CN116115542B

CN116115542B - Fermented emulsion, external preparation containing same and preparation method and application of external preparation

Info

Publication number: CN116115542B
Application number: CN202211638075.3A
Authority: CN
Inventors: 王冬冬; 王昌涛; 李萌
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2022-12-16
Filing date: 2022-12-16
Publication date: 2024-05-07
Anticipated expiration: 2042-12-16
Also published as: CN116115542A

Abstract

The application discloses a fermented emulsion, an external skin preparation containing the fermented emulsion, and a preparation method and application thereof. The preparation method of the fermented emulsion comprises the following steps: step (1): inoculating Saccharomyces cerevisiae into a fermentation substrate comprising rice, vegetable oil and water, fermenting, culturing, and sterilizing to obtain material A; wherein, the mass ratio of the rice to the vegetable oil is (1-10): 1, a step of; step (2): inoculating lactobacillus plantarum into the material A prepared in the step (1), and fermenting, culturing and sterilizing to prepare the fermentation emulsion. Compared with the traditional emulsion, the fermented emulsion prepared by the application still can show good moisturizing, anti-inflammatory and anti-aging effects under the condition that components such as an emulsifying agent, a thickening agent, a surfactant and the like are not required to be added, and has the advantages of good stability, good sensory evaluation, simple preparation process, production cost saving, high use safety and difficult skin allergy.

Description

Fermented emulsion, external preparation containing same and preparation method and application of external preparation

Technical Field

The application belongs to the technical field of cosmetics, and particularly relates to a fermented emulsion, an external skin preparation containing the fermented emulsion, and a preparation method and application of the external skin preparation.

Background

With the rapid development of science and technology, the living conditions of human substances are improved, the aging of population is continuously aggravated, and the active exploration and development of the beauty and skin care products are of great significance for slowing down the aging of the population of society. An important way to keep the skin tender and properly moisten is to use the emulsion with moisturizing effect. At present, moisturizing emulsions in the market are various and have large moisturizing effect difference, and commonly used emulsions are prepared by emulsifying substances such as polyalcohol, basic grease, emulsifying agent, surfactant and the like, but the substances have the defects of low absorption speed by skin, adverse permeation and absorption of active substances, and skin allergy and the like caused by the existing emulsifying agent.

How to develop an emulsion skin care product with good moisturizing effect, easy absorption of active substances by skin and high use safety so as to meet the demands of market aspects is a technical problem which needs to be solved by the technicians in the field.

Disclosure of Invention

The application aims to overcome the technical problems that in the prior art, moisturizing emulsion is prepared by emulsifying substances such as polyalcohol, basic grease, emulsifying agent, surfactant and the like, but the substances are absorbed by skin slowly, are not beneficial to permeation and absorption of active substances, and can cause skin allergy and the like, and provides a fermented emulsion, an external skin preparation containing the fermented emulsion, a preparation method and application thereof. Compared with the traditional emulsion, the fermented emulsion prepared by the application still can show good moisturizing, anti-inflammatory and anti-aging effects under the condition that components such as an emulsifying agent, a thickening agent, a surfactant and the like are not required to be added, and has the advantages of good stability, good sensory evaluation, simple preparation process, production cost saving, high use safety and difficult skin allergy.

The application adopts the following technical scheme to solve the technical problems:

the application provides a preparation method of a fermentation emulsion, which specifically comprises the following steps:

Step (1): inoculating Saccharomyces cerevisiae into a fermentation substrate comprising rice, vegetable oil and water, fermenting, culturing, and sterilizing to obtain material A; wherein the mass ratio of the rice to the vegetable oil is (1-10): 1, a step of;

Step (2): inoculating lactobacillus plantarum into the material A prepared in the step (1), and carrying out fermentation culture and sterilization to prepare the fermentation emulsion.

In step (1), the rice may be a commercially available conventional rice, such as northeast rice.

In the step (1), the rice may further comprise a crushing operation before use, and the crushed grain size may be 50-100 mesh.

In the step (1), the vegetable oil may include at least one of macadamia nut seed oil, peony seed oil, white pool seed oil and prinsepia utilis royle oil.

In the step (1), the mass ratio of the water to the rice may be (10 to 200): 1, preferably (20 to 100): 1, more preferably (20 to 60): 1, e.g. 25:1.

In the step (1), the mass of the rice and the vegetable oil is preferably (3 to 8): 1, more preferably (3 to 5): 1.

In step (1), the fermentation substrate may also be subjected to sterilization procedures conventionally included in the art prior to use.

Wherein the conditions and methods of said sterilization of said fermentation substrate may be those conventional in the art for such procedures and may generally be high temperature sterilization.

When the sterilization of the fermentation substrate is performed by the high temperature sterilization method, the sterilization temperature may be a temperature which is conventional in the art for such operations, preferably 110 to 125 ℃, more preferably 115 to 121 ℃.

When the sterilization of the fermentation substrate is performed by the high temperature sterilization method, the sterilization time may be a time conventional in the art for such an operation, preferably 20 to 60 minutes, more preferably 20 to 40 minutes, for example 30 minutes.

Wherein the fermentation substrate may further comprise a cooling operation, typically to room temperature, after the sterilization operation.

In the step (1), the Saccharomyces cerevisiae may be Saccharomyces cerevisiae with a preservation number of CGMCC No.17452 preserved in China general microbiological culture Collection center.

In the step (1), the Saccharomyces cerevisiae may be added in the form of a Saccharomyces cerevisiae liquid having a viable count of 10 ⁵～10⁹ CFU/mL, preferably 10 ⁶～10⁸ CFU/mL, according to the conventional method in the art.

The amount of said Saccharomyces cerevisiae inoculated per unit volume of said fermentation substrate in step (1) may be conventional in the art, preferably 5X 10 ³～5×10⁷ CFU/mL, more preferably 5X 10 ⁴～5×10⁶ CFU/mL.

In step (1), the fermentation culture may be carried out in a shake incubator according to conventional practice in the art, wherein the rotation speed of the shaker in the shake incubator may be 150 to 300r/min, preferably 150 to 250r/min, for example 200r/min.

In step (1), the fermentation time may be 24 to 120 hours, preferably 48 to 100 hours, for example 96 hours.

In step (1), the temperature of the fermentation culture may be 25 to 38 ℃, preferably 25 to 32 ℃, for example 30 ℃.

In step (1), the conditions and methods of sterilization may be conventional in the art and may generally be high temperature sterilization.

In step (1), when the sterilization is performed by the high temperature sterilization method, the sterilization temperature may be a temperature which is conventional in the art for such an operation, preferably 90 to 125 ℃, more preferably 90 to 110 ℃, for example 100 ℃.

In step (1), when the sterilization is performed by the high temperature sterilization method, the sterilization time may be a time conventional in the art for such an operation, preferably 20 to 60 minutes, more preferably 20 to 40 minutes, for example 30 minutes.

In the step (1), the sterilization operation may further include an operation of cooling to room temperature.

In the step (2), the lactobacillus plantarum may include lactobacillus plantarum purchased from the China industry microbiological culture Collection center, with a accession number of CICC 20261.

In step (2), the lactobacillus plantarum may be added as a lactobacillus plantarum bacterial solution, as is conventional in the art, and the concentration of the lactobacillus plantarum in the lactobacillus plantarum bacterial solution may be 10 ⁶～10¹⁰ CFU/mL, preferably 10 ⁷～10⁹ CFU/mL, for example 10 ⁸ CFU/mL.

The amount of lactobacillus plantarum inoculated per unit volume of the material a in step (2) may be conventional in the art, preferably 10 ⁴～10⁸ CFU/mL, more preferably 10 ⁵～10⁷ CFU/mL, for example 10 ⁶ CFU/mL.

In step (2), the fermentation culture may be stationary fermentation conventionally used in the art.

In the step (2), the fermentation culture time may be 12 to 24 hours, preferably 12 to 18 hours.

In step (2), the temperature of the fermentation culture may be 30 to 45 ℃, preferably 35 to 45 ℃, for example 40 ℃.

In step (2), the conditions and methods of sterilization may be conventional in the art and may generally be high temperature sterilization.

In step (2), when the sterilization is performed by the high temperature sterilization method, the sterilization temperature may be a temperature which is conventional in the art for such an operation, preferably 90 to 125 ℃, more preferably 90 to 110 ℃, for example 100 ℃.

In step (2), when the sterilization is performed by the high temperature sterilization method, the sterilization time may be a time conventional in the art for such an operation, preferably 20 to 50 minutes, more preferably 25 to 35 minutes, for example 30 minutes.

In the step (2), the sterilization operation may further include cooling and/or centrifugation, and collecting supernatant.

Wherein the cooling may be to room temperature, as is conventional in the art.

The rotational speed of the centrifugation may be a rotational speed conventional in the art, preferably 3000 to 9000r/min, more preferably 4000 to 6000r/min, for example 4800r/min.

Wherein the radius of the centrifugation may be a radius conventional in this type of operation in the art, preferably 8-15 cm.

The centrifugation time may be a time conventional in the art, preferably 10 to 40min, more preferably 20 to 40min, for example 30min.

Wherein the centrifugation may be followed by a secondary sterilization and/or mixing with a preservative.

The conditions and methods of the secondary sterilization may be those conventional in the art for such procedures and may generally be high temperature sterilization.

When the secondary sterilization is performed by the high temperature sterilization method, the temperature of the secondary sterilization may be a temperature which is conventional in the art for such operations, preferably 100 to 125 ℃, more preferably 115 to 121 ℃.

When the secondary sterilization is performed by the high temperature sterilization method, the time for the secondary sterilization may be a time conventional in the art for such an operation, preferably 20 to 40 minutes, more preferably 25 to 35 minutes, for example 30 minutes.

The temperature of the mixing during mixing with the preservative may be a temperature conventional in the art for such operations, preferably 50 to 80 ℃, more preferably 70 to 80 ℃.

In mixing with the preservative, the preservative may include p-hydroxyacetophenone and/or 1, 2-hexanediol as is conventional in the art.

When the preservative comprises the p-hydroxyacetophenone and the 1, 2-hexanediol, the mass percentage of the p-hydroxyacetophenone to the supernatant prepared after the centrifugation can be 0.1-1%, and the mass percentage of the 1, 2-hexanediol to the supernatant prepared after the centrifugation can be 0.1-1%; preferably, the p-hydroxyacetophenone accounts for 0.5% of the mass of the supernatant obtained after the centrifugation, and the 1, 2-hexanediol accounts for 0.5% of the mass of the supernatant obtained after the centrifugation.

The application also provides a fermented emulsion, which is prepared by the preparation method of the fermented emulsion.

The application also provides an application of the fermented emulsion in preparing external skin preparations, wherein the fermented emulsion is directly used as a product, an additive or a substrate.

In some embodiments, the fermented emulsion may be used as at least one of a moisturizing active ingredient, an anti-inflammatory active ingredient, and an anti-aging active ingredient in the skin external agent.

Wherein the anti-inflammatory active ingredient may be an anti-inflammatory active ingredient having an effect of inhibiting the production of IL-1 beta inflammatory factor by cells;

wherein the anti-aging active ingredient may be an anti-aging active ingredient having an effect of promoting elastin production.

The application also provides a skin external agent comprising the fermented emulsion as described above.

In some embodiments, the external preparation for skin may further include an active ingredient conventionally used in the art, and may generally include at least one of a moisturizing active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient, and an anti-oxidation active ingredient.

In some embodiments, the skin external preparation may include, but is not limited to, a mask, essence, or toner as is conventional in the art.

In some embodiments, the fermented emulsion may comprise 5% to 99% by mass of the skin external agent, preferably 60% to 99%.

In some embodiments, the room temperature, normal temperature, generally refers to 15-40 ℃.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the application.

The reagents and materials used in the present application are commercially available.

The application has the positive progress effects that: compared with the traditional emulsion, the fermented emulsion prepared by the application still can show good moisturizing, anti-inflammatory and anti-aging effects under the condition that components such as an emulsifying agent, a thickening agent, a surfactant and the like are not required to be added, and has the advantages of good stability, good sensory evaluation, simple preparation process, production cost saving, high use safety and difficult skin allergy.

Drawings

The application may be better understood by reference to the following description taken in conjunction with the accompanying drawings. The accompanying drawings, which are included to provide a further illustration of the preferred embodiments of the application and together with a further understanding of the principles and advantages of the application, are incorporated in and constitute a part of this specification.

Wherein:

FIG. 1 is a graph showing the relative expression levels of the intracellular inflammatory factor IL-1β after the treatment of damaged HaCaT cells with the products prepared in examples 1 to 4 and comparative examples 1 to 5;

FIG. 2 is a graph showing the comparison of anti-aging activities of the products prepared in examples 1 to 4 and comparative examples 1 to 5;

FIG. 3 is a graph showing the comparison of the stability of the products prepared in examples 1 to 4 and comparative examples 1 to 5 after 4 weeks of storage at normal temperature, -20℃and 50℃respectively.

Detailed Description

The application is further illustrated by means of the following examples, which are not intended to limit the scope of the application. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

In the following examples and comparative examples, macadamia nut seed oil was purchased from the company of the oil industry, cross-joint;

In the following examples and comparative examples, peony seed oil was purchased from Kangpu biotechnology Co., ltd in lotus city;

in the following examples and comparative examples, prinsepia utilis oil was purchased from Yunnan English biotechnology Co., ltd;

In the following examples and comparative examples, the white pool flower seed oil was purchased from Jiangxi Xinsen natural vegetable oil limited;

In the following examples and comparative examples, saccharomyces cerevisiae (Saccharomyces cerevisiae) YWY-1 was used, the preservation unit was China general microbiological culture Collection center (CGMCC), the address was North Star Xiyu No.1, no. 3, the post code was 100101, the preservation date was 2019, 03, 27 days, and the preservation number was CGMCC No.17452.

The preparation method of the saccharomyces cerevisiae bacterial liquid comprises the following steps: inoculating Saccharomyces cerevisiae (CGMCC No. 17452) into YPD solid culture medium for streak activation, and culturing in a 28 ℃ incubator for 48 hours to obtain single colony; the single colony obtained by the inoculating loop is picked in YPD liquid culture medium and cultured in a shake incubator with the temperature of 28 ℃ and the rotating speed of 180 r/min.

In the following examples and comparative examples, lactobacillus plantarum was purchased from the China center for Industrial microorganism culture Collection, with the accession number CICC 20261. The lactobacillus plantarum bacterial liquid is prepared by adopting sterile water for dispersion.

In comparative example 3 below, streptococcus thermophilus was Streptococcus thermophilus available from Zhengzhou and Synbiotics engineering Co., ltd, product number HH-ST 08. Sterile water is adopted for dispersion to prepare streptococcus thermophilus bacterial liquid.

Example 1

Step (1): weighing 15g of rice powder with 100 meshes, 3g of macadamia nut seed oil and 300mL of deionized water, mixing, sterilizing at 121 ℃ for 30min, and cooling to room temperature after sterilization to obtain a fermentation substrate;

Inoculating Saccharomyces cerevisiae liquid (CGMCC No. 17452) into the above prepared fermentation substrate; the method comprises the steps of fermenting the saccharomyces cerevisiae in a shaking incubator at 30 ℃ for 96 hours, wherein the number of viable bacteria in the saccharomyces cerevisiae liquid is 10 ⁷ CFU/mL, the adding amount of the saccharomyces cerevisiae liquid is 15mL, the rotating speed of a shaking table in the shaking incubator is 200r/min, sterilizing the obtained fermentation emulsion in a high-temperature sterilizing pot at 100 ℃ for 30min after fermentation culture is finished, and stopping the saccharomyces cerevisiae fermentation; cooling to room temperature after sterilization to obtain a material A;

Step (2): inoculating lactobacillus plantarum bacterial liquid (CICC 20261) into the material A prepared in the step (1), wherein the viable count in the lactobacillus plantarum bacterial liquid is 10 ⁸ CFU/mL, the adding amount of the lactobacillus plantarum bacterial liquid is 3mL, standing and fermenting for 18h in a 40 ℃ incubator, sterilizing in a high-temperature sterilizing pot with the temperature of 100 ℃ for 30min after fermentation and culturing are finished, stopping lactobacillus plantarum fermentation, cooling to room temperature after sterilization, centrifuging again, centrifuging for 30min under the condition that the centrifugal radius is 15cm and 4800r/min, obtaining supernatant, performing secondary sterilization on the supernatant, sterilizing for 30min in a sterilizing pot with the temperature of 100 ℃, avoiding pollution caused by other miscellaneous bacteria in the centrifuging process, and cooling to room temperature after sterilization is finished, thus obtaining the fermented emulsion.

Example 2

Compared with example 1, the difference is only that the macadamia nut seed oil in the step (1) is replaced by the peony seed oil with the same quantity, and other condition parameters are the same as those in example 1, so as to prepare the fermented emulsion.

Example 3

Compared with example 1, the difference is only that in the step (1), the macadamia nut seed oil is replaced by prinsepia utilis royle oil and white pool flower seed oil, wherein the total mass of the oil is not changed to 3g, the mass ratio of the prinsepia utilis royle oil to the white pool flower seed oil is 1:1, and other condition parameters are the same as in example 1, so that the fermented emulsion is prepared.

Example 4

The process for preparing a fermentation substrate in step (1) differs from example 1 only in that other conditions and parameters are the same as in example 1, and a fermented emulsion is prepared;

The preparation method of the fermentation substrate specifically comprises the following steps: weighing 12g of rice powder with 100 meshes, 4g of macadamia nut seed oil and 300mL of deionized water, mixing, sterilizing at 121 ℃ for 30min, and cooling to room temperature after sterilization to obtain a fermentation substrate.

Comparative example 1

In comparison with example 1, the only difference is that no stepwise fermentation is carried out, comprising the following steps:

weighing 15g of rice powder with 100 meshes, 3g of macadamia nut seed oil and 300mL of deionized water, mixing, sterilizing at 121 ℃ for 30min, and cooling to room temperature after sterilization to obtain a fermentation substrate;

15mL of Saccharomyces cerevisiae bacterial liquid (CGMCC No. 17452) and 3mL of lactobacillus plantarum bacterial liquid (CICC 20261) are added into the prepared fermentation substrate; wherein the number of viable bacteria in the saccharomyces cerevisiae bacterial liquid is 10 ⁷ CFU/mL, and the number of viable bacteria in the lactobacillus plantarum bacterial liquid is 10 ⁸ CFU/mL; fermenting in a shaking incubator at 30deg.C for 96 hr at a rotation speed of 200r/min, sterilizing the obtained fermented emulsion in a high-temperature sterilizing pot at 100deg.C for 30min after fermentation culture, and stopping strain fermentation; sterilizing, and cooling to room temperature.

Comparative example 2

Compared with example 1, the difference is that the macadamia nut seed oil is not added to the fermentation substrate in the step (1), and after the step (2) is finished, the product is mixed with 3g of macadamia nut seed oil, and other condition parameters are the same as those in example 1, and the method specifically comprises the following steps:

step (1): weighing 15g of rice powder with the mesh number of 100, mixing with 300mL of deionized water, sterilizing at 121 ℃ for 30min, and cooling to room temperature after sterilization to obtain a fermentation substrate;

Step (2): inoculating lactobacillus plantarum bacterial liquid (CICC 20261) into the material A prepared in the step (1), wherein the viable count in the lactobacillus plantarum bacterial liquid is 10 ⁸ CFU/mL, the addition amount of the lactobacillus plantarum bacterial liquid is 3mL, standing and fermenting for 18h in a 40 ℃ incubator, sterilizing in a high-temperature sterilizing pot with the temperature of 100 ℃ for 30min after fermentation and culturing are finished, stopping lactobacillus plantarum fermentation, cooling to room temperature after sterilization, centrifuging again, centrifuging for 30min under the condition that the centrifugal radius is 15cm and 4800r/min, obtaining supernatant, performing secondary sterilization on the supernatant, sterilizing for 30min in a sterilizing pot with the temperature of 100 ℃ a, avoiding pollution caused by other miscellaneous bacteria in the centrifuging process, and cooling to room temperature after sterilization is finished;

step (3): and (3) uniformly mixing the cooled material in the step (2) with 3g of macadimia nut seed oil, and homogenizing for 10min by using a homogenizer 12000 r/min.

Comparative example 3

The difference from example 1 is that the Lactobacillus plantarum bacterial solution (CICC 20261) in step (2) was replaced with 3mL of Streptococcus thermophilus bacterial solution HH-ST08 (Zhengzhou and He Biotechnology Co., ltd.) and the number of viable bacteria in Streptococcus thermophilus bacterial solution was 10 ⁸ CFU/mL, and the other conditions were the same as in example 1.

Comparative example 4

The process for preparing a fermentation substrate in step (1) differs from example 1 only in that other conditions and parameters are the same as in example 1;

the preparation method of the fermentation substrate specifically comprises the following steps: weighing 20g of rice powder with 100 meshes, 1g of macadamia nut seed oil and 300mL of deionized water, mixing, sterilizing at 121 ℃ for 30min, and cooling to room temperature after sterilization to obtain a fermentation substrate.

Comparative example 5

The difference compared to example 1 is that the operation of step (2) is not carried out, and other condition parameters are the same as example 1, i.e., material A obtained in step (1) of example 1.

Effect example 1: safety evaluation

The human body patch test is mainly used for detecting the irritation of cosmetics or raw materials. The products prepared in examples and comparative examples were subjected to a human closed patch test according to cosmetic hygiene Specification (2015) for evaluation of skin irritation.

1. Test object:

According to the requirements of the diagnostic standard and the treatment principle of the cosmetic contact dermatitis, a subject is selected, and the subject with high physique sensitivity, which has influence on the judgment of the result, such as scar, bright red mole, and the like, on the part to be detected of the skin cannot participate in the test. The trial selected 30 appropriate volunteers, with age ranging from 18 to 60 years of age at random.

2. Experimental method

0.2ML of the product prepared in the above example or comparative example was dropped onto a filter paper sheet, respectively, and the filter paper sheet was placed in a plaque tester. A blank control was set, i.e., distilled water as the sample solvent in an amount equivalent to that of the sample was added to the control plaque assay well. The test period lasted 24h. For the accuracy, credibility and science of the test results, the volunteer could not remove the plaque tester or contact the test site with water as required during the test. After 24 hours, the plaque tester was removed, and after standing for 30 minutes, the skin reaction was observed after waiting for the disappearance of the indentation, and then after 24 hours, the skin reaction was observed, and the skin adverse reaction grading standard is shown in table 1.

TABLE 1 skin adverse reaction grading criteria

3. Test results

The results are shown in Table 2, from which; the results of the sensitization test of the fermented emulsion prepared in the embodiments 1 to 4 are all negative reactions, which shows that the fermented emulsion prepared in the application has good safety and tolerance and does not bring adverse reaction to human body. The products prepared in comparative examples 1 to 5 were relatively poor in safety in use, and adverse reactions occurred in some subjects.

TABLE 2

Effect example 2: moisture retention evaluation

According to the cosmetic moisturizing efficacy evaluation guidelines, the moisture content of the skin is represented by the moisture content of the horny layer, 30 volunteers meeting the conditions are screened to participate in the test, and the moisturizing effects of the products prepared in the examples and the comparative examples on the skin of the volunteers are tested in an environment with the temperature of 22+/-2 ℃ and the humidity of 40% -60%.

The test method is as follows:

After cleaning the skin for 15min, measuring the background value of the forearms on both sides (skin without smearing products), taking the normal skin with the area of 3.5 multiplied by 3.5CM from the inner sides of the forearms on both sides of the test subject, and measuring the skin moisture content of the tested part after 5min, 20min and 1 h by adopting a skin moisture measuring probe Corneometer CM 825. Cutting the mask cloth into 3X 3cm, respectively attaching the mask cloth to corresponding mark of forearm, dripping the product prepared in the embodiment or comparative example on the mask cloth by using a rubber head dropper, taking down the mask cloth after 15min, testing skin moisture content after 5min, 20min and 60min respectively, calculating the lifting value of the moisture content compared with the background value, and the lifting value result is shown in Table 3.

TABLE 3 Table 3

Skin moisture content elevation value	5min(C.U.)	20min(C.U.)	60min(C.U.)
				Example 1	11.1	5.8	2.2
Example 2	12.6	6.5	3.1
				Example 3	10.5	4.6	1.8
Example 4	9.7	3.8	2
				Comparative example 1	6.7	1.6	-2.3
Comparative example 2	5.8	1.7	0.3
				Comparative example 3	7.4	2.6	0.6
Comparative example 4	7.1	1.9	-0.8
				Comparative example 5	5.9	1.3	-1.2

The results show that the fermented emulsions prepared in examples 1 to 4 all have good water supplementing effect. The moisturizing effect of the products prepared in the comparative examples 1 to 5 is significantly different from that of the fermented emulsion prepared in the examples, and the products prepared in the comparative examples 1,4 and 5 have no moisturizing effect and the skin water content is lower than the background value after 60 minutes of use, which indicates that the products prepared in the three groups of comparative examples have very quick skin water loss and cannot achieve the long-term moisturizing effect.

Effect example 3: determination of inflammatory factor (IL-1. Beta.) (repair cells)

Taking logarithmic phase HaCaT cells, inoculating the cell suspension on a 6-hole cell culture plate at the density of 25 ten thousand cells/mL, adding 2mL of cell suspension into each hole, and culturing for 12h; after cells were irradiated with UVB at 40mJ/cm ², the supernatants were blotted off. The blank group and the model group are added with 2mL of DMEM solution, the experimental group is added with 2mL of DMEM solution containing samples (the volume percentage of the samples is 0.1 percent) to treat cells for 24 hours, the supernatant is sucked and removed after the treatment, the cells are washed for 2 to 3 times by PBS, then the cells are treated by cell lysate, and the cells are centrifuged for 10 minutes at the temperature of 10000r/min and 4 ℃, and the supernatant is taken to obtain cell lysate supernatant. Taking 20 mu L of cell lysate and detecting the total protein content in a sample by using a BCA kit; measurement of inflammatory factors the experimental procedure was followed by measurement of OD values at 450nm according to the ELISA kit instructions.

BCA kit procedure:

1. sample pretreatment: according to the usage amount, preparing lysate according to the ratio of RIPA to PMSF of 100:1, removing the culture solution, cleaning one side by PBS, adding the lysate according to the ratio of 200 mu L of lysate added per cell of a 6-well plate, and fully contacting the lysate with cells under the condition of blowing the number by a liquid transfer device.

2. Post-treatment: centrifuging the cracked sample 10000r for 10min, and taking the supernatant to perform subsequent protein concentration determination.

3. BCA working fluid was prepared in a ratio of 50:1 of the reagent (A) to the reagent (B).

4. Mu.L of the protein sample to be tested was added to a 96-well plate.

5. 200. Mu.L of the prepared BCA working solution was added to each well, and the mixture was left at 37℃for 60 minutes.

6. Absorbance was measured at 562 nm.

7. And calculating the protein concentration of the sample according to the standard curve and the dilution times.

ELISA kit detection steps:

1. Reagent preparation

1) And (3) reagent temperature return: firstly, the kit is warmed up 30min before the experiment, the sample to be tested is placed at room temperature, concentrated washing liquid is put into a water bath at 37 ℃ until the crystallization is completely dissolved if crystallization occurs.

2) Preparing a washing liquid: the use volume of the diluted washing liquid is calculated in advance, and then the 20-time concentrated washing liquid is diluted into 1-time application liquid by deionized water, and the unused concentrated washing liquid is put into 4 ℃ for preservation.

3) Sample gradient dilution: 1mL of a standard/sample diluent (SR 1) was added to the lyophilized standard, and the mixture was allowed to stand for 15 minutes, and after complete dissolution, was gently mixed (concentration 1000 pg/mL). Then 500 μl of standard/sample diluent (SR 1) was added to each of the remaining 6 tubes, and the dilution was performed in a multiple ratio according to the following concentrations: 500. 250, 125, 62.5, 31.25, 15.62, 0pg/mL, 1000pg/mL as standard curve peak concentration, standard/sample dilution (SR 1) as standard curve zero point. The reconstituted stock solution (concentration 1000 pg/mL) of the standard substance is discarded or split-charged according to the need according to the amount of one time, and stored in a refrigerator at-80 ℃.

4) Biotinylated antibody working solution: the amount required for the test was calculated in advance, and the 100-fold antibody concentrate was diluted 1-fold with the test diluent (SR 2) and applied to the working solution (thoroughly mixed before dilution) and added to the reaction well within 30 minutes.

5) Enzyme conjugate working solution: the enzyme conjugate was diluted 1-fold with enzyme conjugate diluent (SR 3) and used within 30min after dilution by diluting the 40-fold concentrated enzyme conjugate to the working fluid (centrifugation prior to dilution) in the amounts required for each test.

6) The washing method comprises the following steps: and (3) throwing out the liquid in the enzyme-labeled plate hole, beating the liquid on the absorbent paper, adding 300 mu L/hole of washing liquid into a washing bottle, standing for 30s, throwing out the liquid in the enzyme-labeled plate hole, and beating the liquid on the absorbent paper.

2. Detection step

1) 30Min before the experiment, the kit is taken out, the temperature is restored to the room temperature, and the plate is washed three times and dried before the standard substance/sample is added.

2) 100. Mu.L of standard/sample was added to the wells, the plates were closed and incubated at 37℃for 90min, the plates were beaten and washed 4 times.

3) 100. Mu.L of biotinylated antibody working solution was added to the wells, and after sealing the plates, incubated at 37℃for 60min, plates were beaten and washed 4 times.

4) 100. Mu.L of enzyme conjugate working solution was added to the wells, and after sealing the plates, incubated at 37℃for 30min, plates were beaten and washed 5 times.

5) 50 Mu L of chromogenic substrate is added into the reaction hole, and the plate is sealed and then developed for 15min in a 37 ℃ incubator in a dark place.

6) Add 50. Mu.L of stop solution and immediately measure OD (within 5 min) at 450nm using an ELISA reader.

The release level of IL-1β was calculated based on the OD values obtained by the above test, and the results are shown in Table 4 and FIG. 1, and the release amount of each inflammatory factor required BCA content correction.

Calculating the expression quantity A of inflammatory factors according to a standard curve, and correcting by using the protein content B to obtain C=A/B, wherein the relative expression quantity=C/C _{Blank group}; standard curve: y=0.0042x+0.1443.

In fig. 1, p < 0.05, indicating a statistical difference compared to the model group; * P < 0.01, indicating significant statistical differences compared to model group, significant decreases; * P < 0.001, indicating a very significant statistical difference compared to the model group, a very significant decrease; ^### p < 0.001, indicating a very significant statistical difference compared to the blank, a very significant increase.

TABLE 4 Table 4

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Effect example 4: elastin (ELN) assay

Taking human skin fibroblasts in a logarithmic growth phase, inoculating 50 ten thousand per hole into a 6-hole plate, and culturing overnight in a 5% CO ₂ environment at 37 ℃ in an incubator; the culture medium is replaced in the experimental group, and 2mL of sample diluted by the culture medium is added, which is equivalent to adding the liquid to be detected with the final concentration of 5% by volume; 2mL of new culture medium is replaced by the model group and the blank group, and the culture medium is placed in an incubator at 37 ℃ and 5% CO ₂ environment for culturing for 24 hours; after the culture, the model group and the experimental group except the blank group are irradiated by UVA for continuous culture for 24 hours, the supernatant is sucked and removed after the culture, the culture is washed twice by PBS, the lysate is adopted for treatment, the cell lysate is obtained, and the cell lysate is obtained after centrifugation for 10 minutes under the condition of 4 ℃ and 12000 rpm.

1.1 Preparation of standard substance: taking a standard substance from a human Elastin (ELN) enzyme-linked immunosorbent assay kit produced by CUSABIO company, centrifuging at 6000rpm for 30S, dissolving with 1mL of sample diluent, repeatedly sucking and beating 5 times with a gun head aiming at the bottom of a freezing tube, fully and uniformly mixing to obtain a standard substance S7, sequentially arranging 7 EP tubes (S0-S6) with the concentration of 1.5mL, respectively adding 350 mu L of sample diluent, sucking 350 mu L of standard substance S7 into a first centrifuge tube (S6), and lightly blowing and beating uniformly. From S6, 350. Mu.L was pipetted into a second EP tube (S5), gently blown up uniformly, and so on to double dilution of the standard, S0 being the sample dilution.

B. washing working solution: concentrated washing liquid according to the proportion of 1: dilution was performed 25 times with deionized water.

C. biotin-labeled antibody working solution: diluting biotin-labeled antibody liquid with biotin-labeled antibody diluent at a ratio of 1:100, adding 990 μl biotin-labeled antibody diluent into 10 μl biotin-labeled antibody, mixing gently, and mixing for 10min.

D. Horseradish peroxidase labeled avidin working solution: diluting horseradish peroxidase-labeled avidin with horseradish peroxidase-labeled avidin in a ratio of 1:100, adding 990 μl horseradish peroxidase-labeled avidin into 10 μl horseradish peroxidase-labeled avidin, mixing gently, and preparing within 10 minutes.

1.2 Specific Experimental procedures

A. The reagents are moved to room temperature (18-25 ℃) for balancing for at least 30min, and the reagents are prepared for standby according to the method.

B. Sample adding: and respectively arranging standard holes and sample holes to be tested. 100 mu L of standard substance or sample to be tested (blank group, model group or experimental group test solution configured as above) is added into each hole, the mixture is gently shaken and shaken uniformly, covered with a plate, and incubated for 2 hours at 37 ℃.

C. the liquid in the holes is discarded, and the liquid is dried without washing.

D. 100 mu L of biotin-labeled antibody working solution was added to each well, and a new plate was attached thereto, followed by incubation at 37℃for 1 hour.

E. the liquid in the wells was discarded, the plate was washed 3 times, each time with 2min of immersion, 200. Mu.L of each well, and the plate was spun dry.

F. 100 mu L of horseradish peroxidase avidin working solution is added to each well, a new patch is covered, and the mixture is incubated for 1 hour at 37 ℃.

G. The liquid in the holes is discarded, the plate is dried by spin-drying and washed 5 times. Soaking for 2 minutes, 200 mu L/hole, and spin-drying.

H. And adding 90 mu L of substrate solution into each hole in sequence, and developing for 15-30 min at 37 ℃ in a dark place.

I. The reaction was terminated by adding 50. Mu.L of a stop solution to each well in sequence.

J. The OD of each well was measured sequentially with a microplate reader at a wavelength of 450nm within 5min after termination of the reaction.

K. And (3) calculating: calculating a standard curve and a regression equation according to the concentration and the OD value, wherein the standard curve and the regression equation are specifically y=3.7/[ 1+ (x/74.26) ^-1.11 ] +0.039; and calculating the elastin content in the sample according to the regression equation and the OD value of the sample, wherein the result is shown in table 5 and fig. 2 (in fig. 2, p < 0.05 indicates that the elastin content is statistically different from the model group, p < 0.01 indicates that the elastin content is significantly increased compared with the model group, p < 0.001 indicates that the elastin content is significantly increased compared with the model group, ^### p < 0.001 indicates that the elastin content is significantly decreased compared with the blank group).

TABLE 5

As can be seen from the results in Table 5, the products prepared in examples 1 to 4 of the present application have ideal anti-aging effects, and the anti-aging effects are significantly higher than those of the products prepared in comparative examples 1 to 5; after the human skin fibroblasts treated by the products prepared in examples 1 to 4 of the application are irradiated by UVA, the elastin content is closer to that of a blank control group, and the ideal anti-aging effect is shown.

Effect example 5: sensory evaluation

Sensory evaluation (moisture retention, tackiness, gloss, etc.) was performed on the products produced in examples 1 to 4 and comparative examples 1 to 5. The results of scoring (on a scale of 5, 1: very bad, 2: bad, 3: normal, 4: good, 5: very good) on items concerning the feel of use of the products produced using the above examples or comparative examples were scored for 24 men and women, respectively, and were shown in Table 6.

TABLE 6

According to the results in Table 6, compared with the products prepared in comparative examples 1 to 5, the products prepared in examples 1 to 4 have more ideal moisturizing effect and small viscosity, and are not easy to generate oily light after use; from a finishing point of view, the user has a higher evaluation of the products produced in examples 1 to 4.

Effect example 5: stability investigation

The stability of the products prepared in the above examples and comparative examples was examined at normal temperature, -20℃and 50℃respectively, with a period of 4 weeks, and the results are shown in FIG. 3.

After 4 weeks of stability investigation, the product prepared in the comparative example 1 is unstable at 50 ℃ and-20 ℃ and has layering phenomenon; the product prepared in comparative example 2 is unstable under different conditions and has layering phenomenon; the product prepared in comparative example 3 is unstable under different conditions and has layering phenomenon; the products prepared in examples 1 to 4 and comparative examples 4 to 5 were stable under different conditions.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While the application has been disclosed by the foregoing description of specific embodiments thereof, it will be appreciated that those skilled in the art may devise various modifications, adaptations, or equivalents of the application within the spirit and scope of the appended claims. Such modifications, improvements, or equivalents are intended to be included within the scope of this application as claimed.

Claims

1. A method for preparing a fermented emulsion, comprising the steps of:

step (2): inoculating lactobacillus plantarum into the material A prepared in the step (1), and carrying out fermentation culture and sterilization to prepare the fermentation emulsion;

Wherein, in the step (1), the saccharomyces cerevisiae is the saccharomyces cerevisiae with the preservation number of CGMCC NO.17452 preserved in China general microbiological culture collection center; in the step (2), the lactobacillus plantarum is lactobacillus plantarum with the preservation number of CICC 20261 and preserved in China center for industry microbiological culture collection center.

2. The method of preparing a fermented emulsion according to claim 1, wherein the method of preparing a fermented emulsion satisfies at least one of the following conditions:

In the step (1), the rice further comprises a crushing operation before use, and the particle size after crushing is 50-100 meshes;

In the step (1), the vegetable oil comprises at least one of macadamia nut seed oil, peony seed oil, white pool flower seed oil and prinsepia utilis royle oil;

In the step (1), the mass ratio of the water to the rice is (10-200): 1, a step of;

in the step (1), the mass ratio of the rice to the vegetable oil is (3-8): 1, a step of;

In the step (1), the saccharomyces cerevisiae is added in the form of a saccharomyces cerevisiae liquid, and the number of viable bacteria in the saccharomyces cerevisiae liquid is 10 ⁵～10⁹ CFU/mL;

in step (1), the amount of the Saccharomyces cerevisiae inoculated per unit volume of the fermentation substrate is 5X 10 ³～5×10⁷ CFU/mL;

in the step (1), the fermentation culture is carried out in a shake incubator, and the rotating speed of a shaking table in the shake incubator is 150-300 r/min;

in the step (1), the fermentation culture time is 24-120 hours;

In the step (1), the temperature of the fermentation culture is 25-38 ℃;

In the step (1), the sterilization method is a high-temperature sterilization method;

In the step (1), the sterilization operation further comprises the operation of cooling to room temperature.

3. The method of preparing a fermented emulsion according to claim 2, wherein the method of preparing a fermented emulsion satisfies at least one of the following conditions:

in the step (1), the mass ratio of the water to the rice is (20-100): 1, a step of;

In the step (1), the mass ratio of the rice to the vegetable oil is (3-5): 1, a step of;

In the step (1), the saccharomyces cerevisiae is added in the form of a saccharomyces cerevisiae liquid, and the number of viable bacteria in the saccharomyces cerevisiae liquid is 10 ⁶～10⁸ CFU/mL;

In step (1), the amount of the Saccharomyces cerevisiae inoculated per unit volume of the fermentation substrate is 5X 10 ⁴～5×10⁶ CFU/mL;

In the step (1), the fermentation culture is carried out in a shake incubator, and the rotating speed of a shaking table in the shake incubator is 150-250 r/min;

in the step (1), the fermentation culture time is 48-100 hours;

in the step (1), the temperature of the fermentation culture is 25-32 ℃;

in the step (1), when the high-temperature sterilization method is adopted for the sterilization, the sterilization temperature is 90-125 ℃; when the high-temperature sterilization method is adopted for the sterilization, the sterilization time is 20-60 min.

4. A method of preparing a fermented emulsion according to claim 3, wherein the method of preparing a fermented emulsion satisfies at least one of the following conditions:

in the step (1), the mass ratio of the water to the rice is (20-60): 1, a step of;

In the step (1), when the high-temperature sterilization method is adopted for the sterilization, the sterilization temperature is 90-110 ℃;

in the step (1), when the high-temperature sterilization method is adopted for the sterilization, the sterilization time is 20-40 min.

5. The method of preparing a fermented emulsion according to claim 1, wherein in step (1), the fermentation substrate further comprises a sterilization operation prior to use.

6. The method for producing a fermented emulsion according to claim 1, wherein in the step (1), the method for sterilizing the fermentation substrate is a high-temperature sterilization method.

7. The method of preparing a fermented emulsion according to claim 6, wherein the method of preparing a fermented emulsion satisfies at least one of the following conditions:

when the high-temperature sterilization method is adopted to sterilize the fermentation substrate, the sterilization temperature is 110-125 ℃;

When the high-temperature sterilization method is adopted to sterilize the fermentation substrate, the sterilization time is 20-60 min;

the fermentation substrate further comprises an operation of cooling to room temperature after the operation of sterilizing.

8. The method of preparing a fermented emulsion according to claim 7, wherein the method of preparing a fermented emulsion satisfies at least one of the following conditions:

When the high-temperature sterilization method is adopted to sterilize the fermentation substrate, the sterilization temperature is 115-121 ℃;

when the high-temperature sterilization method is adopted to sterilize the fermentation substrate, the sterilization time is 20-40 min.

9. The method for producing a fermented emulsion according to any one of claims 1 to 8, wherein the method for producing a fermented emulsion satisfies at least one of the following conditions:

in the step (2), lactobacillus plantarum is added in the form of lactobacillus plantarum bacterial liquid, and the concentration of the lactobacillus plantarum in the lactobacillus plantarum bacterial liquid is 10 ⁶～10¹⁰ CFU/mL;

In the step (2), the number of lactobacillus plantarum inoculated in the material A per unit volume is 10 ⁴～10⁸ CFU/mL;

In the step (2), the fermentation culture method is standing fermentation;

In the step (2), the fermentation culture time is 12-24 hours;

In the step (2), the temperature of the fermentation culture is 30-45 ℃;

in the step (2), the sterilization method is a high-temperature sterilization method.

10. The method of preparing a fermented emulsion according to claim 9, wherein the method of preparing a fermented emulsion satisfies at least one of the following conditions:

in the step (2), lactobacillus plantarum is added in the form of lactobacillus plantarum bacterial liquid, and the concentration of the lactobacillus plantarum in the lactobacillus plantarum bacterial liquid is 10 ⁷～10⁹ CFU/mL;

In the step (2), the number of lactobacillus plantarum inoculated in the material A per unit volume is 10 ⁵～10⁷ CFU/mL;

in the step (2), the fermentation culture time is 12-18 hours;

in the step (2), the temperature of the fermentation culture is 35-45 ℃;

In the step (2), when the high-temperature sterilization method is adopted for the sterilization, the sterilization temperature is 90-125 ℃; when the high-temperature sterilization method is adopted for the sterilization, the sterilization time is 20-50 min.

11. The method for preparing a fermented emulsion according to claim 9, wherein when the sterilization is performed by the high temperature sterilization method, the temperature of the sterilization is 90 to 110 ℃; when the high-temperature sterilization method is adopted for the sterilization, the sterilization time is 25-35 min.

12. The method for preparing a fermented emulsion according to claim 1, wherein in the step (2), the step of sterilizing further comprises the steps of cooling and/or centrifuging, and collecting the supernatant.

13. The method of preparing a fermented emulsion according to claim 12, wherein step (2) satisfies at least one of the following conditions: the cooling is to cool to room temperature;

the rotational speed of the centrifugation is 3000-9000 r/min;

The radius of the centrifugation is 8-15 cm;

The centrifugation time is 10-40 min.

14. The method of claim 13, wherein step (2) satisfies at least one of the following conditions:

The rotational speed of the centrifugation is 4000-6000 r/min;

The centrifugation time is 20-40 min.

15. The method of any one of claims 12 to 14, wherein in step (2), the centrifugation step is followed by a secondary sterilization step and/or a preservative mixing step.

16. The method of claim 15, wherein at least one of the following conditions is satisfied in step (2):

the secondary sterilization method is a high-temperature sterilization method;

In the process of mixing with the preservative, the mixing temperature is 50-80 ℃;

during mixing with the preservative, the preservative comprises p-hydroxyacetophenone and/or 1, 2-hexanediol.

17. The method of claim 16, wherein step (2) satisfies at least one of the following conditions:

When the high-temperature sterilization method is adopted for the secondary sterilization, the temperature of the secondary sterilization is 100-125 ℃;

when the high-temperature sterilization method is adopted for the secondary sterilization, the time of the secondary sterilization is 20-40 min;

In the process of mixing with the preservative, the mixing temperature is 70-80 ℃;

When the preservative comprises the p-hydroxyacetophenone and the 1, 2-hexanediol, the mass percentage of the p-hydroxyacetophenone to the supernatant fluid prepared after centrifugation is 0.1-1%, and the mass percentage of the 1, 2-hexanediol to the supernatant fluid prepared after centrifugation is 0.1-1%.

18. The method of claim 17, wherein step (2) satisfies at least one of the following conditions:

when the high-temperature sterilization method is adopted for the secondary sterilization, the temperature of the secondary sterilization is 115-121 ℃;

when the high-temperature sterilization method is adopted for the secondary sterilization, the time of the secondary sterilization is 25-35 min;

when the preservative comprises the p-hydroxyacetophenone and the 1, 2-hexanediol, the mass percentage of the p-hydroxyacetophenone to the supernatant obtained after the centrifugation is 0.5%, and the mass percentage of the 1, 2-hexanediol to the supernatant obtained after the centrifugation is 0.5%.

19. A fermented emulsion prepared by the method of preparing a fermented emulsion according to any one of claims 1 to 18.

20. Use of a fermented emulsion according to claim 19 as a direct product, as an additive or as a substrate in the preparation of a skin external preparation.

21. The use according to claim 20, wherein the fermented emulsion is used as at least one of a moisturizing active ingredient, an anti-inflammatory active ingredient and an anti-aging active ingredient in the skin external agent.

22. A skin external preparation comprising the fermented emulsion according to claim 19.

23. The external skin preparation according to claim 22, wherein at least one of the following conditions is satisfied:

the skin external preparation further comprises at least one of a moisturizing active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient and an anti-oxidation active ingredient;

The skin external agent comprises a facial mask, essence or toner;

the mass percentage of the fermented emulsion in the skin external agent is 5-99%.

24. The external preparation for skin according to claim 23, wherein the fermented emulsion accounts for 60 to 99% by mass of the external preparation for skin.