Preparation method of blackcurrant pomace anthocyanin with anti-obesity and anti-aging activity

Abstract

The invention provides a preparation method of blackcurrant pomace anthocyanin with anti-obesity and anti-aging activity. According to the method, the blackcurrant pomace is taken as a raw material, the blackcurrant pomace anthocyanin is efficiently extracted by adopting a low-temperature continuous phase change extraction (CPE) technology, and the blackcurrant pomace anthocyanin is separated and purified by utilizing polyamide resin, so that the extraction rate and the purity of the blackcurrant pomace anthocyanin are remarkably improved, and the blackcurrant pomace waste is fully utilized. Besides, caenorhabditis elegans is used as a model, in-vivo anti-obesity and anti-aging activity evaluation is carried out on the purified blackcurrant pomace anthocyanin, and it is found that the blackcurrant pomace anthocyanin has excellent anti-obesity and anti-aging effects.

Description

Technical Field

[0001] This invention belongs to the field of anthocyanin extraction technology. More specifically, it relates to a method for preparing anthocyanins from blackcurrant pomace that have anti-obesity and anti-aging activities. Background Technology

[0002] Blackcurrant, also known as blackcurrant or black bean fruit, is abundant in Northeast my country. Its fruit has a higher anthocyanin content than blueberries and exhibits greater bioactivity. The main processed products of blackcurrants are juice and various fermented products, which generate a large amount of pomace. This pomace contains 75% anthocyanins. Therefore, the efficient separation and purification of anthocyanins from blackcurrant pomace to obtain high-purity anthocyanins is an important research direction. However, there are currently no reports on the extraction of anthocyanins from blackcurrant pomace.

[0003] Currently, common methods for extracting anthocyanins include supercritical carbon dioxide extraction and alkaline water extraction. However, supercritical carbon dioxide extraction requires high temperature and pressure, which places high demands on equipment, and the high temperature may cause anthocyanin degradation. Alkaline water extraction has high leaching capacity, but it contains more impurities, which is not conducive to purification, and it also produces a large amount of acid and alkali solutions, causing environmental pollution.

[0004] Therefore, developing a method that can significantly improve the extraction rate and purity of anthocyanins from blackcurrant pomace is crucial for fully utilizing blackcurrant pomace. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a method for preparing anthocyanins from blackcurrant pomace. By combining low-temperature continuous phase change extraction with polyamide resin adsorption, the extraction rate and purity of anthocyanins from blackcurrant pomace are significantly improved, thus achieving full utilization of blackcurrant pomace waste.

[0006] The primary objective of this invention is to provide a method for preparing anthocyanins from blackcurrant pomace.

[0007] A second objective of this invention is to provide anthocyanins from blackcurrant pomace prepared by the above method.

[0008] A third objective of this invention is to provide the application of the above-mentioned blackcurrant pomace anthocyanins in the preparation of anti-obesity products.

[0009] The fourth objective of this invention is to provide the application of the above-mentioned blackcurrant pomace anthocyanins in the preparation of anti-aging products.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a method for preparing anthocyanins from blackcurrant pomace, comprising the following steps: S1. Low-temperature continuous phase change extraction was performed on blackcurrant pomace to obtain a crude extract of anthocyanins from blackcurrant pomace; wherein the extraction temperature was 50–70 °C, the extraction time was 100–140 min, and the extraction solvent was 60% ( v / v ) ~ 80% v / v The bulk density of the blackcurrant pomace in the ethanol solution is 0.3–0.5 kg / L. S2. The crude extract of anthocyanins from blackcurrant pomace was loaded onto a polyamide resin column to adsorb the anthocyanins from the blackcurrant pomace; wherein the concentration of the crude extract of anthocyanins from blackcurrant pomace was 0.12–1.32 mg / mL, the mesh size of the polyamide resin was 30–100 mesh, the loading flow rate was 1–3 mL / min, and the loading volume was 1–2 BV; S3. After removing impurities from the polyamide resin column adsorbed with anthocyanins from blackcurrant pomace, use 30% ( v / v ) ~ 70% v / v Eluting was performed using an ethanol solution.

[0011] The term "bulk density" refers to the mass of a unit volume of product. In this invention, the bulk density of the blackcurrant pomace is adjusted by applying external force to press the blackcurrant pomace. The term "BV" is based on the volume of polyamide resin in the chromatography column (represented by 1 BV), and the corresponding volume is measured in multiples of the volume (such as the sample volume).

[0012] Preferably, the pressure of the low-temperature continuous phase change extraction in S1 is 0.18 to 0.22 MPa, and most preferably 0.2 MPa.

[0013] Preferably, the pH of the extraction solvent in S1 is 1.8 to 2.2, and most preferably 2. It can be adjusted, for example, by hydrochloric acid.

[0014] Preferably, the extraction temperature in S1 is 60 °C.

[0015] Preferably, the extraction time in S1 is 100 min.

[0016] Preferably, the extraction solvent in S1 is 70% ( v / v Ethanol solution.

[0017] Preferably, the bulk density of the blackcurrant pomace in S1 is 0.3 kg / L.

[0018] Preferably, the adsorption time in S2 is 30 to 180 min, more preferably 30 to 60 min, and most preferably 60 min.

[0019] Preferably, the concentration of the crude anthocyanin extract from blackcurrant pomace in S2 is 0.95 mg / mL.

[0020] Preferably, the concentration of the crude anthocyanin extract from blackcurrant pomace in step S2 is adjusted by using an ethanol solution, for example, 95% ( v / v Ethanol solution.

[0021] Preferably, the polyamide resin in S2 has a mesh size of 30 to 60 mesh.

[0022] Preferably, the sample loading rate in S2 is 1 mL / min.

[0023] Preferably, the sample loading volume in S2 is 2 BV.

[0024] Preferably, the impurity removal in S3 is performed by elution with water.

[0025] Preferably, the endpoint of the impurity removal process in S3 is that the effluent is transparent and colorless.

[0026] Preferably, the concentration of the ethanol solution in S3 is 50% ( v / v ) ~ 70% v / v ).

[0027] Preferably, the elution in S3 is gradient elution.

[0028] More preferably, the gradient elution is: first using 45% ( v / v ) ~ 55% v / v Elute with ethanol solution, then use 65% ( v / v ) ~75% v / v Eluting was performed using an ethanol solution.

[0029] More preferably, the gradient elution is: first using 45% ( v / v ) ~ 55% v / v Elute with ethanol solution for 2.5–3.5 BV, then use 65% ( v / v ) ~75% v / v Elute with ethanol solution at 2.5–3.5 BV.

[0030] Most preferably, the gradient elution is performed by first using 50% ( v / v Elute 3 BV with ethanol solution, then use 70% ( v / v Elute with ethanol solution for 3 BV.

[0031] Preferably, after elution, the eluted product is further dried, for example, by freeze-drying.

[0032] The blackcurrant pomace anthocyanins prepared by the above method have high purity (63.43%) and excellent anti-obesity and anti-aging effects. Therefore, the blackcurrant pomace anthocyanins prepared by the above method, and their application in the preparation of anti-obesity and anti-aging products, should be within the scope of protection of this invention.

[0033] The present invention has the following beneficial effects: This invention uses blackcurrant pomace as raw material and employs low-temperature continuous phase change extraction (CPE) technology to efficiently extract anthocyanins from the pomace. Polyamide resin is then used for separation and purification, significantly improving the extraction rate and purity of the anthocyanins and achieving full utilization of blackcurrant pomace waste. Furthermore, using *Caenorhabditis elegans* as a model, this invention evaluates the in vivo anti-obesity and anti-aging activities of the purified blackcurrant pomace anthocyanins, revealing excellent anti-obesity and anti-aging effects. Attached Figure Description

[0034] Figure 1 The content changes of anthocyanins in blackcurrant pomace were observed during continuous phase change extraction at different extraction temperatures.

[0035] Figure 2 The content of anthocyanins in blackcurrant pomace undergoes continuous phase change extraction at different extraction times.

[0036] Figure 3 The content changes of anthocyanins in blackcurrant pomace were obtained by continuous phase change extraction at different ethanol concentrations.

[0037] Figure 4 The content changes of anthocyanins extracted from blackcurrant pomace under different bulk densities through continuous phase change extraction.

[0038] Figure 5 The effect of different adsorption times on the adsorption effect.

[0039] Figure 6 The effect of different sample loading rates on the adsorption effect.

[0040] Figure 7 The effect of different sample volumes on the adsorption effect.

[0041] Figure 8 The effect of different eluent concentrations on elution efficiency (extraction rate).

[0042] Figure 9 This indicates the survival status of nematodes during the toxicity test.

[0043] Figure 10 The results are from Oil Red O staining.

[0044] Figure 11 This is the result of TG content determination.

[0045] Figure 12 The results are from the detection of reactive oxygen species using fluorescent probes.

[0046] Figure 13This is the result of the detection of superoxide dismutase (SOD) content.

[0047] Figure 14 This is the result of the test for malondialdehyde (MDA) content.

[0048] Figure 15 These are the results of the life test. Detailed Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0050] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0051] I. Materials and Methods (1) Experimental materials Blackcurrant pomace: provided by Xinjiang Gobi Fruit Fragrance Agricultural Development Co., Ltd., and ground to 40-60 mesh using a pulverizer, and stored at -20 ℃. Polyamide resin: provided by Shanghai Yuanye Biotechnology Co., Ltd. Caenorhabditis elegans: N2 wild type, hermaphroditic, provided by the Center for Genetics (CGC, UniTroloxrsity of Minnesota); Escherichia coli OP50: provided by the Nematode Laboratory of the College of Food Science, South China Agricultural University.

[0052] (2) Experimental reagents Oil Red O dye: Nanjing Jiancheng Biotechnology Research Institute; Triglycerides (TG) reagent kit: Nanjing Jiancheng Bioengineering Institute, A110-1-1; H2DCFDA Reactive Oxygen Specie (ROS) Probe: Ombio Biotechnology (Shanghai) Co., Ltd.; Superoxide dismutase (SOD) kit: Nanjing Jiancheng Bioengineering Institute, A001-3-2; Malondialdehyde (MDA) reagent kit: Nanjing Jiancheng Bioengineering Institute, A003-4-1.

[0053] (3) Major instruments and equipment 0.01% electronic balance: AL104, Mettler Toledo Instruments Ltd.; 1 / 1000 electronic balance: PL203, Mettler Toledo Instruments Ltd.; Electric heating drying oven: DHG-970, Shanghai Qixin Scientific Instruments Co., Ltd.; Digital display electric thermostatic water bath: DF-101S, Gongyi Yuhua Instrument Co., Ltd.; Continuous phase change extraction apparatus: 3L, South China Agricultural University; Ultraviolet spectrophotometer: UV-3010, Hitachi High Technology Corporation, Japan; Multifunctional microplate reader: Enspire 2003, pE Corporation, USA; Rotary evaporator: R204B3, Shanghai Shensheng Technology Co., Ltd.; Water bath oscillator: HZQ-C, Changzhou Aohua Instrument Factory; High-speed refrigerated centrifuge: 5417R, Eppendorf GmbH, Germany; Vacuum freeze dryer: FD-1PF, Beijing Detianyou Instrument Co., Ltd.; pH meter: DELTA320, Mettler Toledo Instruments (Shanghai) Co., Ltd.; Inverted biological microscope: XSP-15C, Shanghai Changfang Optical Instrument Co., Ltd.

[0054] II. Determination of Anthocyanin Content The anthocyanin content in the sample was determined by pH differential method according to the literature “Nie Jingwen, Yu Jing, Zheng Xin, et al. Determination of anthocyanin content in plant-derived agricultural products [J]. Agricultural Product Quality and Safety, 2024, (2): 31-35.”

[0055] III. Data Statistics and Analysis All data were measured at least three times. SPSS 23.0 statistical software was used to perform analysis of variance on the data, and the results are expressed as mean ± standard deviation. Origin 8.0 was used for plotting.

[0056] Example 1: Low-temperature continuous phase change extraction (CPE) process for anthocyanins from blackcurrant pomace I. Single-factor experimental method 500 g of blackcurrant pomace with a bulk density of 0.3–0.7 kg / L was placed in an extraction vessel. The extraction pressure was set to 0.2 MPa, the extraction temperature to 30–70 ℃, and the extraction time to 60–140 min. The extraction solution was prepared at 50% ( v / v ) ~ 90% v / vEthanol was used as the extractant (pH was adjusted to 2.0 with hydrochloric acid). The extracted blackcurrant pomace anthocyanin extract was then placed in an analytical vessel. The analytical temperature was the same as the extraction temperature. The extractant was heated and depressurized to change phase to gas. The pulp and residue were separated without filtration to obtain a crude extract of blackcurrant pomace anthocyanins.

[0057] (1) Extraction temperature Add 500 g of blackcurrant pomace to the extraction vessel. The designed bulk density of the blackcurrant pomace is 0.3 kg / L, and the extraction time is 90 min. After closing the lid, introduce a solution with a pH of 2.0 and a concentration already adjusted to 60%. v / v An ethanol solution of blackcurrant pomace was used to extract anthocyanins at temperatures of 30, 40, 50, 60, and 70 °C to study the changes in anthocyanin content during continuous phase change extraction from blackcurrant pomace at different temperatures.

[0058] (2) Extraction time Add 500 g of blackcurrant pomace to the extraction vessel. The designed bulk density of the blackcurrant pomace is 0.3 kg / L, the extraction temperature is 50 ℃, and after closing the lid, introduce a solution with a pH of 2.0 and a concentration already adjusted to 60%. v / v An ethanol solution of blackcurrant pomace was extracted for 60, 80, 100, 120, and 140 min, respectively, to study the changes in anthocyanin content in blackcurrant pomace under different extraction times.

[0059] (3) Ethanol concentration 500 g of blackcurrant pomace was added to the extraction vessel. The designed bulk density of the blackcurrant pomace was 0.3 kg / L, the extraction temperature was 50 ℃, and the extraction time was 90 min. After closing the lid, a pH of 2.0 and a concentration of 50% were introduced. v / v ), 60% v / v ), 70% v / v ), 80% v / v ), 90% v / v The study investigated the changes in anthocyanin content in blackcurrant pomace extracted by continuous phase change extraction under different ethanol concentrations using an ethanol solution.

[0060] (4) Bulk density of blackcurrant pomace 500 g of blackcurrant pomace was added to the extraction vessel. The bulk density of the blackcurrant pomace was designed to be 0.3, 0.4, 0.5, 0.6, and 0.7 kg / L, respectively. The extraction temperature was 50 ℃, and the extraction time was 90 min. After closing the lid, pH 2.0 solution with a concentration of 60% was introduced. v / v An ethanol solution was used to study the changes in anthocyanin content in blackcurrant pomace extracted by continuous phase change under different bulk densities.

[0061] II. Results of Single-Factor Experiments The results are as follows Figures 1-4 As shown, where, Figure 1 The changes in anthocyanin content in blackcurrant pomace extracted under different extraction temperatures via continuous phase change extraction are shown. Figure 2 The changes in anthocyanin content in blackcurrant pomace extracted by continuous phase change at different extraction times are shown. Figure 3 The changes in anthocyanin content from blackcurrant pomace extracted by continuous phase change at different ethanol concentrations are shown. Figure 4 The content changes of anthocyanins extracted from blackcurrant pomace under different bulk densities through continuous phase change extraction.

[0062] It is evident that, among the single-factor variables, all four variables showed a trend of first increasing and then decreasing in the extraction yield of anthocyanins from blackcurrant pomace. Furthermore, the extraction yield was highest when the extraction temperature was 60 ℃, the extraction time was 120 min, and the ethanol concentration was 70%. v / v When the bulk density was 0.4 kg / L, the extraction yield of anthocyanins from blackcurrant pomace reached its highest level, at 5.61, 5.39, 5.35, and 5.39 mg / g pomace, respectively.

[0063] III. Orthogonal Experiment Method Based on the results of the aforementioned single-factor experiments, a four-factor, three-level orthogonal experiment was designed using the three optimal levels of bulk density, extraction temperature, extraction time, and ethanol concentration, as shown in Table 1.

[0064] Table 1

[0065] IV. Results of Orthogonal Experiments The results are shown in Tables 2 and 3.

[0066] Table 2

[0067] Table 3

[0068] According to Table 2, the process with the highest anthocyanin extraction yield from blackcurrant pomace was: extraction at 0.2 MPa pressure and pH 2.0, at an extraction temperature of 60 ℃, an extraction time of 100 min, and an ethanol concentration of 70%. v / v ), bulk density 0.4 kg / L.

[0069] However, as shown in Table 3, bulk density has no significant effect on anthocyanin extraction. Therefore, a bulk density of 0.3 kg / L should be selected according to production needs to save raw materials as much as possible.

[0070] Based on the results in Tables 2 and 3, the optimal continuous phase change extraction process for anthocyanins from blackcurrant pomace can be determined as follows: under conditions of 0.2 MPa pressure and pH 2.0, the extraction temperature is 60 ℃, the extraction time is 100 min, and the ethanol concentration is 70%. v / v ), bulk density 0.3 kg / L.

[0071] V. Verification Experiment Verification experiments were conducted under the optimal process determined by orthogonal experiments, and the extraction yield of anthocyanins from blackcurrant pomace was 6.63 mg / g pomace.

[0072] Example 2: Polyamide resin separation and purification process for anthocyanins from blackcurrant pomace I. Preparation of Crude Anthocyanin Extract from Blackcurrant Pomace Weigh 500 g of blackcurrant pomace and extract anthocyanins from the blackcurrant pomace according to the optimal process described in Example 1. The crude extract of blackcurrant pomace anthocyanins was stored at -20 ℃ in the dark.

[0073] II. Pretreatment, Regeneration, and Determination of Indicators of Polyamide Resin The pretreatment and regeneration methods of polyamide resin, as well as the calculation formulas for adsorption capacity, adsorption rate and desorption rate, were all based on the literature "Luo Xuguang. Identification of Flavonoid Components, Study on Anti-inflammatory Activity and Development of Jam in Guangfo Shou [D]. South China Agricultural University, 2020."

[0074] III. Effect of different ethanol concentrations on adsorption efficiency Add 2.0 g of pretreated and dried 30-60 mesh polyamide resin to a 200 mL Erlenmeyer flask, then add 30 mL of crude blackcurrant pomace anthocyanin extract (prepared with 95% ( v / v The blackcurrant pomace was diluted with ethanol solution to a final concentration of anthocyanins of 0.12, 0.37, 0.66, 0.95, and 1.32 mg / mL, respectively. After shaking in a constant temperature shaker at 25 ℃ and 200 r / min for 24 h, the filtrate and residue were collected by filtration.

[0075] The content of anthocyanins in blackcurrant pomace in the filtrate was determined, and then the adsorption capacity and adsorption rate of polyamide resin for blackcurrant pomace anthocyanins were calculated.

[0076] After washing away any unadsorbed material from the filter residue (polyamide resin) with distilled water, transfer the residue to a 200 mL Erlenmeyer flask and add 30 mL of 95% ( v / v The ethanol solution was used for desorption, and the mixture was shaken in a constant temperature shaker at 25 ℃ and 200 r / min for 24 h. The filtrate was collected by filtration, and the content of anthocyanins in blackcurrant pomace in the filtrate was determined. The desorption rate of anthocyanins in blackcurrant pomace by polyamide resin was then calculated.

[0077] The results are shown in Table 4.

[0078] Table 4

[0079] It is evident that when the concentration of the crude anthocyanin extract from blackcurrant pomace is in the range of 0.12–0.95 mg / mL, the adsorption capacity of the polyamide resin increases significantly with increasing concentration (P<0.05), rising from 1.50 mg / g to 18.30 mg / g, an increase of 10.2 times. Subsequently, as the concentration of the crude anthocyanin extract from blackcurrant pomace increases to 1.05 mg / mL, the change in adsorption capacity becomes less significant (P>0.05). Conversely, the desorption rate decreases significantly with increasing anthocyanin concentration (P<0.05), decreasing from an initial 54.65% to 47.26%, a decrease of 7.36%. Therefore, considering all factors, a crude anthocyanin extract concentration of 0.95 mg / mL from blackcurrant pomace is most suitable for the process of this invention.

[0080] IV. Effect of different polyamide resin mesh sizes on adsorption efficiency 2.0 g of pretreated and dried 30-60 mesh and 60-100 mesh polyamide resin were added to 200 mL Erlenmeyer flasks respectively, followed by 30 mL of crude blackcurrant pomace anthocyanin extract (prepared with 95% ( v / v The blackcurrant pomace anthocyanin was diluted with ethanol solution to a final concentration of 0.95 mg / mL. After shaking in a constant temperature shaker at 25 ℃ and 200 r / min for 24 h, the filtrate and residue were collected by filtration. The adsorption capacity, adsorption rate, and desorption rate of the polyamide resin for blackcurrant pomace anthocyanins were determined according to the method in "Effect of Different Ethanol Concentrations on Adsorption Effect".

[0081] The results are shown in Table 5.

[0082] Table 5

[0083] It is evident that the adsorption capacity, adsorption rate, and desorption rate of the 30-60 mesh polyamide resin are all higher than those of the 60-100 mesh polyamide resin, indicating that the 30-60 mesh polyamide resin is most suitable for the process of this invention.

[0084] V. Effect of different adsorption times on adsorption efficiency Add 2.0 g of pretreated and dried 30-60 mesh polyamide resin to a 200 mL Erlenmeyer flask, then add 30 mL of crude blackcurrant pomace anthocyanin extract (prepared with 95% ( v / vThe blackcurrant pomace anthocyanin was diluted with ethanol solution to a final concentration of 0.95 mg / mL and shaken in a constant-temperature shaker at 25 ℃ and 200 r / min for 180 min. Samples were taken at 10, 20, 30, 40, 50, 60, 70, 80, 90, 120, 150, and 180 min, and the filtrate was collected. The adsorption capacity of the polyamide resin for blackcurrant pomace anthocyanins was determined according to the method in "Effect of Different Ethanol Concentrations on Adsorption Effect".

[0085] The results are as follows Figure 5 As shown, the adsorption capacity first increases and then plateaus with the extension of time. It reaches an adsorption capacity of 27.14 mg / g at 30 min and is basically saturated (reaching 30.61 mg / g) at 60 min, indicating that the adsorption time of 30 to 60 min is most suitable for the process of this invention.

[0086] VI. Effect of different sample loading rates on adsorption efficiency 7.0 g of pretreated 30-60 mesh polyamide resin was mixed with 95% ( v / v After wetting with ethanol solution, the crude extract of anthocyanins from blackcurrant pomace (prepared with 95% ethanol solution) was packed into a chromatography column (255 mm × 420 mm, 35 mL). The extraction was carried out at flow rates of 1, 2, and 3 mL / min. v / v The blackcurrant pomace anthocyanin concentration C0 was diluted with ethanol solution to a final concentration C0 of 0.95 mg / mL before loading. The volume of resin in the column was defined as 1 BV, and the volume of the eluent was measured in multiples of this volume. Eluent was collected every 1 BV, and the anthocyanin content C in the eluent was determined. Finally, the anthocyanin loss rate was calculated based on the ratio of the anthocyanin content C to the initial concentration C0 (C / C0 × 100%).

[0087] The results are as follows Figure 6 As shown, the anthocyanin loss rate increases with increasing sample loading rate, indicating that a sample loading rate of 1 mL / min is most suitable for the process of this invention.

[0088] VII. Effect of different sample loading volumes on adsorption efficiency 7.0 g of pretreated 30-60 mesh polyamide resin was mixed with 95% ( v / v After wetting with ethanol solution, the sample was packed into a chromatography column (255 mm × 420 mm, 35 mL). The crude extract of anthocyanins from blackcurrant pomace (prepared with 95% ethanol solution) was processed at a flow rate of 1 mL / min. v / vThe blackcurrant pomace anthocyanin concentration C0 was diluted with ethanol solution to a final concentration C0 of 0.95 mg / mL before loading. The volume of resin in the column was defined as 1 BV, and the volume of the eluent (i.e., the loading volume) was measured using multiples of this volume. Eluent was collected every 1 BV, and the anthocyanin content C in the eluent was determined. Finally, the anthocyanin loss rate was calculated based on the ratio of the anthocyanin content C to the initial concentration C0 (C / C0 × 100%).

[0089] The results are as follows Figure 7 As shown, the loss rate increases slowly (≤5%) with increasing sample volume from 1 to 2 BV, increases significantly with increasing sample volume from 2 to 10.5 BV (reaching 66.36% at 10.5 BV), and does not change significantly with increasing sample volume from >10.5 BV (i.e., polyamide resin adsorption is approaching saturation). This indicates that a sample volume of 1 to 2 BV is more suitable for the process of this invention, with 2 BV being optimal.

[0090] 8. Effect of different eluent concentrations on elution efficiency (extraction rate) 7.0 g of pretreated 30-60 mesh polyamide resin was mixed with 95% ( v / v After wetting with ethanol solution, pack the sample into a chromatography column (255 mm × 420 mm, 35 mL), and proceed with the extraction of 2 BV blackcurrant pomace anthocyanin crude extract (prepared with 95% ethanol solution) at a flow rate of 1 mL / min. v / v The sample was added after diluting it with ethanol solution to a final concentration of 0.95 mg / mL of anthocyanins from blackcurrant pomace.

[0091] When the anthocyanin adsorption reaches saturation, rinse the resin with distilled water to remove impurities (wash away water-soluble impurities that could not be adsorbed) until the outflowing liquid becomes transparent and colorless, then add 30% of 3 BV. v / v ) Ethanol solution → 50% of 3 BV ( v / v ) Ethanol solution → 70% of 3 BV ( v / v Gradient elution was performed using ethanol solution at a flow rate of 1 mL / min. The volume of resin in the column was defined as 1 BV, and the volume of the eluent was measured in multiples of this volume. The eluent was collected every 1 BV, and the anthocyanin content in the eluent was determined.

[0092] The results are as follows Figure 8 As shown in the image, the anthocyanins in blackcurrant pomace are mostly within the 50% range. v / v ) ethanol solution and 70% ( v / v When ethanol solution was used, the elution was relatively concentrated, indicating that after the anthocyanins in the blackcurrant pomace were completely extracted, 50% ( v / v ), 70% v / v Eluting with an ethanol solution can maximize elution.

[0093] IX. Effects of different elution gradients on elution efficiency (purity) 20.0 g of pretreated 30-60 mesh polyamide resin was mixed with 95% ( v / v After wetting with ethanol solution, pack the sample into a chromatography column (255 mm × 420 mm, 110 mL), and proceed with the extraction of 2 BV blackcurrant pomace anthocyanin crude extract (prepared with 95% ethanol solution) at a flow rate of 1 mL / min. v / v The sample was added after diluting it with ethanol solution to a final concentration of 0.95 mg / mL of anthocyanins from blackcurrant pomace.

[0094] When anthocyanin adsorption reaches saturation, the resin is rinsed with distilled water to remove impurities (washing away unadsorbed water-soluble impurities) until the effluent becomes transparent and colorless. The resin is then divided into two groups. Group A is prepared at 30% of 3 BV. v / v ) Ethanol solution → 50% of 3BV ( v / v ) Ethanol solution → 70% of 3 BV ( v / v Group B was eluted with a gradient of ethanol solution; Group B was eluted with 50% of 3 BV ( v / v ) Ethanol solution → 70% of 3 BV ( v / v Gradient elution was performed using ethanol solution, with an elution flow rate of 1 mL / min for both groups.

[0095] The eluent from each group was collected and summarized, and the volume V and anthocyanin content C were determined. The eluent was then dried and weighed M. The purity of anthocyanins was calculated according to "purity (%) = CV / M × 100%". Crude anthocyanin extract from uneluted blackcurrant pomace was used as a blank control. The results are shown in Table 6.

[0096] Table 6

[0097] As can be seen, compared with the blank control, the purity of both group A and group B was significantly increased, and group B had the highest purity and was also more solvent-efficient.

[0098] Example 3: Anti-obesity and anti-aging activities of anthocyanins from blackcurrant pomace I. Cultivating Escherichia coli Escherichia coli OP50 was evenly spread on LB (Lysogeny Broth) solid medium and incubated at 37 °C until single colonies were observed. Single colonies were then picked and transferred to culture flasks containing LB broth liquid medium and cultured with shaking at 37 °C and 200 rpm until OD (October Expiratory Scale) was reached. 600Once the value reaches 0.4, it is then inoculated onto NGM (Nematode Growth Medium) plates for later use.

[0099] II. Preparation of Reagents (1) 1 mol / L potassium phosphate buffer: Add 10.839 g of KH2PO4 and 3.569 g of K2HPO4 to distilled water and mix well to make the total volume of the system reach 100 mL. Then adjust the pH to 6.0 with 1% KOH solution and H3PO4 solution.

[0100] (2) M9 buffer: Add 0.6 g of Na2HPO4, 0.3 g of KH2PO4, 0.5 g of NaCl and 0.025 g of MgSO4·7H2O to distilled water and mix well so that the total volume of the system reaches 100 mL. Then sterilize it in a steam sterilizer at 121 °C for 15 min.

[0101] (3) Liquefaction solution: Dissolve 0.2 g of NaOH in 8 mL of distilled water, then add 2 mL of 5% NaOCl solution and mix well.

[0102] III. Subculturing, Culture, and Synchronization of Caenorhabditis elegans The oviposition period of *Caenorhabditis elegans* was combined with 10 9 CFU / mL *E. coli* OP50 culture was inoculated onto NGM (Nematode Growth Medium) plates and incubated at 20 °C for 3 days. Adult nematodes and eggs on the NGM plates suitable for lysis were repeatedly washed three times with 1 mL M9 buffer. The washed M9 buffer was transferred to a 2 mL sterile centrifuge tube, 1 mL lysis buffer was added, and the plates were shaken for 5 min to break up the nematodes. The plates were then centrifuged at 3000 rpm for 1 min, the supernatant was discarded, and 1 mL M9 buffer was added to wash away the precipitated eggs. This process of centrifugation and M9 buffer washing was repeated twice. 0.3 mL of M9 buffer containing the precipitate was collected, mixed well, and pipette-added to a sterile area of ​​the NGM plate. After incubation at 20 °C for 48 h, the nematode fertilized eggs had mostly hatched and developed into L4-stage nematodes, thus completing the nematode synchronization process.

[0103] IV. Toxicity Testing The anthocyanins from blackcurrant pomace obtained in Group B of Example 2 were added to 95% ( v / vThe nematodes were inoculated with ethanol solutions at final concentrations of 0.125, 0.25, 0.5, and 1 mg / mL, and then streaked onto NGM plates. After inoculation with L4 stage nematodes, the plates were incubated at 20 °C for 48 h. The number of surviving and dead nematodes was recorded. The survival rate was calculated using the formula: "Survival rate (%) = (Number of surviving nematodes / Total inoculation amount) × 100%". The results are as follows: Figure 9 As shown in the image, blackcurrant pomace anthocyanins exhibit high safety against nematodes.

[0104] V. Experimental Grouping and Processing Blank control group: 400 μL of 10 9 CFU / mL Escherichia coli OP50 culture was added to 20 mLNGM medium that had been sterilized and incubated at 20 ℃ for 24 h. Then, 20-30 synchronized nematodes with good growth and uniform size were inoculated and incubated at 20 ℃ for another 48 h. The surface of the NGM medium was then rinsed with M9 buffer and the M9 buffer containing nematodes was collected.

[0105] Model group: 400 μL of 10 9 CFU / mL Escherichia coli OP50 bacterial suspension and 400 μL of 1 mol / L sterile glucose aqueous solution were added to 20 mL of sterilized NGM medium. After incubation at 20 °C for 24 h, 20–30 synchronized nematodes of good growth and uniform size were inoculated. The medium was then incubated at 20 °C for another 48 h. The surface of the NGM medium was then rinsed with M9 buffer, and the M9 buffer containing nematodes was collected.

[0106] Low-dose group (0.25 mg / mL): The blackcurrant pomace anthocyanins obtained from group B in Example 2 were added to 10 9 Take 400 μL of CFU / mL E. coli OP50 bacterial suspension (to achieve a final concentration of 0.25 mg / mL anthocyanins in blackcurrant pomace in the bacterial suspension), and add it together with 400 μL of 1 mol / L sterile glucose aqueous solution to 20 mL of sterilized NGM medium. Incubate at 20 °C for 24 h, then inoculate with 20–30 synchronized nematodes that are in good growth condition and uniform in size. Continue to incubate at 20 °C for 48 h, then rinse the surface of the NGM medium with M9 buffer and collect the M9 buffer containing nematodes.

[0107] Medium-dose group (0.5 mg / mL): The blackcurrant pomace anthocyanins obtained from group B in Example 2 were added to 10 9Add 400 μL of CFU / mL E. coli OP50 bacterial suspension (to achieve a final concentration of 0.5 mg / mL of anthocyanins from blackcurrant pomace in the bacterial suspension) and 400 μL of 1 mol / L sterile glucose aqueous solution to 20 mL of sterilized NGM medium. Incubate at 20 °C for 24 h. Then inoculate with 20–30 synchronized nematodes that are in good growth condition and uniform in size. Continue to incubate at 20 °C for 48 h. Rinse the surface of the NGM medium with M9 buffer and collect the M9 buffer containing nematodes.

[0108] High-dose group (1.0 mg / mL): The blackcurrant pomace anthocyanins obtained from group B in Example 2 were added to 10 9 Add 400 μL of CFU / mL E. coli OP50 bacterial suspension (to achieve a final concentration of 1.0 mg / mL of anthocyanins from blackcurrant pomace in the bacterial suspension) and 400 μL of 1 mol / L sterile glucose aqueous solution to 20 mL of sterilized NGM medium. Incubate at 20 °C for 24 h. Then, inoculate with 20–30 synchronized nematodes that are in good growth condition and uniform in size. Continue to incubate at 20 °C for 48 h. Finally, rinse the surface of the NGM medium with M9 buffer and collect the M9 buffer containing nematodes.

[0109] VI. Oil Red O Staining One hundred nematodes were selected from each group and placed into 1.5 mL centrifuge tubes. The surface of the nematodes was washed three times with M9 buffer to remove residual E. coli OP50 (each wash was performed by centrifuging at 1500 rpm for 1 min). The tubes were then fixed with 4% paraformaldehyde at 25 °C for 30 min, followed by three freeze-thaw cycles at -80 °C, centrifuged at 1500 rpm for 3 min, and then washed three times with M9 buffer (each wash was performed by centrifuging at 1500 rpm for 1 min). The samples were then stained with Oil Red O at 25 °C for 30 min, and the accumulation of lipid droplets within the nematodes was observed under a microscope and photographed.

[0110] The results are as follows Figure 10 As shown in the figure, the model group was darker in color than the blank control group, indicating that the obesity model was successfully constructed. Compared with the model group, the color of each dose of blackcurrant pomace anthocyanin group was lighter, and the higher the dose, the lighter the color, indicating that blackcurrant pomace anthocyanin can effectively inhibit fat accumulation in nematodes and show an anti-obesity effect, especially the high dose group of 1.0 mg / mL, which showed the best effect.

[0111] VII. Determination of TG Content 3000 nematodes were selected from each group and homogenized into a homogenization tube. The E. coli OP50 residue on the surface of the nematodes was washed three times with M9 buffer (each wash was performed by centrifugation at 1500 rpm for 1 min). After that, fresh M9 buffer was added and the nematodes were homogenized. After centrifugation at 3000 rpm for 10 min at 4 ℃, the supernatant obtained by centrifugation was transferred to a 1.5 mL tube. Finally, the triglyceride (TG) content in the nematodes was detected according to the instructions of the triglyceride (TG) kit.

[0112] The results are as follows Figure 11 As shown in the figure, compared with the blank control group, the TG content in the model group was significantly increased, indicating that the obesity model was successfully established. Compared with the model group, the TG content in each dose group of blackcurrant pomace anthocyanin was significantly reduced (low, medium, and high dose groups were reduced by 37.7%, 43.45%, and 49.87%, respectively), and even slightly lower than the blank control group. Moreover, the higher the dose, the lower the TG content, indicating that blackcurrant pomace anthocyanin can effectively inhibit fat accumulation in nematodes and exhibit an anti-obesity effect, especially the high dose group of 1.0 mg / mL, which showed the best effect.

[0113] VIII. Detection of Reactive Oxygen Species by Fluorescent Probes Fifty nematodes were selected from each group and placed into 1.5 mL centrifuge tubes. The E. coli OP50 residue on the surface of the nematodes was washed three times with M9 buffer (each wash was performed by centrifuging at 1500 rpm for 1 min). Then, under light-protected conditions, H2DCFDA reactive oxygen species (ROS) probe was added to the tubes to a final concentration of 5 μg / mL. The tubes were then incubated at 23 ℃ for 30 min. Finally, the nematodes were anesthetized with sodium azide (30 mM), and the fluorescence intensity of the nematodes was observed under a fluorescence microscope and photographed.

[0114] The results are as follows Figure 12 As shown in the figure, the fluorescence intensity of the model group was significantly enhanced compared with the blank control group, indicating that the aging model was successfully constructed. Compared with the model group, the fluorescence intensity of each dose of blackcurrant pomace anthocyanin group was significantly weaker, even weaker than the blank control group. Moreover, the higher the dose, the weaker the fluorescence intensity, indicating that blackcurrant pomace anthocyanin can effectively reduce the production of ROS in nematodes and exhibit anti-aging effects, especially the high-dose group of 1.0 mg / mL, which showed the best effect.

[0115] IX. Detection of Antioxidant Enzyme Content 3000 nematodes were selected from each group and homogenized into a homogenization tube. The E. coli OP50 residue on the surface of the nematodes was washed three times with M9 buffer (each wash was performed by centrifugation at 1500 rpm for 1 min). After that, fresh M9 buffer was added and the nematodes were homogenized. After centrifugation at 3000 rpm for 10 min at 4 ℃, the supernatant was transferred to a 1.5 mL tube. Finally, the superoxide dismutase (SOD) content in the nematodes was detected according to the instructions of the superoxide dismutase (SOD) kit and the malondialdehyde (MDA) content in the nematodes was detected according to the instructions of the malondialdehyde (MDA) kit.

[0116] The results are as follows Figures 13-14 As shown. Among them, Figure 13 The results are for the detection of superoxide dismutase (SOD) content. Figure 14 The results show the detection of malondialdehyde (MDA) content. As can be seen, compared with the model group, the SOD content of each dose of blackcurrant pomace anthocyanin group was significantly increased (89.0% and 114.88% higher in the medium and high dose groups, respectively), and the MDA content was significantly decreased (24.27%, 39.17%, and 58.27% lower in the low, medium, and high dose groups, respectively). This indicates that blackcurrant pomace anthocyanin can effectively increase SOD content and effectively reduce MDA content, exhibiting an anti-aging effect, especially with the high dose group (1.0 mg / mL) showing the best effect.

[0117] 10. Life Test Thirty nematodes were selected from each group and transferred to a new container containing 400 μL of 10... 9 In NGM medium containing CFU / mL *E. coli* OP50 bacterial suspension, the survival days of nematodes were counted from the day of transfer, with the transfer day considered as day 0 of the experiment. During the first four days, due to the high egg-laying rate, the nematodes were transferred to fresh NGM medium daily until the end of the egg-laying period on the fourth day. Afterward, the nematodes were transferred every other day to ensure sufficient food. Nematode mortality was observed every other day, and their lifespan was recorded and the average lifespan calculated. The criteria for nematode death were: no obvious movement or swallowing activity, and no reaction to light touch.

[0118] The results are as follows Figure 15 As shown in Table 7.

[0119] Table 7

[0120] It is evident that, compared with the blank control group, the average lifespan of the model group was significantly shortened, indicating that the aging model was successfully constructed. Compared with the model group, the average lifespan of each dose of blackcurrant pomace anthocyanin group was significantly prolonged (P<0.05), even longer than that of the blank control group. Moreover, the higher the dose, the longer the average lifespan, indicating that blackcurrant pomace anthocyanin can effectively prolong the lifespan of nematodes and exhibit anti-aging effects, especially the high-dose group of 1.0 mg / mL, which showed the best effect.

[0121] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing anthocyanins from blackcurrant pomace, characterized in that, Includes the following steps: S1. Low-temperature continuous phase change extraction was performed on blackcurrant pomace to obtain a crude extract of anthocyanins from blackcurrant pomace; wherein the extraction temperature was 50–70 °C, the extraction time was 100–140 min, and the extraction solvent was 60% ( v / v ) ~ 80% v / v The bulk density of the blackcurrant pomace in the ethanol solution is 0.3–0.5 kg / L. S2. The crude extract of anthocyanins from blackcurrant pomace was loaded onto a polyamide resin column to adsorb the anthocyanins from the blackcurrant pomace; wherein the concentration of the crude extract of anthocyanins from blackcurrant pomace was 0.12–1.32 mg / mL, the mesh size of the polyamide resin was 30–100 mesh, the loading flow rate was 1–3 mL / min, and the loading volume was 1–2 BV; S3. After removing impurities from the polyamide resin column adsorbed with anthocyanins from blackcurrant pomace, use 30% ( v / v ) ~ 70% v / v Eluting was performed using an ethanol solution.

2. The preparation method according to claim 1, characterized in that, The pressure for the low-temperature continuous phase change extraction described in S1 is 0.18–0.22 MPa.

3. The preparation method according to claim 1, characterized in that, The pH of the extraction solvent described in S1 is 1.8 to 2.

2.

4. The preparation method according to claim 1, characterized in that, The adsorption time for S2 is 30–180 min.

5. The preparation method according to claim 1, characterized in that, The impurity removal described in S3 is achieved by elution with water.

6. The preparation method according to claim 1, characterized in that, The concentration of the ethanol solution described in S3 is 50% ( v / v ) ~ 70% v / v ).

7. The preparation method according to claim 1, characterized in that, The elution described in S3 is gradient elution.

8. Blackcurrant pomace anthocyanins prepared by the method according to any one of claims 1 to 7.

9. The use of the blackcurrant pomace anthocyanin according to claim 8 in the preparation of anti-obesity products.

10. The use of the blackcurrant pomace anthocyanin according to claim 8 in the preparation of anti-aging products.

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