Preparation method and application of quinoa bran husk enzymatic hydrolysate

Abstract

This invention discloses a method for preparing quinoa bran husk enzymatic hydrolysate and its application in exfoliating skincare products and cosmetics. The method involves preparing quinoa bran husk enzymatic hydrolysate through homogenization, preparation of the hydrolysate, and post-treatment. This invention uses quinoa bran husk as a raw material and employs a simple liquid enzymatic hydrolysis technique to process it, resulting in the hydrolysate. The obtained hydrolysate can enhance the expression of endogenous exfoliation-related factors in the skin's keratinocytes, thus achieving exfoliation by strengthening endogenous keratinocyte exfoliation, exhibiting excellent exfoliating effects. Furthermore, this application uses quinoa bran husk as a single, safe plant-derived ingredient, eliminating the need for compounding multiple raw materials into a single composition, making the preparation process relatively simple and convenient.

Description

Technical Field

[0001] This invention relates to the field of skin exfoliation treatment, and in particular to a method for preparing quinoa bran husk enzymatic hydrolysate and its application. Background Technology

[0002] The stratum corneum of human skin is composed of dead keratinocytes. As normal skin keratinocytes constantly differentiate and renew to form a new stratum corneum, the outermost layer of keratinocytes is constantly shed. The normal skin renewal cycle is about 28 days. However, when skin metabolism becomes irregular, skin ages, or is affected by environmental factors, the rate of keratinocyte shedding slows down. This results in an excess of dead keratinocytes that should have shed remaining on the skin's surface, leading to thickened keratinocytes, rough skin, clogged pores, and inflammation. Furthermore, nutritional imbalances can reduce skin's softness and even cause wrinkles.

[0003] Currently, skincare products on the market that achieve exfoliation effects can be broadly categorized into two types based on their mechanism of action: chemical exfoliation and physical exfoliation. The first type includes acidic products, which use the low pH of alpha-hydroxy acids to peel away old keratinocytes from the skin's surface, or the slow release of beta-hydroxy acids to dissolve keratinized substances; or products containing proteases, which use active enzymes to break down old keratin proteins on the skin's surface. For example, invention patent CN113520893A discloses an exfoliating composition and exfoliating gel. The exfoliating composition includes the following components in parts by weight: gluconolactone 1-5 parts, supramolecular salicylic acid 0.5-2 parts, glycolic acid 1-4 parts, hydroxyethylpiperazine ethanesulfonic acid 2-8 parts, and 10-hydroxydecanoic acid 0.5-2 parts. This composition contains various organic acids, which can exfoliate and regulate sebum secretion. This patent achieves the effect of promoting the shedding of the stratum corneum by combining different organic acids.

[0004] For example, invention patent CN114939082A discloses a skincare composition with exfoliating, spot-fading, and moisturizing effects, mainly comprising: papain, hydroxyethylpiperazine ethanesulfonic acid, carrageenan powder, water, and other ingredients; it exfoliates by combining multiple ingredients to achieve a combination of two mechanisms: softening the stratum corneum and enzymatic hydrolysis of proteins. This patent utilizes the combination of protease, hydroxyethylpiperazine ethanesulfonic acid, and other ingredients to form an acidic substance that uses enzymatic hydrolysis to loosen and dissolve the keratin proteins of the skin, thereby promoting the exfoliation of the stratum corneum.

[0005] The second type of products mostly contain exfoliating particles. During use, mechanical friction abrades the aged stratum corneum of the skin, promoting its exfoliation. For example, invention patent CN 115531264 B discloses an oil-controlling, moisturizing, exfoliating cleansing product and its preparation method. The exfoliating cleansing product is a scrub containing 10-30% by weight of solid abrasive. The solid abrasive is a combination of exfoliating particles and quinoa seed coat powder. The exfoliating particles are selected from one or more of sea salt, sucrose, and hydrated silica. The quinoa seed coat powder is prepared by drying quinoa seed coat at 40-45℃, low-temperature pulverizing, and taking the portion with a particle size of 100% less than 200μm as quinoa seed coat powder. This patent utilizes quinoa particles as exfoliating particles, achieving the effect of abrading and exfoliating the aged stratum corneum through mechanical friction on the skin.

[0006] Therefore, the mechanisms of traditional exfoliation are achieved through physical friction, chemical acid hydrolysis, or enzymatic hydrolysis. The compositions mainly consist of enzymes and organic acids, which dissolve or loosen keratin proteins through enzymatic or acidic action on the stratum corneum, thereby promoting the shedding of aged keratinocytes. The raw materials are often complex blends of multiple ingredients, some requiring specific formulation procedures, making the process relatively complicated and inconvenient.

[0007] Therefore, there is an urgent need to develop a component that can promote the shedding of the stratum corneum from an endogenous mechanism. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing quinoa bran enzymatic hydrolysate and its application. This invention promotes cuticle shedding from an endogenous mechanism, and the ingredients are simple and pure, the process is simple, and the operation is convenient.

[0009] The technical solution of the present invention: a method for preparing quinoa bran enzymatic hydrolysate, comprising the following steps:

[0010] S1. Preparation of Quinoa Husk Homogenization

[0011] Weigh out a certain amount of quinoa hull powder and mix it thoroughly with 10 to 50 times the weight of pure water to obtain a quinoa hull homogenate.

[0012] S2, Preparation of Quinoa Bran Hull Enzymatic Hydrolysate

[0013] Add 0.02–0.4% of the first amylase of quinoa bran powder and stir at 37–72°C for 1.2–1.5 h to obtain enzymatic hydrolysate A;

[0014] Continue to add 0.01-0.3% of protease and 0.3-1.2% of quinoa hull powder by weight of wheat bran hull powder to the above enzymatic hydrolysate A, and stir and enzymatically hydrolyze at 60-66℃ for 1-1.5h to obtain enzymatic hydrolysate B;

[0015] The enzymatic hydrolysate B is passed through an 80-400 mesh filter. After sterilization, the filtrate is cooled to room temperature and then passed through a 200-500 mesh filter again. The resulting filtrate is the crude enzymatic hydrolysate of quinoa bran.

[0016] S3, Post-processing

[0017] The crude quinoa husk enzymatic hydrolysate was filtered through diatomaceous earth at a concentration of 5-10% of the crude quinoa husk enzymatic hydrolysate. The filtrate was allowed to stand at 4-8℃ for 4-10 hours to settle, and then centrifuged at 6000-10000 rpm and 4℃ for 10-20 minutes. The supernatant was filtered through a 0.11-0.22 μm filter membrane to obtain the quinoa husk enzymatic hydrolysate.

[0018] In the aforementioned method for preparing quinoa husk enzymatic hydrolysate, after step S3, the quinoa husk enzymatic hydrolysate is concentrated according to usage requirements.

[0019] In the aforementioned method for preparing quinoa husk enzymatic hydrolysate, in step S1, the particle size of the quinoa husk powder is 40-80 mesh.

[0020] In the aforementioned method for preparing quinoa husk enzymatic hydrolysate, the first amylase is a mesophilic α-amylase, the protease is a neutral protease, and the second amylase is maltose amylase.

[0021] In the aforementioned method for preparing quinoa husk enzymatic hydrolysate, in step S2, the enzymatic hydrolysate B passes through a first filter screen with a mesh size of 100-300.

[0022] In the aforementioned method for preparing quinoa husk enzymatic hydrolysate, in step S2, the sterilization temperature is 121°C and the time is 20 min.

[0023] In the aforementioned method for preparing quinoa bran enzymatic hydrolysate, in step S3, the centrifugation speed is 7500–9500 rpm.

[0024] In the aforementioned method for preparing quinoa husk enzymatic hydrolysate, the quinoa husk powder is derived from one or more of various colored quinoa husk powders.

[0025] Application of quinoa bran hydrolysate in exfoliating skin care products.

[0026] In the aforementioned application of quinoa bran hydrolysate in exfoliating skincare products, the amount of quinoa bran hydrolysate added to the exfoliating skincare products is 2% of the product's weight.

[0027] A cosmetic product containing quinoa bran hydrolysate.

[0028] Compared with existing technologies, this invention uses quinoa husks as raw material and employs a simple liquid enzymatic hydrolysis technique to process the quinoa husks into an enzymatic hydrolysate. The resulting quinoa husk hydrolysate can enhance the expression of endogenous exfoliation-related factors in the skin's keratinocytes, thus achieving an exfoliating effect by strengthening endogenous keratinocyte exfoliation, exhibiting excellent exfoliating properties. Furthermore, this application uses quinoa husks as a single, safe plant-based ingredient for processing, eliminating the need for compounding multiple raw materials into a composition, making the preparation process relatively simple and convenient. This application improves the utilization rate of whole quinoa while giving quinoa husk processing products new uses. Attached Figure Description

[0029] Figure 1 These are diagrams showing the morphological changes of the stratum corneum in the blank group (A), the positive control group (B), and the sample group (C).

[0030] Figure 2 This is an immunofluorescence assay image of SPINK5; the left side of the image represents the blank control, and the right side represents the sample group.

[0031] Figure 3 This is a DSC1 immunofluorescence assay image; the left side of the image represents the blank control, and the right side represents the sample group.

[0032] Figure 4 This is a DSG1 immunofluorescence assay image. The left side of the image represents the blank control, and the right side represents the sample group. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0034] Example. A method for preparing quinoa bran husk enzymatic hydrolysate, comprising the following steps:

[0035] S1. Preparation of Quinoa Husk Homogenization

[0036] Weigh out quinoa husk powder (particle size 40-80 mesh) and mix it thoroughly with 10-50 times its weight of pure water to obtain quinoa husk homogenate.

[0037] S2, Preparation of Quinoa Bran Hull Enzymatic Hydrolysate

[0038] Add 0.02–0.4% (by weight) of α-amylase to wheat bran hull powder, and stir at 37–72°C for 1.2–1.5 h to obtain enzymatic hydrolysate A.

[0039] Continue adding 0.01–0.3% (by weight) of neutral protease and 0.3–1.2% (by weight) of quinoa husk powder to the above enzymatic hydrolysate A, and stir and hydrolyze at 60–66°C for 1.0–1.5 h to obtain enzymatic hydrolysate B. Pass enzymatic hydrolysate B through an 80–400 mesh (optimally 100–300 mesh) filter to remove large particles of precipitate, then sterilize (121°C, 20 min), cool to room temperature, and pass through a 200–500 mesh filter again. The resulting filtrate is the quinoa husk enzymatic hydrolysate.

[0040] S3, Post-processing

[0041] After enzymatic hydrolysis, the quinoa husk hydrolysate is filtered through diatomaceous earth at a concentration of 5-10% of the hydrolysate mass. The resulting filtrate is allowed to stand at 4-8°C for 4-10 hours to settle, and then centrifuged at 6000-10000 rpm (optimal 7500-9500 rpm) at 4°C for 10-20 minutes. The supernatant is then filtered through a 0.11-0.22 μm filter membrane. The resulting filtrate is the quinoa husk hydrolysate.

[0042] The sources of the selected quinoa husks are not limited to red quinoa, black quinoa, white quinoa, or other types of quinoa, or combinations thereof.

[0043] Example 1

[0044] Specific process:

[0045] 1. Weigh 100g of red quinoa hull powder and mix thoroughly with 3.0L of pure water. Add 0.4% (by weight of red quinoa hull powder) of Ban 480L medium-temperature amylase and hydrolyze in a 72℃ water bath with stirring for 1.5h. Continue to add 0.3% (by weight of red quinoa hull powder) of Neutase 0.8L neutral protease and Maltogenase 2XL maltose amylase and hydrolyze in a 66℃ water bath with stirring for 1.0h.

[0046] 2. The obtained enzymatic hydrolysate is filtered through a 300-mesh filter to remove large particles, then sterilized (121℃, 20 min), cooled to room temperature, and filtered through a 300-mesh filter again. The resulting filtrate is the quinoa bran husk enzymatic hydrolysate.

[0047] 3. The quinoa husk enzymatic hydrolysate was filtered through diatomaceous earth at a concentration of 10% of the enzymatic hydrolysate mass. The resulting filtrate was allowed to stand at 4-8℃ for 8 hours to settle, and then centrifuged at 9000 rpm at 4℃ for 15 minutes. The supernatant was then filtered through a 0.22 μm filter membrane. The resulting filtrate is the red quinoa husk enzymatic hydrolysate 1.

[0048] Example 2

[0049] Specific process:

[0050] 1. Weigh 120g of red quinoa hull powder, mix thoroughly with 3.0L of pure water, add 0.3% (by weight) of Ban 480L medium-temperature amylase from the red quinoa hull powder, and hydrolyze by stirring in a 37℃ water bath for 1.2h. Continue by adding 0.05% (by weight) of FFG-0657 neutral protease and 1.2% (by weight) of FDG-2250 maltose amylase from the red quinoa hull powder, and hydrolyze by stirring in a 65℃ water bath for 1.5h.

[0051] 2. The obtained enzymatic hydrolysate is filtered through a 200-mesh filter to remove large particles, then sterilized (121℃, 20 min), cooled to room temperature, and then filtered through a 100-mesh filter again. The resulting filtrate is the quinoa bran husk enzymatic hydrolysate.

[0052] 3. Centrifuge the quinoa husk enzymatic hydrolysate at 10,000 rpm and 4°C for 20 min, then filter the supernatant through a 0.22 μm filter membrane. The resulting filtrate is red quinoa husk enzymatic hydrolysate 2.

[0053] Example 3

[0054] Specific process:

[0055] 1. Weigh 120g of black quinoa bran powder, mix thoroughly with 3.0L of pure water, add 0.04% (w / w) of FFG-0659 amylase, and hydrolyze by stirring in a 40℃ water bath for 1.3h. Continue to add 0.02% (w / w) of S10013 neutral protease and 0.9% (w / w) of S-M100-L maltose amylase, and hydrolyze by stirring in a 60℃ water bath for 1.5h.

[0056] 2. The obtained enzymatic hydrolysate is filtered through a 100-mesh filter to remove large particles, then sterilized (121℃, 20 min), cooled to room temperature, and then filtered through a 200-mesh filter again. The resulting filtrate is the quinoa bran husk enzymatic hydrolysate.

[0057] 3. The quinoa husk enzymatic hydrolysate was filtered through diatomaceous earth at a concentration of 8% of the enzymatic hydrolysate mass. The filtrate was allowed to stand at 4-8℃ for 9 hours to settle, and then centrifuged at 8500 rpm at 4℃ for 20 minutes. The supernatant obtained was the black quinoa husk enzymatic hydrolysate 3.

[0058] Example 4

[0059] Specific process:

[0060] 1. Weigh 110g of quinoa bran powder, mix thoroughly with 2.5L of pure water, add 0.02% (by weight) of S-D1W-LD amylase from the quinoa bran powder, and hydrolyze by stirring in a 40℃ water bath for 1.3h. Continue by adding 0.01% (by weight) of SNbase-NP neutral protease from the quinoa bran powder, and hydrolyze by stirring in a 65℃ water bath for 1.2h.

[0061] 2. The obtained enzymatic hydrolysate is filtered through a 100-mesh filter to remove large particles, then sterilized (121℃, 20 min), cooled to room temperature, and then filtered through a 300-mesh filter again. The resulting filtrate is the quinoa bran husk enzymatic hydrolysate.

[0062] 3. The quinoa husk enzymatic hydrolysate was filtered through diatomaceous earth at a concentration of 7% of the enzymatic hydrolysate mass. The resulting filtrate was allowed to stand at 4-8℃ for 10 hours to settle, and then centrifuged at 10,000 rpm at 4℃ for 15 minutes. The supernatant was then filtered through a 0.22 μm filter membrane. The resulting filtrate is the white quinoa husk enzymatic hydrolysate.

[0063] Experimental Example 1: Determination of Active Components in Quinoa Enzymatic Hydrolysate

[0064] The flavonoid and total phenol content of the samples prepared in Examples 1, 3, and 4 were determined, and the differences in active substances before and after enzymatic hydrolysis were compared.

[0065] Experimental methods:

[0066] The flavonoid content was determined according to the operating procedure of the Plant Flavonoid Content Detection Kit (Sangon Biotech (Shanghai) Co., Ltd. D799280-0100).

[0067] Determination of total polyphenol content

[0068] Weigh 0.1 g of gallic acid standard, add 10 mL of methanol and bring the volume to 100 mL. Dilute different working solution concentrations (10, 20, 30, 40, 50 μg / mL) and prepare 7.5% sodium carbonate solution. Follow the procedure in the worksheet below. After all systems are added, shake for 1 min, incubate in a 70℃ water bath for 30 min, and take 200 μL into a 96-well plate for measurement at 750 nm.

[0069]

[0070] According to the method for determining flavonoid content, the standard curve for rutin is y = 0.5386x - 0.0483, R0 2 =0.9938; In the method for determining polyphenol content, the standard curve for gallic acid is y = 0.0048x + 0.0008, R 2 =0.9998.

[0071] Experimental results:

[0072] Example 1: Flavonoid and Total Polyphenol Content of Samples

[0073]

[0074] Example 3: Flavonoid and Total Polyphenol Content of Samples

[0075]

[0076] Example 4: Flavonoid and Total Polyphenol Content of Samples

[0077]

[0078] Note: P < 0.05 indicates a significant difference, and P < 0.01 indicates a highly significant difference.

[0079] Experiments confirmed that polyphenols and flavonoids are present in quinoa husks of different colors. Furthermore, in the preparation of the enzymatic hydrolysates of Examples 1, 3, and 4, there were significant differences in flavonoid content before and after enzymatic hydrolysis. Specifically, the flavonoid content in the hydrolysate group increased by 11.9%, 15.7%, and 17.9% compared to the group before hydrolysis, respectively. Similarly, there were significant differences in total polyphenol content between the samples before and after enzymatic hydrolysis. The total polyphenol content in the hydrolysate group increased by 15%, 6.7%, and 18.1% compared to the group before hydrolysis, respectively, indicating that enzymatic hydrolysis increased the content of flavonoids and total polyphenols in the quinoa hydrolysate.

[0080] The model used in the following experimental tests was a 3D epidermal skin model. Keratinocytes were provided by Guangdong Boxi Biotechnology Co., Ltd.; the material used in the sample group was the red quinoa husk enzymatic hydrolysate 1 from Example 1.

[0081] Experimental Example 2: Total Protein Content and Stratum Corneum Morphology Test under the EpiKutis Model

[0082] Experimental methods:

[0083] 1) The experiment was divided into groups according to the positive control group, blank control group and sample group, and the model was transferred to a 6-well plate.

[0084] 2) The blank control group received no treatment. The positive control group received the corresponding concentration of working solution. For the sample group, the working solution was evenly distributed on the model surface and incubated in a CO2 incubator (37℃, 5% CO2) for 3 days, with the drug administered once a day for a total of 3 times. After the last administration and incubation, the sample was rubbed in a circular motion, washed, and the washing solution was collected for later use.

[0085] Total protein content was determined according to the BCA protein assay kit instructions. A model used for stratum corneum morphology detection was fixed with 4% paraformaldehyde for 24 hours, followed by H&E staining. The experiment was conducted according to the protocol shown in the table below. Images were taken and analyzed using a microscope.

[0086]

[0087] Experimental results:

[0088] Total protein content test results:

[0089]

[0090] Note: When performing statistical analysis using the t-test method, the significance of the PC group, the sample group, and the BC group is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0091] The experimental results showed that, compared with the blank control group, the total protein content in the cleansing solution of the 3D epidermal skin model treated with 4% salicylic acid in the positive control group was significantly increased, indicating that the keratinization model was valid and proving the effectiveness of the positive control in this test. Compared with the blank group, the total protein content in the sample groups at a concentration of 5% was significantly increased, with an increase rate of 116.67%.

[0092] Results of changes in stratum corneum morphology as follows Figure 1 As shown in AC: blank group (BC), sex control group (PC), and sample group.

[0093] Compared to the control group, the live cell layer in the positive control group was significantly thinner, indicating damage to the live cells, and the stratum corneum became looser with keratin shedding. Similarly, compared to the control group, after 5% of the samples treated with Example 1 showed a thinner live cell layer in the skin model, with no significant damage to the live cells, while the stratum corneum became looser with keratin shedding, thus promoting exfoliation.

[0094] Experimental Example 3: Immunofluorescence assay in the EpiKutis model

[0095] Experimental methods

[0096] The model used for detection (same as in Experiment 2) was fixed with 4% paraformaldehyde. After 24 hours of fixation, immunofluorescence detection was performed, and images were taken and analyzed under a microscope. Figure 2-4 As shown.

[0097] Experimental results:

[0098] SPINK5 Immunofluorescence Analysis Results

[0099]

[0100] Note: Integrated optical density (IOD) reflects the SPINK5 content. When performing statistical analysis using the t-test method, significance compared to group BC is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0101] DSC1 immunofluorescence analysis results

[0102]

[0103] Note: Integrated optical density (IOD) reflects the DSC1 content. When performing statistical analysis using the t-test method, significance compared to group BC is indicated by *, P-value < 0.05 is indicated as *, and P-value < 0.01 is indicated as **.

[0104] DSG1 immunofluorescence analysis results

[0105]

[0106] Note: Integrated optical density (IOD) reflects the DSG1 content. When performing statistical analysis using the t-test method, significance compared to group BC is indicated by *, P-value < 0.05 is indicated as *, and P-value < 0.01 is indicated as **.

[0107] In the experimental results, compared with the blank group, the contents of SPINK5, DSC1 and DSG1 in the sample group after treatment with 5% of Example 1 were significantly reduced, indicating that the sample had an inhibitory effect on the expression of desmosomes and intercellular adhesion proteins, with inhibition rates of 85.00%, 40.00% and 55.00%, respectively.

[0108] Example 4: Expression level test of relevant genes in a cell model

[0109] Cytotoxicity test, test method:

[0110] 1) Cell seeding: After cell resuscitation, when the cell seeding rate reaches about 60%, the cells are seeded into 96-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2).

[0111] 2) Experimental Groups: The experiment included a zeroing group, a solvent control group, a positive control group, and a sample group. In the sample group, each sample (using the red quinoa husk enzymatic hydrolysate 1 from Example 1) was prepared at 8 concentrations, with 3 replicates at each concentration. Drug administration was initiated when the cell seeding rate in the 96-well plate reached 50%-60%. The solvent control group received 200 μL of culture medium per well; the positive control group received 200 μL of culture medium containing 10% DMSO per well; the sample group received 200 μL of culture medium containing the corresponding concentration of the sample per well; the zeroing group received no cell seeding, only 200 μL of cell culture medium. After drug administration, the 96-well plates were placed in a CO2 incubator 4 / 13 (37℃, 5% CO2) and incubated for 24 h.

[0112] 5) Detection: After culturing cells for 24 hours, discard the supernatant, add MTT working solution (0.5 mg / mL), and incubate at 37°C in the dark for 4 hours. After incubation, discard the supernatant, add 150 μL LDMSO to each well, and read the OD value at 490 nm.

[0113] 6) Calculation of relative cell viability: According to the formula, relative cell viability (%) = (OD of sample well - OD of zeroing well) / (OD of solvent control well - OD of zeroing well × 100%).

[0114] The results of the relative cell viability test are shown in the table below.

[0115]

[0116] Based on MTT and morphological results, the sample from Example 1, which was based on keratinocytes, was considered to have no significant cytotoxicity at a concentration of 0.625%.

[0117] Gene relative expression level test, test method:

[0118] The sample group was supplemented with the sample from Example 1 at a drug concentration of 0.625% (v / v), while the blank group was supplemented with an equal volume of cell culture medium. The specific operating steps are as follows:

[0119] 1) Cell seeding: After cell resuscitation, when the cell plating rate reaches about 60%, seed the cells into 6-well plates and incubate overnight in a CO2 incubator (37℃, 5% CO2).

[0120] 2) The relative expression levels of KLK7, KLK5, and Caspase-14 were tested using qRT-PCR at a drug concentration of 0.625%. 2 mL of culture medium was added to each well in the blank control group, and 2 mL of culture medium containing the corresponding concentration of the sample was added to each well in the sample group. After drug administration, the 6-well plate was placed in a CO2 incubator (37℃, 5% CO2) for 24 h.

[0121] 3) Gene expression detection: RNA was extracted, reverse transcribed into cDNA, and then detected by real-time quantitative PCR. -△△CT The method is used to calculate the results.

[0122] 6) Calculation of upregulation rate: Upregulation rate (%) = (sample group - blank control group) / (blank control group) × 100%.

[0123] Results and statistical analysis: Results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0124] Experimental results:

[0125] KLK5 gene testing results

[0126]

[0127] KLK7 gene testing results

[0128]

[0129] Caspase14 gene detection results

[0130]

[0131] Note: The above results use 2 -△△CT When calculating the amplification fold of mRNA in the BC group using the t-test method, the amplification fold of the BC group was normalized. The significance of the comparison with the BC group is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0132] In the experimental results, compared with the blank group, after adding 0.625% (v / v) concentration of the treatment as the sample group in Example 1, the relative expression levels of KLK5, KLK7, and Caspase14 were significantly upregulated. The upregulation rates were 149.00%, 172.00%, and 350.00%, respectively.

[0133] Overall conclusion:

[0134] The test results based on the 3D epidermal skin model showed that, compared with the blank control group, the sample in Example 1, after being treated at a concentration of 5% (v / v) for 3 days, exhibited a significant increase in the content of exfoliated protein. Since the stratum corneum is composed of nucleated dead cells, primarily consisting of protein and lipids, the experiment, by detecting the content of exfoliated protein, reflects the sample's effectiveness in removing dead skin cells.

[0135] Based on this model, compared with the blank control group, the inhibition rates of SPINK5, DSG1, and DSC1 were significantly upregulated. Among them, SPINK5 encodes the protein LEKTI, an inhibitor of kazal-type 5 serine protease in lymphoepithelial cells, which can target SCCE / KLK7 and SCTE / KLK5, thereby regulating keratin shedding. DSC1 (desmoin 1) is a calcium-dependent glycoprotein constituting the adhesion protein of intercellular junctions, and DSG1 (desmosome core glycoprotein 1) is a major component of desmosomes, contributing to the mechanical protective function of the stratum corneum. The inhibition of these proteins in the sample indicates a reduction in intercellular adhesion and junctional capacity, which helps activate related proteases and has a certain effect on promoting keratin shedding.

[0136] Based on keratinocytes, compared with the blank control group, after treatment at a concentration of 0.625% (v / v) for 24 hours, the expression levels of keratinizing enzymes KLK5, KLK7, and Caspase-14 were significantly upregulated. KLK5 and KLK7 both belong to the serine protease family. KLK5 is a key regulatory enzyme in keratin shedding, while KLK7 hydrolyzes desmosome core protein, allowing keratinocytes to reach the stratum corneum and shed. Caspase-14 is a member of the cysteine-aspartate specific protease Caspase family, exhibiting skin tissue specificity. Its main function is to participate in terminal cell differentiation, forming a complete stratum corneum. Therefore, the significant upregulation of KLK5, KLK7, and Caspase-14 genes in the experimental results indicates that the sample, at the corresponding concentration, can upregulate the expression levels of endogenous keratinizing oxidases KLK7, KLK5, and Caspase-14 genes, increasing the content of exfoliated proteins and achieving the exfoliation effect.

[0137] In summary, the results indicate that the sample, at the corresponding concentration, can upregulate the expression levels of endogenous keratinogenic enzymes KLK7, KLK5, and Caspase-14, promoting keratin shedding and increasing the content of exfoliated proteins; at the same time, it can reduce the content of SPINK5, DSC1 (adenosin 1), and DSG1 (desmosome core glycoprotein 1), thereby achieving the exfoliation effect.

[0138] Application Example 1: Moisturizing Lotion

[0139] Manufacturing process: (Ensure all equipment and containers are clean and dry)

[0140] A. Put all the raw materials of phase A (phases A01-A12 in the formula below) into the operating container, heat to 80℃, and stir until completely uniform;

[0141] B. Add all raw materials of phase B (phases B01-B09 in the formula below) into the operating container, heat to 78°C, and stir until completely homogeneous;

[0142] C. Add phase B to phase A and mix thoroughly.

[0143] D. Add the C phase raw materials (CO1-CO3 in the formula below) at 42℃ and stir until completely homogeneous; if a filter is required, a dry 100-mesh filter must be used.

[0144] Moisturizing Lotion Formula

[0145]

[0146]

[0147] Application 2: Serum

[0148] Preparation process: (Ensure all equipment and containers are clean and dry)

[0149] A. Put all the raw materials of phase A (phases A01-A12 in the formula below) into the operating container, heat to 80℃, and stir until completely uniform;

[0150] B. Add phase B raw materials (phases B01-B02 in the formula below) at 40℃ and stir until completely homogeneous; if a filter is required, a dry 100-mesh filter must be used.

[0151] serum formula

[0152]

[0153]

Claims

1. The application of quinoa bran enzymatic hydrolysate in the preparation of exfoliating skincare products, characterized in that, The preparation method of the quinoa bran enzymatic hydrolysate includes the following steps: S1. Preparation of Quinoa Husk Homogenization Weigh out a certain amount of quinoa hull powder and mix it thoroughly with 10 to 50 times the weight of pure water to obtain a quinoa hull homogenate. S2, Preparation of Quinoa Bran Hull Enzymatic Hydrolysate Add 0.02-0.4% of the first amylase of quinoa bran powder and stir at 37-72℃ for 1.2-1.5 h to obtain enzymatic hydrolysate A; Continue to add 0.01-0.3% of quinoa hull powder by weight of protease and 0.3-1.2% of quinoa hull powder by weight of second amylase to the above enzymatic hydrolysate A, and stir and enzymatically hydrolyze at 60-66℃ for 1-1.5 h to obtain enzymatic hydrolysate B; The enzymatic hydrolysate B is passed through an 80-400 mesh filter. After sterilization, the filtrate is cooled to room temperature and then passed through a 200-500 mesh filter again. The resulting filtrate is the crude enzymatic hydrolysate of quinoa bran. S3, Post-processing The crude quinoa husk enzymatic hydrolysate was filtered through diatomaceous earth at a concentration of 5-10% of the crude quinoa husk enzymatic hydrolysate. The filtrate was allowed to stand at 4-8℃ for 4-10 h for cold sedimentation, and then centrifuged at 6000-10000 rpm and 4℃ for 10-20 min. The supernatant was filtered through a 0.11-0.22 μm filter membrane. The resulting filtrate was the quinoa husk enzymatic hydrolysate. The first amylase used was mesophilic α-amylase, the protease used was neutral protease, and the second amylase used was maltose amylase.

2. The application of the quinoa bran enzymatic hydrolysate according to claim 1 in the preparation of exfoliating skincare products, characterized in that: After step S3, the quinoa bran hydrolysate is concentrated according to the usage requirements.

3. The application of the quinoa bran enzymatic hydrolysate according to claim 1 in the preparation of exfoliating skincare products, characterized in that: In step S1, the particle size of the quinoa husk powder is 40-80 mesh.

4. The application of the quinoa bran enzymatic hydrolysate according to claim 1 in the preparation of exfoliating skincare products, characterized in that: In step S2, the enzyme hydrolysate B passes through a first filter with a mesh size of 100-300.

5. The application of the quinoa bran enzymatic hydrolysate according to claim 1 in the preparation of exfoliating skincare products, characterized in that: In step S2, the sterilization temperature is 121℃ and the time is 20 min.

6. The application of the quinoa bran enzymatic hydrolysate according to claim 1 in the preparation of exfoliating skin care products, characterized in that: In step S3, the centrifugation speed is 7500~9500 rpm.

7. The application of the quinoa bran enzymatic hydrolysate according to claim 1 in the preparation of exfoliating skincare products, characterized in that: The quinoa hull powder is derived from one or more of various types of quinoa hull powder.

8. The application of the quinoa bran enzymatic hydrolysate according to claim 1 in the preparation of exfoliating skin care products, characterized in that: The amount of quinoa bran hydrolysate added to exfoliating skincare products is 2% of the product's weight.

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