Fermented rice bran lipid extract having moisturizing efficacy, and preparation method and application thereof

Abstract

The application discloses a fermented rice bran lipid extract with moisturizing efficacy and a preparation method and application thereof. The preparation method comprises the following steps: sterilizing rice bran, adding 3-5 times of sterile water by weight of the rice bran, and then inoculating lactic acid bacteria and yeast seed liquid to perform fermentation; the lactic acid bacteria is Lactobacillus casei; the yeast is one of Pichia amicola, Kluyveromyces marxianus and Candida alcoholiphila; the obtained fermentation system is added with ethanol for ultrasonic-assisted extraction; the obtained mixture is suction filtered, rotary evaporated, and freeze-dried to obtain the fermented rice bran lipid extract. According to the method, the content of fatty acyl and glycerol lipid in the product after fermentation is significantly reduced, glycerophospholipid and sphingolipid are enriched, and especially, the content of ceramide in the sample fermented by the yeast and the lactic acid bacteria is obviously increased. The fermented rice bran lipid extract sample has high abilities of scavenging free radicals and chelating ferric ions in vitro, promotes HaCaT cell proliferation, and has excellent moisturizing activity.

Description

Technical Field

[0001] This invention relates to functional fermented rice bran lipid extracts, specifically to a method for preparing ceramide-rich lipid extracts from rice bran through co-fermentation with yeast and lactic acid bacteria, and its application. The fermented rice bran lipid extracts are mainly used for skin moisturizing; this invention belongs to the field of fermentation engineering technology. Background Technology

[0002] The decline in skin structure and function is caused by a combination of factors, primarily including physiological aging, environmental damage, and improper skincare. With age, the secretion of lipids in aging skin decreases, the proliferation capacity of keratinocytes declines, the barrier function weakens, and the skin's water-holding capacity diminishes. Furthermore, unsuitable environmental temperatures, humidity, and ultraviolet radiation can all damage the skin, leading to dryness. In addition, incorrect skincare methods can also cause dry and sensitive skin, further reducing lipid secretion and damaging the skin barrier. Currently, common moisturizers mainly include polyols and polysaccharides. Although they have good water solubility, hygroscopicity, and skin affinity, their long-term moisturizing effect is poor, and they are easily affected by environmental humidity. Therefore, developing a safe and effective functional ingredient for skincare and moisturizing is essential.

[0003] Human skin can be described as having a "brick wall and mortar" structure. The "brick wall" mainly represents keratinocytes, while the "mortar" refers to lipids in the intercellular spaces, of which 50% is ceramides, with a smaller portion consisting of cholesterol and fatty acids. The "brick wall" and "mortar" together form a strong epidermal structure, limiting the loss of water from inside and outside cells to maintain the cell's barrier function. This barrier is not only a physical barrier but also an immune barrier, a chemical barrier, a microbial barrier, and a pigment barrier. While reducing skin moisture loss, it also maintains pH balance, resists chemical penetration, maintains a stable skin microbiome, and provides photoprotection through pigmentation. Therefore, functional active lipid ingredients in skincare products can not only improve skin's water-holding capacity but also enhance its barrier function. Ceramides are the most abundant lipid in the intercellular space and are natural moisturizing factors. Exogenous ceramides can also be used as cosmetic supplements. Ceramides are mainly derived from artificial synthesis, animal and plant extraction, and microbial fermentation. Plant-derived ceramides are considered safer and less expensive. Existing research on plant-derived ceramides has been conducted. Chinese invention patent applications 202210385862.5 and 201610350666.9 disclose different enzymatic hydrolysis and extraction methods for the efficient extraction and purification of ceramides from rice bran. Chinese invention patent 202210385862.5 requires cumbersome steps such as enzymatic hydrolysis and petroleum ether extraction, and the organic reagents pose safety concerns. Chinese invention patent 201610350666.9 requires enzymatic hydrolysis, microwave countercurrent extraction, concentration, organic solvent extraction, silica gel chromatography adsorption separation, and concentration and drying. The enzymatic hydrolysis time is relatively long, and the organic solvent used is ethyl acetate, which is volatile and has relatively low safety.

[0004] Studies have shown that microbial fermentation is a commonly used method for preparing ceramides in recent years. Typically, Pichia pastoris and Saccharomyces cerevisiae are fermented under specific conditions to obtain tetraacetyl phytosphingosine (TAPS), which is then deacetylated to obtain phytosphingosine, and finally fatty acids are added to synthesize ceramides and other substances. For example, Chinese invention patent application 201910248114.0 discloses a method for extracting and preparing ceramides from rice bran and chestnuts using yeast fermentation. This method involves drying, removing impurities from, and pulverizing rice bran and chestnut shells, sieving them to obtain the raw material matrix, preparing a fermentation medium, inoculating with yeast, homogenizing and breaking down the cell walls, separating, filtering, and decolorizing the mixture, and then selectively separating the filtrate using continuous chromatography to obtain ceramides. While yeast itself can produce ceramide precursors through fermentation, facilitating the extraction of ceramides from the raw materials, this technology requires a specially prepared fermentation medium and complex steps such as homogenization and slurry preparation. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a fermented rice bran lipid extract with moisturizing effects. This fermented rice bran lipid extract is rich in a variety of active lipid components and has strong in vitro and cellular moisturizing activities. The in vitro moisturizing rate of the extract can reach 70-80%, and the moisture absorption rate can reach more than 12%. It can increase the survival rate of dry and damaged cells by 25-35%, and also has good in vitro antioxidant function. The IC50 value of DPPH free radical scavenging rate is 18-25 μg / mL, and the ABTS free radical scavenging rate is 8-10 μg / mL.

[0006] Another object of the present invention is to provide the application of the aforementioned fermented rice bran lipid extract with moisturizing effect in the preparation of a naturally extracted moisturizing agent.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for preparing a fermented rice bran lipid extract with moisturizing effects includes the following steps:

[0009] (1) The strain was inoculated and cultured in a culture medium to prepare a seed culture; after sterilizing rice bran, sterile water was added in 3 to 5 times the weight of the rice bran, and then lactic acid bacteria and yeast seed culture were inoculated for fermentation; the lactic acid bacteria was Lactobacillus casei M8, with accession number GDMCC NO: 64441; the yeast was one of the following three: Pichia amenthionina Y, with accession number CGMCC NO: 10183; Kluyveromyces marxianus SP-1, with accession number GDMCC NO: 64440; Candida ethanolica ATW1, with accession number GDMCC NO: 61360;

[0010] (2) Add ethanol to the fermentation system obtained in step (1) and extract with ultrasound assistance;

[0011] (3) The mixture obtained in step (2) was filtered, rotary evaporated, and freeze-dried to obtain fermented rice bran lipid extract.

[0012] To further achieve the purpose of this invention, preferably, in step (1), the fermentation temperature is 30~37℃ and the fermentation time is 5~7 days.

[0013] Preferably, in step (1), the inoculation amount of yeast and lactic acid bacteria is 1 to 5% of the total volume of the rice bran and water mixture.

[0014] Preferably, in step (2), the volume concentration of ethanol is 90-95% ethanol; g and mL are used as the units of mass and volume, respectively, and the ratio of rice bran mixture to ethanol is 1:4-6; the temperature of ultrasonic-assisted extraction is 45-55℃ and the time is 40-50 min.

[0015] Preferably, in step (3), the filtration is performed using a circulating water multi-purpose vacuum pump at a temperature of 25~30℃ for 5~15 minutes.

[0016] Preferably, in step (3), the temperature of the rotary evaporation is 30-45°C.

[0017] Preferably, in step (1), the rice bran is edible rice bran.

[0018] A fermented rice bran lipid extract with moisturizing effect is prepared by the above-mentioned preparation method; the fermented rice bran lipid extract has an in vitro moisturizing rate of 70-80% and a moisture absorption rate of more than 12%.

[0019] The application of the fermented rice bran lipid extract with moisturizing effects in the preparation of natural moisturizing and anti-inflammatory skin care products.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1) This invention uses Pichia amenthionina Y or Kluyveromyces marxianus SP-1 or Candida ethanolica ATW1 to co-ferment with Lactobacillus casei M8. The yeast used in this invention contains sphingosine, a precursor to ceramide, which is beneficial for the synthesis of ceramide.

[0022] 2) SP-1, Y and ATW1 of the present invention have strong lipase activity and stronger lipid metabolism ability, which makes the extract rich in lipid components. Cholesterol and ceramide are the main components of intercellular lipids, which can be linked with cell surface proteins through ester bonds to play a role in cell adhesion, and covalently bind with water molecules to play a role in cell barrier and moisturizing. Moreover, the rich lipid components can play a good role in moisturizing and water-locking, and the effect is more stable and lasting.

[0023] 3) The lipid extract obtained by this invention has in vitro moisturizing and hygroscopic properties, and can protect and repair dry and damaged HaCaT cells by significantly improving cell survival rate, and promote the expression of moisturizing-related protein mRNA. In addition, it also has strong in vitro antioxidant activity.

[0024] 4) The lactic acid bacteria M8 used in this invention can utilize glucose, cellobiose, starch and carboxymethyl cellulose as the sole carbon source, respectively. There is no need to add additional carbon source or other nutrients to rice bran. This invention does not require cumbersome steps such as homogenization and cell wall breaking, making it safer and more efficient.

[0025] 5) The extract of this invention is extracted with ethanol without the use of other organic reagents, making the extract safer.

[0026] 6) The raw material of this invention is rice bran, which is abundant and helps to improve the bioavailability of this difficult-to-utilize by-product. The extraction process is simple, easy to operate, and has low processing costs, and has good industrialization prospects.

[0027] 7) The rice bran used in this invention is a plant-derived ingredient that is highly effective, safe, green, healthy and environmentally friendly, which is beneficial for its application as a moisturizing ingredient in skin care and cosmetic products.

[0028] 8) Existing technologies require the preparation of additional fermentation culture medium and complex steps such as cell wall breaking and homogenization. This invention does not require the preparation of fermentation culture medium, nor does it require the addition of additional carbon sources or other nutrients to rice bran. Moreover, this invention inoculates lactic acid bacteria during fermentation, and the cellulase and amylase produced by them hydrolyze the cell wall of rice bran, which is more convenient and safer.

[0029] 9) This invention can not only obtain more ceramides, but also rich fatty acids, glycerophospholipids, and sterol lipids, which are the main components of intercellular lipids and have moisturizing effects. This invention also obtains functional lipids such as hydroxy fatty acids and acylcarnitine, which can better promote the skin care effect of the sample. Attached Figure Description

[0030] Figure 1 The diagram shows the types and relative contents of sphingolipids in the examples and comparative examples.

[0031] Figure 2 The diagram shows the repair effect of HaCaT cells damaged by drying in the examples and comparative examples.

[0032] Figure 3 The diagram shows the protective effect of drying-damaged HaCaT cells in the examples and comparative examples.

[0033] Figure 4 The diagram shows the FLG gene status of HaCaT cells damaged by drying in the examples and comparative examples.

[0034] Figure 5 The diagram shows the IVL gene status of HaCaT cells damaged by drying in the examples and comparative examples. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the implementation of the present invention is not limited thereto.

[0036] This invention utilizes *Kluyveromyces marxianus* SP-1 isolated from fermented bean curd blanks, *Candida ethanolica* ATW1 isolated from fermented bean curd slurry, and *Pichia amenthionina* Y and *Lactobacillus casei* M8 strains isolated from fermented bean curd slurry. These three strains all grow well in the rice bran system. Lactic acid bacteria can produce acid to lower the pH of the system and are rich in various enzymes, which facilitates the decomposition of rice bran cell walls, releasing internal active substances. Furthermore, yeast fermentation can improve the bioavailability of sphingosine, a precursor to ceramides, thereby enhancing the moisturizing activity of the fermented extract. The strains used in this invention are those isolated and preserved by the applicant, as detailed below:

[0037] Pichia amenthionina Y is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, accession number CGMCC NO: 10183, deposited on December 15, 2014.

[0038] Kluyveromyces marxianus SP-1 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China. The accession number is GDMCC NO: 64440, and the deposit date is March 21, 2024.

[0039] Candida ethanolica ATW1 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCCNO: 61360 and deposit date of December 11, 2020.

[0040] Lactobacillus casei M8 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC NO: 64441 and deposit date of March 21, 2024.

[0041] Rice bran is rich in starch and cellulose. Fermentation by lactic acid bacteria facilitates the decomposition of rice bran cell walls, making its active ingredients more readily soluble. Furthermore, the conversion of starch and other sugars by lactic acid bacteria allows yeast to grow better in the fermentation system. The fats in rice bran are mostly unsaturated fatty acids such as oleic acid and linoleic acid, and also contain bioactive substances such as vitamin E, sterols, and oryzanol. The metabolic enzymes and lipid metabolism capabilities of yeast are beneficial for the biotransformation of lipid components in rice bran, improving their bioavailability and bioactivity. The applicant discovered that *Lactobacillus casei* M8 contains enzymes that can hydrolyze large molecules such as cellulose and starch. During fermentation, the breakdown of carbohydrates produces lactic acid, lowering the pH of the system, which is beneficial for the decomposition of rice bran cell walls and the dissolution of free ceramides. Additionally, the small-molecule sugars produced during M8 fermentation can provide a carbon source for yeast fermentation. The *Pichia pastoris* Y, *Kluyveromyces martensii* SP1, and *Candida albicans* ATW1 of this invention have strong lipid metabolism capabilities, which are more conducive to the metabolism and transformation of lipids in the entire fermentation system. Moreover, sphingosine, a precursor to ceramide, has been detected by lipidomics technology and is beneficial to the synthesis of ceramide. Thus, it is possible to combine microbial fermentation and plant extraction to obtain fermented rice bran lipid extracts with a relatively higher ceramide content.

[0042] Therefore, the technical solution adopted by the present invention is as follows:

[0043] A method for preparing a fermented rice bran lipid extract with moisturizing effects includes the following steps:

[0044] (1) The strain was inoculated and cultured in a culture medium to prepare a seed liquid; after sterilizing rice bran, sterile water was added in 3 to 5 times the weight of the rice bran, and then lactic acid bacteria and yeast seed liquid were inoculated for fermentation; the lactic acid bacteria was Lactobacillus casei M8, with accession number GDMCC NO: 64441; the yeast was one of the following three: Pichia amenthionina Y, with accession number CGMCC NO: 10183; Kluyveromyces marxianus SP-1, with accession number GDMCC NO: 64440; Candida ethanolica ATW1, with accession number GDMCC NO: 61360;

[0045] (2) Add ethanol to the fermentation system obtained in step (1) and extract with ultrasound assistance;

[0046] (3) The mixture obtained in step (2) was filtered, rotary evaporated, and freeze-dried to obtain fermented rice bran lipid extract.

[0047] The fermentation, sterilization, ultrasonic-assisted ethanol extraction, filtration, rotary evaporation, and freeze drying methods described above can be obtained from existing technologies according to the present invention.

[0048] The test results of this invention demonstrate that the lipid extract obtained from rice bran after co-fermentation with yeast and lactic acid bacteria has good moisturizing effects, can improve the survival rate of HaCaT cells damaged by dryness, and the relative expression levels of filin and epidermal protein mRNA. Simultaneously, this lipid extract also exhibits strong in vitro antioxidant capacity, including ABTS and DPPH free radical scavenging ability and the ability to reduce ferric iron. The co-fermentation of lactic acid bacteria and yeast can enrich and diversify the lipid components of the fermentation product, thereby obtaining an extract with moisturizing, antioxidant, and other biological activities. Moisturizing and antioxidant properties are fundamental to all skincare product efficacy, and the two are interconnected. Moisturizing helps lock in moisture and maintain the skin barrier function, protecting cells from free radical damage. Antioxidants can alleviate oxidative stress damage, maintain skin homeostasis, and also benefit skin water retention and hydration.

[0049] In the following embodiments:

[0050] (1) MRS medium (for lactic acid bacteria culture) and YPG medium (for yeast culture): are prepared by dissolving 1% yeast extract, 2% peptone and 2% glucose in 100mL of distilled water and sterilizing at 121℃ for 15min.

[0051] (2) Lipidomics analysis: UPLC-Q Exactive ultra-high performance liquid chromatography-tandem high resolution mass spectrometry and ACQUITY UPLC BEH C18 (1.7μm 100mm*2.1mm) column were used. Chromatographic separation was performed using binary elution. Mobile phase A was acetonitrile:water = 6:4, with 10 mmol / L ammonium formate and 0.1% formic acid added. Mobile phase B was isopropanol:acetonitrile = 9:1, with 10 mmol / L ammonium formate and 0.1% formic acid added. The column temperature was 40℃, the flow rate was 0.3 mL / min, and the injection volume was 2 μL. The elution gradient conditions were as follows: 0–2.0 min, 20–30% solvent B; 2.0–6.0 min, 30–65% solvent B; 6.0–12.0 min, 60–85% solvent B; 12.0–16.0 min, 85–95% solvent B; 16.0–16.1 min, 95–20% solvent B; 16.1–18.0 min, 20% solvent B. Each sample was acquired once in both positive and negative ion modes. During acquisition, a QC scan was performed every 10 samples, and the quality difference between QC scans was used to correct for systematic errors in the entire batch. The raw mass spectrometry data was converted into readable mzXML data using Proteowizard's MSConvert software. Peak extraction was performed using XCMS software, and peak extraction quality control was performed. The extracted substances were annotated with additive ions using CAMERA, and then identified using metaX software. Identification was performed using primary mass spectrometry information, and secondary mass spectrometry information was matched with an in-house standard database.

[0052] (3) In vitro antioxidant detection methods:

[0053] DPPH, ABTS free radical scavenging capacity, and ferric ion reducing capacity are common methods for detecting the in vitro antioxidant capacity of substances and are applicable to fat-soluble substances.

[0054] The sample was prepared into a high-concentration stock solution using DMSO, and then diluted with DMSO to the required concentration during the experiment.

[0055] DPPH free radical scavenging ability

[0056] Accurately weigh 5 mg of DPPH and dissolve it in 50 mL of anhydrous ethanol as a stock solution. Dilute with anhydrous ethanol to a working solution of 0.075 mg / mL for the experiment. Add 180 μL of DPPH working solution and 20 μL of sample to each well of a 96-well plate. Incubate at room temperature in the dark for 30 min, then measure the absorbance at 517 nm using a microplate reader. The free radical scavenging ability of the sample is expressed as the IC50 value.

[0057] ABTS free radical scavenging ability

[0058] Accurately weigh 0.0384 g ABTS and 0.0067 g K₂S₂O₈ and dissolve them separately in 10 mL of distilled water. Mix them 1:1 and let them stand at room temperature in the dark for 16–24 h to obtain the ABTS stock solution. For experiments, dilute the stock solution 16 times with distilled water. Prepare the ABTS working solution fresh for each use. Add 180 μL of ABTS working solution to each well of a 96-well plate, and add 20 μL of sample to each well. After reacting for 180 min, measure the absorbance of the reaction system at 734 nm.

[0059] Fe 3+ Reducing accuracy (FRAP)

[0060] Prepare 300 mmol / L, pH 3.6 acetate buffer, 20 mmol / mL FeCl3·6H2O solution, and 10 mmol / L TPTZ solution using 40 mmol / L HCl solution. Mix FeCl3·6H2O solution, TPTZ solution, and acetate buffer in a 1:1:10 ratio to obtain the FRAP solution. Dissolve and dilute with DMSO to prepare a sample concentration of 16 mg / mL. Add 180 μL of FRAP solution and 20 μL of sample to each well of a 96-well plate, incubate at 37°C for 10 min, and measure the absorbance at 593 nm using a microplate reader. Dilute tocopherol (Trolox) with anhydrous ethanol to prepare a series of standard solutions with gradient concentrations. The standard determination procedure is the same as above. Plot a standard curve with concentration on the x-axis and corresponding absorbance on the y-axis. Substitute the sample OD values ​​into the curve to calculate the corresponding Trolox equivalent.

[0061] (4) In vitro moisturizing and hygroscopic testing methods

[0062] The weighing method is a common way to measure the moisturizing effect of cosmetics or skin care products. It is convenient and quick, and can intuitively show the moisturizing and hygroscopic effects of the sample.

[0063] External moisturizing properties

[0064] Saturated potassium carbonate solution and dry silica gel were placed separately in a sealed desiccator to maintain a relative humidity of 43% and an extremely dry environment. The desiccator was then placed in a constant temperature incubator at 25°C. Weighing bottles were washed and dried to constant weight. 100 mg of each sample was weighed into an open weighing bottle, and 40% distilled water was added to each sample. Samples were weighed at 0, 4, 8, 12, 24, 36, 48, 60, and 72 hours. Glycerol was used as a control. Three replicates were set up for each sample. The sample moisture retention rate (Rr) was calculated using the following formula:

[0065]

[0066] External hygroscopicity

[0067] Saturated potassium carbonate and ammonium sulfate solutions were placed in sealed glass desiccators, respectively, to achieve relative humidity levels of 43% and 81%. The desiccators were then placed in a constant temperature incubator at 25°C. Weighing bottles were washed and dried to constant weight. 100 mg of sample was weighed into each open weighing bottle, and the sample mass was measured at 0, 4, 8, 12, 24, 36, 48, 60, and 72 hours. Glycerol was used as a positive control, and each sample was tested in triplicate. The moisture absorption rate (Ra) of the sample was calculated using the following formula:

[0068]

[0069] (5) Method for detecting the survival rate of HaCaT cells damaged by drying:

[0070] The repair effect of the sample on desiccation-damaged HaCaT cells

[0071] HaCaT cells were seeded into 96-well plates at a seeding rate of 8000 cells / well and incubated in a CO2 incubator for 12 h until cell adhesion. Subsequently, drying and modeling were performed. After a certain drying time, 100 μL of the test sample prepared with DMEM was added and the cells were incubated for 24 h. Then, 10 μL of MTT solution was added to each well, and the plates were incubated for 4 h. After removing the MTT solution, 100 μL of DMSO was added to each well, and the plates were slowly shaken on a shaker to dissolve the cells. The absorbance was measured at 490 nm.

[0072] Protective effect of the sample on desiccation-damaged HaCaT cells

[0073] HaCaT cells were seeded into 96-well plates at a seeding rate of 8000 cells / well and incubated in a CO2 incubator for 12 h until cell adhesion occurred. The culture medium was aspirated, and 100 μL of the test sample prepared in DMEM medium was added. After culturing for 24 h, the culture medium was aspirated, and the cells were subjected to drying damage treatment. Cell viability was then determined using the MTT assay, following the same method as above.

[0074] (6) Method for determining the relative expression levels of HaCaT cell polyfilament and epidermal protein mRNA:

[0075] Total RNA extraction from HaCaT cells

[0076] Total RNA was extracted using a column chromatography method. Cell lysis: according to (5) HaCaT cells were cultured in 6-well plates. Before lysis, the culture medium was completely aspirated, and 500 μL of RTL Lysis Buffer was added to each well of the 6-well plate. The lysate was collected with a cell scraper and transferred to a 1.5 mL sterile enzyme-free centrifuge tube. Sample homogenization: Homogenize the sample 5-10 times using a pipette; Adjusting the binding conditions: Add 0.7 times the volume of Buffer SW2 to the lysis buffer, and pipette 5-10 times; Column adsorption: Pack the purification column into the collection tube, transfer all the mixture into the column, and centrifuge at 12000 × g for 1 min; Protein removal: Discard the filtrate, put the column back into the collection tube, add 700 μL Buffer RW1, and centrifuge at 12000 × g for 1 min; Desalting: Discard the filtrate, reassemble the column into the collection tube, add 700 μL of Buffer SW1, and centrifuge at 12000 × g for 1 min; Drying: Discard the filtrate, put the column back into the collection tube, centrifuge at 12000 × g for 2 min to dry the column; Elution: Transfer the column to a 1.5 mL centrifuge tube and add 30 μL of RNase-free water to the center of the column membrane. Incubate at room temperature for 2 minutes. Centrifuge at 12,000 × g for 1 minute. Discard the column and store the RNA at -80°C.

[0077] Reverse transcription to synthesize cDNA

[0078] Add 1 μg of the extracted RNA and 4 μL of gDNA clearer to a 0.2 mL RNase-free PCR tube, then add RNase-free ddH2O to a final volume of 12 μL. Use a pipette to gently mix the above mixture, briefly centrifuge, and then incubate in a PCR instrument at 42°C for 2 minutes. Then cool on ice for later use. Add 8 μL of 2.5×Prescript to the reaction solution from the previous step. After gently mixing with RT ProMix and briefly centrifuging, place the mixture in a PCR instrument and perform reverse transcription at 25°C for 5 min, 50°C for 15 min, and 85°C for 2 min. After the reaction, store the synthesized cDNA at -20°C.

[0079] Real-time quantitative PCR detection

[0080] Prepare the following reaction system according to the kit: Add 10 μL of 2×Robust SYBR GreenqPCR ProMix, 0.5 μL each of 10 μM forward and reverse primers, and a certain volume of Nuclease-Free Water to the reaction tube. Mix well and then aliquot into the wells of an eight-tube strip. Add the diluted sample cDNA to a total volume of 20 μL. Immediately after preparation, run the system on a real-time quantitative PCR instrument. The PCR program is as follows: pre-denaturation at 95℃ for 10 min, followed by 40 cycles at 95℃ for 5 s, and then 40 cycles at 60℃ for 20 s. Use 2... -ΔΔCt The method calculates the fold change in gene expression and normalizes the Ct values ​​of the target genes FLG and IVL to the internal reference gene ACTB.

[0081] Table 1 Primer synthesis sequences

[0082]

[0083] In the following figures, Control represents the normal cell group without any treatment, Model represents the model cell group, Glycerol represents the positive control group treated with 25 μg / mL, and the other groups represent the corresponding samples treated with 25 μg / mL.

[0084] Example 1: Fermentation of rice bran with Kluyveromyces marxianus SP-1 and Lactobacillus casei M8

[0085] Step 1: Strain Preparation. Take a 50 mL Erlenmeyer flask, add 20 mL of YPG medium, and sterilize at 121 °C for 15 min. After cooling, dispense 5 mL into 10 mL glass tubes, and inoculate with 1% (v / v) Kluyveromyces marxianus SP-1. Incubate at 30 °C for 24 h to obtain the seed culture. Take a 50 mL Erlenmeyer flask, add 20 mL of MRS medium, and sterilize at 121 °C for 15 min. After cooling, dispense 5 mL into 10 mL glass tubes, and inoculate with 1% (v / v) Lactobacillus casei M8. Incubate at 37 °C for 12 h.

[0086] Step 2: Rice bran preparation. Take edible rice bran, dry it thoroughly, weigh 20 g into a 100 mL Erlenmeyer flask, sterilize at 115℃ for 30 min, cool, add 80 mL of sterile water, and mix thoroughly.

[0087] Step 3: Fermenting rice bran. Based on the total volume of the rice bran system prepared in Step 2, inoculate with 1% Kluyveromyces marxianus SP-1 seed culture and 1% Lactobacillus casei M8 seed culture, ensuring that the final bacterial count of both bacteria in the rice bran fermentation system reaches 1×10⁻⁶. 6 The concentration was 100 cfu / mL, and then the sample was incubated at 32°C for 7 days.

[0088] Step 4: Lipid extraction. After fermentation, 90% ethanol was added to the rice bran at a material-to-liquid ratio of 1:4. Extraction was performed at 45°C with ultrasonic assistance for 50 minutes. The mixture was then filtered while hot, and the filtrate was rotary evaporated at 45°C to constant weight, yielding a yellowish-brown viscous liquid. This liquid was freeze-dried for 1 day to obtain a brownish viscous paste-like solid. This extract was named RB-SP1+M8.

[0089] Step 5: Types and relative contents of lipids and sphingolipids, as well as their in vitro antioxidant and moisturizing activities. Figure 1 The composition and relative content of sphingolipids in fermented rice bran were determined using non-targeted lipidomics. The relative content of sphingolipids in RB-SP1+M8 increased. Sphingolipids were classified into five types: acidic glycosphingolipids, neutral glycosphingolipids, ceramides, sphingomyelin, and sphingosine, with ceramides being the predominant type. The following data were obtained using the in vitro antioxidant assays described above: the IC50 values ​​for scavenging DPPH and ABTS free radicals by RB-SP1+M8 were 8.02 and 18.90 μg / mL, respectively, and the FRAP was 168.97 μg Trolox / mL. The following data were obtained using the in vitro moisturizing and hygroscopic methods described above: after 72 h, the moisturizing rates of RB-SP1+M8 in a 43% relative humidity and a dry environment were 76.57% and 72.43%, respectively; the hygroscopic rates at 43% and 81% relative humidity were 13.1% and 43.6%, respectively.

[0090] Step 6: Test on the repair effect of the sample on HaCaT cells damaged by drying. HaCaT cells were seeded into 96-well plates at a seeding rate of 8000 cells / well and incubated in a CO2 incubator for 12 h until cell adhesion. Then, drying modeling was performed. After 15 min of drying, 100 μL of the test sample prepared with DMEM was added and the cells were incubated for 24 h. 10 μL of MTT solution was added to each well, and the cells were incubated for 4 h. The MTT solution was then discarded, and 100 μL of DMSO was added to each well. The cells were then slowly shaken on a shaker to dissolve the cells, and the absorbance was measured at 490 nm. The untreated HaCaT cells had 100% viability, and the cells treated with drying for 15 min were used as the Model group. Figure 2Cell viability in different groups was determined using the MTT assay described above. Cell viability after treatment with different samples was higher than that in the Model group, with RB-SP1+M8 showing a cell viability of 90.57%. This demonstrates that RB-SP1+M8 can repair the decrease in cell viability caused by desiccation damage.

[0091] Step 7: Test of the protective effect of the sample on HaCaT cells damaged by drying. HaCaT cells were seeded into 96-well plates at a seeding rate of 8000 cells / well and incubated in a CO2 incubator for 12 h until cell adhesion. The culture medium was discarded, and 100 μL of the test sample prepared in DMEM medium was added. After culturing for 24 h, the culture medium was discarded, and the cells were subjected to drying treatment. The Control group had 100% cell viability, and the cells treated for 15 min after drying served as the Model group. Figure 3 The results were obtained based on the MTT method described above. Figure 3 It can be seen that the cell survival rate of the RB-SP1+M8 treatment group can reach 79.87%, and RB-SP1+M8 also has a certain protective effect on desiccation-damaged cells.

[0092] Step 8: Assay for the relative expression levels of HaCaT cell polyfilamentin and epidermal protein mRNA. Seed 1×10⁶ cells into 6-well plates. 5 HaCaT cells were cultured in a 37°C cell culture incubator for 12 h, and then cultured for another 24 h in a medium containing RB-SP1+M8. After drying and modeling, the cells were lysed to extract RNA, which was then reverse transcribed into cDNA for real-time quantitative PCR detection. Figure 4 The results were obtained through the above real-time quantitative PCR assay. Figure 4 It can be seen that the relative expression level of FLG mRNA decreased after drying treatment, but after treatment with 25 μg / mL RB-SP1+M8, the relative expression level of FLG mRNA increased significantly, reaching 5.69 times that of the Model group. Figure 5 The results were obtained by the above real-time quantitative PCR assay. Figure 5 It was found that the IVL gene expression level in the Control group was 1, while the expression level in the Model group after drying treatment was 0.61, indicating that drying downregulated the relative expression level of IVL mRNA. Similarly, after treatment with 25 μg / mL RB-SP1+M8, the gene expression level was 4.60 times that of the Model group, significantly increasing IVL gene expression. Therefore, it is speculated that RB-SP1+M8 can alleviate dryness and moisturize the skin by improving skin barrier function.

[0093] Example 2: Fermentation of rice bran by Pichia amenthionina Y and Lactobacillus casei M8

[0094] Step 1: Strain Preparation. Take a 50 mL Erlenmeyer flask, add 20 mL of YPG medium, and sterilize at 121℃ for 15 min. After cooling, dispense 5 mL into 10 mL glass tubes, and inoculate with *Pichia aamenthionina* Y at 2% (v / v). Incubate at 30℃ for 24 h to obtain the seed culture. Take a 50 mL Erlenmeyer flask, add 20 mL of MRS medium, and sterilize at 121℃ for 15 min. After cooling, dispense 5 mL into 10 mL glass tubes, and inoculate with *Lactobacillus casei* M8 at 2% (v / v). Incubate at 37℃ for 12 h.

[0095] Step 2: Rice bran preparation. Take edible rice bran and dry it thoroughly. Weigh 30 g into a 100 mL Erlenmeyer flask, sterilize at 115℃ for 30 min, cool, add 150 mL of sterile water, and mix thoroughly.

[0096] Step 3: Fermenting rice bran. Inoculate the rice bran system with 5% Pichia aamenthionina Y seed culture and 5% Lactobacillus casei M8 seed culture, ensuring both strains reach a final count of 1×10⁻⁶ in the fermentation system. 6 The concentration was 100 cfu / mL, and then the sample was incubated at 35°C for 6 days.

[0097] Step 4: Lipid extraction. After fermentation, 95% ethanol was added to the rice bran at a material-to-liquid ratio of 1:5. Extraction was performed at 50°C with ultrasonic assistance for 45 min. The mixture was then filtered while hot, and the filtrate was rotary evaporated at 35°C to constant weight, yielding a yellowish-brown viscous liquid. This liquid was freeze-dried for 2 days to obtain a brownish viscous paste-like solid. This extract was named RB-Y+M8.

[0098] Step 5: Testing of lipid and sphingolipid types, relative contents, and in vitro antioxidant and moisturizing activities. (e.g.) Figure 1The composition and relative content of sphingolipids in fermented rice bran were determined using non-targeted lipidomics. The content of sphingolipids was also increased in RB-Y+M8, including acidic glycosphingolipids, neutral glycosphingolipids, ceramides, sphingomyelin, and sphingosine, with ceramides having the largest proportion. The following data were obtained using the above in vitro antioxidant assay: the IC50 values ​​for scavenging DPPH and ABTS free radicals by RB-Y+M8 were 9.27 and 22.16 μg / mL, respectively, and the FRAP was 167.86 μg Trolox / mL. The following data were obtained using the above in vitro moisturizing and hygroscopic methods: after 72 h of experimentation, the moisturizing rate of RB-Y+M8 in a 43% relative humidity and a dry environment was 77.29% and 74.07%, respectively; the hygroscopic rates at 43% and 81% relative humidity were 14.6% and 40.3%, respectively.

[0099] Step 6: Test on the repair effect of the sample on HaCaT cells damaged by drying. HaCaT cells were seeded into 96-well plates at a seeding rate of 8000 cells / well and incubated in a CO2 incubator for 12 h until cell adhesion. Then, drying modeling was performed. After 15 min of drying, 100 μL of the test sample prepared with DMEM was added and the cells were incubated for 24 h. 10 μL of MTT solution was added to each well, and the cells were incubated for 4 h. The MTT solution was then discarded, and 100 μL of DMSO was added to each well. The cells were then slowly shaken on a shaker to dissolve the cells, and the absorbance was measured at 490 nm. The untreated HaCaT cells had 100% viability, and the cells treated with drying for 15 min were used as the Model group. Figure 2 Cell viability in different groups was determined using the MTT assay described above. The RB-Y+M8 group showed a cell viability of 91.03%, significantly improving the survival rate of desiccation-damaged cells.

[0100] Step 7: Test of the protective effect of the sample on HaCaT cells damaged by drying. HaCaT cells were seeded into 96-well plates at a seeding rate of 8000 cells / well and incubated in a CO2 incubator for 12 h until cell adhesion. The culture medium was aspirated, and 100 μL of the test sample prepared in DMEM medium was added. After culturing for 24 h, the culture medium was aspirated, and the cells were subjected to drying treatment. Cell viability was then determined using the MTT assay. The untreated HaCaT cells had a viability of 100%, while the cells treated with drying for 15 min were designated as the Model group. Figure 3 The results were obtained based on the MTT method described above. Figure 3 It can be seen that the cell survival rate of the RB-Y+M8 treatment group can reach 83.85%, which can protect HaCaT to a certain extent and reduce the impact of drying on its cell survival rate.

[0101] Step 8: Assay for the mRNA expression levels of HaCaT cell fimbriae and epidermal proteins. Seed 1×10⁶ cells into 6-well plates. 5 HaCaT cells were cultured in a 37°C cell culture incubator for 12 h, and then cultured for another 24 h in a medium containing RB-Y+M8. After drying and modeling, the cells were lysed to extract RNA, which was then reverse transcribed into cDNA for real-time quantitative PCR detection. Figure 4 The results were obtained through the above real-time quantitative PCR assay. Figure 4 It can be seen that the relative expression level of FLG mRNA decreased after drying treatment. After treatment with 25 μg / mL RB-Y+M8, the relative expression level of FLG mRNA increased significantly, reaching 20.20 times that of the Model group and far exceeding the normal level of cells, reaching 12.14 times that of the Control group. Figure 5 The results were obtained by the above real-time quantitative PCR assay. Figure 5 It was found that the expression level of the Model group was 0.61 after drying treatment, indicating that drying would downregulate the relative expression level of IVL mRNA. Similarly, after treatment with 25 μg / mL RB-Y+M8, the gene expression level was 3.98 times that of the Model group, which significantly increased the expression of the IVL gene.

[0102] Example 3: Fermentation of rice bran with Candida ethanolica ATW1 and Lactobacillus casei M8

[0103] Step 1: Strain Preparation. Take a 50 mL Erlenmeyer flask, add 20 mL of YPG medium, and sterilize at 121℃ for 15 min. After cooling, dispense 5 mL into 10 mL glass tubes, and inoculate with *Candida ethanolica* ATW1 at 5% (v / v). Incubate at 30℃ for 24 h to obtain the seed culture. Take a 50 mL Erlenmeyer flask, add 20 mL of MRS medium, and sterilize at 121℃ for 15 min. After cooling, dispense 5 mL into 10 mL glass tubes, and inoculate with *Lactobacillus casei* M8 at 5% (v / v). Incubate at 37℃ for 24 h.

[0104] Step 2: Rice bran preparation. Take edible rice bran, dry it thoroughly, weigh 25 g into a 100 mL Erlenmeyer flask, sterilize at 121℃ for 15 min, cool, add 75 mL of sterile water, and mix thoroughly.

[0105] Step 3: Fermenting rice bran. Inoculate the rice bran system with 2% Candida ethanolica ATW1 seed culture and 2% Lactobacillus casei M8 seed culture, ensuring both strains reach a final count of 1×10⁻⁶. 6 The concentration was 1 cfu / mL, and then the sample was incubated at 37°C for 5 days.

[0106] Step 4: Lipid extraction. After fermentation, 95% ethanol was added to the rice bran at a material-to-liquid ratio of 1:3. Extraction was performed at 55℃ with ultrasonic assistance for 40 min. The mixture was then filtered while hot, and the filtrate was rotary evaporated at 30℃ to constant weight, yielding a yellowish-brown viscous liquid. This liquid was freeze-dried for 2 days to obtain a brownish viscous paste-like solid. This extract was named RB-ATW1+M8.

[0107] Step 5: Testing the types and relative amounts of lipids and sphingolipids, as well as their in vitro antioxidant and moisturizing activity. (For example...) Figure 1 This study used non-targeted lipidomics to determine the composition and relative content of sphingolipids in fermented rice bran. Figure 1 As shown, the content of sphingolipids was also increased in RB-ATW1+M8, including acidic glycosphingolipids, neutral glycosphingolipids, ceramides, sphingomyelin, and sphingosine, with ceramides having the largest proportion. The following data were obtained using the above in vitro antioxidant assay: the IC50 values ​​for scavenging DPPH and ABTS free radicals by RB-ATW1+M8 were 9.00 and 30.97 μg / mL, respectively, and the FRAP was 170.41 μg Trolox / mL. The following data were obtained using the above in vitro moisturizing and hygroscopic methods: after 72 h of experiment, the moisturizing rate of RB-ATW1+M8 in a relative humidity of 43% and a dry environment was 71.89% and 72.44%, respectively; the hygroscopic rates at relative humidity of 43% and 81% were 13.3% and 41.4%, respectively.

[0108] Comparative example: Unfermented rice bran

[0109] Step 1: Rice bran preparation. Thoroughly dry edible rice bran, accurately weigh 25g into a 100mL Erlenmeyer flask, sterilize at 121℃ for 30 min, cool, add 100mL of sterile water, and mix thoroughly. Then place under the same conditions as fermented rice bran and let stand for 7 days.

[0110] Step 2: Lipid extraction. Add 95% ethanol to rice bran at a material-to-liquid ratio of 1:5, and extract with ultrasound at 45°C for 45 min. Filter while hot, and rotary evaporate the filtrate at 35°C to constant weight, obtaining a yellowish-brown viscous liquid. Freeze-dry for 1 day to obtain a brownish viscous paste-like solid. Record as UFRB.

[0111] Step 3: Types and relative contents of lipids and sphingolipids, and their in vitro antioxidant and moisturizing activities. For example... Figure 1 This study used non-targeted lipidomics to determine the composition and relative content of sphingolipids in fermented rice bran. Figure 1 As shown, the ceramide content of UFRB was relatively lower than that of other fermentation groups. The following data were obtained using the above in vitro antioxidant assay: the IC50 values ​​for scavenging DPPH and ABTS free radicals by UFRB were 29.72 and 27.57 μg / mL, respectively, and the FRAP value was 147.38 μg Trolox / mL. The following data were obtained using the above in vitro humidification and moisture absorption methods: after reaching equilibrium, the humidification rates of UFRB in a 43% relative humidity and a dry environment were 75.64% and 72.71%, respectively; the moisture absorption rates at 43% and 81% relative humidity were 12.4% and 39.7%, respectively.

[0112] Step 4: Test of the repair effect of the sample on desiccation-damaged HaCaT cells. HaCaT cells were seeded into 96-well plates at a seeding rate of 8000 cells / well and incubated in a CO2 incubator for 12 h until cell adhesion. Subsequently, desiccation modeling was performed. After 15 min of desiccation, 100 μL of UFRB prepared with DMEM was added and cultured for 24 h. Then, 10 μL of MTT solution was added to each well, and the plates were incubated for 4 h. After incubation, the MTT solution was discarded, and 100 μL of DMSO was added to each well. The plates were then gently shaken on a shaker to dissolve the cells, and the absorbance was measured at 490 nm. The cell viability in the Control group was 100%, and the cell viability in the Model group was 50.82%. Figure 2 Cell viability in different groups was determined using the MTT assay described above. The UFRB group showed a cell viability of 60.27%, which improved the survival rate of desiccation-damaged cells, but the difference was not statistically significant.

[0113] Step 5: Test of the protective effect of the sample on HaCaT cells damaged by drying. HaCaT cells were seeded into 96-well plates at a seeding rate of 8000 cells / well and incubated in a CO2 incubator for 12 h until cell adhesion. The culture medium was aspirated, and 100 μL of the test sample prepared in DMEM medium was added. After culturing for 24 h, the culture medium was aspirated, and the cells were subjected to drying treatment. Cell viability was then determined using the MTT assay. The model group consisted of HaCaT cells with 100% viability without drying treatment and cells that underwent drying treatment for 15 min. Figure 3 The result was obtained by the MTT method described above. Figure 3 It can be seen that the cell survival rate of the UFRB treatment group can reach 70.08%, which can significantly increase the survival rate of desiccation-damaged HaCaT cells, but the effect is not as obvious as that of RB-SP1+M8 and RB-Y+M8.

[0114] Step 8: Assay for the mRNA expression levels of HaCaT cell fimbriae and epidermal proteins. Seed 1×10⁶ cells into 6-well plates. 5 HaCaT cells were cultured in a 37°C cell culture incubator for 12 h, then cultured for another 24 h in a medium containing UFRB. After drying and modeling, the cells were lysed to extract RNA, which was then reverse transcribed into cDNA for real-time quantitative PCR detection. Figure 4 The results were obtained through the above real-time quantitative PCR assay. Figure 4 It can be seen that the relative expression level of FLG mRNA decreased after drying treatment, while the relative expression level of FLG mRNA increased after treatment with 25 μg / mL UFRB, which was 13.15 times that of the Model group and 7.86 times that of the Control group. Figure 5 The results were obtained by the above real-time quantitative PCR assay. Figure 5 It was found that the expression level of IVL mRNA in the Model group was 0.61 after drying treatment, indicating that drying reduces the relative expression level of IVL mRNA. After treatment with 25 μg / mL UFRB, the gene expression level was 2.52 times that of the Model group, significantly increasing the expression of the IVL gene.

[0115] A comparison of the comparative and exemplary embodiments revealed that co-fermenting rice bran with Kluyveromyces marxianus SP-1, Pichia amenthionina Y, or Candida ethanolica ATW1 and Lactobacillus casei M8 in the embodiments yielded a fermented rice bran lipid extract with moisturizing effects. The extract of the present invention showed significantly higher levels of sphingolipids and ceramides compared to the comparative embodiments. Furthermore, other active lipid components in the present invention also exert moisturizing effects, repairing and protecting dry and damaged cells, and increasing the mRNA expression levels of genes related to skin barrier function proteins such as filin and epidermal proteins. The extract of the present invention also exhibits strong in vitro scavenging capabilities for DPPH and ABTS free radicals, as well as the ability to reduce ferric iron, thus providing both moisturizing and antioxidant / anti-aging benefits.

[0116] In summary, this invention utilizes one of the following fermentation methods—Kluyveromyces marxianus SP-1, Pichia amenthionina Y, or Candida ethanolica ATW1—co-fermented with Lactobacillus casei M8. The lactic acid bacteria and yeast work synergistically during fermentation. The lactic acid bacteria utilize and break down large sugar molecules such as starch and cellulose in rice bran into smaller molecules, which facilitates the decomposition of the rice bran cell walls. The yeast utilizes the small sugar molecules produced by the lactic acid bacteria, as well as the extracellular polysaccharides produced by the lactic acid bacteria, thus promoting better yeast growth. Compared to Chinese Invention Patent 201910248114.0, which employs complex steps such as homogenization and cell wall disruption, this method is more convenient, safe, and pollution-free. Furthermore, the active metabolites produced by the lactic acid bacteria during fermentation further enhance the bioactivity of the sample. Yeast's strong lipid metabolism ability can transform lipids in rice bran, including cholesterol and ceramides, which are major components of intercellular lipids. Furthermore, the extract contains a richer variety of ceramides than that in invention patent 201910248114.0, and also exhibits greater diversity and higher content of other lipid-active components. It also possesses stronger HaCaT cell moisturizing activity and in vitro antioxidant activity. This invention uses rice bran as a raw material, which is abundant, inexpensive, and requires no complex processing or procedures. Moreover, the product prepared by this invention is safe, efficient, and environmentally friendly, showing great promise for industrialization and application in skincare products.

[0117] The embodiments of the present invention are not limited to the above-described examples. Any changes, modifications, substitutions, combinations, simplifications, etc., made without departing from the spirit and principle of the present invention are equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A method for preparing a fermented rice bran lipid extract with moisturizing effects, characterized in that... It includes the following steps: (1) The strain was inoculated and cultured in a culture medium to prepare a seed culture; after sterilizing rice bran, sterile water was added in 3 to 5 times the weight of the rice bran, and then lactic acid bacteria and yeast seed culture were inoculated for fermentation; the lactic acid bacteria was Lactobacillus casei M8, with accession number GDMCC NO: 64441; the yeast was one of the following three: Pichia amenthionina Y, with accession number CGMCC NO: 10183; Kluyveromyces marxianus SP-1, with accession number GDMCC NO: 64440; Candida ethanolica ATW1, with accession number GDMCC NO: 61360; the fermentation temperature was 30~37℃, and the fermentation time was 5~7 days; (2) Add ethanol to the fermentation system obtained in step (1) and extract with ultrasound assistance; (3) The mixture obtained in step (2) was filtered, rotary evaporated, and freeze-dried to obtain fermented rice bran lipid extract.

2. The method for preparing a fermented rice bran lipid extract with moisturizing effect according to claim 1, characterized in that, In step (1), the inoculation amount of yeast and lactic acid bacteria is 1 to 5% of the total volume of the rice bran and water mixture.

3. The method for preparing a fermented rice bran lipid extract with moisturizing effect according to claim 1, characterized in that, In step (1), the sterilization temperature is 115~121℃ and the sterilization time is 15~30min.

4. The method for preparing a fermented rice bran lipid extract with moisturizing effect according to claim 1, characterized in that, In step (2), the volume concentration of ethanol is 90-95% ethanol; g and mL are used as the units of mass and volume, respectively; the ratio of rice bran mixture to ethanol is 1:4-6; the temperature of ultrasonic-assisted extraction is 45-55℃ and the time is 40-50 min.

5. The method for preparing a fermented rice bran lipid extract with moisturizing effect according to claim 1, characterized in that, In step (3), the filtration is performed using a circulating water multi-purpose vacuum pump at a temperature of 25~30℃ for 5~15 minutes.

6. The method for preparing a fermented rice bran lipid extract with moisturizing effect according to claim 1, characterized in that, In step (3), the temperature of the rotary evaporation is 30-45°C.

7. The method for preparing a fermented rice bran lipid extract with moisturizing effect according to claim 1, characterized in that, In step (1), the rice bran is edible rice bran.

8. A fermented rice bran lipid extract with moisturizing effects, characterized in that... It is prepared by the preparation method described in any one of claims 1-7; the fermented rice bran lipid extract has an in vitro moisturizing rate of 70-80% and a moisture absorption rate of more than 12%.

9. The use of the fermented rice bran lipid extract with moisturizing effect as described in claim 8 in the preparation of natural moisturizing skin care products.

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