Method for extracting antioxidant components from aaronia berry fruit and uses thereof

Abstract

The application provides an extraction method and application of antioxidant components in aronia melanocarpa fruits, and the extraction method comprises the following steps: (1) juicing and concentrating aronia melanocarpa to obtain aronia melanocarpa juice; (2) adding a composite enzyme into the aronia melanocarpa juice, and performing enzymolysis under ultrasonic waves; after the enzymolysis is completed, the aronia melanocarpa juice is extracted, filtered, and stratified by standing, the supernatant is collected, concentrated, and freeze-dried to obtain aronia melanocarpa crude extract; and (3) performing aqueous two-phase extraction on the aronia melanocarpa crude extract, centrifuging after filtration, concentrating and drying the supernatant to obtain the antioxidant components in the aronia melanocarpa fruits. The composite enzyme is used for enzymolysis, and ultrasonic waves are used for assistance, so that the antioxidant components in the aronia melanocarpa are released; and the aqueous two-phase extraction technology is further used, so that the antioxidant components in the aronia melanocarpa are effectively extracted. The antioxidant components in the aronia melanocarpa fruits extracted by the application can reach a hydroxyl radical clearance rate of 70.2% at a concentration of 3.2 mg / mL.

Description

Technical Field

[0001] This invention belongs to the field of plant component extraction technology, specifically relating to a method and application for extracting antioxidant components from arugula fruit. Background Technology

[0002] Aronia melanocarpa, also known as wild cherries, is scientifically named *Aronia melanocarpa*, belonging to the genus *Sorbus* of the family Rosaceae. Native to the wet forests and swamps of eastern North America, it gained widespread use in the 18th century due to its excellent taste, color, and aroma in the production of wine, jams, spices, and tea. Since 1989, my country has introduced eight varieties, and today the country possesses relatively abundant cultivation resources. In 2018, my country included it in its list of new food ingredients, and it is currently cultivated in Inner Mongolia and Northeast China.

[0003] Aronia berries are rich in polyphenols, sugars, organic acids, minerals, vitamins, and various trace elements, with polyphenol content being the highest among fruits. In recent years, research on their functional properties has increased. Their juice has strong antioxidant properties and also exhibits anti-cancer, anti-inflammatory, urinary tract infection prevention, diabetes treatment, liver protection, and obesity-treating effects. Extracts from aronia berries are significantly effective in preventing and treating cardiovascular and cerebrovascular diseases. Regular consumption of aronia berries can regulate bodily functions, control blood pressure, and slow down aging.

[0004] CN111825647A discloses a method for extracting anthocyanins from *Acer palmatum*, comprising the following steps:

[0005] (1) Raw material cleaning; (2) Juicing and separation; (3) Extraction; (4) Column adsorption and ethanol elution; (5) Vacuum concentration to a relative density of 1.12-1.15, adjusting the pH of the concentrated paste to 2.5-2.6 with citric acid, and then spray drying to obtain powder; (6) Adding stabilizers to the powder obtained in step (5) to obtain argan anthocyanins, wherein the stabilizers include ascorbic acid, vitamin E and bamboo leaf antioxidants. The anthocyanins prepared by this method have stable content, avoiding the problem of anthocyanin content reduction during storage, and can be stored at room temperature for more than two years.

[0006] CN104447666A discloses a method for extracting anthocyanins from aronia berries. Using aronia berries as raw material, the berries are first homogenized, and then the anthocyanins are extracted using an ethanol-acetic acid-water solvent with ultrasonic assistance. After extraction, the residue is removed by vacuum filtration, and the crude extract is removed by rotary evaporation to obtain a concentrated anthocyanin solution. Impurities are removed using distilled water, and then the anthocyanins are eluted with an ethanol solution. The eluent is then subjected to ultra-high pressure treatment, and impurities are extracted with ethyl acetate. After standing and separation, the aqueous phase is collected. The aronia berry anthocyanin aqueous phase solution is then freeze-dried under vacuum to obtain anthocyanin powder. This process is simple, pollution-free, and yields high-purity anthocyanins, fully utilizing the nutritional value of aronia berries. Furthermore, the macroporous resin is easy and rapid to activate, allowing for reuse and reducing costs.

[0007] CN103013936A discloses a compound enzyme preparation comprising the following components: 7-15 wt% cellulase, 7-15 wt% hemicellulase, 7-15 wt% protease, and 55-79 wt% pectinase. It also discloses a method for extracting anthocyanins using a compound enzyme, comprising the following steps: (1) preparing fresh fruit pulp and adjusting the solid-liquid ratio with water; (2) stirring the fruit pulp obtained in step (1) at low speed in a sealed container, controlling the temperature between 40-60℃ and the pH value between 3-6, adding the above-mentioned compound enzyme preparation, and enzymatically hydrolyzing for 1-8 hours; the dosage of the compound enzyme preparation is 1 mg / g-3 mg / g; (3) after enzymatic hydrolysis, removing the fruit residue from the fruit pulp to obtain a clear juice; (4) after purification by chromatographic chromatography of the clear juice, spray drying to obtain the anthocyanin product.

[0008] It is evident that current methods for extracting active substances from natural plants mostly involve washing or leaching with organic solvents, which consume large amounts of solvent and suffer from low extraction efficiency. Furthermore, existing technologies for extracting from aronia berries primarily focus on anthocyanins. However, aronia berries contain a variety of active ingredients with strong antioxidant properties. Therefore, developing a method for extracting antioxidant components from aronia berries that possesses good biological activity and application value is a key research focus in this field. Summary of the Invention

[0009] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method and application for extracting antioxidant components from aronia berries, and the extracted product can have good antioxidant properties and wide application value.

[0010] To achieve this objective, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a method for extracting antioxidant components from argan fruit, the extraction method comprising:

[0012] (1) Juice the aralia elata and concentrate the juice to obtain aralia elata juice;

[0013] (2) Add a compound enzyme to the aronia juice and perform enzymatic hydrolysis under ultrasound. After the enzymatic hydrolysis is completed, extract, filter, stand and separate, collect the supernatant, concentrate and freeze dry to obtain crude aronia extract.

[0014] (3) The crude extract of the aronia berry was subjected to aqueous two-phase extraction, filtered and centrifuged, and the supernatant was collected, concentrated and dried to obtain the antioxidant components in the aronia berry fruit.

[0015] This invention employs a two-phase aqueous extraction technology, a highly efficient and green bio-separation technique. Based on the difference in solubility of substances between two phases, it achieves efficient separation and extraction of active ingredients. Furthermore, the extraction conditions are mild, with low interfacial tension and minimal organic solvent residue, allowing for the effective extraction of antioxidants from arugula. Specific complex enzymes are used for enzymatic hydrolysis, which degrades the cell wall and promotes the dissolution of intracellular substances. Simultaneously, ultrasound is used to enhance the cavitation effect, further damaging the plant cell wall and promoting the release of phenolic substances.

[0016] Preferably, the content of soluble solids in the aralia juice in step (1) is ≥40%, for example, it can be 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, etc.

[0017] Preferably, the amount of the compound enzyme added is 0.3-0.6 U / g, for example, 0.35 U / g, 0.4 U / g, 0.45 U / g, 0.5 U / g, 0.55 U / g, etc.

[0018] Preferably, the complex enzyme includes pectinase and cellulase.

[0019] Preferably, the mass ratio of pectinase to cellulase is 1:(0.5-1), for example, it can be 1:0.6, 1:0.7, 1:0.8, 1:0.9, etc.

[0020] Preferably, the enzymatic hydrolysis temperature is 30-40℃ (e.g., 32℃, 34℃, 36℃, 38℃, etc.), the ultrasonic power is 450-550W (e.g., 460W, 470W, 480W, 490W, etc.), and the time is 1.5-2.5h, e.g., 1.6h, 1.8h, 2h, 2.2h, 2.4h, etc.

[0021] Preferably, during the enzymatic hydrolysis, the mass ratio of arugula juice to water is 1:(4-8), for example, it can be 1:4, 1:5, 1:6, 1:7, etc.

[0022] Preferably, in step (2), the reagent used for extraction is an aqueous ethanol solution.

[0023] Preferably, the ethanol aqueous solution contains 25-35% ethanol by mass, for example, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, etc., and is preferably 30%.

[0024] Preferably, the extraction reagent used in the aqueous two-phase extraction is a combination of aqueous ethanol solution and aqueous ammonium sulfate solution.

[0025] Preferably, the extraction reagent contains 30-38% ethanol by mass, for example, 32%, 34%, 36%, 38%, etc., and preferably 35%.

[0026] Preferably, the mass percentage of ammonium sulfate in the extraction reagent is 20-30%, for example, it can be 22%, 24%, 26%, 28%, etc., and preferably 25%.

[0027] Preferably, ultrasonic extraction is used to assist the extraction during the aqueous two-phase extraction. The power of the ultrasonic waves is 400-500W, such as 420W, 440W, 460W, 480W, etc.

[0028] Preferably, the filter paper used in step (3) has a pore size of 1-3 μm, such as 1.2 μm, 1.4 μm, 1.6 μm, 1.8 μm, 2 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, etc.

[0029] Preferably, the extraction method includes:

[0030] (1) Juice and concentrate the aralia elata to obtain aralia elata juice, wherein the soluble solids content in the aralia elata juice is ≥40%;

[0031] (2) Add 0.3-0.6 U / g of compound enzyme to the aronia juice and perform enzymatic hydrolysis at 30-40℃ and 450-550W ultrasound for 1.5-2.5h. During enzymatic hydrolysis, the mass ratio of aronia juice to water is 1:(4-8). After enzymatic hydrolysis, extract with 25-35% ethanol aqueous solution, filter, stand for separation, collect the supernatant, concentrate, freeze dry to obtain crude aronia extract.

[0032] (3) The crude extract of the aronia berry was subjected to aqueous two-phase extraction. The extraction reagent used was a combination of aqueous ethanol solution and aqueous ammonium sulfate solution, wherein the mass percentage of ethanol was 30-38% and the mass percentage of ammonium sulfate was 20-30%. Ultrasonic extraction was performed to assist extraction. The ultrasonic power was 400-500W. After filtration through a pore size of 1-3μm, the extract was centrifuged, and the supernatant was collected, concentrated, and dried to obtain the antioxidant components in the aronia berry fruit.

[0033] Secondly, the present invention provides an antioxidant component in the fruit of the aralia elata, wherein the antioxidant component in the fruit of the aralia elata is extracted using the extraction method described in the first aspect.

[0034] Thirdly, the present invention provides an application of the antioxidant components in the aralia elata fruit as described in the second aspect in beverages, foods or health products.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention employs a two-phase aqueous extraction technique, taking advantage of the different solubilities of antioxidant components in aronia berries between two phases to achieve efficient separation and extraction of active ingredients. The extraction conditions are mild, with low interfacial tension and minimal organic solvent residue, ensuring effective extraction of antioxidant components from aronia berries. Specific complex enzymes are used for enzymatic hydrolysis, further employing a combination of pectinase and cellulase to effectively degrade cell walls and promote the dissolution of intracellular substances. Simultaneously, ultrasound is used to enhance the cavitation effect, increasing the degree of damage to plant cell walls and promoting the release of phenolic substances. The antioxidant components extracted from aronia berries using this invention achieve a hydroxyl radical scavenging rate of 70.2% at a concentration of 3.2 mg / mL. Attached Figure Description

[0037] Figure 1 This is a comparison image of the beverages after the light exposure test in Test Example 4. Detailed Implementation

[0038] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0039] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not limited to those elements and may also include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0040] "Optional" or "any one" means that the matter or event described thereafter may or may not occur, and the description includes both the possibility that the event will occur and the possibility that the event will not occur.

[0041] The indefinite articles “a” and “an” preceding an element or component of this invention do not impose any limitation on the quantity (i.e., number of occurrences) of the element or component. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers only to the singular form.

[0042] The terms "one embodiment," "some embodiments," "exemplary," "specific example," or "some examples," etc., used in this invention refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this document, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example.

[0043] The reagents or instruments used in the following examples are from the following sources:

[0044] Immortelle: Originating in Dalian, Liaoning Province;

[0045] The compound enzyme was purchased from Shandong Longda Biotechnology Co., Ltd., and the mass ratio of pectinase to cellulase was 2:1.

[0046] Example 1

[0047] This embodiment provides an antioxidant component from the fruit of the aralia elata, and its extraction method is as follows:

[0048] (1) After cleaning and removing impurities from the aronia berries, crush and juice them, and concentrate the aronia berry juice with a Brix (soluble solids) of 45%.

[0049] (2) Add 0.4 U / g of compound enzyme to the aralia juice from step (1) and perform enzymatic hydrolysis at 35°C and 500W ultrasound for 2 hours. Add 5 times the amount of water during enzymatic hydrolysis. After the enzymatic hydrolysis is completed, extract with 30% ethanol aqueous solution for 0.5 hours, then filter, let the filtrate stand to separate into layers, collect the supernatant, concentrate under reduced pressure, and freeze dry to obtain crude aralia extract.

[0050] (3) The crude extract of the aronia was subjected to aqueous two-phase extraction. During extraction, the concentration of ethanol in the system was controlled to be 35% and the concentration of ammonium sulfate to be 25%. After filtration through a 2μm pore size, the extract was centrifuged, and the supernatant was collected for vacuum concentration and drying to obtain the antioxidant components in the aronia fruit.

[0051] Example 2

[0052] This embodiment provides an antioxidant component from the fruit of the aralia elata, and its extraction method is as follows:

[0053] (1) After cleaning and removing impurities from the aronia berries, crush and juice them, and concentrate the aronia berry juice with a Brix (soluble solids) of 50%.

[0054] (2) Add 0.3 U / g of compound enzyme to the aralia juice from step (1) and perform enzymatic hydrolysis at 40℃ and 450W ultrasound for 2.3 h. Add 6 times the amount of water during enzymatic hydrolysis. After the enzymatic hydrolysis is completed, extract with 30% ethanol aqueous solution for 0.5 h, then filter, let the filtrate stand to separate into layers, collect the supernatant for vacuum concentration and freeze drying to obtain crude aralia extract.

[0055] (3) The crude extract of the aronia berry was subjected to aqueous two-phase extraction, with the ethanol concentration controlled at 30% and the ammonium sulfate concentration controlled at 30%. After filtration through a 1.5 μm pore size, the extract was centrifuged, and the supernatant was collected for vacuum concentration and drying to obtain the antioxidant components in the aronia berry fruit.

[0056] Example 3

[0057] This embodiment provides an antioxidant component from the fruit of the aralia elata, and its extraction method is as follows:

[0058] (1) After cleaning and removing impurities from the aronia berries, crush and juice them, and then concentrate the aronia berry juice with a Brix (soluble solids) of 55%.

[0059] (2) Add 0.6 U / g of compound enzyme to the aralia juice from step (1) and enzymatically hydrolyze it at 30°C and 550W ultrasound for 1.8 h. Add 5 times the amount of water during enzymatic hydrolysis. After enzymatic hydrolysis, extract with 30% ethanol aqueous solution for 0.5 h, then filter, let the filtrate stand to separate into layers, collect the supernatant, concentrate under reduced pressure, and freeze dry to obtain crude aralia extract.

[0060] (3) The crude extract of the aronia berry was subjected to aqueous two-phase extraction, with the concentration of ethanol in the system controlled at 38% and ammonium sulfate at 20%. After filtration through a 2.5 μm pore size, the extract was centrifuged, and the supernatant was collected for vacuum concentration and drying to obtain the antioxidant components in the aronia berry fruit.

[0061] Example 4

[0062] This embodiment provides an antioxidant component from aralia elata fruit. The only difference between this embodiment and Example 1 is that the amount of compound enzyme added is adjusted to 0.2 U / g. All other raw materials, dosages, and extraction methods are the same as in Example 1.

[0063] Example 5

[0064] This embodiment provides an antioxidant component from aralia elata fruit. The only difference between this embodiment and Example 1 is that the amount of compound enzyme added is adjusted to 0.7 U / g. All other raw materials, dosages, and extraction methods are the same as in Example 1.

[0065] Example 6

[0066] This embodiment provides an antioxidant component from aralia elata fruit. The only difference between this embodiment and Example 1 is that the enzymatic hydrolysis time is adjusted to 1 hour. All other raw materials, dosages, and extraction methods are the same as in Example 1.

[0067] Example 7

[0068] This embodiment provides an antioxidant component from aralia elata fruit. The only difference between this embodiment and Example 1 is that the enzymatic hydrolysis time is adjusted to 3 hours. All other raw materials, dosages, and extraction methods are the same as in Example 1.

[0069] Example 8

[0070] This embodiment provides an antioxidant component from aralia elata fruit. The only difference between this embodiment and Example 1 is that the ultrasonic power in step (2) is adjusted to 400W. The other raw materials, dosages, and extraction methods are the same as in Example 1.

[0071] Example 9

[0072] This embodiment provides an antioxidant component from aronia berries. The only difference between this embodiment and Example 1 is that the ultrasonic power in step (2) is adjusted to 600W. The other raw materials, dosages, and extraction methods are the same as in Example 1.

[0073] Example 10

[0074] This embodiment provides an antioxidant component from aralia elata fruit. The only difference between this embodiment and Example 1 is that the concentration of the ethanol aqueous solution in step (3) is adjusted to 25%. The other raw materials, dosages, and extraction methods are the same as in Example 1.

[0075] Example 11

[0076] This embodiment provides an antioxidant component from aralia elata fruit. The only difference between this embodiment and Example 1 is that the concentration of the ethanol aqueous solution in step (3) is adjusted to 40%. The other raw materials, dosages, and extraction methods are the same as in Example 1.

[0077] Example 12

[0078] This embodiment provides an antioxidant component from aralia elata fruit. The only difference between this embodiment and Example 1 is that the concentration of the ammonium sulfate aqueous solution in step (3) is adjusted to 15%. The other raw materials, dosages, and extraction methods are the same as in Example 1.

[0079] Example 13

[0080] This embodiment provides an antioxidant component from aralia elata fruit. The only difference between this embodiment and Example 1 is that the concentration of the ammonium sulfate aqueous solution in step (3) is adjusted to 35%. The other raw materials, dosages, and extraction methods are the same as in Example 1.

[0081] Comparative Example 1

[0082] This comparative example provides an antioxidant component from arugula fruit. The only difference between this example and Example 1 is that the complex enzyme is replaced with an equal amount of pectinase. All other raw materials, dosages, and extraction methods are the same as in Example 1.

[0083] Comparative Example 2

[0084] This comparative example provides an antioxidant component from arugula fruit. The only difference between this example and Example 1 is that the complex enzyme is replaced with an equal amount of cellulase. All other raw materials, dosages, and extraction methods are the same as in Example 1.

[0085] Comparative Example 3

[0086] This comparative example provides an antioxidant component from aralia elata fruit, which differs from Example 1 only in step (3). Step (3) of this comparative example is as follows:

[0087] The crude extract of Aristolochia rubra was subjected to aqueous two-phase extraction, with the ethanol concentration in the system controlled at 35% during extraction. After filtration through a 2μm pore size, the extract was centrifuged, and the supernatant was collected, concentrated under reduced pressure, and dried to obtain the antioxidant components in the Aristolochia rubra fruit.

[0088] Comparative Example 4

[0089] This comparative example provides an antioxidant component from aralia elata fruit, which differs from Example 1 only in step (3). Step (3) of this comparative example is as follows:

[0090] The crude extract of Aristolochia rubra was subjected to aqueous two-phase extraction, with the ammonium sulfate concentration in the system controlled at 25% during extraction. After filtration through a 2μm pore size, the extract was centrifuged, and the supernatant was collected, concentrated under reduced pressure, and dried to obtain the antioxidant components in the Aristolochia rubra fruit.

[0091] Comparative Example 5

[0092] This comparative example provides an antioxidant component in aralia elata fruit. The only difference between this example and Example 1 is that step (2) is not performed. The aralia elata juice obtained in step (1) is directly subjected to the aqueous two-phase extraction in step (3). The other raw materials, dosages and extraction methods are the same as in Example 1.

[0093] Test Example 1

[0094] Extraction rate

[0095] The determination method followed the Folin-Ciocalteu method, using gallic acid as the standard. Different concentrations of standard solutions were reacted with folin-phenol and disodium bicarbonate. A linear regression equation was established based on the absorbance values ​​at 760 nm: Y = 0.0263X + 0.0401(R²). 2 =0.9993), each sample was measured an average of 3 times, and the extraction rates of antioxidant components in the examples and comparative examples are shown in Table 1.

[0096] Test Example 2

[0097] DPPH-Free Radical Scavenging Experiment

[0098] A 96-well plate was used, with three replicates per group, and the total volume was 200 μL. For the sample group: appropriate amounts of the examples and comparative samples were dissolved in 100 μL of distilled water to achieve a final concentration of 3.2 mg / mL. 100 μL of 0.1 mM DPPH solution was then added to the reaction system. For the control group: 100 μL of distilled water was used, followed by the addition of 100 μL of 0.1 mM DPPH solution. After the reaction system was constructed, it was shaken in the dark for 10 min, and the absorbance at 520 nm was measured using a microplate reader.

[0099] The clearance rate is calculated as follows: Clearance rate (%) = [(A0-Ax) / A0] × 100%, where A0 is the absorbance of the control group and Ax is the absorbance of the sample group. A higher clearance rate indicates a better antioxidant effect.

[0100] The results of the DPPH free radical scavenging test are shown in Table 1.

[0101] Test Example 3

[0102] Hydroxyl radical scavenging experiment

[0103] Main reagents: salicylic acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd.), ferrous sulfate (analytical grade, Sinopharm Chemical Reagent Co., Ltd.), anhydrous ethanol (analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.), hydrogen peroxide.

[0104] Main equipment: Spectra Max M5 Molecular Device (multifunctional microplate reader)

[0105] Experimental Methods: Sample groups (examples and comparatives) were prepared with antioxidant components diluted to 3.2 mg / mL, and a negative control group was also included. 0.1 parts ferrous sulfate, 0.1 parts ethanol, 0.1 parts salicylic acid, 0.1 parts sample or negative control, 1 part deionized water, and 0.1 parts hydrogen peroxide were added sequentially to 1.5 mL centrifuge tubes. The tubes were tightly capped and shaken thoroughly by inverting. The tubes were incubated at 37°C for 15 min. The solutions were then transferred to 96-well plates, with three replicates per group. The absorbance at 510 nm was measured using a microplate reader.

[0106] The hydroxyl radical scavenging rate is calculated using the following formula:

[0107] Hydroxyl radical scavenging rate % = {[A0 - (AX - AX0)] / A0} × 100%, where A0 is the absorbance of the negative control group, Ax is the absorbance of the sample group, and Ax0 is the absorbance of the sample background. The test results are shown in Table 1.

[0108] Table 1

[0109] sample Extraction rate (%) DPPH removal rate (%) Hydroxyl radical scavenging rate (%) Example 1 12.2 68.1 70.2 Example 2 11.9 65.3 67.3 Example 3 11.8 65.3 67.8 Example 4 11.4 65.0 66.2 Example 5 11.9 64.9 67.3 Example 6 10.2 50.9 58.1 Example 7 11.8 65.4 60.8 Example 8 11.6 65.3 63.2 Example 9 12.0 65.6 63.1 Example 10 11.4 65.1 63.7 Example 11 11.7 65.2 63.5 Example 12 9.8 53.2 55.2 Example 13 12.0 68.0 64.3 Comparative Example 1 10.3 50.3 56.1 Comparative Example 2 8.9 49.2 52.4 Comparative Example 3 6.2 40.8 46.3 Comparative Example 4 4.6 38.6 42.1 Comparative Example 5 7.2 46.2 48.4

[0110] According to the table data, when the extraction method provided by this invention is used to extract the antioxidant components of *Aristolochia debilis*, both the extraction rate and antioxidant activity can be guaranteed. Examples 1 and 4-5 show that the highest extraction rate is achieved when the amount of compound enzyme added is 0.3-0.6 U / g. Examples 1 and 6-7 show that an enzymatic hydrolysis time of 1.5-2.5 h yields the best results; too short a time results in insufficient hydrolysis, inadequate cell wall destruction, and incomplete release of phenolic acids; too long a time significantly improves the overall effect but also prolongs the reaction time, which is not conducive to subsequent continuous operations. Examples 1 and 8-9 show that the optimal power for auxiliary ultrasound during enzymatic hydrolysis is 450-550 W; too low a power results in insufficient cavitation effect of ultrasound on the cell wall, while too high a power affects the overall activity of the dissolved substances. Examples 4-5 show that... As shown in Examples 1 and 10-11, the optimal concentration of the ethanol-water solution for aqueous two-phase extraction is 30-38%. If the concentration is too low, the phase separation ability is poor, the two phases are unstable, and the aqueous two-phase solution formed at this time has poor permeability to the plant cell wall, making it difficult for active substances to precipitate. If the concentration is too high, salt precipitation will occur in the lower phase, affecting the precipitation of active substances. As shown in Examples 1 and 12-13, the optimal concentration of ammonium sulfate for aqueous two-phase extraction is 20-30%. If the concentration is too low, the binding ability of the lower phase to water is weak, resulting in a relatively low concentration of ethanol in the upper phase, which is not conducive to the precipitation of antioxidants. If the concentration is too high, salt precipitation will occur in the aqueous two-phase system, reducing the precipitation rate of active substances.

[0111] As shown in Example 1 and Comparative Examples 1-2, the enzymatic hydrolysis using a compound enzyme yields the best results. Using a single pectinase or cellulase will affect the extraction rate and further impact the antioxidant properties of the product. Therefore, the use of a compound enzyme is considered. As shown in Example 1 and Comparative Examples 3-4, the present invention uses aqueous two-phase extraction to extract antioxidant components from arugula fruit. If a single solvent is used, the extraction rate will decrease significantly, and the antioxidant properties of the product will also be adversely affected. Aqueous two-phase extraction can improve the overall antioxidant activity of the extract. As shown in Example 1 and Comparative Example 5, if arugula juice is directly subjected to aqueous two-phase extraction without enzymatic hydrolysis, the extraction rate will decrease significantly, and the antioxidant properties will also decrease significantly. Enzymatic hydrolysis can promote the full release of antioxidants.

[0112] Test Example 4

[0113] A pink lemon juice beverage was prepared by adding 10% pure lemon juice and 1% concentrated pink lemon juice. 1.0% of the antioxidant components from *Aristolochia debilis* (prepared in Example 1), a combined antioxidant (antioxidant components from *Aristolochia debilis* prepared in Example 1 and vitamin C mixed in a 1:1 ratio), or vitamin C were added, designated as 1#, 2#, and 3# respectively. A control sample without added antioxidants was used. The samples were placed under light for 30 days. The original samples were stored refrigerated and protected from light. The flavor of the product was simultaneously evaluated, and the flavor evaluation criteria are shown in Table 2. The sensory evaluation, residual oxygen content, soluble solids, and acidity test results are shown in Table 3. A comparison chart of the tested beverages is shown below. Figure 1 As shown.

[0114] Table 2

[0115] Sensory items standard Score Appearance The color is translucent and glossy, ranging from pink to light red; a small amount of aggregate at the bottom of the bottle is permissible. 20 aroma The lemon scent is fresh, natural, and uplifting, with no unpleasant odor. 30 taste It has a perfect balance of sweet and sour, a juicy flavor, a natural taste without any off-flavors, a clean finish, and a slight astringency. 50

[0116] Table 3

[0117] sample Sensory score Residual oxygen level (mg / L) Soluble solids (%) acidity(%) Original sample 100 3.6 11.5 0.54 control sample 40 0 11.6 0.55 1# 95 0 11.5 0.54 2# 98 0 11.5 0.55 3# 94 0 11.5 0.55

[0118] According to the table data, the sensory evaluation of the beverage was superior when the antioxidant components and vitamin C from the aronia berry fruit provided in Example 1 were added. The overall flavor of the sample with added aronia berry extract was closer to the original sample, with a more natural and pure overall style. Adding vitamin C resulted in a slightly unpleasant oxidized flavor, which deteriorated significantly with extended shelf life. Furthermore, [the text abruptly ends here]. Figure 1 It is known that adding the antioxidant components of aronia berries can ensure that the product maintains a good flavor and color under light conditions, ensure the stability of the product's flavor and color during the shelf life, maintain the overall color with an effect comparable to the original sample, and make the overall final product ingredients cleaner.

[0119] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for extracting antioxidant components from argan fruit, characterized in that, The extraction method is as follows: (1) Juice the aralia elata and concentrate it to obtain aralia elata juice; (2) Add a compound enzyme to the aralia elata juice and perform enzymatic hydrolysis under ultrasound. After enzymatic hydrolysis, extract, filter, allow to stand for separation, collect the supernatant, concentrate, and freeze-dry to obtain crude aralia elata extract. The compound enzyme is pectinase and cellulase. The reagent used for extraction is an aqueous ethanol solution. The temperature during enzymatic hydrolysis is 30-40℃, the ultrasound power is 450-550W, and the time is 1.5-2.5 h. The amount of compound enzyme added is 0.3-0.6 U / g. The mass percentage of ethanol in the aqueous ethanol solution is 25-35%. (3) The crude extract of the aralia elata is subjected to aqueous two-phase extraction, filtered and centrifuged, and the supernatant is collected, concentrated and dried to obtain the antioxidant components in the aralia elata fruit; the aqueous two-phase extraction uses a combination of aqueous ethanol solution and aqueous ammonium sulfate solution; the mass percentage of ethanol in the extraction reagent is 30-38%; the mass percentage of ammonium sulfate in the extraction reagent is 20-30%.

2. The extraction method according to claim 1, characterized in that, The content of soluble solids in the aralia juice in step (1) is ≥40%.

3. The extraction method according to claim 1, characterized in that, During the enzymatic hydrolysis, the mass ratio of aronia berry juice to water is 1:(4-8).

4. The extraction method according to claim 1, characterized in that, The aqueous two-phase extraction is performed with ultrasonic assistance, and the ultrasonic power is 400-500 W.

5. The extraction method according to claim 1, characterized in that, The filter paper used in step (3) has a pore size of 1-3 μm.

6. The extraction method according to claim 1, characterized in that, The extraction method is as follows: (1) Juice the aralia elata and concentrate it to obtain aralia elata juice, wherein the soluble solids content of the aralia elata juice is ≥40%; (2) Add 0.3-0.6 U / g of compound enzyme to the aronia juice and perform enzymatic hydrolysis at 30-40℃ and 450-550 W ultrasound for 1.5-2.5 h. During enzymatic hydrolysis, the mass ratio of aronia juice to water is 1:(4-8). After enzymatic hydrolysis, extract with 25-35% ethanol aqueous solution, filter, stand for separation, collect the supernatant, concentrate, freeze dry to obtain crude aronia extract. (3) The crude extract of the aronia berry was subjected to aqueous two-phase extraction. The extraction reagent was a combination of aqueous ethanol solution and aqueous ammonium sulfate solution, wherein the mass percentage of ethanol was 30-38% and the mass percentage of ammonium sulfate was 20-30%. Ultrasonic extraction was performed to assist extraction. The ultrasonic power was 400-500 W. After filtration through a pore size of 1-3 μm, the extract was centrifuged, and the supernatant was collected, concentrated, and dried to obtain the antioxidant components in the aronia berry fruit.

7. An antioxidant component from aronia berry fruit, characterized in that, The antioxidant components in the *Agerospermum jasminoides* fruit were extracted using the extraction method described in any one of claims 1-6.

8. The application of the antioxidant components in the aralia elata fruit as described in claim 7 in beverages, foods or health products.

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