Preparation method of vinasse repairing peptide essence with rice vinasse as main raw material

Abstract

The invention discloses a preparation method of vinasse repairing peptide essence with rice vinasse as a main raw material, and belongs to the related technical field of food, skin care products and the like, and the preparation method comprises the following steps: S1, detecting basic indexes of a rice vinasse stock solution; s2, filtering the rice wine lees in the S1 to obtain rice wine lees supernate; s3, carrying out molecular sieve interception on the vinasse extract obtained by filtration in S2, and filtering to obtain the vinasse small-molecule repair peptide; s4, putting the human skin fiber cells into a culture medium for cell culture to obtain passage cells; s5, performing a toxicity experiment on the subculture cells obtained by culture in the step S4 to calculate the survival rate of the cells; s6, selecting several groups of cells with good survival rate in S5, adding vinasse small molecule repair peptide for cell scratch repair, and calculating the scratch repair rate of each group of cells; and S7, according to the result of S6, further compounding the vinasse small-molecule repair peptide to obtain vinasse repair peptide essence. The product can be applied to skin nourishing and moisturizing and skin barrier repairing, and high-valued comprehensive utilization of the rice wine residues is achieved.

Description

Technical Field

[0001] The invention relates to the technical fields related to food, skin care products and the like, and in particular to a method for preparing distiller's grains repair peptide essence using rice distiller's grains as a main raw material. Background Art

[0002] Skin is the first physiological barrier of the human body. Healthy skin can resist external damage and sunlight, and also has nourishing, moisturizing and regulating functions. Due to work, living environment, work and rest patterns and skin care habits, the skin barrier is easily damaged, making the skin's own defense ability insufficient, which in turn leads to a series of skin problems such as oily skin, sensitivity, inflammation, aging, etc.

[0003] Beneficial ingredients that balance the water and oil content of the skin include active peptides, which have the function of repairing the skin barrier and restoring skin health, and have broad market prospects. At present, most studies on natural active peptides focus on evaluating their biological activity through in vitro tests, while research reports on combining bioactive substances with cosmetic preparation are relatively limited. Using modern scientific and technological methods to modify natural ingredients or extract active ingredients from natural products, they are compounded into various cosmetics while ensuring safety, stability and good usability.

[0004] Rice is a cereal grain of the Gramineae family and one of the three major food crops in the world. It contains a high content of starch, about 65%-72%, and 7%-8% protein. In addition, rice also contains a small amount of fat, cellulose and B vitamins. At present, the comprehensive utilization of rice resources in my country is low, and the research level is lagging behind, resulting in the economic value of rice not being fully demonstrated. Rice wine is one of the ways to process and utilize rice. During the production process, a large amount of fermentation base material is produced, which can also be called rice wine lees.

[0005] According to research, rice wine lees are currently being developed for use as feed and fertilizer, as well as for the extraction and separation of active substances such as phytic acid, melanoidins and peptides in wine lees. At the same time, rice wine lees are rich in nutrients, and the functional factors mainly include acids, alcohols, phenols, esters, aldehydes and ketones, active peptides, amino acids, polysaccharides and pyrazine compounds. A large number of studies have shown that various functional factors can be obtained from rice wine lees through different extraction methods. However, there is no systematic and comprehensive understanding and in-depth research on the extraction and application of active repair peptides in wine lees. From the advantages of high safety, economical source, and no toxic side effects, the study of the application of wine lees in the field of food and cosmetics has become a focus. Summary of the invention

[0006] 1. Technical issues to be solved

[0007] In view of the deficiencies in the prior art, the present invention provides a method for preparing rice wine lees repair peptide essence using rice wine lees as the main raw material, which solves the problems raised in the above-mentioned background technology.

[0008] (II) Technical solution

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0010] A method for preparing distiller's grains repair peptide essence using rice distiller's grains as main raw material comprises the following steps:

[0011] S1: Physical and chemical index testing of raw materials: basic index testing of rice wine lees stock solution, including ash, ethanol, and protein;

[0012] S2: Coarse filtration: The rice wine lees stock solution in S1 is first filtered through a 100-mesh gauze and then filtered through a 400-mesh gauze to remove macromolecular particles;

[0013] S3: Purification: centrifuge the lees extract obtained by filtration in S2, and filter it with an ultrafiltration tube to obtain lees small molecule repair peptides;

[0014] S4: Human skin fibroblast culture: human skin fibroblasts were placed in DMEM medium for cell culture, and when the cells grew to 80% to 90%, they were passaged to obtain passage cells;

[0015] S5: Cytotoxicity experiment: The passage cells obtained by culture in S4 were equally divided into several groups, and the lees small molecule repair peptide obtained in S3 was added to the passage cells obtained in S4 after being prepared according to the first concentration group, and then the cells were cultured separately, and then the optical density of each group of cells was measured at 450nm on an ELISA reader, and the survival rate of each group of cells was calculated according to the OD value of each well;

[0016] S6: Cell scratch repair: Take several groups of cells with high survival rates in S5 and add them to the lees small molecule repair peptide prepared according to the second concentration group to perform cell scratch repair. Then use an inverted microscope to observe the migration degree of each group of cells and take pictures, and calculate the scratch repair rate of each group of cells;

[0017] S7: Product compounding: Take the wine lees small molecule repair peptide and its concentration corresponding to the highest cell scratch repair rate in S6 and compound them with tangerine peel extract to obtain the wine lees repair peptide essence.

[0018] Furthermore, the concentrations of the first concentration group of the vinasse small molecule repair peptide in S5 were 0, 50, 100, 250, 500, 1000, and 2000 μmol / L, respectively.

[0019] Furthermore, the concentrations of the vinasse small molecule repair peptide in S6 according to the second concentration group were 100, 500, and 1000 μg / mL, respectively.

[0020] Furthermore, the wine lees repair peptide essence includes the following components by weight percentage: 0.18% sodium hyaluronate; 0.32% acrylic acid (ester); 6% glycerol; 5.8% 1,3-butylene glycol; 0.22% serine; 0.28% arginine; 0.2% niacinamide; 0.4% preservative, 0.1% wine lees small molecule repair peptide, 2% tangerine peel extract, and the balance is deionized water;

[0021] The lees small molecule repair peptide and its concentration corresponding to the highest cell scratch repair rate of S6 are used.

[0022] Furthermore, the preservative comprises polyol and glyceryl caprylate, wherein the mass ratio of polyol to glyceryl caprylate is 13:18.

[0023] Furthermore, the acrylic acid (ester) is acrylic acid (ester) / C10-30 alkyl acrylate cross-linked polymer.

[0024] After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0025] 1. In order to improve the resource utilization rate and economic value of rice wine lees, a wine lees repair peptide essence with moisturizing and repairing effects on the skin is developed from it. The present invention uses rice wine lees as raw materials and obtains a wine lees polypeptide through a variety of separation and purification methods. After measuring its repair effect on human skin fibroblasts, the wine lees repair peptide essence product is evaluated for efficacy through a human efficacy evaluation test, and the effects of the product on nourishing degree, collagen fiber, skin elasticity, dermis moisture, etc. are evaluated respectively, and the effect is good.

[0026] 2. The present invention uses rice wine lees as raw materials, and obtains a wine lees repair peptide through molecular sieve and component detection. The active repair peptide has the characteristics of artificial synthesis, safety and non-toxicity. The results of the activity determination of the wine lees repair peptide show that the repair peptide has the effects of moisturizing the skin and repairing the skin barrier in vitro, can be applied to the fields of food and cosmetics, and is conducive to improving the comprehensive utilization rate of rice wine lees. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a bar graph showing the survival rate of HSF cells at different sample concentrations of ≤3K Da lees small molecule repair peptides in the cytotoxicity test.

[0028] Figure 2 This is a bar graph of HSF cell migration rates at different sample concentrations for cell scratch repair using ≤3K Da lees small molecule repair peptides.

[0029] Figure 3 This is a comparison chart of cell scratch repair with ≤3K Da lees small molecule repair peptides before and after 24 hours of cell scratch repair with different sample concentrations.

[0030] Figure 4 Photo of the CBS Skin Analyzer.

[0031] Figure 5 This is the finished product of the lees repair peptide essence.

[0032] Figure 6 These are the skin oil test results for different lees repair peptide compound products.

[0033] Figure 7 This is the skin oil content status of the L7 group after 20 days of use.

[0034] Figure 8 These are the test results of skin collagen fibers in products formulated with different wine lees repair peptides.

[0035] Fig. 9 This is the state of skin collagen fibers in the L7 group after 20 days of use.

[0036] Fig.10 These are the skin elasticity test results of different wine lees repair peptide compound products.

[0037] Fig.11 This is the skin moisture test status of the L6 group after 20 days of use.

[0038] Fig.12 These are the skin moisture test results of different lees repair peptide compound products.

[0039] Fig.13 This is the skin moisture content status of the L6 group after 20 days of use. DETAILED DESCRIPTION

[0040] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Unless otherwise specified, the reagents, methods and equipment used in the examples are conventional reagents, methods and equipment in the art.

[0042] The rice wine lees raw materials used in the following examples are all from Guangdong Shunde Distillery Co., Ltd.

[0043] Electronic balance was purchased from Mettler-Toledo, Sweden.

[0044] The electric constant temperature air drying oven was purchased from Shanghai Qixin Scientific Instrument Co., Ltd., China.

[0045] The microplate reader was purchased from PerkinElmo, USA.

[0046] The digestion furnace was purchased from Shanghai Fiber Inspection Instrument Co., Ltd.

[0047] The nitrogen analyzer was purchased from Shanghai Fiber Inspection Instrument Co., Ltd.

[0048] DF-101S heat collecting constant temperature heating magnetic stirrer was purchased from Gongyi Yuhua Instrument Co., Ltd.

[0049] Muffle furnace was purchased from Guangzhou Shenhua Biotechnology Co., Ltd.

[0050] HPLC chromatogram was purchased from Shimadzu.

[0051] Skin analyzer, purchased from CBS.

[0052] Example

[0053] A method for preparing distiller's grains repair peptide essence using rice distiller's grains as main raw material comprises the following steps:

[0054] S1: Physical and chemical index testing of raw materials: basic index testing of rice wine lees stock solution, including ash, ethanol and protein;

[0055] The ash determination method refers to "GB 5009.4-2016 National Food Safety Standard Determination of Ash in Food", and the protein determination method refers to "GB 5009.5-2016 National Food Safety Standard Determination of Protein in Food".

[0056] The above test results are shown in Table 1.

[0057] Table 1

[0058] Test items Detection Methods Content (by dry weight) protein Kjeldahl method 28.07％ Ethanol Weight loss method (80℃ rotation for 6h) 3.70％ Ash National Standard Law 22.03％

[0059] S2: Coarse filtration: First, pass the rice wine lees in S1 through a 100-mesh gauze to obtain the filtrate; then pass it through a 400-mesh gauze to obtain the filtrate to obtain the rice wine lees supernatant;

[0060] S3: Purification:

[0061] The lees extract obtained by filtration of S2 was filtered through a 50K Da ultrafiltration tube, centrifuged at 4000 rpm for 15 min, the supernatant was taken, and then filtered through 10K Da and 3K Da ultrafiltration tubes in sequence to obtain lees small molecule repair peptides. The lees small molecule repair peptides with ≤3K Da were used for subsequent experiments;

[0062] S4: Culture of human skin fibroblasts: The cells were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics (penicillin and streptomycin), and placed in an incubator at 37°C and 5% CO2 for static culture. When the cells grew to about 80% to 90%, they were passaged. The old culture medium was removed, and the cells were rinsed twice with PBS. 1 to 2 mL of 0.25% trypsin was added. When the cytoplasm shrank and the intercellular space increased, DMEM medium containing 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin) was added to terminate the digestion. The passaged cells were placed in an incubator for continued culture.

[0063] S5: Cytotoxicity test: In a 96-well cell culture plate, culture S4 until 80% of the cells are digested, centrifuged, counted, and the cell concentration is adjusted to 1×10 with DMEM medium containing 10% serum. 5 / mL, add 100μL cell suspension to each well, set 6 duplicate wells in each group, and culture in a 37℃, 5% CO2 incubator overnight. Discard the culture medium in the 96-well plate, and add 100μL of lees small molecule repair peptide with concentrations of 0, 50, 100, 250, 500, 1000, and 2000μmol / L respectively. After continuing to culture for 24h, add 10μL of CCK-8 solution to each well, and continue to incubate the culture plate in a 37℃, 5% CO2 incubator for 2h. Then, place it on an ELISA instrument to measure the optical density value of each well at 450nm, that is, the OD value, and calculate the survival rate of the cells after sample intervention based on the OD value of each well.

[0064] Cytotoxicity experiments were performed using ≤3KDa lees small molecule repair peptides at 0, 50, 100, 250, 500, 1000, and 2000 μmol / L. The survival rates of HSF cells at different sample concentrations of ≤3KDa lees small molecule repair peptides were as follows: Figure 1 As shown;

[0065] Cell survival rate % = (OD experimental group - OD blank control group) / (OD negative control group - OD blank control group) × 100%;

[0066] S6: Cell scratch repair: From the sample concentrations of S5, three groups of sample concentrations, low, medium and high, were selected according to the survival rate for cell scratch repair. Then, an inverted microscope was used to observe the degree of cell migration in each group and take pictures, and the scratch repair rate of each group of cells was calculated.

[0067] First, use a marker pen to mark the back of the 6-well plate. Draw horizontal lines at equal distances (0.5-1 cm) in each well, with at least 3 lines crossing each well. Select cells in the logarithmic growth phase and adjust the concentration to 1×10 5 / mL were evenly inoculated in a 6-well plate. After the cells covered the bottom of the plate, they were quickly scratched at an average equal distance in the vertical direction according to the marked line using a sterile 200μL pipette tip, rinsed with PBS, and floating cells were removed. The culture medium was replaced with DMEM culture medium without FBS, and 100, 500, and 1000μg / mL of lees small molecule repair peptides were added as sample treatment groups. A blank control group (CK group) was also set up and cultured for 24 hours. An inverted microscope was used to observe the degree of cell migration and take pictures, that is, the cell migration status at 0h. The cells were then placed in a 37°C, 5% CO2 incubator and cultured for 24 hours, and then samples were taken and photographed for recording. The cell migration status was recorded under a microscope at 4x, and the migration distance was analyzed using Image J software;

[0068] Cell scratch repair rate % = (0h scratch width - scratch width after culture) / 0h scratch width × 100%.

[0069] After treating cells with various concentrations of lees small molecule repair peptides ≤3K Da, the migration rates of all sample groups were significantly higher than those of the blank group (P < 0.05), and the 1000 μg / mL peptide had the most significant effect on promoting HSF migration, with a cell migration rate of 68.40%. Figure 2 and Figure 3 shown.

[0070] The above results show that the lees small molecule repair peptides with a molecular weight of ≤3KDa have a repair effect on the HSF cell damage model and have the potential for skin repair. In the future, the lees small molecule repair peptides with a molecular weight of ≤3KDa will continue to be used for further compounding.

[0071] S7: Product compounding: Take the wine lees small molecule repair peptide and its concentration corresponding to the highest cell scratch repair rate in S6 and compound them with tangerine peel extract to obtain the wine lees repair peptide essence.

[0072] The wine lees repair peptide essence includes the following raw materials in 100% by weight percentage: 0.18% sodium hyaluronate; 0.32% acrylates / C10-30 alkyl acrylate cross-linked polymer; 6% glycerol; 5.8% 1,3-butylene glycol; 0.22% serine; 0.28% arginine; 0.2% niacinamide; 0.13% polyol (preservative); 0.18% glyceryl caprylate (preservative); and different proportions of wine lees small molecule repair peptides and tangerine peel extract in Table 2 and the remaining proportion of deionized water are added.

[0073] Five essences were sampled, among which the component contents of lees small molecule repair peptide and tangerine peel extract are shown in Table 2, with the labels L0, L5, L6, L7, and L8 respectively. Figure 8 shown.

[0074] Table 2

[0075]

[0076]

[0077] Results test:

[0078] Five volunteers with the same skin condition, aged between 20 and 40 years old, were selected. The five volunteers used L0, L5, L6, L7, and L8 once a day for 20 days.

[0079] 1. Use CBS skin analyzer to measure the oil content on the skin surface, and obtain the corresponding data after scanning and integrating the calculation. The results show that compound products L6 and L7 can reduce the oil content of the skin (red area decreases), and have a good oil control effect. However, other compound products cannot significantly reduce the oil content of the skin. Figure 6 , Figure 7 shown.

[0080] 2. Use CBS skin analyzer to measure the collagen fibers on the skin surface, and obtain the corresponding data after scanning and integrating the instrument. The results show that products L5, L6 and L7 with rice wine lees can effectively supplement skin collagen and increase collagen content. Among them, L7 product has the most significant effect, increasing the collagen content from 68% to 78%. Collagen fiber results, such as Figure 8 , Fig. 9 shown.

[0081] 3. Use CBS skin analyzer to measure the elasticity of the skin surface, and obtain the corresponding data after scanning and integrating the instrument. The results show that products L5, L6, and L7 with added wine lees can effectively improve elasticity, among which L6 has the best effect, which changes the network structure of skin elastic fibers from the original 25% to 45%. Elasticity results, such as Fig.10 , Fig.11 shown.

[0082] 4. Use CBS skin analyzer to measure the moisture content of the dermis layer of the skin, and obtain the corresponding data after scanning and integrating the instrument. The more blue points, the higher the moisture content of the skin. The results show that products L5, L6, L7, and L8 with added wine lees can effectively increase the moisture content of the skin, and the effect of L2 is the most obvious, increasing the moisture content of the skin from the original 3% to 5%. Skin moisture results, such as Fig.12 and Fig.13 shown.

[0083] From the above, it can be seen that this patent conducts cytotoxicity and repair tests on the rice wine lees small molecule repair peptides obtained by extraction, enzymatic hydrolysis and purification after testing the physical and chemical indicators of the rice wine lees stock solution.

[0084] By adding tangerine peel extracts in different proportions and compounding the obtained distiller's grains repair peptide essence, through skin testing, it was found that distiller's grains repair peptide has the effect of balancing water and oil and repairing the skin barrier, and a distiller's grains repair peptide essence product with obvious and stable product repair function was prepared.

[0085] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing distiller's grains repair peptide essence using rice distiller's grains as the main raw material, characterized in that: The following steps are involved: S1: Physical and chemical index testing of raw materials: basic index testing of rice wine lees stock solution, including ash, ethanol and protein; S2: Coarse filtration: First, pass the rice wine lees in S1 through a 100-mesh gauze to obtain the filtrate; then pass it through a 400-mesh gauze to obtain the filtrate to obtain the rice wine lees supernatant; S3: Purification: centrifuge the lees extract obtained by filtration in S2, and filter it with an ultrafiltration tube to obtain lees small molecule repair peptides; S4: Human skin fibroblast culture: human skin fibroblasts were placed in DMEM medium for cell culture, and when the cells grew to 80% to 90%, they were passaged to obtain passage cells; S5: Cytotoxicity experiment: The passage cells obtained by culture in S4 were equally divided into several groups, and the lees small molecule repair peptide obtained in S3 was added to the passage cells obtained in S4 after being prepared according to the first concentration group, and then the cells were cultured separately, and then the optical density of each group of cells was measured at 450nm on an ELISA reader, and the survival rate of each group of cells was calculated according to the OD value of each well; S6: Cell scratch repair: Take several groups of cells with high survival rates in S5 and add them to the lees small molecule repair peptide prepared according to the second concentration group to perform cell scratch repair. Then use an inverted microscope to observe the migration degree of each group of cells and take pictures, and calculate the scratch repair rate of each group of cells; S7: Product compounding: Take the wine lees small molecule repair peptide and its concentration corresponding to the highest cell scratch repair rate in S6 and compound them with tangerine peel extract to obtain the wine lees repair peptide essence.

2. The method for preparing the lees repair peptide essence using rice wine lees as the main raw material according to claim 1, characterized in that: The concentrations of the first concentration group of vinasse small molecule repair peptide in S5 were 0, 50, 100, 250, 500, 1000, and 2000 μmol / L, respectively.

3. The method for preparing the lees repair peptide essence using rice wine lees as the main raw material according to claim 1, characterized in that: The concentrations of the vinasse small molecule repair peptide in S6 according to the second concentration group were 100, 500, and 1000 μg / mL respectively.

4. The method for preparing the lees repair peptide essence using rice wine lees as the main raw material according to claim 1, characterized in that: The wine lees repair peptide essence comprises the following components by weight percentage: 0.18% sodium hyaluronate; 0.32% acrylic acid (ester); 6% glycerol; 5.8% 1,3-butylene glycol; 0.22% serine; 0.28% arginine; 0.2% niacinamide; 0.31% preservative, 0.1% wine lees small molecule repair peptide, 2% tangerine peel extract, and the balance is water; The lees small molecule repair peptide and its concentration corresponding to the highest cell scratch repair rate of S6 are used.

5. The method for preparing the lees repair peptide essence using rice wine lees as the main raw material according to claim 4, characterized in that: The preservative comprises polyol and glyceryl caprylate, wherein the mass ratio of the polyol to glyceryl caprylate is 13:

18.

6. The method for preparing the lees repair peptide essence using rice wine lees as the main raw material according to claim 4, characterized in that: The acrylic acid (ester) is acrylic acid (ester) / C10-30 alkyl acrylate cross-linked polymer.

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