Wine lees extract with anti-aging effect, preparation method thereof and cosmetic

Abstract

The application relates to a distiller's grains extract with anti-aging efficacy, a preparation method thereof and a cosmetic. The method comprises the following steps: extracting the fat-soluble components from distiller's grains powder to obtain a first extract and first distiller's grains; extracting the alcohol-soluble components from the first distiller's grains to obtain a second extract and second distiller's grains; and extracting the water-soluble components from the second distiller's grains to obtain a third extract. In the technical scheme, the fat-soluble components in the distiller's grains are extracted first, and then the alcohol extraction and water extraction are sequentially adopted, so that various active substances in the distiller's grains are greatly extracted, the characteristic components of the distiller's grains extract are enriched, and the resource utilization of the distiller's grains is greatly expanded. The distiller's grains extract contains the characteristic components of the distiller's grains extract, such as linoleic acid, oleic acid, maltol, eugenol, alpha-caryophyllene, alpha-terpineol, gamma-aminobutyric acid, goose myo-peptide, plant sphingosine, dihydrosphingosine, lactic acid and glycolic acid, so that the efficacy of the distiller's grains extract is greatly enriched.

Description

Technical Field

[0001] This application relates to a sake lees extract with anti-aging effects, its preparation method, and cosmetics. Background Technology

[0002] Sake lees are solid waste from the sake brewing industry. They are characterized by high water content, high acid content, and easy spoilage. They are often treated inefficiently and extensively, such as being made into low-value fertilizers or animal feed, or being directly landfilled, resulting in a huge waste of resources and a serious threat to environmental safety. Summary of the Invention

[0003] The purpose of this application is to provide a sake lees extract with anti-aging effects, its preparation method, and a cosmetic product.

[0004] In a first aspect, this application provides a method for preparing a sake lees extract with anti-aging effects, comprising:

[0005] The lipophilic components of the sake lees powder were extracted to obtain the first extract and the first sake lees.

[0006] The alcohol-soluble components of the first lees were extracted to obtain the second extract and the second lees.

[0007] The second batch of sake lees was subjected to water-soluble component extraction to obtain the third extract.

[0008] In the above technical solution, by first extracting the fat-soluble components from the lees, and then sequentially using alcohol extraction and water extraction, the various active substances in the lees are extracted to a great extent, enriching the characteristic components of the lees extract and greatly expanding the resource utilization of lees.

[0009] Furthermore, in the above technical solution, by sequentially extracting the fat-soluble components, alcohol-soluble components, and water-soluble components from sake lees, a sake lees extract containing characteristic components such as linoleic acid, oleic acid, maltol, eugenol, α-caryophyllene, α-terpineol, γ-aminobutyric acid, anserine, phytosphingosine, dihydrosphingosine, lactic acid, and glycolic acid can be obtained, greatly enriching the efficacy of sake lees extract. Applying this sake lees extract to cosmetics can effectively enhance the antioxidant and anti-aging effects of cosmetics.

[0010] In other embodiments of this application, the extraction of fat-soluble components from sake lees powder includes:

[0011] Alkanes were used as the first extractant to extract sake lees powder;

[0012] Optionally, the first extractant includes n-butane.

[0013] In other embodiments of this application, the extraction of fat-soluble components from sake lees powder includes:

[0014] Extraction was carried out at an extraction pressure of 0.2 MPa to 0.8 MPa and an extraction temperature of 30°C to 50°C.

[0015] Optionally, the extraction time is 60 min to 120 min;

[0016] Optionally, the extraction of fat-soluble components from the sake lees powder is performed using a continuous phase change extraction device; the desorption temperature is 40℃~60℃; and the flow rate is optionally 20L / h~60L / h.

[0017] In other embodiments of this application, the first sake lees is subjected to alcohol-soluble component extraction, including:

[0018] Alcohol was used as the second extractant to extract the first batch of sake lees;

[0019] Optionally, the second extractant includes ethanol.

[0020] In other embodiments of this application, the first sake lees is subjected to alcohol-soluble component extraction, including:

[0021] Extraction was carried out at an extraction pressure of 0.2 MPa to 0.8 MPa and an extraction temperature of 60°C to 80°C.

[0022] Optionally, the extraction time is 60 min to 120 min;

[0023] Optionally, the extraction of alcohol-soluble components from the first lees is performed using a continuous phase change extraction device; the desorption temperature is 40℃~60℃; and the flow rate is optionally 20L / h~60L / h.

[0024] In other embodiments of this application, the second sake lees is subjected to water-soluble component extraction, including:

[0025] Water was used as the third extractant to extract the second batch of sake lees.

[0026] In other embodiments of this application, the second sake lees is subjected to water-soluble component extraction, including:

[0027] Extraction was carried out at an extraction pressure of 0.2 MPa to 0.8 MPa and an extraction temperature of 60°C to 80°C.

[0028] Optionally, the extraction time is 60 min to 120 min;

[0029] Optionally, the extraction of alcohol-soluble components from the second lees is performed using a continuous phase change extraction device; the desorption temperature is 40℃~60℃; and the flow rate is optionally 20L / h~60L / h.

[0030] In other embodiments of this application, the preparation of sake lees powder includes:

[0031] After freeze-drying the sake lees, they are pulverized and sieved.

[0032] Optionally, freeze drying includes: vacuum drying at -45℃ to -35℃ for 20 to 30 hours;

[0033] Optionally, pulverizing and sieving includes: passing through a 40-100 mesh sieve and collecting the material passing through the sieve.

[0034] Secondly, this application provides a sake lees extract, which is prepared by the sake lees extract preparation method provided in the first aspect above;

[0035] The sake lees extract includes: first extract, second extract and third extract.

[0036] In other embodiments of this application, the characteristic components of the first extract include: linoleic acid, oleic acid, maltol, eugenol, α-caryophyllene, and α-terpineol.

[0037] In other embodiments of this application, the characteristic components of the second and third extracts include: γ-aminobutyric acid, anserine, phytosphingosine, dihydrosphingosine, lactic acid, and glycolic acid.

[0038] Thirdly, this application provides a cosmetic product, including the sake lees extract provided in the first or second aspect above. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 The relationship between the dilution factor of the extracts obtained in Examples 1-3 and their in vitro antioxidant activity is shown. The DPPH(a) and ABTS(b) free radical scavenging rates of each extract in Example 1; the DPPH(c) and ABTS(d) free radical scavenging rates of each extract in Example 2; and the DPPH(e) and ABTS(f) free radical scavenging rates of each extract in Example 3 are also shown.

[0041] Figure 2The relationship between the dilution factor of alcohol extracts obtained under different process parameters and their in vitro antioxidant activity;

[0042] Figure 3 The relationship between the dilution factor of alcohol extracts obtained from different processes and their in vitro antioxidant activity;

[0043] Figure 4 The effect of different extracts on the survival curves of nematodes under H2O2 oxidative stress;

[0044] Figure 5 The effect of different extracts on the survival curves of nematodes under PQ oxidative stress;

[0045] Figures 6a-6d Representative spectra for the analysis of characteristic components. Total ion chromatogram of alkyl extract analyzed by gas chromatography-mass spectrometry (GC-MS). Figure 6a ); Total ion chromatogram of alkyl extract analyzed by headspace solid-phase microextraction-gas chromatography-mass spectrometry ( Figure 6b ); Total ion chromatogram of alcohol extract analyzed by liquid chromatography-mass spectrometry (LC-MS) Figure 6c ); Total ion chromatogram of alcohol extract analyzed by gas chromatography-mass spectrometry (GC-MS) Figure 6d );

[0046] Figure 7 Image of the alkyl extract obtained in Example 1;

[0047] Figure 8 Here is an image of the alcohol extract obtained in Example 1;

[0048] Figure 9 The image shows the water extract obtained in Example 1. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are some embodiments of this application, but not all embodiments.

[0050] Therefore, the following detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0051] The inventors discovered that sake lees contains various high-value components that contribute to anti-aging activity, such as amino acids, polypeptides, polysaccharides, and organic acids. Applying these active ingredients to cosmetics as green and natural active ingredients not only yields green and natural cosmetics but also enables the resource utilization of sake lees.

[0052] However, the extraction of active substances from sake lees is quite difficult. Current common methods can only extract a small amount of active ingredients from sake lees, resulting in low utilization of sake lees. This single extraction method is almost impossible to achieve effective industrial application, thus significantly hindering the industrial application of sake lees.

[0053] Moreover, there is currently no solution on the market that can extract as much of the abundant active substances from sake lees as possible.

[0054] This application provides a method for preparing a sake lees extract with anti-aging effects, comprising:

[0055] The lipophilic components of the sake lees powder were extracted to obtain the first extract and the first sake lees.

[0056] The alcohol-soluble components of the first lees were extracted to obtain the second extract and the second lees.

[0057] The second batch of sake lees was subjected to water-soluble component extraction to obtain the third extract.

[0058] In the above technical solution, by first extracting the fat-soluble components from the lees, and then sequentially using alcohol extraction and water extraction, the various active substances in the lees are extracted to a great extent, enriching the characteristic components of the lees extract and greatly expanding the resource utilization of lees.

[0059] Furthermore, in the above-mentioned technical solution, by sequentially extracting the fat-soluble components, alcohol-soluble components, and water-soluble components from sake lees, a sake lees extract containing characteristic components such as linoleic acid, oleic acid, maltol, eugenol, α-caryophyllene, α-terpineol, γ-aminobutyric acid, anserine, phytosphingosine, dihydrosphingosine, lactic acid, and glycolic acid can be obtained, greatly enriching the efficacy of sake lees extract. Currently, no similar products are available on the market. Applying this sake lees extract to cosmetics can effectively enhance the antioxidant and anti-aging effects of cosmetics.

[0060] It should also be noted that the inventors have discovered that changing the extraction order (e.g., sequentially extracting water-soluble components, alcohol-soluble components, and fat-soluble components) or (sequentially extracting alcohol-soluble components, water-soluble components, and fat-soluble components) or (sequentially extracting alcohol-soluble components, fat-soluble components, and water-soluble components) or (sequentially extracting water-soluble components, fat-soluble components, and alcohol-soluble components) all result in a decrease in the characteristic components of the obtained sake lees extract.

[0061] Furthermore, in some embodiments of this application, the preparation method of sake lees extract with anti-aging effects includes the following steps:

[0062] Step S1: Prepare sake lees powder.

[0063] In some embodiments of this application, the preparation of sake lees powder includes:

[0064] After freeze-drying the sake lees, they are pulverized and sieved.

[0065] Further optionally, in some embodiments of this application, the above-mentioned freeze drying includes: vacuum drying at -45°C to -35°C for 20 to 30 hours.

[0066] For example, in some embodiments of this application, the freeze drying described above includes: a range of -45°C, -42°C, -40°C, -38°C, -37°C, -36°C, -35°C or any two of the aforementioned temperature values; and vacuum drying for 20h, 22h, 25h, 26h, 28h, 30h or any two of the aforementioned time periods.

[0067] Further optionally, in some embodiments of this application, pulverizing and sieving includes: passing through a 40-100 mesh sieve and collecting the material passing through the sieve.

[0068] Crushing sake lees to the aforementioned particle size facilitates subsequent extraction of fat-soluble, alcohol-soluble, and water-soluble components, thereby increasing the yield of sake lees extract.

[0069] Step S2: Extract the fat-soluble components from the sake lees powder.

[0070] In some embodiments of this application, the extraction of fat-soluble components from sake lees powder includes:

[0071] Alkanes were used as the first extractant to extract sake lees powder.

[0072] Furthermore, in some embodiments of this application, the first extractant includes n-butane.

[0073] In other optional embodiments of this application, the first extractant described above may include other alkanes, such as butane.

[0074] Furthermore, in some embodiments of this application, the extraction of fat-soluble components from the sake lees powder includes:

[0075] Extraction was carried out at an extraction pressure of 0.2 MPa to 0.8 MPa and an extraction temperature of 30°C to 50°C.

[0076] In the above technical solution, extraction is carried out at an extraction pressure of 0.2MPa to 0.8MPa and an extraction temperature of 30℃ to 50℃, which can effectively improve the yield of fat-soluble components.

[0077] Exemplary, in some embodiments of this application, the above-described extraction of fat-soluble components from sake lees powder includes:

[0078] Extraction was performed at pressures of 0.2 MPa, 0.25 MPa, 0.3 MPa, 0.35 MPa, 0.4 MPa, 0.45 MPa, 0.5 MPa, 0.55 MPa, 0.6 MPa, 0.65 MPa, 0.7 MPa, 0.75 MPa, 0.8 MPa, or any two of the aforementioned values.

[0079] Exemplary, in some embodiments of this application, the above-described extraction of fat-soluble components from sake lees powder includes:

[0080] Extraction was performed at temperatures of 30°C, 32°C, 35°C, 38°C, 40°C, 42°C, 45°C, 48°C, 50°C, or any two of the aforementioned values.

[0081] Furthermore, in some embodiments of this application, the above-mentioned extraction of fat-soluble components from sake lees powder includes an extraction time of 60 min to 120 min.

[0082] For example, in some embodiments of this application, the extraction of fat-soluble components from sake lees powder includes extraction for a time of 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min or any two of the aforementioned values.

[0083] Furthermore, in some embodiments of this application, the above-mentioned extraction of fat-soluble components from sake lees powder is performed using low-temperature continuous phase change extraction technology.

[0084] Low-temperature continuous phase change extraction technology combines the principles of supercritical and subcritical extraction, using green and clean solvents. The entire process is closed, oxygen-free, and continuous, enabling safe and efficient extraction and retention of active ingredients. Based on this, this application utilizes low-temperature continuous phase change extraction technology to fully extract sake lees, providing a safe and effective raw material for anti-aging cosmetics.

[0085] Further, alternatively, in some embodiments of this application, the extraction of fat-soluble components from the sake lees powder is performed using a continuous phase change extraction apparatus.

[0086] Further optionally, in some embodiments of this application, when extracting the fat-soluble components from the sake lees powder using a continuous phase change extraction device, the desorption temperature is 40°C to 60°C; optionally, the flow rate is 20L / h to 60L / h.

[0087] The above-mentioned technical solution can extract active substances from wine lees more gently, which is beneficial to improving the yield of active substances.

[0088] For example, in some embodiments of this application, when extraction is performed using a continuous phase change extraction apparatus, the resolution temperature is 40°C, 42°C, 45°C, 48°C, 50°C, 52°C, 55°C, 58°C, 60°C, or a temperature within the range of any two of the aforementioned values.

[0089] For example, in some embodiments of this application, when extraction is performed using a continuous phase change extraction apparatus, the flow rate is 20 L / h, 22 L / h, 25 L / h, 28 L / h, 30 L / h, 32 L / h, 35 L / h, 38 L / h, 40 L / h, 42 L / h, 45 L / h, 48 L / h, 50 L / h, 55 L / h, 60 L / h, or a range between any two of the aforementioned values.

[0090] Step S3: Extract the alcohol-soluble components from the first batch of sake lees.

[0091] In some embodiments of this application, the first sake lees is subjected to alcohol-soluble component extraction, including:

[0092] Alcohol was used as the second extractant to extract the first batch of sake lees.

[0093] In some embodiments of this application, the second extractant includes ethanol.

[0094] Further optionally, in some embodiments of this application, the second extractant described above comprises an aqueous ethanol solution with a mass fraction of 70% to 85%.

[0095] For example, in some embodiments of this application, the second extractant described above includes an aqueous ethanol solution with a mass fraction of 70%, 72%, 75%, 76%, 78%, 80%, 82%, 84%, 85%, or any two of the aforementioned values.

[0096] Furthermore, in some embodiments of this application, the first sake lees are subjected to extraction of alcohol-soluble components, including:

[0097] Extraction was carried out at an extraction pressure of 0.2 MPa to 0.8 MPa and an extraction temperature of 60°C to 80°C.

[0098] In the above technical solution, extraction is carried out at an extraction pressure of 0.2MPa to 0.8MPa and an extraction temperature of 60℃ to 80℃, which is beneficial to improving the yield of alcohol-soluble components.

[0099] Exemplary, in some embodiments of this application, the first sake lees is subjected to alcohol-soluble component extraction, including:

[0100] Extraction is performed at an extraction pressure of 0.2 MPa, 0.3 MPa, 0.4 MPa, 0.5 MPa, 0.6 MPa, 0.7 MPa, 0.8 MPa, or any two of the aforementioned values.

[0101] Exemplary, in some embodiments of this application, the first sake lees is subjected to alcohol-soluble component extraction, including:

[0102] Extraction is performed at temperatures of 60°C, 62°C, 65°C, 68°C, 70°C, 72°C, 75°C, 78°C, 79°C, 80°C, or any two of the aforementioned values.

[0103] Furthermore, in some embodiments of this application, the first sake lees are subjected to extraction of alcohol-soluble components, including an extraction time of 60 min to 120 min.

[0104] For example, in some embodiments of this application, the extraction of alcohol-soluble components from the first sake lees includes extraction for a time of 60 min, 65 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min or any two of the aforementioned values.

[0105] Furthermore, in some embodiments of this application, the first sake lees are subjected to extraction of alcohol-soluble components using low-temperature continuous phase change extraction technology.

[0106] Further, optionally, and exemplary, in some embodiments of this application, the first sake lees are subjected to extraction of alcohol-soluble components using a continuous phase change extraction apparatus.

[0107] Further optionally, in some embodiments of this application, when using a continuous phase change extraction apparatus for extraction, the desorption temperature is 40°C to 60°C.

[0108] For example, in some embodiments of this application, extraction is performed at temperatures of 40°C, 42°C, 45°C, 48°C, 50°C, 52°C, 55°C, 58°C, 60°C, or any two of the aforementioned values.

[0109] Further optionally, in some embodiments of this application, when using a continuous phase change extraction apparatus for extraction, the flow rate is 20 L / h to 60 L / h.

[0110] For example, in some embodiments of this application, extraction is performed at flow rates of 20 L / h, 22 L / h, 25 L / h, 28 L / h, 30 L / h, 32 L / h, 35 L / h, 38 L / h, 40 L / h, 42 L / h, 45 L / h, 48 L / h, 50 L / h, 55 L / h, 58 L / h, 60 L / h, or any range between the aforementioned two values.

[0111] Step S4: Extract water-soluble components from the second batch of sake lees.

[0112] Furthermore, in some embodiments of this application, the second sake lees are subjected to water-soluble component extraction, including:

[0113] Water was used as the third extractant to extract the second batch of sake lees.

[0114] Furthermore, in some embodiments of this application, the second sake lees are subjected to water-soluble component extraction, including:

[0115] Extraction was carried out at an extraction pressure of 0.2 MPa to 0.8 MPa and an extraction temperature of 60°C to 80°C.

[0116] In the above technical solution, extraction at an extraction pressure of 0.2MPa to 0.8MPa and an extraction temperature of 60℃ to 80℃ is beneficial to improving the yield of water-soluble components.

[0117] Further optionally, and exemplary, in some embodiments of this application, extraction is performed at an extraction pressure of 0.2 MPa, 0.3 MPa, 0.4 MPa, 0.5 MPa, 0.6 MPa, 0.7 MPa, 0.8 MPa, or any two of the aforementioned pressure values.

[0118] Further optionally, and exemplary, in some embodiments of this application, extraction is performed at an extraction temperature of 60°C, 62°C, 65°C, 68°C, 70°C, 72°C, 75°C, 78°C, 80°C, or any two of the aforementioned temperature values.

[0119] Furthermore, in some embodiments of this application, the second sake lees are subjected to water-soluble component extraction, including:

[0120] The extraction time is 60 min to 120 min.

[0121] Within the aforementioned extraction time, the water-soluble components in the second lees can be effectively extracted, and the yield of the water-soluble components can be increased.

[0122] Further optionally, and exemplaryly, in some embodiments of this application, the extraction time described above is 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min, 100 min, 110 min, 120 min, or a range between any two of the aforementioned time values.

[0123] Furthermore, in some embodiments of this application, the second sake lees are subjected to water-soluble component extraction using low-temperature continuous phase change extraction technology.

[0124] Further, optionally, and exemplary, in some embodiments of this application, the second sake lees are subjected to water-soluble component extraction using a continuous phase change extraction apparatus.

[0125] Further optionally, in some embodiments of this application, when the second sake lees is extracted for water-soluble components using a continuous phase change extraction device, the extraction temperature includes: a desorption temperature of 40°C to 60°C.

[0126] For example, in some embodiments of this application, extraction is performed at temperatures of 40°C, 42°C, 45°C, 48°C, 50°C, 52°C, 55°C, 58°C, 60°C, or any two of the aforementioned values.

[0127] Further optionally, in some embodiments of this application, when the second sake lees is extracted for water-soluble components using a continuous phase change extraction apparatus, the flow rate is 20 L / h to 60 L / h.

[0128] For example, in some embodiments of this application, extraction is performed at flow rates of 20 L / h, 22 L / h, 25 L / h, 28 L / h, 30 L / h, 32 L / h, 35 L / h, 38 L / h, 40 L / h, 42 L / h, 45 L / h, 48 L / h, 50 L / h, 55 L / h, 58 L / h, 60 L / h, or any range between the aforementioned two values.

[0129] Some embodiments of this application provide a cosmetic product prepared by the method for preparing sake lees extract provided in any of the foregoing embodiments.

[0130] Furthermore, in some embodiments of this application, the aforementioned sake lees extract includes: a first extract, a second extract, and a third extract.

[0131] Furthermore, in some embodiments of this application, the characteristic components of the first extract described above include: linoleic acid, oleic acid, maltol, eugenol, α-caryophyllene, and α-terpineol.

[0132] Furthermore, in some embodiments of this application, the first extract described above has a typical and rich sake lees aroma.

[0133] Furthermore, in some embodiments of this application, the characteristic components of the second and third extracts described above include: γ-aminobutyric acid, anserine, phytosphingosine, dihydrosphingosine, lactic acid, and glycolic acid.

[0134] Furthermore, in some embodiments of this application, the second and third extracts described above have a light and refreshing aroma.

[0135] Furthermore, in some embodiments of this application, the above-mentioned sake lees extract has a high DPPH and ABTS free radical scavenging capacity and can significantly improve the antioxidant stress capacity of nematodes, thereby delaying the aging and death process of nematodes. It can be seen that the sake lees extract of this application has anti-aging effects.

[0136] Furthermore, some embodiments of this application provide a cosmetic product including the sake lees extract provided in any of the foregoing embodiments.

[0137] In the above technical solution, by including the aforementioned sake lees extract in the cosmetics, it is beneficial to improve the antioxidant and anti-aging effects of the cosmetics.

[0138] The features and performance of this application will be further described in detail below with reference to embodiments:

[0139] Example 1

[0140] A method for preparing sake lees extract is provided, comprising the following steps:

[0141] S1. The wet sake lees are freeze-dried at -40℃ for 24 hours until the moisture content is ≤10%, and then pulverized to 40 mesh to obtain dry sake lees powder.

[0142] S2. Weigh 1500g of the sake lees powder obtained in step S1 and add it to the 3L extraction chamber of a continuous phase change extraction apparatus (i.e., raw material bulk density 0.5kg / L). Use n-butane as the extraction solvent and extract for 90min at an extraction pressure of 0.8MPa, an extraction temperature of 50℃, a desorption temperature of 60℃, and a flow rate of 60L / h. The first extract is obtained directly by separating it from the solvent through the desorption chamber. The defatted sake lees powder after extraction is the first sake lees.

[0143] S3. Weigh 550g of the first sake lees obtained in step S2 and add it to a 3L extraction vessel (i.e., raw material bulk density 0.18kg / L). Use 80% ethanol as the extraction solvent and extract for 90min at an extraction pressure of 0.8MPa, an extraction temperature of 80℃, a desorption temperature of 60℃, and a flow rate of 20L / h. The second extract is then concentrated to 5L by evaporation using the desorption vessel. The sake lees obtained after extraction are called the second sake lees.

[0144] S4. In addition, 550g of the second sake lees obtained in step S3 is added to a 3L extraction vessel (i.e., raw material bulk density 0.18kg / L). Water is used as the extraction solvent. Extraction is carried out for 90min under the conditions of extraction pressure of 0.8MPa, extraction temperature of 80℃, desorption temperature of 60℃, and flow rate of 20L / h. The third extract is obtained by evaporation and concentration to 5L through the desorption vessel.

[0145] The obtained sake lees extract was analyzed:

[0146] (1) Calculation of alkyl extract yield: Alkyl extract yield (%) = Alkyl extract mass (g) / Distillery lees mass (g) × 100%

[0147] (2) Methods for analyzing total sugar content:

[0148] Accurately measure 0.2 mL of sample solution into a stoppered test tube, add distilled water to 2 mL, add 1 mL of 5% phenol, shake well, add 5 mL of concentrated sulfuric acid, shake well, let stand for 10 min, incubate in a 40℃ water bath for 15 min, and measure the absorbance at a wavelength of 490 nm. Calculate the total sugar content of the sample according to the regression equation of the glucose standard curve. Repeat the measurement 3 times for each sample.

[0149] (3) Methods for analyzing total amino acid content:

[0150] Accurately measure 0.5 mL of sample solution into a stoppered test tube, add distilled water to 2 mL, add 1 mL of 1.5% ninhydrin solution and shake well, add 1 mL of phosphate buffer solution, shake well, incubate in a 90℃ water bath for 15 min, remove and immediately cool to room temperature, add distilled water to 10 mL, and measure the absorbance value at a wavelength of 570 nm. Calculate the total amino acid content according to the regression equation of the L-arginine standard curve. Repeat the measurement 3 times for each sample.

[0151] (4) Total protein content analysis method:

[0152] The extract was diluted proportionally to prepare the reagent 1 working solution according to the BCA kit instructions. The reagent 1 working solution was then mixed with reagent 2 at a ratio of 50:1 to prepare the working solution. Reagent 3 was a 524 μg / mL protein standard solution. Samples were added according to the instructions, the plate was gently shaken to mix, and incubated at 37°C for 30 min. The absorbance of each well was read using a microplate reader at a wavelength of 562 nm. The total protein content was calculated based on the regression equation of the protein standard curve. Each sample was measured three times.

[0153] (5) Total phenol content analysis method:

[0154] Accurately pipette 0.5 mL of the extract into a test tube, add distilled water to 5 mL, add 1 mL of Folin-Ciocateu reagent and 4 mL of 10% Na2CO3 solution, shake well and let it develop color at room temperature for 60 min. Measure the absorbance at 765 nm. Calculate the polyphenol content according to the regression equation of the gallic acid standard curve. Repeat the measurement 3 times for each sample.

[0155] The test results are as follows:

[0156] The first extract (alkyl extract) obtained was 19.20g, with a yield of 1.28%. The contents of total sugar, total amino acids, total protein and total phenol were 14.16±2.39mg / g, 7.06±0.51mg / g, 2.39±0.34mg / g and 0.69±0.01mg / g, respectively.

[0157] The total sugar, total amino acid, total protein, and total phenol contents of the second extract (ethanol extract) were 46.13±0.83 mg / g, 87.49±0.84 mg / g, 59.41±1.20 mg / g, and 3.20±0.01 mg / g, respectively.

[0158] The total sugar, total amino acid, total protein, and total phenol contents of the third extract (water extract) were 45.52±0.56 mg / g, 53.62±6.30 mg / g, 40.77±0.48 mg / g, and 1.41±0.10 mg / g, respectively.

[0159] Example 2

[0160] A method for preparing sake lees extract is provided, comprising the following steps:

[0161] S1. The wet sake lees are freeze-dried at -40℃ for 24 hours until the moisture content is ≤10%, and then pulverized to 40 mesh to obtain dry sake lees powder.

[0162] S2. Weigh 1500g of the sake lees powder obtained in step S1 and add it to the 3L extraction chamber of a continuous phase change extraction apparatus (i.e., raw material bulk density 0.5kg / L). Use n-butane as the extraction solvent and extract for 60min at an extraction pressure of 0.2MPa, an extraction temperature of 30℃, a desorption temperature of 60℃, and a flow rate of 60L / h. The first extract is obtained by separating it from the solvent through the desorption chamber. The defatted sake lees powder after extraction is the first sake lees.

[0163] S3. Weigh 550g of the first sake lees obtained in step S2 and add it to a 3L extraction vessel (i.e., raw material bulk density 0.18kg / L). Use 80% ethanol as the extraction solvent and extract for 60 minutes at an extraction pressure of 0.2MPa, an extraction temperature of 60℃, a desorption temperature of 60℃, and a flow rate of 30L / h. The second extract is then concentrated to 5L by evaporation using a desorption vessel. The sake lees obtained after extraction are called the second sake lees.

[0164] S4. Additionally, 550g of the second sake lees obtained in step S3 is added to a 3L extraction vessel (i.e., raw material bulk density 0.18kg / L). Water is used as the extraction solvent, and extraction is carried out for 60min under the conditions of extraction pressure of 0.2MPa, extraction temperature of 60℃, desorption temperature of 60℃, and flow rate of 30L / h. The third extract is then concentrated to 5L by evaporation through the desorption vessel.

[0165] The obtained sake lees extract was tested using the same method as in Example 1; the results are as follows:

[0166] The first extract (alkyl extract) obtained was 10.34 g, with a yield of 0.70%. The contents of total sugar, total amino acids, total protein and total phenol were 24.81±3.29 mg / g, 6.24±0.13 mg / g, 2.31±0.39 mg / g and 1.05±0.30 mg / g, respectively.

[0167] The total sugar, total amino acid, total protein, and total phenol contents of the second extract (ethanol extract) were 33.75±0.65 mg / g, 17.36±0.41 mg / g, 21.57±1.31 mg / g, and 2.04±0.03 mg / g, respectively.

[0168] The total sugar, total amino acid, total protein, and total phenol contents of the third extract (water extract) were 29.51±0.19 mg / g, 12.30±1.17 mg / g, 12.11±2.04 mg / g, and 1.15±0.08 mg / g, respectively.

[0169] Example 3

[0170] A method for preparing sake lees extract is provided, comprising the following steps:

[0171] S1. Freeze-dry the wet sake lees at -40℃ for 24 hours until the moisture content is ≤10%, then pulverize to 40 mesh to obtain dry sake lees powder.

[0172] S2. Weigh 1500g of the sake lees powder obtained in step S1 and add it to the 3L extraction chamber of a continuous phase change extraction apparatus (i.e., raw material bulk density 0.5kg / L). Use n-butane as the extraction solvent and extract for 120min at an extraction pressure of 0.5MPa, an extraction temperature of 40℃, a desorption temperature of 60℃, and a flow rate of 60L / h. The first extract is obtained by separating it from the solvent through the desorption chamber. The defatted sake lees powder after extraction is the first sake lees.

[0173] S3. Weigh 550g of the first lees obtained in step S2 and add it to a 3L extraction vessel (i.e., raw material bulk density 0.18kg / L). Use 80% ethanol as the extraction solvent and extract for 120min at an extraction pressure of 0.5MPa, an extraction temperature of 70℃, a desorption temperature of 60℃, and a flow rate of 15L / h. The second extract is then concentrated to 5L by evaporation using a desorption vessel. The lees obtained after extraction are called the second lees.

[0174] S4. In addition, 550g of the second lees obtained in step S3 is filled into a 3L extraction vessel (i.e., raw material bulk density 0.18kg / L), and water is used as the extraction solvent. The extraction is carried out for 120min under the conditions of extraction pressure of 0.5MPa, extraction temperature of 70℃, desorption temperature of 60℃, and flow rate of 15L / h. The third extract is then concentrated to 5L by evaporation through the desorption vessel.

[0175] The obtained sake lees extract was tested using the same method as in Example 1; the results are as follows:

[0176] The first extract (alkyl extract) obtained was 16.80g, with a yield of 1.12%. The contents of total sugar, total amino acids, total protein and total phenol were 27.29±3.62mg / g, 6.87±0.14mg / g, 2.54±0.43mg / g and 1.16±0.33mg / g, respectively.

[0177] The total sugar, total amino acid, total protein, and total phenol contents of the second extract (ethanol extract) were 37.13±0.71 mg / g, 19.10±0.45 mg / g, 23.72±1.44 mg / g, and 2.24±0.04 mg / g, respectively.

[0178] The total sugar, total amino acid, total protein, and total phenol contents of the third extract (water extract) were 32.47±0.21 mg / g, 13.53±1.29 mg / g, 13.32±2.24 mg / g, and 1.26±0.08 mg / g, respectively.

[0179] Experimental Example 1

[0180] The in vitro antioxidant activity of the sake lees extracts prepared in Examples 1-3 was investigated.

[0181] The in vitro antioxidant activity of extracts obtained under three different process parameters (Example 1, Example 2, Example 3) was compared. Each extract was diluted 1 to 64 times, and vitamin C (1 mg / mL) was compared simultaneously. The relationship between the dilution factor of the extract and the free radical scavenging rate was determined.

[0182] The results are as follows Figure 1 As shown, the scavenging abilities of various sake lees extracts and vitamin C against DPPH free radicals exhibit a clear dose-response relationship. The ethanol extract, even diluted to 1 / 8 of its original volume, maintains a high antioxidant capacity similar to that of vitamin C (1 mg / mL). Simultaneously, the extracts show a higher ABTS free radical scavenging rate than those against DPPH free radicals, and even after high dilution, their antioxidant capacity remains highly similar to that of vitamin C (1 mg / mL). Each extract maintains a near 100% scavenging rate even when diluted to 1 / 4 of its original volume, with the ethanol and water extracts maintaining a scavenging rate exceeding 80% even after further dilution.

[0183] The ethanol extract exhibited a higher free radical scavenging rate compared to the alkyl and water extracts, and maintained a leading level throughout the dilution process. Therefore, the in vitro antioxidant activity of the ethanol extracts obtained under three different process parameters (Example 1, Example 2, and Example 3) was further compared.

[0184] Depend on Figure 2 The data on DPPH and ABTS free radical scavenging rates of the ethanol extracts obtained with different process parameters show that the process parameters in Example 1 are more suitable for extraction. The resulting ethanol extract exhibits a high free radical scavenging rate, maintaining a scavenging rate of over 80% for both free radicals even at an 8-fold dilution, comparable to the effect of 1 mg / mL vitamin C. Furthermore, it maintains a significantly higher scavenging rate than the extracts from Examples 2 and 3 at progressively increasing dilution ratios. This indicates that, under the currently tested process parameters, the process parameters in Example 1 are the most suitable for extracting substances with high antioxidant activity.

[0185] Comparative Example 1

[0186] A method for preparing sake lees extract is provided, comprising the following steps:

[0187] Wet sake lees were freeze-dried at -40℃ for 24 hours until the moisture content was ≤10%, and then pulverized to 40 mesh to obtain dry sake lees powder. 550g of the above-mentioned dried undefatted sake lees powder was packed into a 3L extraction vessel (i.e., raw material bulk density 0.18kg / L), and extracted with 80% ethanol as the extraction solvent at an extraction pressure of 0.8MPa, an extraction temperature of 80℃, a desorption temperature of 60℃, and a flow rate of 20L / h for 90min. The extract was then concentrated to 5L by evaporation using a desorption vessel.

[0188] Comparative Example 2

[0189] A method for preparing sake lees extract is provided, comprising the following steps:

[0190] 1600g of undefatted wet sake lees was packed into a 3L extraction vessel. 80% ethanol was used as the extraction solvent. Extraction was carried out for 90min at an extraction pressure of 0.8MPa, an extraction temperature of 80℃, a desorption temperature of 60℃, and a flow rate of 20L / h. The extract was then concentrated to 5L by evaporation through the desorption vessel.

[0191] Experiment Example 2

[0192] The in vitro antioxidant activity of the sake lees extracts prepared in Example 1, Comparative Example 1, and Comparative Example 2 was investigated.

[0193] The test was conducted using the method described in Experiment Example 1 above.

[0194] The results are as follows Figure 3 As shown, the extracts obtained from the three process flows of Example 1, Comparative Example 1, and Comparative Example 2 exhibit a significant dose-response relationship in DPPH and ABTS free radical scavenging rates. The process flow of Example 1, which involves freeze-drying pretreatment, butane extraction of lipid-soluble components, and finally low-temperature continuous phase change extraction according to appropriate parameters, yielded an extract with higher DPPH and ABTS free radical scavenging rates. Even after 8-fold dilution, the free radical scavenging rate remained above 80%, indicating the best in vitro antioxidant activity.

[0195] Comparative Example 3

[0196] A method for preparing sake lees extract is provided, comprising the following steps:

[0197] The ultrasonic-assisted solvent extraction method for extracting distillers' lees includes the following steps:

[0198] Weigh 2g of sake lees powder into an Erlenmeyer flask, with a solid-liquid ratio of 1:10 (g:mL), add 20mL of 80% ethanol, and sonicate at 55℃ and 600W (20kHz) for 45min. After cooling, centrifuge at 4000r / min for 10min, and collect the supernatant to obtain the ethanol extract.

[0199] Experimental Example 3

[0200] The total amino acid, total protein, and total sugar content of the sake lees extracts prepared in Example 1 and Comparative Example 3, as well as their in vitro antioxidant activity, were investigated. The results are shown in Table 1.

[0201] Table 1

[0202]

[0203] Note: When comparing items in the same column, different letters indicate a significant difference (p<0.05), while the same letters indicate no significant difference (p>0.05).

[0204] As can be seen from the test results in Table 1 above, compared with the two extraction methods, the content of main components in the alcohol extract prepared in Example 1 is higher than that in Comparative Example 3, especially the total amino acid and total sugar content, which are significantly higher, indicating that the extraction effect of the method in Example 1 of this application is more complete. In addition, the alcohol extract of Example 1 exhibits a higher antioxidant effect, and its scavenging rate of DPPH and ABTS free radicals is significantly higher than that of the extract obtained by ultrasound-assisted solvent extraction in Comparative Example 1. This is because the method of this application is a mild and environmentally friendly extraction technology. Under the extraction pressure and temperature conditions used, the active ingredients are not destroyed during extraction and retention. Therefore, the extraction technology of this application has good feasibility and applicability in extracting anti-aging active ingredients.

[0205] Experiment Example 4

[0206] Characteristic component analysis was performed on the three different polar components obtained in Example 1: alkyl extract, alcohol extract, and water extract.

[0207] (1) Gas chromatography-mass spectrometry analysis of alkyl extract

[0208] S1. Fatty acid methyl esterification: Performed in accordance with GB 5009.168-2016 "Determination of fatty acids in food";

[0209] S2. Chromatographic conditions: A DB-5 capillary column (30m × 0.25mm × 0.25μm film thickness) was used with helium as the carrier gas in splitless mode. The injection port and transfer line temperatures were 250℃ and 280℃, respectively. The temperature program was as follows: initial column oven temperature 40℃, hold for 2 min, increase to 180℃ at a rate of 8℃ / min, hold for 2 min, increase to 250℃ at a rate of 3℃ / min, hold for 10 min; column flow rate 1 mL / min, injection volume 1 μL.

[0210] S3. Mass spectrometry conditions: Electron impact (EI) source, electron energy 70 eV, ion source temperature 230℃, quadrupole temperature 150℃, solvent delay 15.5 min, scan mass range 30~500 m / z.

[0211] Test results are shown Figure 6a And Table 2, Figure 6a This is the total ion chromatogram of the alkyl extract analyzed by gas chromatography-mass spectrometry.

[0212] Table 2 Fatty acid composition and relative content of alkyl extracts

[0213]

[0214] Eight peaks were identified in the alkyl extract, and the relative contents of each component were determined using the peak area normalization method. The alkyl extract was rich in unsaturated fatty acids, accounting for 65.20% of the total fatty acids, especially linoleic acid, which accounted for 53.83%. The content of saturated fatty acids was low, at only 34.80%, mainly palmitic acid (28.50%). Linoleic acid is often referred to as "beauty acid" in cosmetics and is frequently used as a nutritional additive. It is an excellent skin moisturizer because a lack of essential linoleic acid can lead to dryness, thickened stratum corneum, and slowed skin repair. Meanwhile, oleic acid has a similar structure to skin fatty acids, thus promoting penetration and increasing the protective function of the stratum corneum, thereby improving skin moisture content. Palmitoleic acid, as an omiga-7 fatty acid, can effectively improve skin pigmentation. It is lost from the skin with age, and timely and effective replenishment helps delay skin aging.

[0215] (2) Headspace solid-phase microextraction-gas chromatography-mass spectrometry analysis of alkyl extracts

[0216] S1. Add 0.1 g of the alkyl extract to a 20 mL headspace vial. Before analysis, equilibrate the vial at 60 °C for 20 min. Extract the volatile compounds using a DVB / CAR / PDMS three-phase solid-state extraction needle (50 / 30 μm). Insert the extraction needle into the headspace vial, extending the solid-phase microextraction fiber tip, and allow adsorption at 60 °C for 30 min. After adsorption, quickly insert the extraction needle into the injection port and desorb at 230 °C for 5 min. All operations were performed using a fully automated sample introduction device.

[0217] S2. Chromatographic conditions: TG-WAX MS polar column (60m×0.25mm); injection port temperature 250℃; carrier gas 99.99% pure helium, flow rate 1mL / min; initial temperature 40℃, hold for 3min, increase to 60℃ at 10℃ / min, hold for 5min, then increase to 190℃ at 3℃ / min, hold for 5min, and finally increase to 240℃ at 10℃ / min, hold for 10min.

[0218] S3. Mass spectrometry conditions: electron ionization source temperature is 230℃, transfer line temperature is 240℃, and mass scan range is 33~500m / z.

[0219] Test results are shown Figure 6b And Table 3; Figure 6b The total ion chromatogram of the alkyl extract is obtained by headspace solid-phase microextraction-gas chromatography-mass spectrometry.

[0220] Table 3. Composition and relative content of volatile compounds in alkyl extracts.

[0221]

[0222]

[0223] A total of 73 volatile compounds were identified in the alkyl extract, including various odor compounds. Phenylene alcohol had the highest content (47.29%), a substance with a soft, pleasant, and long-lasting rose aroma, widely used in food flavorings, cosmetics, and soaps. Ethyl decanoate (17.93%) was the second most abundant, possessing a coconut-like aroma and primarily used in the formulation of food flavorings.

[0224] Alkyl extracts contain other aroma components in relatively high amounts. Ethyl octanoate (6.69%) is a strong aroma agent found in mead and is also an important component in strong-aroma baijiu (Chinese liquor), with a pineapple-like aroma. 2,3-Butanediol (2.23%) has fruity and creamy aroma characteristics and can be used as a moisturizer and antibacterial agent in cosmetics. γ-Butyrolactone (2.15%) also has a fruity aroma and possesses antibacterial, antioxidant, and anti-inflammatory biological activities. In the food and fragrance industries, it is mainly used as a fragrance and flavor enhancer, and in the cosmetics industry, it is mainly used as a fragrance and preservative. Ethyl 3-methylbutyrate (2.01%) is mainly used in the formulation of apple, pineapple, and various fruit-type fragrances, and acts as a top note agent in daily chemical products. Ethyl laurate (7.65%) has a peanut aroma and is a good fixative. Ethyl myristate (5.55%) can be used in daily chemical fragrances, often used in the formulation of violet-type fragrances, and is also a good fixative.

[0225] Alkyl extracts also contain other active ingredients beneficial to the skin. D-glucuronide, a major cosmetic additive, nourishes the skin and delays aging. Maltol and eugenol are also potential raw materials in antioxidant cosmetics. α-Caryophyllene, α-terpineol, linalool, and 2-methoxy-4-vinylphenol are substances with beneficial anti-inflammatory and antibacterial effects.

[0226] (3) Liquid chromatography-mass spectrometry analysis of alcohol extract and water extract

[0227] S1. The extract was lyophilized and dissolved in methanol. Protein removal from the sample: An appropriate amount of sample was diluted with an equal volume of water, then mixed with an equal volume of 5% sulfosalicylic acid, allowed to stand for 1 hour, and centrifuged at 12000 r / min. The supernatant was filtered through a 0.22 μm aqueous filter and placed in a liquid chromatography vial.

[0228] S2. Chromatographic conditions: Agilent EC-C18 column (4.6 mm × 50 mm, 1.8 μm); column temperature 30 ℃; flow rate 0.2 mL / min; injection volume 10 μL; mobile phase A is water (0.1% formic acid); mobile phase B is acetonitrile (0.1% formic acid).

[0229] S3. Mass spectrometry conditions: Electrospray positive ionization source mode (ESI+) was used, and the scanning mode was multiple reaction monitoring (MRM); nebulizer gas pressure was 30 psi; drying gas temperature was 300 ℃; drying gas flow rate was 9 L / min; capillary voltage was 4000 V.

[0230] Test results are shown Figure 6c And Table 4. Figure 6c This is the total ion chromatogram of the alcohol extract analyzed by liquid chromatography-mass spectrometry.

[0231] Table 4. Material composition and relative content of alcohol extract and water extract.

[0232]

[0233]

[0234] LC-MS analysis of the alcohol and water extracts revealed the presence of various protein amino acids, such as L-tyrosine (5.69%), L-phenylalanine (4.89%), L-leucine (4.53%), L-arginine (3.28%), and L-aspartic acid (0.88%). Notably, it contained two types of sphingosine (phytosphingosine and D-erythro-dihydrosphingosine), with relative contents ranging from 5.06% to 5.84%, representing high-value antioxidants in sake lees extract. Additionally, a dipeptide (Lys-Gln) and a tripeptide (Leu-Arg-Lys) were also found, with relative contents ranging from 3.10% to 4.74%, both exhibiting good antioxidant activity. Furthermore, a coumarin compound (4-hydroxycoumarin) was also present, with a relative content ranging from 3.31% to 4.89%, which has been found to possess antioxidant and anti-aging activities in recent years.

[0235] Phytosphingosine is a precursor to phospholipids and also an important lipid component of the human epidermis. It plays a crucial role in maintaining moisture and barrier function. With age, the amount of phytosphingosine in the skin gradually decreases, leading to dry and rough skin. Topical application of phytosphingosine can effectively maintain skin suppleness and protect the dermis. Phytosphingosine also has antibacterial, anti-inflammatory, and anti-aging effects. Dihydrosphingosine has ceramide-like effects, inhibiting sebum secretion and providing skin care benefits. It also has anti-hair loss and hair care effects, improving scalp health and positively impacting hair growth.

[0236] (4) Gas chromatography-mass spectrometry analysis of alcohol extract

[0237] S1: Dissolve and filter the freeze-dried extract with methanol, and transfer it to a 50 mL volumetric flask and make up to volume; then accurately pipette 2 mL of the filtrate into a 10 mL volumetric flask and make up to volume with methanol to obtain the test solution.

[0238] S2. Chromatographic conditions: The column was a 5% benzyl silane quartz capillary column (30m×250μm×0.25μm); the injection port temperature was 280℃; the initial column temperature was 50℃, held for 2 min, then increased to 280℃ at a rate of 10℃ / min, and held for 15 min; the interface temperature was 280℃; the carrier gas was helium, with a flow rate of 1.5 mL / min; the injection volume was 1.0 μL (split ratio of 100:1).

[0239] S3. Mass spectrometry conditions: EI source, 230℃, tetrode temperature 150℃, electron energy 70eV, automatic tuning and correction mode, mass range 10~700m / z, scan rate 0.5s / s, solvent delay 3.1min.

[0240] Test results are shown Figure 6d Total ion chromatogram of alcohol extract analyzed by gas chromatography-mass spectrometry.

[0241] The composition and relative content of the sago lees extract were obtained by searching the standard mass spectrometry library and conducting manual analysis through literature review, and by calculating the peak area normalization method. The results are shown in Table 5.

[0242] Table 5. Material composition and relative content of alcohol extracts

[0243]

[0244] Lactic acid and glycolic acid are among the cosmetic active ingredients that deserve special attention. These organic acids belong to the AHA (alpha-hydroxy acid) family and are naturally found in milk, certain fruits and vegetables (such as tomatoes or guavas), and wine. Due to their small molecular weight, AHAs are rapidly absorbed by the skin and have moisturizing effects. High concentrations of AHAs can promote the shedding of keratinocytes, thus aiding in new skin regeneration. They can also stimulate the production of collagen and elastin, and effectively improve wrinkles caused by sun damage, rough skin, and pigmentation.

[0245] It should be noted that the above detection methods are for detecting volatile components, while the aroma (volatile components) of alcohol extracts is richer, and therefore, they have a better antioxidant effect than water extracts.

[0246] (5) Amino acid analysis of alcohol and water extracts

[0247] S1. Take an appropriate amount of sample and dilute it with an equal volume of water, then mix it with an equal volume of 5% sulfosalicylic acid. After standing for 1 hour, centrifuge at 12000 r / min. Filter the supernatant through a 0.22 μm aqueous filter and put it into a liquid phase injection bottle.

[0248] S2. Analysis was performed using a fully automated amino acid analyzer. The specific sample loading and detection conditions were as follows: the chromatographic column was a sulfonic acid cation exchange column (4.6 mm × 60 mm), the injection volume was 20 μL; the detector was a tungsten lamp detector; the mobile phase was lithium citrate PF buffer, the flow rate was 0.35 mL / min for the elution pump and 0.30 mL / min for the derivatization pump; the reaction column temperature was 135℃; the detection wavelength was 570 nm for channel 1 and 440 nm for channel 2.

[0249] The test results are shown in Table 6.

[0250] Table 6. Amino acid composition and content of alcoholic and aqueous extracts

[0251]

[0252]

[0253] As shown in the table above, the sake lees extract is rich in free amino acids. Both extracts contain 20 protein amino acids, including 8 essential amino acids. Five other non-protein amino acids and one dipeptide were also detected, including some rare amino acids such as γ-aminobutyric acid and anserine.

[0254] In the protein amino acids, the essential amino acids in the alcohol extract and water extract accounted for 35.79% and 32.10% of the total amino acids, respectively, which is close to the 40% EAA / TAA ratio proposed by FAO / WHO. Hydrophobic amino acids accounted for a high proportion (48.28–49.07%). These amino acids help promote the formation of highly hydrophobic peptides and can enhance the ability of substances to scavenge free radicals through various pathways, such as acting as hydrogen donors and fully utilizing their chelating properties with metal ions (such as Fe2+ and Cu2+). Acidic and basic amino acids also play important roles in antioxidation; their charges directly determine the extract's chelating ability with metal ions, accounting for 17.7–21.22% and 10.34–6.63%, respectively. These three types of amino acids, highly correlated with antioxidation, accounted for 76.13–77.11% of the total protein amino acids, which can explain the antioxidant activity of this extract to some extent.

[0255] Alanine has the highest content (around 1.44%). As a precursor to carnosine, it is converted into carnosine molecules upon ingestion, slowing down lipid oxidation and enhancing the body's antioxidant capacity. Following this are glutamic acid, leucine, and aspartic acid. Glutamic acid and aspartic acid are important excitatory neurotransmitters in the central nervous system and are also the main source of ATP in mammalian intestinal cells. They can be converted into other nutrients through decarboxylation or transamination, exerting health effects in the body. Studies have shown a positive correlation between the content of glutamic acid and aspartic acid and antioxidant capacity.

[0256] In addition, other trace amino acids also contribute to the antioxidant activity of the extract. Cystathione is a metabolic intermediate of sulfur-containing amino acids, participating in the interconversion of cysteine ​​and methionine. Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system, possessing good water solubility and thermal stability. It acts as a regulator of oxidative metabolites, reducing the accumulation of reactive oxygen species intermediates and protecting organisms from oxidative damage and peroxidative death. Ornithine acts as an antioxidant, is easily oxidized by free radicals, and participates in the urea cycle along with citrulline and arginine. Citrulline can act as a skin conditioning agent and moisturizer, scavenging hydroxyl radicals. Arginine also combats free radicals and improves skin hydration. Anserine, along with carnosine and serpentine, is collectively known as a histidine dipeptide, possessing water solubility and strong antioxidant and anti-aging functions, and is used as a natural antioxidant in the food industry.

[0257] Experimental Example 5

[0258] Anti-aging effects of sake lees extract

[0259] The anti-aging activity of the sake lees extract obtained in Example 1 was evaluated using the nematode elegans model, and its effect on prolonging the lifespan of nematodes under oxidative stress damage was tested.

[0260] The nematode elegans is easy to manipulate and observe, has a simple structure, a short life cycle, a clear genetic background, high homology with humans, and complete gene sequencing. It has played a significant role in many studies in the field of anti-aging, and its use as an animal model has been widely recognized.

[0261] Oxidative stress, referring to the excessive production of reactive oxygen species (ROS) in the body, is considered a key factor in nematode aging. The oxidative stress resistance of nematodes is strongly correlated with their lifespan, with lifespan increasing with improved oxidative stress resistance. Hydrogen peroxide (H₂O₂) effectively mimics the biological process of free radical-induced apoptosis and has become an important tool for preparing cellular oxidative damage models. Paraquat (PQ) can induce intracellular oxidative stress, leading to excessive ROS production and mitochondrial dysfunction, and is widely used in experiments investigating mitochondrial oxidative damage. Therefore, treating nematodes with different extracts and then exposing them to H₂O₂ or PQ-induced oxidative stress environments, while recording the nematode survival status, can reflect the anti-aging activity of the extracts.

[0262] (1) Experiment on H2O2-induced oxidative stress damage

[0263] After nematode synchronization, L4 stage larvae were selected and transferred to the control and sample groups. After 3 days of drug treatment, 30-50 nematodes from each culture medium were exposed to H2O2-NGM medium for oxidative stress induction intervention. Three parallel plates were set up for the experiment, and the number of nematode survivors, escapees, and deaths was recorded every 30 minutes until all nematodes died, obtaining the nematode survival curve under H2O2 oxidative stress.

[0264] The results are as follows Figure 4 As shown, compared with the blank group, the survival curves of each sample group showed a significant rightward shift (P < 0.0001), indicating that each extract significantly improved the resistance of nematodes to H2O2 oxidative stress damage and effectively prolonged their lifespan. The average lifespan of the blank group was 2.5 h, while the average lifespans of the alcohol extract group, water extract group, alkyl extract group, and positive control group (3 mM astaxanthin) group were 3.81, 3.23, 3.62, and 3.61 h, respectively, representing increases of 52.50%, 29.20%, 45.00%, and 44.38% compared to the blank group. This indicates that the various polarity extracts of sake lees can significantly enhance the survival ability of nematodes. Meanwhile, the anti-aging activity of the alcohol and alkyl extracts was not significantly different from that of 3 mM astaxanthin (p > 0.05), with the alcohol extract showing the best effect.

[0265] (2) PQ-induced oxidative stress injury experiment

[0266] After nematode synchronization, L4 stage larvae were selected and transferred to the control and sample groups. After 3 days of drug treatment, 50-60 nematodes from each culture medium were exposed to NGM medium containing PQ for oxidative stress induction intervention. Three parallel plates were set up for the experiment. The number of surviving, escaping, and dying nematodes was recorded approximately every 24 hours until all nematodes died, thus obtaining the survival rate of nematodes at different time points under PQ oxidative stress.

[0267] The results are as follows Figure 5 As shown in the results, similar to those of the H2O2 oxidative stress experiment, the survival curves of each sample group showed a significant rightward shift compared to the blank group (P < 0.001), indicating that extracts of different polarities of sake lees could prolong the survival time of nematodes under PQ oxidative damage conditions. The average lifespan of the blank group was 1.29 days, while the average lifespans of the alcohol extract group, water extract group, alkyl extract group, and positive control group (3 mM astaxanthin) group were 1.81, 1.57, 1.79, and 1.82 days, respectively. The average survival time of nematodes in the alcohol extract group, water extract group, and alkyl extract group was extended by 40.10%, 21.57%, 39.49%, and 41.09%, respectively. The effects of the alcohol extract and alkyl extract were more significant, comparable to those of 3 mM astaxanthin (p > 0.05), indicating that they can effectively enhance the resistance of nematodes to oxidative stress, thereby prolonging their lifespan.

[0268] Table 7 below shows a summary of basic information about the three extracts from Example 1.

[0269] Table 7

[0270]

[0271] The images of the alkyl extract, alcohol extract, and water extract in Table 7 above are provided in the instruction manual. Figures 7-9 .

[0272] In summary, the sake lees extract prepared by the low-temperature continuous phase change extraction method in this application has high content of major chemical components, good in vitro antioxidant activity, and is rich in anti-aging active ingredients, thus possessing anti-aging effects. Furthermore, a single set of equipment solves the comprehensive utilization of both water-soluble and fat-soluble components of sake lees, making it green, safe, and highly efficient, suitable for industrial production applications.

[0273] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing a distiller's grains extract having an anti-aging effect, characterized by, The method comprises the following steps: extracting the fat-soluble components from the wine lees powder to obtain a first extract and a first wine lees; extracting the alcohol-soluble components from the first wine lees to obtain a second extract and a second wine lees; extracting the water-soluble components from the second wine lees to obtain a third extract; the characteristic components of the first extract include linoleic acid, oleic acid, maltol, eugenol, alpha-caryophyllene and alpha-terpineol; the first extract has typical and rich wine lees aroma; the characteristic components of the second extract and the third extract include gamma-aminobutyric acid, opheline, phytosphingosine, dihydrosphingosine, lactic acid and glycolic acid; the second extract and the third extract have light and refreshing aroma; the step of extracting the fat-soluble components from the wine lees powder comprises: extracting the wine lees powder by using an alkane as a first extractant; the first extractant comprises n-butane; the step of extracting the alcohol-soluble components from the first wine lees comprises: extracting the first wine lees by using an alcohol as a second extractant; the second extractant comprises ethanol; the step of extracting the water-soluble components from the second wine lees comprises: extracting the second wine lees by using water as a third extractant; the sequence of the extraction of the fat-soluble components, the extraction of the alcohol-soluble components and the extraction of the water-soluble components is irreversible, and the first extract, the second extract and the third extract jointly constitute the wine lees extract; the step of extracting the fat-soluble components from the wine lees powder comprises: extracting at an extraction pressure of 0.2 MPa to 0.8 MPa and an extraction temperature of 30 ℃ to 50 ℃; the step of extracting the alcohol-soluble components from the first wine lees comprises: extracting at an extraction pressure of 0.2 MPa to 0.8 MPa and an extraction temperature of 60 ℃ to 80 ℃; the step of extracting the water-soluble components from the second wine lees comprises: extracting at an extraction pressure of 0.2 MPa to 0.8 MPa and an extraction temperature of 60 ℃ to 80 ℃.

2. The method according to claim 1, wherein the extraction time of the step of extracting the fat-soluble components from the wine lees powder is 60 min to 120 min.

3. The method according to claim 1, wherein the step of extracting the fat-soluble components from the wine lees powder is performed by using a continuous phase change extraction device; the desorption temperature is 40 ℃ to 60 ℃; and the flow rate is 20 L / h to 60 L / h.

4. The method according to claim 1, wherein the extraction time of the step of extracting the alcohol-soluble components from the first wine lees is 60 min to 120 min.

5. The method according to claim 1, wherein the step of extracting the alcohol-soluble components from the first wine lees is performed by using a continuous phase change extraction device; the desorption temperature is 40 ℃ to 60 ℃; and the flow rate is 20 L / h to 60 L / h. ​ ​ ​ ​ 6.The method of claim 1, wherein the second distiller's grains are extracted for water-soluble components for 60 min to 120 min. 7.The method of claim 1, wherein the second distiller's grains are extracted for water-soluble components using a continuous phase change extraction device, at a temperature of 40 ℃ to 60 ℃ and a flow rate of 20 L / h to 60 L / h. 8.The method of any one of claims 1 to 7, wherein the moisture content of the distiller's grains powder is less than or equal to 10% by mass. 9.The method of claim 8, wherein the freeze-drying is performed at a temperature of -45 ℃ to -35 ℃ for 20 h to 30 h. 10.The method of claim 8, wherein the powdering and sieving is performed by passing the freeze-dried distiller's grains through a 40 mesh to 100 mesh sieve to obtain the sieved material. 11.An extract of distiller's grains having anti-aging effects, prepared by the method of any one of claims 1 to 10. 12.The extract of claim 11, wherein the extract is composed of the first extract, the second extract, and the third extract. 13.The extract of claim 12, wherein the first extract comprises linoleic acid, oleic acid, maltol, eugenol, α-caryophyllene, and α-terpineol. 14.The extract of claim 12 or 13, wherein the second extract and the third extract comprise γ-aminobutyric acid, carnosine, phytosphingosine, dihydrosphingosine, lactic acid, and glycolic acid. 15.A food or beverage composition comprising the extract of claim 11. ​ ​ 11. A vinasse extract, characterized in that, ​ ​ ​ ​ 12. A cosmetic product, characterized by, ​

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