CN111467286B

CN111467286B - Millet compound fermentation product and preparation method and application thereof

Info

Publication number: CN111467286B
Application number: CN202010414963.1A
Authority: CN
Inventors: 马道品; 李华发; 周广华; 陈文瀚; 陈文刚
Original assignee: Wanjing Chuangke Guangzhou Biotechnology Co ltd
Current assignee: Wanjing Chuangke Guangzhou Biotechnology Co ltd
Priority date: 2020-05-15
Filing date: 2020-05-15
Publication date: 2022-07-01
Anticipated expiration: 2040-05-15
Also published as: CN111467286A

Abstract

The invention provides a millet composite fermentation product and a preparation method and application thereof, wherein the preparation method comprises the following steps: inoculating the fermentation bacteria to a fermentation substrate consisting of millet extract, sophora fruit and soybean peptide for fermentation culture, and then performing sterilization treatment to obtain the millet composite fermentation product. The prepared novel millet composite fermentation raw stock has good antiallergic effect and good HaCa T keratinocyte anti-inflammatory and relieving effect.

Description

Millet compound fermentation product and preparation method and application thereof

Technical Field

The disclosure relates to the technical field of fermentation, in particular to a millet compound fermentation product and a preparation method and application thereof.

Background

Millet (milla. germanica) is also called millet and millet. The millet has high nutritive value and comprehensive and balanced nutrition, and mainly contains carbohydrate, protein, amino acid, fat, fatty acid, vitamin, mineral substances and the like; wherein, the content of carbohydrate is 72.0 percent to 79.5 percent, the main component is starch (about 59.4 percent to 70.2 percent), and 80 percent to 85 percent of the main component is amylopectin; the protein content is 4.88% -15.58%, and the millet protein is a rich source of methionine, isoleucine, leucine, phenylalanine and other essential amino acids; millet contains abundant VB1, VB2 and nicotinic acid; the content of mineral substances is about 2.5-3.5%, such as sodium, calcium, magnesium, phosphorus and potassium; millet contains organic selenium. Millet has the effects of clearing heat and quenching thirst, and can relieve symptoms of spleen and stomach qi weakness, indigestion and the like. The millet can also treat halitosis, and relieve beriberi to a certain extent. In addition, millet has skin caring effect.

The fructus sophorae is a common Chinese medicinal material in China, and is a fruit of Sophora japonica (Sophora japonica L.) belonging to leguminosae, which is also called as follows: fructus Sophorae, semen Phaseoli vulgaris, and fructus Sophorae. The main components comprise flavone and isoflavone, alkaloid, triterpenoid saponin, amino acid, phospholipid compound, etc.; wherein, the flavone and isoflavone compound is one of the main active ingredients in the sophora fruit.

The soybean peptide is a multifunctional peptide substance extracted from soybeans, has the common protein nutrition effect, also has good nutritional characteristics, physiological functions and processing characteristics, and is a very promising functional food raw material. Due to the unique processing performance, the method has wide application prospect in various fields such as food production and the like. For example, it can be used in medical food, protein beverage, nutrition-enriched food for the elderly, sports food, and weight-reducing food, and can also be used as fermentation promoter, and in addition, it can be used in nutritional cosmetics.

Fermentation refers to a process in which people prepare microbial cells themselves, or direct metabolites or secondary metabolites, by virtue of the life activities of microorganisms under aerobic or anaerobic conditions, and is widely used in the food industry, the biological and chemical industries. The extraction of nutrients by fermentation technology has been reported, but the extraction effect is very different according to the fermentation substrate and the fermentation process conditions. The method depends on researchers to continuously explore more fermentation substrate combinations and fermentation methods to prepare more products with better effects so as to meet the requirements of consumers. At present, the report that the product is extracted by fermenting millet, sophora fruit and soybean peptide together and is applied to cosmetics does not exist.

Disclosure of Invention

The following presents a simplified summary of the disclosure in order to provide a basic understanding of some aspects of the disclosure. It should be understood that this summary is not an exhaustive overview of the disclosure. It is not intended to identify key or critical elements of the disclosure or to delineate the scope of the disclosure. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is discussed later.

In view of the above-mentioned defects of the prior art, the present disclosure aims to provide a millet composite fermentation product, a preparation method and an application thereof, and the millet composite fermentation product provided by the present disclosure has good anti-allergy performance.

According to one aspect of the present disclosure, there is provided a method for preparing a millet composite fermentation product, comprising: inoculating the zymophyte into a fermentation substrate consisting of millet extract, sophora fruit and soybean peptide for fermentation culture, and then carrying out sterilization treatment to obtain the millet composite fermentation product.

The zymophyte adopted in the method is probiotics, so that a new skin environment can be maintained, the ecological balance and comfort of the skin can be improved, a microbial protective film is strengthened, and the skin microcirculation is promoted. Preferably, the probiotic is a lactic acid bacterium; specifically, it is selected from one or more of Lactobacillus buchneri (CGMCC 1.15607), Lactobacillus delbrueckii subsp. Bulgaricus (CGMCC 1.16075), and Bifidobacterium bifidum (CGMCC 1.5029); preferably, the concentration of the lactic acid bacteria seed liquid used for inoculation to the fermentation substrate is 10⁷-10¹⁰CFU/ml (e.g., 10)⁸CFU/ml、10⁹CFU/ml)。

The fermentation substrate in the present disclosure is prepared by mixing and sterilizing sophora fruit, soybean peptide and millet extract. Namely, the millet extract is used for replacing water adopted by a conventional fermentation substrate.

The millet extract adopted in the disclosure is clear liquid or dry powder thereof obtained by processing millet by a hot water extraction method; preferably, the method for preparing the millet extract used in the present disclosure is as follows: adding a proper amount of millet into water, wherein the mass ratio of the millet to the water is 1: 25-1: 100 (such as 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 and the like), heating to 90-100 ℃ (such as 92 ℃, 94 ℃, 96 ℃, 98 ℃ and the like) to extract for 1.5-2.5h (such as 1.8h, 2h, 2.2h and the like), and then separating and taking supernatant to obtain the millet extract used in the disclosure, wherein the millet extract is rich in active component oligopeptides extracted from the millet and can improve skin health, and the preferable amount is 99.2-99.89 wt%, namely the amount of the millet extract accounts for 99.2-99.89% of the total weight of fermentation substrates.

In the above method for preparing millet extract, the mass ratio of millet to water is preferably 1: 25-1: 50 (such as 1:30, 1:35, 1:40, 1:45, etc.).

In the above preparation method of millet extract, centrifugation is preferably adopted, and the centrifugation speed is 4000-.

The fructus sophorae used in the present disclosure is a dried mature fruit of sophora japonica, a leguminous plant. The pagodatree fruit contains many microelements such as rutin, pagodatree flower diol, glucose, glucuronic acid and tannin, and can improve skin immunity to allergy and irritation; preferably, the amount is 0.1-0.3%, i.e., 0.1-0.3% (e.g., 0.15%, 0.2%, 0.25%, etc.) of the total weight of the fermentation substrate. Experiments prove that the effective effect cannot be reflected when the dosage ratio of the fructus sophorae is too low, the whole color of the fermentation liquor obtained when the dosage ratio of the fructus sophorae is too high is darker, the skin is hyperpigmented, and the skin allergic reaction is easily caused.

The soybean peptide adopted in the disclosure is formed by decomposing macromolecular soybean protein into small molecular fragments consisting of 2 to 10 amino acids by using biotechnology enzyme, and has the effects of resisting oxidation, promoting cell proliferation and maintaining beauty and keeping young. Preferably, the amount is 0.01-0.5%, i.e., the soybean peptide is 0.01-0.5% (e.g., 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.45%, etc.) of the total weight of the fermentation substrate. Experiments prove that the soybean peptide cannot reflect the corresponding effect when the dosage ratio is too low, the effect is improved to a limited extent when the dosage ratio is too high, and the cost is increased.

In the above method for preparing a millet composite fermentation product, as a preferred embodiment, the fermentation tubes are Lactobacillus delbrueckii subsp.

Further, the preparation method of the seed liquid comprises the following steps:

and (3) activation: inoculating the strain into a test tube of an MRS liquid culture medium, and activating for 20-48 h at 35-37 ℃;

and (3) purification: carrying out gradient dilution on the activated liquid strain, inoculating the liquid strain into an MRS solid culture medium plate, and carrying out standing culture at 35-37 ℃ for 40-48 h;

liquid culture: taking 2-3 rings of a single colony in the plate, inoculating the single colony in 100mL of MRS liquid culture medium, and activating for 20-48 h at 35-37 ℃;

and (3) amplification culture: taking 10% of the inoculum size to obtain the bacterial liquid, inoculating into MRS liquid culture medium (volume ratio of bacterial liquid to culture medium is 10:100), culturing at 35-37 deg.C for about 10-14 h, and culturing until bacterial amount reaches 10⁷-10¹⁰CFU/mL, obtaining seed liquid.

Wherein, the formula of the MRS liquid culture medium is as follows: 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast powder, 20.0g of glucose and K₂HPO₄·7H₂O2.0 g, NaO 3H₂O5.0 g, triammonium citrate 2.0g, MgSO₄·7H₂0.05g of O, 801.0 mL of Tween, pH6.2, and supplementing water to 1000 mL; the MRS solid culture medium is prepared by adding 1.5% agar powder into the MRS liquid culture medium; the culture medium sterilization conditions were: sterilizing for 15-20min at 115-121 ℃.

In the above method for preparing a composite fermented millet product, as a preferred embodiment, the inoculation ratio of the fermentation tubes, that is, the volume ratio of the seed liquid to the fermentation substrate is 1% to 3% (e.g., 1.2%, 1.5%, 2%, 2.5%, 2.8%, etc.).

In the above method for preparing a millet fermented complex product, as a preferred embodiment, the fermentation culture temperature is 30-50 ℃ (such as 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃ and the like) for 6-10h (such as 6.5h, 7h, 8h, 9h, 9.5h and the like).

In the above method for preparing a composite fermented millet product, as a preferred embodiment, the temperature of the sterilization treatment is 115-121 ℃ (e.g. 116 ℃, 117 ℃, 118 ℃, 119 ℃, 120 ℃) and the time is 15-20min (e.g. 16min, 17min, 18min, 19min, etc.).

In the above method for preparing a millet composite fermented product, as a preferred embodiment, before the sterilization treatment, a separation treatment is further included, and the millet composite fermented product, that is, the millet composite fermented raw stock, is finally obtained. More preferably, the speed of the centrifugation treatment is 3500-8000r/min (such as 4000r/min, 4500r/min, 5000r/min, 5500r/min, 6500r/min, 7000r/min, 7500r/min, etc.), and the time is 20-60min (such as 25min, 30min, 40min, 50min, 55min, etc.).

In the above method for preparing a millet composite fermented product, as a preferred embodiment, the method further comprises drying after the sterilization treatment, and finally obtaining the millet composite fermented product, i.e. the millet composite fermented dry powder.

In the above method for preparing a millet fermented product, the drying treatment may be spray drying, vacuum freeze drying, or the like, as a preferred embodiment.

According to still another aspect of the disclosure, there is also provided a composite fermented millet dry powder prepared by the above preparation method.

According to another aspect of the disclosure, the millet composite fermentation raw stock prepared by the preparation method is also provided.

According to another aspect of the disclosure, the application of the millet composite fermentation dry powder in preparing cosmetics is also provided.

According to another aspect of the disclosure, the application of the millet composite fermentation protoplasm in the preparation of cosmetics is also provided.

The cosmetic can be facial mask, essence or toner.

According to the technical scheme of the embodiment of the disclosure, the novel millet composite fermentation raw stock is prepared by carrying out composite fermentation on the fermentation substrate consisting of the millet extract, the sophora fruit and the soybean peptide, and has good antiallergic effect and anti-inflammatory and relieving effect on HaCa T keratinocytes.

These and other advantages of the present disclosure will become more apparent from the following detailed description of the preferred embodiments of the present disclosure when taken in conjunction with the accompanying drawings.

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 shows the results of skin moisture content (abbreviated as "water content") and skin moisture loss (i.e., percutaneous moisture loss, abbreviated as "water dispersion") tests using the millet complex fermented puree prepared in example 1 and 0.1% hyaluronic acid (0.1% HA);

fig. 2 shows the results of skin moisture content (abbreviated as "moisture content") test using the millet composite fermented puree prepared in example 1 and 0.1% hyaluronic acid (0.1% HA), wherein a represents the change trend of the percentage of moisture content before use at different time points and each area after use, and B represents the median change trend of the moisture content at different time points and different areas;

FIG. 3 shows the results of skin water loss (i.e., percutaneous water loss, abbreviated as "water dispersion") tests using the millet compound fermented puree prepared in example 1 and 0.1% hyaluronic acid (0.1% HA), wherein A represents the variation trend of the percentage of the TEWL value before use at different time points and each area after use, and B represents the median variation trend of the TEWL value at different time points and different areas;

fig. 4 shows the results of a transdermal absorption experiment using the millet composite fermented puree prepared in example 1;

FIG. 5 shows the results of ABTS free radical test for antioxidant scavenging using the millet composite fermentation puree prepared in example 1;

fig. 6 shows photographs taken by a volunteer for skin wrinkle and roughness tests after using the millet composite fermentation broth prepared in example 1, wherein photographs taken for analyzing fine lines and wrinkles are sequentially from top to bottom, and photographs taken before using a sample and 5min, 20min and 60min after using the sample are sequentially from left to right;

FIG. 7 shows the average depth of skin fine lines at the test site before and after the volunteers shown in FIG. 6 used the millet composite fermentation broth prepared in example 1;

fig. 8 shows the average skin wrinkle depths of the volunteers shown in fig. 6 at the test sites before and after using the millet composite fermentation broth prepared in example 1.

Detailed Description

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.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The millet in the following examples is commercially available.

The sophorae fructus in the following examples was purchased from hojorang farm and sideline products purchasing company, Inc., of Bozhou city, Anhui province.

The soybean peptides in the following examples are from Hualong Biotech, Inc., of mountain milk.

The fermentation bacteria in the following examples are lactic acid bacteria, specifically: lactobacillus delbrueckii subsp. Bulgaricus with a collection number of CGMCC1.16075, available from CGMCC. The solution of the present disclosure can also be implemented by using Lactobacillus buchneri (deposited number of CGMCC 1.15607) or Bifidobacterium bifidum (deposited number of CGMCC1.5029) or a combination of any two or three of them according to the content of the embodiments of the present disclosure instead.

Example 1 preparation of millet Compound fermentation puree

1. Activation of strains: lactobacillus delbrueckii subsp. Bulgaricus colony 2 was loop-inoculated into a 100mL tube of MRS liquid medium and activated for 24h at 35 ℃.

2. And (3) strain purification: after the liquid strains are diluted in a gradient way, 200 mu L of the diluted liquid strains are inoculated and coated on an MRS solid culture medium plate, and the mixture is kept stand and cultured for 48 hours at the temperature of 35 ℃.

3. Liquid culture: taking 3 rings of single colony in the plate, inoculating in 100mL MRS liquid culture medium, activating for 48h at 35 ℃.

4. And (3) amplification culture: measuring the bacterial liquid obtained by liquid culture with 10% of inoculation amount, inoculating into 100 mM MRS liquid culture medium, culturing at 37 deg.C for 12 hr until the bacterial amount reaches 10⁸CFU/mL, obtaining seed liquid.

5. Preparation of fermentation substrate

1) Preparing millet supernatant: adding 10g of millet into 500g of water (the mass ratio of the millet to the water is 1:50), stirring and extracting for 2h at 95 ℃, keeping the water content during the extraction, and centrifuging for 30min at 5000r/min after the extraction is finished to obtain supernatant, namely the millet supernatant used in the disclosure;

2) taking 0.6g of sophora fruit and 0.6g of soybean peptide, and 300g of the millet supernatant prepared in the step 1), adding sophora fruit and soybean peptide into the millet supernatant, uniformly mixing, and sterilizing to obtain a fermentation substrate; wherein, the fructus Sophorae accounts for about 0.2%; soybean peptide accounts for about 0.2%; the millet supernatant is about 99.6%.

6. Preparation of millet composite fermentation raw stock

Inoculating 9mL of the zymocyte seed liquid obtained in the step 4 into 300g of fermentation substrate to obtain a fermentation system; fermenting the fermentation system in a shaking table at 45 ℃ for 8 hours to obtain a fermentation product; centrifuging the fermentation product at 5000r/min for 30min, removing precipitate, collecting supernatant, sterilizing at 115 deg.C for 20min to inactivate bacteria, and obtaining sterilized fermentation product, i.e. semen Setariae composite fermentation raw stock.

The millet composite fermentation raw stock prepared in the embodiment 1 is yellowish semitransparent viscous liquid, has the pH value of 5.5-6.5, the viscosity of 150-300cP, the content of soluble solid substances of 1.5-5.0 percent and the total number of colonies of less than 50CFU/ml, and has no pathogenic bacteria detection. According to the cosmetic hygiene standard GB7916-87, the total number of bacteria in the cosmetic is not higher than 1000CFU/ml, so that the fermented extract meets the quality requirement of the cosmetic.

Performing component analysis on the millet composite fermentation raw stock, wherein a protein detection method refers to GB5009.5-2010, a crude polysaccharide detection method refers to GB/T5009.8-2008, a flavone detection method refers to GB/T5009.124-2003, and a total phenol detection method refers to GB/T8313-2008; the results obtained were as follows: the millet composite fermentation raw pulp prepared in the embodiment 1 contains 3.193g/kg of protein, 7.567g/kg of crude polysaccharide, 0.036g/kg of total flavone (counted by rutin) and 0.021g/kg of total phenol.

Example 2 application of millet composite fermentation raw stock as cosmetic

Safety detection of primary millet and millet composite fermentation pulp

The human body patch test is mainly used for detecting the irritation of the final cosmetic product or raw materials. The present disclosure performed a closed patch test on the fermented puree obtained in example 1 according to the cosmetic hygiene code (2015) with the aim of evaluating its potential skin irritation.

1. Test subjects:

in the experiment, 30 suitable volunteers are selected, and the age range is 18-60 years.

2. The test method comprises the following steps:

0.02mL to 0.025mL of a liquid sample (100% millet composite fermentation protoplasm) is dripped on a filter paper sheet, and the filter paper sheet is placed in a spot tester. A blank control was set for each sample and an equal amount of sample solvent (distilled water) was added to the control cuvette well. 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. And removing the spot tester after 24h, standing for 30min, waiting for the indentation to disappear, and observing the reaction of the skin. If the test is negative, it needs to be observed once more at 24h and 48h after the patch test, respectively. The grading standard of adverse skin reactions in the human patch test is shown in Table 1.

TABLE 1 grading Standard of adverse skin reactions

3. And (3) test results:

see table 2. As can be seen from the table: the volunteers all have negative reactions after using the millet composite fermentation raw stock obtained in example 1, which shows that the millet composite fermentation raw stock provided by the invention has safety and does not bring adverse reactions to human bodies.

Table 2, test results of spots on the original millet fermentation broth obtained in example 1

Water content and water dispersion test of millet composite fermentation raw stock

The moisturizing effect of the millet composite fermentation raw stock obtained in example 1, specifically, skin moisture content test and skin moisture loss test, Hyaluronic Acid (HA) is generally regarded as a positive control sample with a good moisturizing effect, and this test uses 10% of millet composite fermentation raw stock (marked as 10% sample, namely 10% aqueous solution prepared from the millet composite fermentation raw stock prepared in example 1) and 0.1% of hyaluronic acid (marked as 0.1% HA, namely 0.1% aqueous solution prepared from hyaluronic acid) to perform a comparative experiment. The method comprises the following specific steps:

1. and (3) testing environment: the temperature is 22 +/-1 ℃; humidity is 50-60%.

2. Test area: skin moisture content test, skin moisture loss test: the left and right forearms.

3. Testing time points: skin moisture content test, skin moisture loss test: before use, 5min, 20min, and 60min after use.

4. An experimental instrument: cornemeter, CM825, Tewameter, TM 300.

5. The test method comprises the following steps:

1)30 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 40-60%. Before detection, cleaning forearms of both sides with facial cleanser, standing for 30min, and drawing normal skin with area of 3.5 × 3.5cm from inner side of forearms of the subject with marking pen. The skin moisture content and the amount of skin moisture loss before use were measured in this order.

2) Cutting the facial masks soaked with the samples into 3 × 3cm, respectively sticking on corresponding marks of the forearm, taking down after 15min, lightly dipping the undried essence on the tested part with cosmetic cotton, and timing.

3) The water content and TEWL value of stratum corneum of 3 parts are tested at 5min, 20min and 60min respectively. Each site measurement was averaged 3 times.

6. And (3) testing results:

fig. 1 is a dispersion graph of skin water content and water dispersion measured by arms of 30 volunteers, and it can be seen from fig. 1 that the change trends of 10% sample (i.e. 10% millet composite fermentation raw stock) and 0.1% HA water content are similar, and the significance of T-Test analysis shows that the water content 5min after use is significantly higher than the background (sample p is 0.017, vs background; HA p is 0.01, vs background), and the water content and the background have no significant difference at other time points.

The data were further analyzed and in FIG. 2, A is a plot of the percentage change trend for the water content of the 10% sample and 0.1% HA and B is a plot of the median change trend for the water content of the 10% sample and 0.1% HA. It can be seen from fig. 2 that the median water content of the sample and HA reached the maximum 5min after use, and both the median water content of the sample and HA showed a decreasing trend as the time after use was increased, and the percentage change of the water content was below 0, indicating that the water content at this time point after use was below the background data, but the sample and HA data trends similarly.

In FIG. 3, A is a graph showing the trend of percentage change in transdermal water loss of 10% sample and 0.1% HA, and B is a graph showing the trend of median change in transdermal water loss of 10% sample and 0.1% HA. As can be seen from FIG. 3, the change law of the percutaneous water loss of the sample and HA is similar, and both of them reach the maximum 5min after use, because the percutaneous water loss is also large due to the large water content; the transdermal water loss gradually decreased with time after use and rebounded at 60min after use, and the percent change of TWEL at this time point was greater than 0, indicating that TWEL at this time point was above the skin background due to the absence of further application of water-locking components (such as oil).

Therefore, the millet composite fermentation raw pulp prepared in the example 1 HAs good water replenishing performance, and the water replenishing performance of 10% of samples is equivalent to that of 0.1% of HA.

Third, percutaneous absorption test

1. Preparation of rat skin

The nude mice are killed after cervical vertebra is removed, the back hairs are quickly shaved off by a shaver, the back skin is peeled off, subcutaneous fat and blood vessels are removed, the nude mice are repeatedly washed to be clean by distilled water, washed by physiological saline for a plurality of times and stored in a refrigerator at minus 80 ℃ for standby (the nude mice are used up within 5 days).

2. In vitro transdermal absorption experiment of Franz diffusion cell

The experimental process comprises the following steps: adding a proper amount of water into a thermostatic bath of the in-vitro infiltration diffusion device. Starting a power supply and a thermostatic bath for magnetic stirring, and setting the water temperature in the thermostatic bath to be 37 +/-0.1 ℃. Fixing the prepared rat skin between two diffusion cells by using an iron clamp, adding 5mL of receiving liquid into a receiving cell of a vertical diffusion cell, putting the receiving cell into a thermostatic bath of an in vitro permeation diffusion device for preheating, and setting the stirring speed of the receiving cell to be 400 r/min. Feeding liquids (10% sample, i.e. 10% aqueous solution prepared from the raw millet fermentation broth prepared in example 1) were added into the feeding pools, and the upper openings were sealed with preservative films. At the beginning of the test, when the samples (i.e. the feeding liquid) permeate for 0, 2, 4, 6, 8, 12 and 24 hours (specific time intervals are determined according to actual samples), 500 mul of samples are respectively taken and placed in a centrifuge tube with a plug, and each sampling is carried out while the same amount of receiving liquid is supplemented into the receiving pool and air bubbles in the pool are removed. The sample content will be determined. Then, the cumulative permeation quantity Q (mg/cm) was calculated according to the following formula²). Wherein the diameter of the bottom of the diffusion cell is 1.50cm, and the contact area of the sample is 1.77cm²。

Wherein Q is the cumulative permeation, S is the transdermal diffusion area, V is the volume of the receiving chamber of the modified Franz diffusion cell, Cn is the concentration of the receiving solution at the nth sampling, Ci is the concentration of the receiving solution at the ith sampling, and 0.5 is the sampling amount. After calculation by the above formula, a plot of accumulated amount versus time is made. A NaCl solution (physiological saline) having a receiving solution concentration of 0.9%; the feed solution was a 10% sample, i.e., the millet composite fermentation broth prepared in example 1 was prepared as a 10% aqueous solution by volume.

3. Results of the experiment

The results of the experiment are shown in FIG. 4. It can be seen from the graph that the transmittance of the sample rapidly increases to 0.65mg/cm from 0 to 2 hours³The transmittance tends to be stable over time, indicating that the sample can smoothly penetrate the skin.

Fourth, repair performance test of HaCaT inflammatory cell model

A HaCaT inflammation model taking LPS as an inducer is established, and the specific method comprises the following steps: selecting HaCaT cells with good growth, controlling the cell number to be 5 × 10⁵one/mL, plated in 6-well plates, 2mL per well, then 5% CO at 37 ℃₂The incubator is overnight, and 100mg/L LPS is selected to stimulate HaCaT cells for 24 h.

Grouping samples: after cell modeling, further acting a 0.05% sample (namely a millet composite fermentation protoplasm aqueous solution with the volume concentration of 0.05%) on a HaCaT inflammatory cell model for 24 hours to serve as a sample group; after cell modeling, taking serum-free cell culture solution DMEM to further act on a HaCaT inflammatory cell model for 24h as a model group; serum-free cell culture solution DMEM is used for replacing 100mg/L LPS in the modeling process, and fresh serum-free cell culture solution DMEM is further used for acting on the cells for 24 hours to serve as a blank group.

Detection indexes are as follows: and qRT-PCR is used for detecting the expression level of inflammatory factor Interleukin-1 alpha (Interleukin-1 alpha, IL-1 alpha). The specific method comprises the following steps: taking the cells subjected to different treatments, operating according to the instructions of the Trizol total RNA extraction reagent, and placing the extracted total RNA in a refrigerator at minus 80 ℃ for later use. Use of

One-Step gDNA Removal and cDNA Synthesis Supermix reverse transcription kit for first strand cDNA Synthesis reaction. According to the gene sequence published in NCBI, primers are designed through PrimerExpress software, ERK1 is used as an internal reference gene, and the relative expression quantity of IL-1 alpha is detected through q RT-PCR. According to

The Top Green qPCR SuperMix kit is operated, the total reaction system is 20 mu L, and the specific reagents and the dosage refer to the kit instruction.

The specific results are shown in Table 3. The relative expression quantity of the IL-1 alpha in the model group is obviously higher than that in the blank group, which shows that the modeling is successful; when 0.05% of the sample acts on inflammatory cells for 24 hours, the relative expression quantity of the IL-1 alpha is remarkably reduced (p is less than 0.05), which indicates that the sample has remarkable anti-inflammatory effect.

TABLE 3

Grouping of cells	Relative expression amount of IL-1 alpha	Significance analysis
			Blank group	1±0.04	——
Model set	6.43±1.04	In comparison with the blank group, p<0.01
			Sample set	3.23±0.87	In comparison with model group, p<0.05

Fifth, test of anti-oxidation free radical scavenging property

1. Experimental methods

Preparing ABTS +. stock solution: preparing 2.45mmol/L potassium persulfate, dissolving ABTS with potassium persulfate to prepare 7mmol/L ABTS stock solution, and standing at room temperature in a dark condition for 12-16 h, wherein the stock solution can be stabilized for 3-4 d.

Preparing an ABTS working solution: 5mL of 7mmol/LABTS and 88. mu.L of 140mmol/L potassium persulfate solution were mixed and left to stand overnight at room temperature in the absence of light to form an ABTS working solution. Before use, the solution is diluted into a working solution by absolute ethyl alcohol, and the absorbance of the working solution at 734nm is 0.70 +/-0.02.

And (3) experimental operation: adding 40 μ L sample solution to be tested (i.e. the prepared millet composite fermentation raw stock aqueous solution with volume concentration of 5%, 10%, 20%, 50% and 100%) into 4mL ABTS working solution, accurately shaking for 30s, and measuring absorbance A sample at 734nm wavelength after reaction for 6 min.

Calculated according to the following formula:

ABTS + clearance/% (1-a sample/0.700) × 100

2. Results of the experiment

See fig. 5 for experimental results. It can be seen from the figure that samples of different concentrations have different ABTS free radical scavenging abilities and exhibit dose dependence, with 50% of the samples having an ability to scavenge ABTS free radicals above 25%. The millet composite fermentation raw stock prepared in example 1 has certain antioxidant performance.

Sixthly, the anti-allergic performance of the product is tested by hyaluronidase inhibition experiment

The hyaluronidase I type anaphylactic reaction participants have strong correlation with inflammation and allergy, researches report that various medicaments for releasing histamine from mast cells can regulate the activity of the hyaluronidase, and some antiallergic medicaments have strong inhibition on the activity of the hyaluronidase, so that the inhibition on the activity of the hyaluronidase is used as an index for researching the antiallergic effect.

See tables 4 and 5 below for experimental methods and results of hyaluronidase inhibition experiments. A sample solution to be detected (namely a pre-prepared millet composite fermentation raw stock aqueous solution with volume concentrations of 5%, 10%, 20%, 50% and 100%).

TABLE 4 Experimental methods for Hyaluronidase inhibition experiments

Hyaluronidase inhibition was calculated according to the following formula, and the results are shown in table 5.

Hyaluronidase inhibition (%) - (C-D) - (A-B)/(C-D) × 100%

In the formula: OD value of a — (hyaluronidase + sample + sodium hyaluronate) test sample solution;

OD value of blank sample B (acetic acid buffer solution + sample + acetic acid buffer solution);

OD value of C- (hyaluronidase + deionized water + sodium hyaluronate) control solution;

d- (acetate buffer + DI water + acetate buffer) control blank OD value.

As can be seen from table 5, the samples with different concentrations have different hyaluronidase inhibition abilities and show dose dependence, and 50% or more of the samples have hyaluronidase inhibition abilities of 50% or more, which indicates that the millet composite fermentation raw stock prepared in example 1 has good hyaluronidase inhibition abilities.

TABLE 5 results of five samples on hyaluronidase inhibitory ability

Sixth, human skin wrinkle and roughness test

1. Test method

The Antera 3D camera can emit parallel light with various wavelengths, a skin image is constructed by collecting the wavelength of reflected light on the surface of the skin in a detected area, and parameters such as skin wrinkles, textures, pigmentation degrees and the like can be accurately measured according to a built-in complex algorithm.

10 eligible volunteers were selected for testing. The test place has no direct light and no wind, the room temperature is 22-24 ℃, and the humidity is 40-60%. The face is cleaned with facial cleanser before detection, and the face is rested for 30min, the canthus position of the tested person is taken as the tested part, the facial mask soaked with the sample (100% millet composite fermentation raw stock prepared in example 1) is respectively cut into 3 × 3cm, the facial mask is attached to the canthus, the facial mask is taken down after 15min, the non-dried essence of the tested part is lightly stained with cosmetic cotton, the timing is started, and the facial mask is analyzed by Antera 3D facial imaging before use (background), 5min, 20min and 60 min. Changes in fine lines, wrinkles of the canthus skin before and after use were measured.

2. Test results

The test results are shown in fig. 6-8.

FIG. 6 is a graph of analysis of the change in the skin of the canthus during the application of the sample to one of the volunteers, wherein the graph is taken from left to right in the order of before application (background), 5min, 20min and 60min after application of the sample; it can be seen from the figure that the fine lines and wrinkles of the volunteers before use are obvious, and the millet composite fermentation raw stock prepared in example 1 is used for improving the fine lines and wrinkles obviously.

Further, data were captured by software for trend analysis, resulting in fig. 7-8.

FIG. 7 shows the variation trend of average depth of fine lines in the canthus area analyzed by volunteers, from which it can be seen that the average depth of skin fine lines in the same area is 0.05mm at background, and the average depth of skin fine lines 5min, 20min and 60min after application is lower than background, indicating that the sample has anti-fine line ability.

FIG. 8 is the variation trend of the average wrinkle depth in the canthus area analyzed by the volunteers, from which it can be seen that the average skin fine line depth in the same area is 0.07-0.08 mm, and the average skin wrinkle depth 20min and 60min after application is significantly lower than the background, indicating that the sample has anti-wrinkle ability.

In summary, in the embodiments according to the present disclosure, the present disclosure provides the following technical solutions, but is not limited thereto:

scheme 1, a preparation method of a millet composite fermentation product, which is characterized by comprising the following steps: inoculating the zymophyte into a fermentation substrate consisting of millet extract, sophora fruit and soybean peptide for fermentation culture, and then carrying out sterilization treatment to obtain the millet composite fermentation product.

The method according to claim 2 or 1, wherein the fermentation tubes are lactic acid bacteria.

The preparation method according to the embodiment 3 or 2, wherein the fermentation tubes are selected from one or more of the following: lactobacillus buchneri (CGMCC 1.15607), Lactobacillus delbrueckii subsp. bulgaricus (CGMCC 1.16075) and Bifidobacterium bifidum (CGMCC 1.5029).

Scheme 4, the preparation method of any one of schemes 1 to 3, wherein the fermentation substrate is prepared by adding a proper amount of sophora fruit and soybean peptide to the millet extract, mixing and sterilizing.

Scheme 5, the method of any of schemes 1-4, wherein the millet extract is a clear solution obtained by treating millet with a hot water extraction method.

Scheme 6, the preparation method as described in scheme 5, characterized in that the preparation method of the millet extract is as follows: adding a proper amount of millet into water, wherein the mass ratio of the millet to the water is 1: 25-1: 100, heating to 90-100 ℃, extracting for 1.5-2.5h, and then separating to obtain a supernatant.

Scheme 7 and the preparation method as described in scheme 6, wherein in the preparation method of the millet extract, the mass ratio of the millet to water is 1: 25-1: 50.

The preparation method according to scheme 8 or 6 or 7, wherein the millet extract is prepared by centrifugation at 4000-6000r/min for 20-40 min.

Scheme 9, the method of any of schemes 6-8, wherein the millet extract is present in an amount of 99.2% to 99.89% of the total weight of the fermentation substrate.

Scheme 10, the preparation method according to any of schemes 1 to 9, wherein the sophorae fructus accounts for 0.1-0.3% of the total weight of the fermentation substrate.

Scheme 11, the method of any one of schemes 1-10, wherein the soy peptide comprises 0.01-0.5% of the total weight of the fermentation substrate.

The method according to claim 12 or any one of claims 2 to 11, wherein the concentration of the lactic acid bacterium seed solution to be inoculated to the fermentation substrate is 10⁷-10¹⁰CFU/ml, wherein the volume ratio of the seed liquid to the fermentation substrate is 1-3%.

Scheme 13, the preparation process according to any one of schemes 1 to 12, wherein the fermentation bacteria is Lactobacillus delbruuchii subsp.

Scheme 14, the preparation method according to scheme 13, wherein the preparation process of the Lactobacillus delbrueckii subsp.

and (3) expanding culture: and (3) measuring the bacterial liquid obtained in the liquid culture step by using 10% of inoculation amount, inoculating the bacterial liquid into an MRS liquid culture medium, and culturing for 10-14 h at 35-37 ℃ to obtain a seed liquid.

Scheme 15 the preparation method according to scheme 14, wherein the MRS liquid medium formulation is: 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast powder, 20.0g of glucose and K₂HPO₄·7H₂O2.0 g, Nacetate 3H₂O5.0 g, triammonium citrate 2.0g, MgSO₄·7H₂O0.05g, Tween 801.0 mL, pH6.2, make up water1000mL of the solution; the MRS solid culture medium is prepared by adding 1.5% agar powder into the MRS liquid culture medium; the sterilization conditions of the culture medium are as follows: sterilizing for 15-20min at 115-121 ℃.

Scheme 16, the preparation method according to any of the schemes 1-15, characterized in that the temperature of the fermentation culture is 30-50 ℃ and the time is 6-10 h.

Scheme 17, the preparation method of any one of schemes 1 to 15, wherein the temperature of the sterilization treatment is 115 to 121 ℃ and the time is 15 to 20 min.

The method according to claim 18 or any one of claims 1 to 17, further comprising a separation treatment before the sterilization treatment.

Scheme 19, the preparation method as scheme 18, characterized in that, the speed of the centrifugal treatment is 3500-.

Scheme 20, the preparation method according to any one of schemes 1 to 19, further comprising a drying process after the sterilization process, and finally obtaining the millet composite fermented dry powder.

The method according to claim 21 or 20, wherein the drying treatment comprises at least one of the following methods: spray drying and vacuum freeze drying.

Scheme 22, the millet composite fermentation dry powder is characterized by being prepared by the preparation method of scheme 20 or 21.

Scheme 23 discloses a millet composite fermentation raw stock, which is characterized by being prepared by the preparation method of any one of schemes 1-19.

Scheme 24, the use of the millet composite fermentation dry powder as described in scheme 22 in the preparation of cosmetics.

Scheme 25, application of the millet composite fermentation raw stock in preparation of cosmetics as described in scheme 23.

The use of regimen 26, or regimen 24 or 24, wherein the cosmetic is a mask, serum, or toner.

Finally, it is also noted that, in the present disclosure, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, 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. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in the process, method, article, or apparatus that comprises the element.

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 a millet composite fermentation product is characterized by comprising the following steps:

adding appropriate amount of fructus Sophorae and semen glycines peptide into semen Setariae extract, mixing, and sterilizing to obtain fermentation substrate;

inoculating zymophyte into the fermentation substrate for fermentation culture, and then performing sterilization treatment to obtain the millet composite fermentation product;

the millet extract is clear liquid obtained by processing millet by a hot water extraction method; the preparation method of the millet extract comprises the following steps: adding a proper amount of millet into water, wherein the mass ratio of the millet to the water is 1: 25-1: 100, heating to 90-100 ℃, extracting for 1.5-2.5h, and then separating to obtain a supernatant;

the millet extract accounts for 99.2-99.89% of the total weight of the fermentation substrate; the fructus sophorae accounts for 0.1-0.3% of the total weight of the fermentation substrate; the soybean peptide accounts for 0.01-0.5% of the total weight of the fermentation substrate;

the zymocyte is Lactobacillus delbrueckii subspecies bulgaricus (B.delbrueckii)Lactobacillus delbrueckiisubsp. Bulgaricus) CGMCC 1.16075; the concentration of the seed liquid used for inoculation to the fermentation substrate was 10⁷-10¹⁰CFU/ml, wherein the volume ratio of the seed liquid to the fermentation substrate is 1% -3%;

the temperature of the fermentation culture is 30-50 ℃, and the time is 6-10 h.

2. The preparation method of claim 1, wherein in the preparation method of the millet extract, the mass ratio of the millet to the water is 1: 25-1: 50.

3. The method according to claim 1, wherein the millet extract is prepared by centrifugation at 4000-.

4. The method according to claim 1, wherein the Lactobacillus delbrueckii subspecies bulgaricus (B.bulgaricus) (L.bulgaricus)Lactobacillus delbrueckiisubsp. Bulgaricus) seed liquid is prepared as follows:

and (3) purification: carrying out gradient dilution on the activated liquid strain, inoculating the liquid strain into an MRS solid culture medium plate, and carrying out static culture for 40-48 h at 35-37 ℃;

5. The preparation method according to claim 4, wherein the MRS liquid medium formula is as follows: 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast powder and grapeSugar 20.0g, K₂HPO₄·7H₂O2.0 g, NaO 3H₂O5.0 g, triammonium citrate 2.0g, MgSO₄·7H₂0.05g of O, 801.0 mL of Tween, pH6.2, and water is replenished to 1000 mL; the MRS solid culture medium is prepared by adding 1.5% agar powder into the MRS liquid culture medium; the sterilization conditions of the culture medium are as follows: sterilizing for 15-20min at 115-121 ℃.

6. The method according to any one of claims 1 to 5, wherein the temperature of the sterilization treatment is 115 to 121 ℃ and the time is 15 to 20 min.

7. The method according to any one of claims 1 to 5, further comprising a separation treatment before the sterilization treatment.

8. The method according to claim 7, wherein the separation process is a centrifugation process at 3500-8000r/min for 20-60 min.

9. The preparation method according to any one of claims 1 to 5, wherein the sterilization treatment is followed by a drying treatment to obtain the millet composite fermented dry powder.

10. The method of claim 9, wherein the drying process is performed by a method comprising at least one of: spray drying and vacuum freeze drying.

11. The millet composite fermentation dry powder is characterized by being prepared by the preparation method of claim 9 or 10.

12. The millet composite fermentation puree is characterized by being prepared by the preparation method of any one of claims 1-8.

13. The use of the millet composite fermented dry powder of claim 11 for the preparation of cosmetics.

14. The use of the millet composite fermentation puree of claim 12 for preparing cosmetics.

15. Use according to claim 13 or 14, wherein the cosmetic product is a mask, a serum or a toner.