the spot pasting device is torn after 24 hours, and the water loss and the skin heme value of the skin of the area are detected after the spot pasting device is torn for 8 hours; and then smearing a sample on the spot patch area, and performing single coating according to the dosage of (2.0+/-0.1) mg/cm ², wherein after smearing for 2 hours, the moisture loss and the skin heme value of the detection area are detected, and the repairing and reddening effects of the sample are judged.

The negative change rate of skin moisture loss indicates that skin moisture loss is reduced, skin barrier function is restored, and the greater the negative change rate of skin moisture loss indicates that the better the sample repair function.

The negative value of the change rate of the skin heme value indicates that the skin redness is weakened after the sample is used, the sample has a reddening effect, and the larger the negative value of the change rate of the skin heme value is, the better the reddening function of the sample is.

The calculation formula of the change rate:

Skin moisture loss rate of change/skin heme value rate of change (%) = (T _2h-T_0h)/T_0h x 100%

T _2h refers to the skin moisture loss value/skin heme value after 2 hours of use of the sample; t _0h refers to the skin moisture loss value/skin heme value after removal of the patch.

The test results are shown in Table 4.

TABLE 4 Table 4

Group of	Skin moisture loss rate of change (%)	Skin heme value change rate (%)
			Blank essence	-4.7	-3.1
Example 1	-28.4	-35.8
			Example 2	-25.7	-28.9
Example 3	-28.8	-36.0
			Example 4	-25.4	-31.5
Example 5	-27.0	-34.2
			Example 6	-24.8	-29.6
Example 7	-28.7	-31.7
			Example 8	-23.9	-31.1
Example 9	-26.1	-28.5
			Example 10	-22.0	-27.9
Example 11	-24.6	-30.6
			Example 12	-23.2	-26.9
Example 13	-22.7	-28.2
			Comparative example 1	-10.8	-13.6
Comparative example 2	-6.4	-8.1
			Comparative example 3	-17.8	-22.5
Comparative example 4	-9.6	-14.9
			Comparative example 5	-16.1	-21.8
Comparative example 6	-11.6	-23.5
			Comparative example 7	-7.2	-7.5
Comparative example 8	-18.6	-20.2
			Comparative example 9	-17.5	-22.6

The test results show that the absolute values of the skin moisture loss change rate and the skin heme value change rate are larger than those of the blank essence after the skin care essence of the examples and the comparative examples is used, and the final products of the examples and the comparative examples have certain repairing and reddening (reddening improvement) effects. The repair and redness effects of example 1 are superior to those of comparative examples 1,2 and 4, showing that the simultaneous fermentation of soybean milk with lactobacillus and schizophyllum commune gives a better repair and redness effect of the final fermentation product, the fermentation of either fungus or the replacement of schizophyllum commune with saccharomyces cerevisiae, the repair and redness effects of the final product are inferior, and the repair effect of the mixed fermentation of lactobacillus and saccharomyces cerevisiae (comparative example 4) is inferior to that of the fermentation of lactobacillus alone (comparative example 1). The repair and redness effects of example 1 are superior to those of comparative examples 5 to 7, showing that the type of beans of the fermentation raw material has an influence on the repair and redness effects of the final fermentation product of the final product, and the repair and redness effects of the final product are poor by selecting black soybean milk, green bean powder or red bean powder as the fermentation raw material. The repair and redness effects of the final product of example 1 were inferior to those of examples 2 and 3, showing that the addition amount of the soybean milk powder affects the repair and redness effects of the final fermentation product, and that the more the soybean milk powder is added, the better the repair and redness effects are, but as the soybean milk powder is added in an amount exceeding 5%, although the repair and redness effects of the final fermentation product can be increased by further adding the soybean milk powder, but the increase in the repair and redness effects is not significant, and in order to control the production cost, the addition amount of the soybean milk powder is optimally 5% in the present application. The repairing and redness-repairing effects of example 1 are superior to those of examples 4, 5 and comparative example 2, and it is shown that the inoculation ratio of lactobacillus and schizophyllum commune affects the growth and metabolism of strains in fermentation raw materials, further affects the fermentation effect of the final product, and in the initial state of fermentation, inoculating a small proportion of schizophyllum commune into fermentation broth is beneficial to the final fermentation product to produce more repairing active substances. The repairing and redness-trimming effects of examples 1, 8 and 9 are superior to those of comparative example 8, and the fact that the temperature has great influence on the whole fermentation process shows that the optimal fermentation temperatures of lactobacillus and schizophyllum commune are greatly different. The repairing and redness-repairing effects of examples 1, 10 and 11 are superior to those of comparative example 9, which shows that the aeration rate has a great influence on the whole fermentation process, and the aeration rate of the sterile air in the application is controlled to be 0.3-0.8vvm, and the optimal aeration rate is 0.5vvm, so that the repairing and redness-repairing effects of the obtained soybean milk fermentation product are the best.

Efficacy test example 3: relaxation property

The test basis is as follows: the detection is carried out according to the TRPV1 protein content detection method based on capsaicin stimulated keratinocyte.

The test principle is as follows: sensitive skin refers to a state of hyperreactivity of the skin under physiological or pathological conditions, which occurs mainly on the face. TRPV1 is an ion channel receptor, also known as capsaicin receptor, and TRPV1 acts as a heterogeneous receptor widely distributed in the peripheral nervous system and can be directly or indirectly activated by various ligands such as capsaicin, etc., causing intracellular Ca ²⁺ to rise, triggering action potentials. Thus, TRPV1 is closely related to skin burning and pain. The higher the expression level of TRPV1, the higher the neural sensitivity of the skin. Can play a certain role in nerve relief by inhibiting the expression of TRPV 1. When TRPV1 is inhibited after the test object acts, the concentration of intracellular Ca ²⁺ is reduced, so that the test object can be proved to achieve the effect of relieving by inhibiting TRPV 1.

Test sample: the final fermentation products of examples 1-13 and comparative examples 1-9 were prepared as 0.78mg/mL solutions in cell culture broth.

The testing steps are as follows:

(1) Cell inoculation: keratinocytes were incubated overnight at 37℃under 5% CO ₂ in 6-well plates at an seeding density of 1.8X10 ⁵ cells/well.

(2) Sample preparation: each working fluid was prepared according to table 5.

TABLE 5

Grouping	Reagent information	Concentration of	Stimulation conditions
				Blank Control (BC)	Cell culture fluid	/	Does not irritate
Negative Control (NC)	Cell culture fluid	/	CAP(15μM)
				Positive Control (PC)	trans-4-tert-butylcyclonhexanol	15.6μg/mL	CAP(15μM)
Sample group	Examples 1 to 11 or comparative examples 1 to 13	0.781mg/mL	CAP(15μM)

(3) Administration: when the cell plating rate in the 6-hole plate reaches 40% -50%, carrying out group drug administration according to the table, wherein the drug administration amount of each hole is 2mL, and 3 compound holes are arranged in each group, namely placing the 6-hole plate in an incubator (37 ℃ and 5% CO ₂) for incubation and culture for 24 hours after the drug administration is completed;

(4) TRPV1 detection and analysis: fixing with 4% paraformaldehyde for 30min, immunofluorescence detection of capsaicin receptor (TRPV 1) is performed, and the images are taken and analyzed under microscope. The results are shown as MeanSD using GRAPHPAD PRISM plots. Comparisons between groups were performed using t-test statistical analysis.

The inhibition rate calculation method comprises the following steps:

Inhibition (%) = (X-X ₁)/x×100%

Where X is the negative control group relative IOD/cell number average and X ₁ is the sample group relative IOD/cell number average.

The test results are shown in table 6:

TABLE 6

The test results show that the inhibition rate of the TRPV1 expression of the inventive examples is higher than that of the comparative examples, which shows that the soothing effect of the inventive examples is better than that of the comparative examples. The inhibition rate of TRPV1 expression in example 1 is higher than that in comparative examples 1,2 and 4, which shows that the fermentation of soybean milk by lactobacillus and schizophyllum commune simultaneously results in a better relief effect of the final fermentation product, and the relief effect of the final product is poor by selecting one of the bacteria for fermentation or changing the schizophyllum commune into saccharomyces cerevisiae for mixed fermentation. In terms of the soothing effect, the effect of the mixed fermentation of lactobacillus and saccharomyces cerevisiae (comparative example 4) was comparable to that of the fermentation using lactobacillus alone (comparative example 1). The soothing effect of example 1 was superior to that of comparative examples 5 to 7, indicating that the kind of beans of the fermentation material had an effect on the poor soothing effect of the final fermentation product of the final product, and the use of black soybean milk, green bean powder or red bean powder as the fermentation material gave a poor soothing effect of the final product and the use of red bean powder gave the worst soothing effect (comparative example 7). The soothing effect of example 1 is superior to examples 4, 5 and comparative example 2, showing that the inoculation ratio of lactobacillus and schizophyllum commune affects the growth and metabolism of strains in fermentation raw materials, further affects the fermentation effect of the final product, and in the initial state of fermentation, inoculating small proportion of schizophyllum commune into fermentation broth is beneficial to the final fermentation product to produce more repairing active substances, and when the inoculation ratio of schizophyllum commune is excessive, the growth of lactobacillus is affected by excessive initial bacterial load of schizophyllum commune, resulting in less active substances of the final product and poor soothing effect. The soothing effect of examples 1, 8 and 9 is superior to that of comparative example 8, which shows that the temperature has great influence on the whole fermentation process, and the optimal fermentation temperatures of lactobacillus and schizophyllum commune are greatly different. The soothing effect of examples 1, 10 and 11 is superior to that of comparative example 9, which shows that the aeration rate has a great effect on the whole fermentation process, and the aeration rate of the sterile air in the application is controlled to be 0.3-0.8vvm, and the optimal aeration rate is 0.5vvm, so that the soothing effect of the obtained soybean milk fermentation product is the best.

The above embodiments are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present application should be made in the equivalent manner, and the embodiments are included in the protection scope of the present application.

Claims

1. A method for preparing lactobacillus soybean milk fermentation product with tightening and repairing effects, which is characterized by comprising the following steps:

2. The process according to claim 1, wherein the fermentation feedstock in step (1) comprises 3-8% soy milk powder and 92-97% water.

3. The method according to claim 1, wherein the lactobacillus in the step (2) is at least one of lactobacillus plantarum, lactobacillus brevis and lactobacillus rhamnosus.

4. The method according to claim 1, wherein the total inoculum size of the lactobacillus seed liquid and the schizophyllum commune seed liquid in the step (2) is 1-4% of the volume of the fermentation raw material; the volume ratio of the lactobacillus seed liquid to the schizophyllum commune seed liquid is (3-5): 1.

5. The process according to claim 1, wherein the fermentation in step (2) is carried out at a temperature of 32-37 ℃, the sterile air ventilation of the fermentation is 0.3-0.8vvm, and the fermentation time is 60-80h.

6. The process according to claim 1, wherein in step (3),

The filtering is performed by a diatom filter;

The sterilization is to keep the temperature of the crude fermentation product at 80-95 ℃ for 15-30min.

7. The method according to claim 1, wherein the method for producing the lactobacillus seed solution in the step (2) comprises:

8. The method according to claim 1, wherein the method for preparing the schizophyllum commune seed solution in the step (2) comprises:

9. A lactobacillus soymilk fermentation product with tightening and repairing effects, which is prepared by the preparation method according to any one of claims 1-8.

10. Use of the lactobacillus soymilk fermentation product with tightening and repairing effects according to claim 9 for preparing cosmetics.