CN113559045A

CN113559045A - Oat bran fermentation product, skin external preparation containing oat bran fermentation product, and preparation method and application of oat bran fermentation product

Info

Publication number: CN113559045A
Application number: CN202110993230.2A
Authority: CN
Inventors: 史豆豆; 邓朝庆
Original assignee: Zhejiang Chenhai Life Science Co ltd
Current assignee: Zhejiang Chenhai Life Science Co ltd
Priority date: 2021-08-26
Filing date: 2021-08-26
Publication date: 2021-10-29

Abstract

The invention discloses oat bran leavening, a skin external agent containing the oat bran leavening, and a preparation method and application of the skin external agent. The preparation method of oat bran leavening comprises the following steps: inoculating Saccharomyces cerevisiae into fermentation substrate, fermenting, culturing, and sterilizing; wherein the fermentation substrate comprises oat bran. The oat bran fermentation product prepared by the invention has no obvious toxic or side effect on cells, has good whitening effect, water locking effect and antioxidant effect, can be widely applied to the field of skin external preparations, improves the utilization rate of oat bran and realizes the change of fertilizer into treasure.

Description

Oat bran fermentation product, skin external preparation containing oat bran fermentation product, and preparation method and application of oat bran fermentation product

Technical Field

The invention belongs to the technical field of fermentation, and particularly relates to oat bran leavening, a skin external agent containing the oat bran leavening, and a preparation method and application of the oat bran leavening.

Background

Oat bran is the bran coat on the surface layer of malts and has a protection effect on the oats, and the oat bran is generally filtered as waste in the oat processing process. Because the oat bran contains a large amount of water-soluble dietary fibers, non-soluble dietary fibers, oat firming protein, linoleic acid, sapogenin, beta-glucan and other substances, the oat bran has the effects of reducing blood fat, reducing blood sugar, cleaning intestinal tracts and the like, has attracted wide attention in the field of foods in recent years, becomes a favorite food for weight-losing people, but has a fresh report on the application of the oat bran in the field of skin external preparations.

Related reports of using a water extraction method to prepare oat bran extract exist in the prior art, and the prepared product is easy to cause skin allergy and has the defects of cytotoxicity and the like. Therefore, the technology for effectively extracting the effective active ingredients from the oat bran to reduce the skin allergy phenomenon is an urgent technical problem to be solved in the field. The active substance extraction method commonly used in the field of skin external preparations includes water extraction, organic solvent extraction, ultrasonic extraction, microwave extraction, supercritical fluid extraction, microbial fermentation, etc. Since different extraction methods have great influence on the types and yields of active ingredients in the final product and the efficacy of the final product, in the face of such many extraction methods, how to screen the active ingredients more favorably extracted from oat bran for use in skin external preparations remains a technical problem for researchers in the field.

Therefore, there is a need in the art to develop an extraction method for extracting active substances from oat bran, which can be used in the field of skin external preparations, and the extract meets the requirements of high safety and multiple effects, so that the oat bran is fully utilized, and waste materials are changed into valuable materials.

Disclosure of Invention

The invention aims to overcome the defects that in the prior art, the oat bran utilization rate is low, an oat bran extract prepared by a water extraction method is easy to cause skin allergy and high in cytotoxicity in the using process, the existing plant extraction processes are various, and the method for screening out active ingredients which can be extracted from oat bran and are suitable for the field of skin external preparations is still difficult, and the like, and provides an oat bran leavening, a skin external preparation containing the oat bran leavening, and a preparation method and application of the skin external preparation. The oat bran fermentation product prepared by the invention has no obvious toxic or side effect on cells, has good whitening effect, water locking effect and antioxidant effect, can be widely applied to the field of skin external preparations, improves the utilization rate of oat bran and realizes the change of fertilizer into treasure.

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

the invention provides a preparation method of oat bran leavening, which specifically comprises the following steps: inoculating Saccharomyces cerevisiae into fermentation substrate, fermenting, culturing, and sterilizing; wherein the fermentation substrate comprises oat bran.

In some embodiments, the Saccharomyces cerevisiae may be any one or more of Saccharomyces cerevisiae (Saccharomyces cerevisiae) with a collection number of CGMCC No.2.5472, Saccharomyces cerevisiae (Saccharomyces cerevisiae) with a collection number of CGMCC No.2.964, and Saccharomyces cerevisiae (Saccharomyces cerevisiae) with a collection number of CGMCC No. 2.1543.

In some embodiments, the saccharomyces cerevisiae may be added in the form of a saccharomyces cerevisiae bacterial solution according to the conventional method in the art, and the concentration of the saccharomyces cerevisiae in the saccharomyces cerevisiae bacterial solution may be 10⁵-10⁹CFU/mL, preferably 10⁷-10⁹CFU/mL。

Wherein, the mass ratio of the saccharomyces cerevisiae bacterial liquid to the fermentation substrate can be conventional in the field, and is preferably (1-5): 100.

the preparation method of the saccharomyces cerevisiae bacterial liquid can be conventional in the field, and specifically comprises the following steps: the saccharomyces cerevisiae is inoculated in a YPD liquid culture medium and is obtained by fermentation culture.

In the preparation process of the saccharomyces cerevisiae bacterial liquid, the fermentation culture can be performed on a shaking table according to the conventional practice in the art, and the rotation speed of the shaking table can be the conventional practice in the art, and generally can be 150-.

In the preparation process of the saccharomyces cerevisiae bacterial liquid, the time of fermentation culture can be the time of the operation routine in the field, and is preferably 36-48 h.

In the preparation process of the saccharomyces cerevisiae bacterial liquid, the temperature of the fermentation culture can be the temperature which is conventional in the operation in the field, preferably 25-30 ℃, and more preferably 25-28 ℃.

In some embodiments, the oat bran may be a bran conventionally recognized by those skilled in the art as an oat top layer, such as oat bran produced by Zhangkou Avena sativa food Co.

In some embodiments, the oat bran may be conventional in the art, typically 1% to 2% by mass of the fermentation substrate.

In some embodiments, the fermentation substrate may further comprise a solvent and/or glucose.

The solvent may be a solvent conventionally used in the art, preferably water, and more preferably deionized water.

Wherein the mass percentage of the glucose in the fermentation substrate can be conventional in the field, and can be generally 0.5-2%.

In a preferred embodiment, the fermentation substrate consists of the following components in percentage by mass: 1-2 wt% of the oat bran, 0.5-2 wt% of the glucose and the balance of the water.

In some embodiments, the fermentation substrate may further comprise a sterilization operation prior to use. The sterilization conditions and method may be high temperature sterilization conventionally used in the art.

The temperature for sterilization may be a temperature conventional in the art, preferably 95-130 ℃, more preferably 110-130 ℃.

Wherein the time for sterilization can be the time which is conventional in the operation in the field, and is preferably 15-30 min.

Wherein the pressure for sterilization may be a pressure conventional for such operations in the art, preferably 0.09-0.15 MPa.

In some embodiments, the conditions and methods of the fermentation culture can be conventional in the art, and can be performed on a shaker, wherein the rotation speed of the shaker can be conventional in the art, preferably 150-200rpm, and more preferably 170-200 rpm.

In some embodiments, the fermentation culture may be carried out for a period of time conventional in the art for such procedures, preferably from 24 to 72 hours, more preferably from 45 to 48 hours.

In some embodiments, the temperature of the fermentation culture may be a temperature conventional in such procedures in the art, preferably from 20 to 30 ℃, more preferably from 25 to 28 ℃.

In some embodiments, the sterilization conditions and methods may be high temperature sterilization as is conventional in the art.

In some embodiments, the sterilization temperature may be a temperature conventional in such operations in the art, preferably 90-120 ℃, more preferably 100-.

In some embodiments, the time for sterilization may be a time conventional in such operations in the art, preferably 15-30 min.

In some embodiments, the pressure of the sterilization may be a pressure conventional to such operations in the art, preferably 0.09 to 0.15 MPa.

In some embodiments, the operation of fermentation culture may be followed by a further operation of centrifugation.

Wherein, the rotation speed of the centrifugation can be the rotation speed conventional in the operation in the field, preferably 3900-.

Wherein, the time of the centrifugation can be the time which is conventional in the operation in the field, preferably 15-30min, and more preferably 20-30 min.

In some embodiments, the sterilizing operation may be further followed by an operation of mixing with a preservative.

Wherein, during the mixing with the preservative, the temperature of the mixing can be the temperature which is conventional in the operation of the type in the field, and is preferably 70-75 ℃.

Wherein, in the process of mixing with the preservative, the mixing time can be the time which is conventional in the operation of the type in the field, and is preferably 20-30 min.

The type of the preservative can be conventional in the field, and preferably is p-hydroxyacetophenone and/or 1, 2-hexanediol. When the preservative comprises p-hydroxyacetophenone and 1, 2-hexanediol, the mass percentage of the p-hydroxyacetophenone in the sterilized material can be 0.2-0.5%, and the mass percentage of the 1, 2-hexanediol in the sterilized material can be 0.5-2%; preferably, when the preservative comprises p-hydroxyacetophenone and 1, 2-hexanediol, the mass percentage of the p-hydroxyacetophenone in the sterilized material is 0.3% -0.5%, and the mass percentage of the 1, 2-hexanediol in the sterilized material is 1% -2%.

The invention also provides an oat bran ferment prepared by the preparation method of the oat bran ferment.

The invention also provides application of the oat bran leavening as a product, a product additive or a product base in preparing a skin external agent.

In some embodiments, the oat bran ferment may be used as any one or more of a whitening active ingredient, a water-locking active ingredient, and an antioxidant active ingredient in the skin external preparation.

Wherein the whitening active ingredient can be a whitening active ingredient with tyrosinase activity inhibiting effect.

Wherein the antioxidant active ingredient can be an antioxidant active ingredient having an active oxygen scavenging effect.

The invention also provides a skin external agent, which comprises the oat bran leavening.

In some embodiments, the oat bran ferment may be present in the external skin preparation in an amount of 10% to 100%, preferably 90% to 100% by weight.

In some embodiments, the external skin preparation may include a mask, essence, or toner as is conventional in the art.

In some embodiments, the skin external preparation may further include a thickener and/or a moisturizer.

Wherein the thickening agent may comprise hydroxypropylmethyl cellulose as is conventional in the art.

Wherein, the mass percentage of the thickening agent in the skin external agent can be conventional in the field, and can be generally 0.1-0.5%.

Wherein the humectant may comprise 1, 3-propanediol and/or glycerin as is conventional in the art.

Wherein, the mass percentage of the humectant in the skin external preparation can be conventional in the field, and can be generally 3-5%.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

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

The positive progress effects of the invention are as follows: the oat bran leavening is prepared by a microbial fermentation method by selecting oat bran as a fermentation substrate and saccharomyces cerevisiae as a fermentation strain, has good water locking effect, whitening effect and antioxidation effect, has no obvious toxic or side effect on cells, is good in safety and free of irritation to skin, can be used as an excellent raw material of a skin external agent or directly used as the skin external agent, improves the utilization rate of the oat bran, and realizes the change of the fertilizer into the treasure.

Drawings

The disclosure may be better understood by reference to the following description taken in conjunction with the accompanying drawings. The accompanying drawings, which are incorporated in and form a part of this specification, illustrate preferred embodiments of the present disclosure and, together with the detailed description, serve to explain the principles and advantages of the disclosure. Wherein:

FIG. 1 is a graph showing the survival rate of B16 cells after treating B16 cells with oat bran fermentate prepared in examples 1-3 and oat bran aqueous extract prepared in comparative example 1 at different concentrations;

FIG. 2 is a graph comparing the total antioxidant capacity of oat bran fermentates prepared in examples 1-3 with products prepared in comparative examples 1-2;

FIG. 3 is a graph showing the comparison of the active oxygen content in HFF-1 cells after the oat bran fermentate prepared in examples 1-3 and the oat bran aqueous extract prepared in comparative example 1, respectively;

FIG. 4 is a graph showing a comparison of tyrosinase activities in cells treated with oat bran fermentates prepared in examples 1-3 of the present invention and oat bran aqueous extracts prepared in comparative example 1, respectively;

FIG. 5 is a graph showing the comparison of the percutaneous water loss rate of skin after the skin is treated with the essence prepared from oat bran fermentations prepared in examples 1-3 and the essence of a blank control group, respectively.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Exemplary embodiments of the present disclosure will be described hereinafter with reference to the accompanying drawings.

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

The Saccharomyces cerevisiae 1 in the following example 1 is specifically Saccharomyces cerevisiae (Saccharomyces cerevisiae) which is purchased from China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the collection number of CGMCC No. 2.964.

The Saccharomyces cerevisiae 2 in the following example 2 is specifically Saccharomyces cerevisiae (Saccharomyces cerevisiae) which is purchased from China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the collection number of CGMCC No. 2.5472.

The Saccharomyces cerevisiae 3 in the following example 3 is specifically Saccharomyces cerevisiae (Saccharomyces cerevisiae) which is purchased from China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the collection number of CGMCC No. 2.1543.

The following sweet koji (rhizopus) of comparative example 1 was purchased from Angel Yeast Ltd;

in the following examples, the oat bran production company is Zhangkou naked oat sprout food company Limited.

The YPD liquid medium formulation in the following examples was: 2 wt% of glucose, 1 wt% of peptone and 0.5 wt% of yeast extract; the YPD solid culture medium is YPD liquid culture medium added with 2 wt% agar powder.

The preparation method of the saccharomyces cerevisiae 1 bacterial liquid in the following embodiment 1 comprises the following steps:

(1) activation of strains: inoculating Saccharomyces cerevisiae 1 into YPD solid culture medium for streak activation, and culturing in 28 deg.C incubator for 48 hr to obtain single colony;

(2) and (3) expanding culture of strains: inoculating the single colony obtained in the step (1) into 100mL YPD liquid culture medium, and culturing in a shaking table at 28 ℃ and 180rpm for 48h to obtain Saccharomyces cerevisiae 1 bacterial liquid with the concentration of 10⁷CFU/mL。

Compared with the saccharomyces cerevisiae 1 bacterial liquid, the preparation method of the saccharomyces cerevisiae 2 bacterial liquid and the saccharomyces cerevisiae 3 bacterial liquid in the following embodiments only has the difference that the strains inoculated in the step (1) are respectively replaced by the saccharomyces cerevisiae 2 or the saccharomyces cerevisiae 3, and other condition parameters are the same.

Example 1

The preparation method of the fermentation substrate comprises the following steps: adding 6g of oat bran and 1.5g of glucose into 300g of water, and sterilizing at 110 ℃ and 0.1MPa for 30min to obtain an oat bran fermentation substrate;

the concentration obtained by the above method is 10⁷Preparing oat bran leavening by using a CFU/mL saccharomyces cerevisiae 1 bacterial liquid and an oat bran fermentation substrate as raw materials, and specifically comprising the following steps: inoculating 3g of saccharomyces cerevisiae 1 bacterial liquid into 300g of oat bran fermentation substrate, and fermenting and culturing for 45 hours at the temperature of 28 ℃ and under the condition of stirring, wherein the rotating speed of stirring is 170 rpm; transferring the prepared material into a centrifuge, centrifuging at 4500rpm for 20min, collecting supernatant, and sterilizing the supernatant at 100 deg.C and 0.1MPa for 30 min; sterilizing, cooling, and mixing with antiseptic at 70 deg.C for 30 min; in the preservative, the mass percent of the p-hydroxyacetophenone in the sterilized material is 0.3%, and the mass percent of the 1, 2-hexanediol in the sterilized material is 1%, so as to prepare the oat bran ferment.

Example 2

the concentration obtained by the above method is 10⁷Preparing oat bran leavening by using a CFU/mL saccharomyces cerevisiae 2 bacterial liquid and an oat bran fermentation substrate as raw materials, and specifically comprising the following steps: inoculating 3g of saccharomyces cerevisiae 2 bacterial liquid into 300g of oat bran fermentation substrate, and fermenting and culturing for 45 hours at the temperature of 28 ℃ and under the condition of stirring, wherein the rotating speed of stirring is 170 rpm; transferring the prepared material into a centrifuge, centrifuging at 4500rpm for 2min, collecting supernatant, and sterilizing the supernatant at 100 deg.C and 0.1MPa for 30 min; sterilizing, cooling, and mixing with antiseptic at 70 deg.C for 30 min; in the preservative, the mass percent of the p-hydroxyacetophenone in the sterilized material is 0.3%, and the mass percent of the 1, 2-hexanediol in the sterilized material is 1%, so as to prepare the oat bran ferment.

Example 3

the concentration obtained by the above method is 10⁷Preparing oat bran leavening by using a CFU/mL saccharomyces cerevisiae 3 bacterial liquid and an oat bran fermentation substrate as raw materials, and specifically comprising the following steps: inoculating 3g of saccharomyces cerevisiae 3 bacterial liquid into 300g of oat bran fermentation substrate, and fermenting and culturing for 45 hours at the temperature of 28 ℃ and under the condition of stirring, wherein the rotating speed of stirring is 170 rpm; transferring the prepared material into a centrifuge, centrifuging at 4500rpm for 20min, collecting supernatant, and sterilizing the supernatant at 100 deg.C and 0.1MPa for 30 min; sterilizing, cooling, and mixing with antiseptic at 70 deg.C for 30 min; the antiseptic contains 0.3% of p-hydroxyacetophenone and 1, 2-hexanediol, and is prepared from sterilized materialsOat bran fermentate.

Comparative example 1

The preparation method of the oat bran aqueous extract comprises the following steps: adding 6g of oat bran into 300mL of water, heating, stirring and extracting for 2h under the conditions that the temperature is 95 ℃ and the rotating speed is 500rmp, transferring the prepared material into a centrifugal machine, centrifuging for 20min under the condition that the rotating speed is 4500rpm, collecting supernatant, and sterilizing for 30min under the conditions that the temperature is 100 ℃ and the pressure is 0.1 MPa; sterilizing, cooling, and mixing with antiseptic at 70 deg.C for 30 min; in the preservative, the mass percent of the p-hydroxyacetophenone in the sterilized material is 0.3%, and the mass percent of the 1, 2-hexanediol in the sterilized material is 1%, so as to prepare the oat bran aqueous extract.

Comparative example 2

Rehydrating and activating the sweet distiller's yeast: adding water at 27-30 deg.C to 1g of sweet distiller's yeast powder, adding 10g of water, and stirring to obtain sweet distiller's yeast liquid;

the method for preparing the oat bran leavening comprises the following steps of: inoculating 3g of sweet distiller's yeast liquid into 300g of oat bran fermentation substrate, and fermenting and culturing for 45 hours at the temperature of 28 ℃ and under the condition of stirring, wherein the rotating speed of stirring is 170 rpm; transferring the prepared material into a centrifuge, centrifuging at 4500rpm for 20min, collecting supernatant, and sterilizing the supernatant at 100 deg.C and 0.1MPa for 30 min; sterilizing, cooling, and mixing with antiseptic at 70 deg.C for 30 min; in the preservative, the mass percent of the p-hydroxyacetophenone in the sterilized material is 0.3%, and the mass percent of the 1, 2-hexanediol in the sterilized material is 1%, so as to prepare the oat bran ferment.

Effect example 1 safety test

The human body patch test is mainly used for detecting the irritation of the final cosmetic product or raw materials. The products obtained in examples 1 to 3 and comparative examples 1 to 2 were subjected to a closed patch test for human body according to the cosmetic hygiene code (2015) for evaluation of skin irritation.

1. Test subjects:

according to the requirements of 'cosmetic contact dermatitis diagnostic standard and treatment principle', the selected test subject cannot participate in the test, and the test subject and the person with high physique sensitivity who have scars, nevus flammeus and other influences on the result judgment at the part to be tested of the skin cannot participate in the test. In the experiment, 30 suitable volunteers are selected, and the age range is 18-60 years.

2. Experimental methods

0.02mL of each of the products prepared in examples 1-3 and comparative examples 1-2 described above was dropped on a filter paper sheet, and the filter paper sheet was placed in a spot tester. The samples were each provided with a blank control by adding an equal amount of sample solvent distilled water to the control chamber wells. The test period lasted 24 h. In order to ensure the accuracy, credibility and scientificity of test results, the volunteers cannot remove the spot tester or make the tested part contact water according to the requirements during the test. The plaque tester was removed after 24h, left to stand for 30min, and the skin reaction was observed after 24h, after waiting for the disappearance of the indentation. The grading standard of the skin adverse reaction of the body patch test is shown in the table 1.

TABLE 1 grading Standard of adverse skin reactions

3. Test results

See table 2 for results, from which it can be seen; the sensitivity test results of the oat bran fermentations obtained in the embodiments 1-3 of the invention are all negative reactions, which shows that the oat bran fermentations prepared by the invention have safety and do not bring adverse reactions to human bodies. The oat bran aqueous extract prepared in the comparative example 1 individually has weak erythema and relatively poor safety.

TABLE 2

Effect example 2 cytotoxicity test

B16 cells from the center for cell resources of the institute of basic medicine, academy of medical sciences of china) were used in this experiment to verify the cytotoxicity of the products prepared in examples 1 to 3 and comparative example 1.

1. The experimental steps are as follows:

the products of examples 1-3 and comparative example 1 above were each diluted to test solutions having volume percentages of 0.750%, 1.500%, 3.000%, and 6.000%.

B16 cells were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1X 10)⁵U/L penicillin, 100mg/L streptomycin). Cells were grown at 37 ℃ with 5% CO₂In the incubator saturated with humidity, when the cell fusion reached 85% or more, the cells were subjected to digestion with 0.05% trypsin and the digestion reaction was terminated with serum-containing DMEM. Counting with cell counting plate, adjusting cell suspension concentration to 5 × 10⁴one/mL, the cell suspension was inoculated into a 96-well plate at a rate of 100. mu.L/well, at 37 ℃ with 5% CO₂Incubate under conditions overnight. Removing culture solution, adding filter sterilized solution to be tested, making 6 multiple wells for each sample, and culturing for 24 h. After the completion of the culture, 100. mu.L of a mixed solution of MTT solution (5mg/mL) and DMEM (v/v, 1: 5) was added to each well and incubated for 4 hours. The old medium was removed, 150. mu.L of DMSO was added to each well, and the absorbance (denoted A) of the experimental group was read at 490nm after incubation at 37 ℃ for 10 min. The cell-free treated group (cell suspension was replaced with serum-free DMEM, and the rest was the same) was set as a blank control group, and designated as B. The cell control group (the solution to be tested was replaced with serum-free DMEM, and the rest steps were the same) was designated as C.

The calculation formula of the relative survival rate of the cells is as follows:

relative cell survival rate ═ (a-B)/(C-B) × 100%;

the relative survival rates of B16 cells after treating B16 cells with the oat bran fermentates prepared in examples 1-3 of the present invention and the oat bran aqueous extract prepared in comparative example 1 at different concentrations are shown in fig. 1 and table 3; it can be seen from table 3 that the oat bran fermentations prepared in examples 1-3 according to the present invention have significantly reduced cytotoxicity and even cell growth promoting effect, as compared to the oat bran aqueous extract prepared in comparative example 1.

TABLE 3

Effect example 3 evaluation of Total Oxidation resistance

The principle of ABTS method for determining total antioxidant capacity is as follows, ABTS is oxidized into green ABTS under the action of proper oxidant⁺ABTS in the Presence of antioxidants⁺Production of (B) is suppressed, and ABTS is measured at 734nm or 405nm⁺The absorbance can be measured and the total antioxidant capacity of the sample can be calculated. Trolox is an analogue of vitamin E, has the antioxidant capacity similar to that of vitamin E, and is used as a reference for the total antioxidant capacity of other antioxidants. The total antioxidant capacity is detected according to the specification of the total antioxidant capacity detection kit, and the specific test method comprises the following steps:

1. the experimental steps are as follows:

the products of the above examples 1-3 and comparative examples 1-2 were diluted with PBS to a solution to be tested having a volume concentration of 40%; 10mM Trolox standard solution is diluted into 0.1, 0.15, 0.3 and 0.6mM solution to be detected.

200 μ L of ABTS working solution was added to each assay well of the 96-well plate. Adding 10 μ L of distilled water or PBS or other appropriate solution into blank control hole; 10 mu L of Trolox standard solutions with various concentrations are added into the standard curve detection holes; and adding 10 mu L of various samples into the sample detection holes, gently mixing the samples, incubating the mixture at room temperature for 2-6min, and then measuring the absorbance at 734 nm. According to the test result of the standard curve detection hole, fitting a standard curve equation as follows: y-0.6923 x +0.456, R²＝0.9993。

And calculating the total antioxidant capacity of the sample according to the standard curve. If the absorbance measured by the sample is outside the range of the standard curve, the sample is diluted or concentrated appropriately and then measured.

The results of the experiments are shown in Table 4 below and FIG. 2. from Table 4, it can be seen that the oat bran fermentates prepared in examples 1-3 have a stronger total antioxidant capacity than the products prepared in comparative examples 1-2.

The significant difference P in the total antioxidant capacity of examples 1-3 compared to comparative examples 1-2 was calculated and the results are shown in table 4; wherein P >0.05 indicates insignificant variability; 0.01< P <0.05 indicates significant variability; p <0.01 indicates that the variability is very significant. The results show that the total antioxidant capacity of the products prepared in examples 1-3 is significantly improved compared to comparative examples 1-2.

TABLE 4

Effect example 4 testing of antioxidant scavenging reactive oxygen species in vitro

The products prepared in examples 1-3 above were tested for their effect on the active oxygen content in cells. Preparing the products prepared in the examples 1 to 3 into solutions to be tested with the volume percentage of 6.000 percent respectively; collecting cells HFF-1 with good logarithmic phase state, digesting with trypsin, stopping digestion with complete culture medium, and counting; adjusting the cell concentration of the cell suspension to 1X 10⁵one/mL, and added to 6-well plates, 1mL fresh complete medium per well, 1mL cell suspension, 5% CO at 37 ℃₂Culturing in an incubator overnight; sucking culture medium, adding 2mL of the prepared solution to be tested into each well, adding 2mL of basal culture medium into cell control wells, and culturing at 37 deg.C with 5% CO₂Culturing for 48h in an incubator;

after 24h of culture, the culture medium was removed, trypsinized, digestion was stopped with complete medium, and the cell suspension was diluted with PBS (pH7.2-7.4) to a cell concentration of about 100 ten thousand/mL. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifugation was carried out for about 20 minutes (2000-. The supernatant was carefully collected. If precipitate is formed during the storage process, the solution should be centrifuged again.

Detection was performed using a human Reactive Oxygen Species (ROS) ELISA kit.

The kit adopts a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). And adding the specimen, the standard substance and the detection antibody marked by the HRP into the coated micropores previously coated with the human Reactive Oxygen Species (ROS) capture antibody, incubating and thoroughly washing. The color is developed with the substrate TMB, which is converted to blue by the catalysis of peroxidase and to the final yellow color by the action of an acid. The shade of the color is positively correlated with the human Reactive Oxygen Species (ROS) in the sample. The absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader, and the sample concentration was calculated.

Compared with a cell control group, the oat bran leavening prepared in the examples 1-3 has obviously reduced active oxygen content and ideal antioxidant capacity; among them, the oat bran fermentations prepared in examples 1-2 were significantly stronger in antioxidant capacity than comparative example 1, and the results are shown in table 5 and fig. 3.

TABLE 5

Numbering	Sample concentration/active oxygen content IU/mL
		Example 1	2.247
Example 2	2.21
		Example 3	2.39
Comparative example 1	2.337
		CellsControl group	2.47

Effect example 5

The tyrosinase inhibitory activity of the products obtained in examples 1-3 and comparative example 1 was tested. Respectively preparing the oat bran fermentate prepared in examples 1-3 and the oat bran aqueous extract prepared in comparative example 1 into samples to be tested with the concentration of 0.75% by using distilled water;

1. principle of experiment

Tyrosinase is a key enzyme in the process of melanin synthesis, and is closely related to the color of skin. Active substances of the whitening cosmetics popular in the market are tyrosinase inhibitors, and the whitening cosmetics mainly inhibit the synthesis of melanin by inhibiting the activity of tyrosinase so as to play a whitening role. Meanwhile, tyrosinase has a unique dual catalytic function and has a close relation with human body aging. The abnormal over-expression can cause pigmentation diseases of human bodies, and further cause oxidative damage of the bodies. Therefore, the inhibition effect on tyrosinase can reflect the whitening effect of the active ingredients and the activity of resisting the oxidative damage of the organism.

The L-tyrosinase has the maximum absorption wavelength at 475nm, can perform catalytic reaction with a substrate L-tyrosine or L-dopa solution, and can generate inhibition effect on the catalytic reaction after a reagent with the activity inhibition effect on the L-tyrosinase is added into an experimental system. Therefore, the inhibition rate of the sample on the L-tyrosinase activity was evaluated by measuring the absorbance at 475nm of the sample before and after the addition of the enzyme.

2. Experimental methods

Collecting cells with good logarithmic phase state, digesting with trypsin, stopping digestion with complete culture medium, and counting; the cell suspension was adjusted to 10X 10⁴cells/mL, seeded in 96-well plates, 100. mu.L of cell suspension per well, and marginal wells filled with sterile PBS at 37 ℃ with 5% CO₂Culturing overnight in incubator, sucking culture medium, adding different concentrations into each wellThe method comprises the steps of weighing 100 mu L of sample, acting the sample for 48 hours, adding 50 mu L of Trition X-100 solution with the concentration of 1% to crack cells, quickly putting the sample into an ultra-low temperature refrigerator with the temperature of 80 ℃ below zero to be frozen for 1 hour, then thawing the sample at room temperature to release tyrosinase in the cells out of the cells, pre-warming the sample at the temperature of 37 ℃, adding 50 mu L of levodopa solution with the concentration of 1% to react for 2 hours at the temperature of 37 ℃, measuring the absorbance value of each hole at the wavelength of 490nm, setting more than 3 auxiliary holes for each concentration, and averaging.

Tyrosinase activity change ═ (assay well OD value-blank OD value)/(cell control OD value-blank OD value) × 100%.

3. Results of the experiment

See fig. 4 and table 6 for experimental results. As can be seen from FIG. 4 and Table 6, the products obtained in examples 1-3 all have inhibitory effects on tyrosinase, and the inhibitory abilities are different, and the product obtained in example 2 has the most desirable inhibitory effect on tyrosinase. The products prepared in examples 1-3 all have stronger inhibition effect on tyrosinase than comparative example 1, and have very significant difference compared with comparative example 1, and the results are shown in table 6.

TABLE 6

Effect example 5

The products of examples 1-3 were tested for moisture retention. Compounding the oat bran fermentations prepared in the examples 1-3 to obtain No. 1-3 essence, taking No. 4 as an auxiliary material matrix, wherein the specific formula is shown in Table 7;

TABLE 7

1. Experiment design:

and (3) testing environment: the temperature is 22 +/-2 ℃; humidity of 50-60%

Test area: the test site was selected from the medial forearm of both sides.

Testing indexes are as follows: amount of water dispersed through skin.

Testing time points: moisturizing efficacy (arm): after the background test, skin tests are respectively carried out 30min, 1h, 2h, 4h and 8h after the membrane cloth is taken down.

An experimental instrument: the MPA 580.

2. The test method comprises the following steps:

10 eligible volunteers participated in the test. The test place has no direct light and no wind, the room temperature is 22-24 ℃, and the humidity is 50-60%. Before detection, cleaning forearms of both sides with facial cleanser, standing for 30min, drawing 6 normal skin with area of 3.5 × 3.5cm from inner side of forearms of the subject with marking pen, and marking with numbers. The amount of water dispersed through the skin was measured. Cutting the mask cloth into 3 × 3cm, respectively sticking to corresponding mark position of forearm, dripping corresponding mark sample, taking down after 15min, lightly wetting the un-dried essence solution of the test part with cosmetic cotton, and timing. The TEWL value of the horny layer of each part is tested at 30min, 1h, 2h, 4h and 8h respectively. Each site measurement was averaged 3 times.

3. Results of the experiment

The transdermal water loss rate was calculated for 4 samples and the results are shown in figure 5 and table 8. From fig. 5 and table 8, it can be seen that the four products all have the water locking effect, but the water locking effect of the essence liquid nos. 1-3 is obviously superior to that of the essence liquid No. 4.

TABLE 8

Finally, it should be further noted that, in the present invention, 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 disclosure has been disclosed above by the description of specific embodiments thereof, it should be understood that various modifications, improvements or equivalents of the disclosure may be devised by those skilled in the art within the spirit and scope of the appended claims. Such modifications, improvements and equivalents are intended to be included within the scope of the present disclosure as claimed.

Claims

1. A preparation method of oat bran leavening is characterized by comprising the following steps: inoculating Saccharomyces cerevisiae into fermentation substrate, fermenting, culturing, and sterilizing; wherein the fermentation substrate comprises oat bran.

2. The method for preparing oat bran fermentate of claim 1, wherein the Saccharomyces cerevisiae is any one or more of Saccharomyces cerevisiae (Saccharomyces cerevisiae) with a preservation number of CGMCC No.2.5472, Saccharomyces cerevisiae (Saccharomyces cerevisiae) with a preservation number of CGMCC No.2.964, and Saccharomyces cerevisiae (Saccharomyces cerevisiae) with a preservation number of CGMCC No. 2.1543;

and/or the saccharomyces cerevisiae is added in the form of saccharomyces cerevisiae bacterial liquid, and the concentration of the saccharomyces cerevisiae in the saccharomyces cerevisiae bacterial liquid is 10⁵-10⁹CFU/mL；

And/or the oat bran accounts for 1-2% of the fermentation substrate by mass;

and/or, the fermentation substrate further comprises a solvent and/or glucose;

and/or, the fermentation culture is carried out on a shaking table, and the rotation speed of the shaking table is 150-200 rpm;

and/or the fermentation culture time is 24-72 h;

and/or the temperature of the fermentation culture is 20-30 ℃;

and/or the sterilization method is high-temperature sterilization.

3. The method of claim 2, wherein the concentration of the saccharomyces cerevisiae in the saccharomyces cerevisiae bacterial liquid is 10⁷-10⁹CFU/mL；

And/or the mass ratio of the saccharomyces cerevisiae bacterial liquid to the fermentation substrate is (1-5): 100, respectively;

and/or the preparation method of the saccharomyces cerevisiae bacterial liquid comprises the following steps: inoculating the saccharomyces cerevisiae into an YPD liquid culture medium, and performing fermentation culture;

and/or, the solvent in the fermentation substrate is water;

and/or the glucose in the fermentation substrate accounts for 0.5-2% of the fermentation substrate by mass;

and/or, the fermentation culture is carried out on a shaking table, and the rotation speed of the shaking table is 170-200 rpm;

and/or the fermentation culture time is 45-48 h;

and/or the temperature of the fermentation culture is 25-28 ℃;

and/or, when the high-temperature sterilization is adopted for sterilization operation, the sterilization temperature is 90-120 ℃;

and/or the sterilization time is 15-30 min;

and/or the pressure of the sterilization is 0.09-0.15 MPa.

4. The method of preparing oat bran fermentate of claim 3 wherein the solvent in the fermentation substrate is deionized water;

and/or the fermentation substrate consists of the following components in percentage by mass: 1-2 wt% of the oat bran, 0.5-2 wt% of the glucose and the balance of the water;

and/or, when the high-temperature sterilization is adopted for sterilization operation, the sterilization temperature is 100-120 ℃.

5. The method for preparing oat bran ferment of any one of the claims 1 to 4, wherein the fermentation substrate further comprises a sterilization operation before use;

and/or, after the operation of fermentation culture, further comprising the operation of centrifugation;

and/or, the operation of sterilizing further comprises the operation of mixing with a preservative.

6. The method for preparing oat bran fermentate of claim 5 wherein the method of sterilization of the fermentation substrate prior to use is high temperature sterilization; preferably, the temperature for sterilization is 95-130 ℃, more preferably 110-; preferably, the sterilization time is 15-30 min; preferably, the pressure of the sterilization is 0.09-0.15 MPa;

and/or, in the operation of the centrifugation after the fermentation culture, the rotation speed of the centrifugation is 3900-;

and/or, in the operation of the centrifugation after the fermentation culture, the centrifugation time is 15-30min, preferably 20-30 min;

and/or, in the operation of mixing with the preservative after the sterilization, the temperature of the mixing is 70-75 ℃;

and/or, in the operation of mixing with the preservative after the sterilization, the mixing time is 20-30 min;

and/or, in the operation of mixing with a preservative after the sterilization, the preservative comprises p-hydroxyacetophenone and/or 1, 2-hexanediol; when the preservative comprises p-hydroxyacetophenone and 1, 2-hexanediol, the mass percent of the p-hydroxyacetophenone in the sterilized material is 0.2-0.5%, and the mass percent of the 1, 2-hexanediol in the sterilized material is 0.5-2%; preferably, when the preservative comprises p-hydroxyacetophenone and 1, 2-hexanediol, the mass percentage of the p-hydroxyacetophenone in the sterilized material is 0.3% -0.5%, and the mass percentage of the 1, 2-hexanediol in the sterilized material is 1% -2%.

7. An oat bran ferment obtained by the method of preparing an oat bran ferment of any one of claims 1 to 6.

8. Use of the oat bran ferment of claim 7 as a product, product additive or product base in the preparation of a skin external preparation.

9. The use according to claim 8, wherein the oat bran ferment is used as any one or more of a whitening active ingredient, a water-locking active ingredient and an antioxidant active ingredient in the skin external preparation;

preferably, the whitening active ingredient is a whitening active ingredient having the effect of inhibiting tyrosinase activity;

preferably, the antioxidant active ingredient is an antioxidant active ingredient having an active oxygen scavenging effect.

10. An external preparation for skin, comprising the oat bran fermented product of claim 7;

preferably, the oat bran leavening is 10-100% by mass, more preferably 90-100% by mass;

preferably, the skin external agent comprises a mask, essence or toner;

preferably, the skin external agent further comprises a thickening agent and/or a humectant; the thickener is preferably hydroxypropyl methylcellulose; the thickener accounts for 0.1 to 0.5 percent of the mass of the external skin preparation; the humectant is preferably 1, 3-propanediol and/or glycerol; the humectant accounts for 3 to 5 percent of the external skin preparation by mass.