Olive leaf polyphenol extract, polyphenol refined olive oil and methods for their preparation

Abstract

The application provides an olive leaf polyphenol extract, a polyphenol refined olive oil and a preparation method thereof. The olive leaf polyphenol extract without chlorophyll is obtained by means of dark standing treatment, microwave-assisted extraction and chlorophyll removal treatment, and then the olive leaf polyphenol extract is enriched in the refined olive oil to prepare the refined olive oil with high polyphenol content, thereby simultaneously solving the technical problems of low oil yield and high chlorophyll content.

Description

Technical Field

[0001] This invention relates to the field of olive oil preparation technology, specifically to an olive leaf polyphenol extract, polyphenol-refined olive oil, and their preparation methods. Background Technology

[0002] The European Food Safety Authority (EFSA) stated in its 2011 health declaration for all commercially available olive oil that only olive oil containing at least 250 mg of polyphenols per kilogram meets health requirements. However, the polyphenol content in commonly consumed refined olive oil is extremely low, often below 40 mg / kg, far below the level required by the EFSA health declaration.

[0003] Currently, technical solutions for specifically increasing polyphenol content in refined olive oil have issues such as a significant decrease in oil yield or a higher chlorophyll content leading to a darker color. Summary of the Invention

[0004] Therefore, it is necessary to provide a refined olive oil with a high content of polyphenols and a method for its preparation.

[0005] The present invention adopts the following technical solution:

[0006] This invention provides a polyphenol-refined olive oil with a total phenol content greater than 250 mg / kg and a chlorophyll content less than 15 mg / kg. The polyphenol-refined olive oil also contains hydroxytyrosol, luteolin, quercetin, and oleacein.

[0007] This invention provides a method for preparing polyphenol-refined olive oil, comprising the following steps:

[0008] Obtain olive leaf polyphenol extract powder;

[0009] The olive leaf polyphenol extract is mixed with refined olive oil at a mass ratio of 1:15 to 1:40, and then subjected to high-pressure solubilization and / or pulsed ultrasonic solubilization and enrichment treatment. After centrifugation and filtration, refined olive oil with high polyphenol content is obtained.

[0010] In some embodiments, the process conditions for high-pressure dissolution treatment are preferably: pressure 150-250 MPa, treatment time 3-10 min.

[0011] In some embodiments, the preferred process parameters for pulsed ultrasonic dissolution are: ultrasonic power of 450-650W, total ultrasonic treatment time of 2-10min, ultrasonic pulse interval program of 10s working and 5s stopping, ultrasonic frequency of 250-350Hz, and water bath temperature of 30-45℃.

[0012] In some embodiments, preferably, the process conditions for centrifugal filtration are as follows: first, a disc centrifuge is used to remove solid particles by centrifugation at 3500g for 10 minutes, and then the mixture is left to stand in the dark before being filtered using an industrial-grade oil filter.

[0013] In some embodiments, the preparation method of the olive leaf polyphenol extract powder includes the following steps:

[0014] S1, Obtain olive leaf raw material, wash it, and obtain material A;

[0015] S2, mix material A with water, add acid to adjust the pH to 5.5-6.5, carry out the reaction in the dark under stirring conditions, then add alkaline solution to adjust the pH to 6.5-7.0, carry out the reaction in the dark, vacuum dry, and pulverize to obtain reactant B;

[0016] S3, Mix reactant B with extraction solvent, and extract with microwave-assisted extraction to obtain extract C;

[0017] S4, add acid to the extract C to adjust the pH to 2.5-3.5, mix well, then add alkaline solution to adjust the pH to 9.5-10.0, let stand at low temperature to precipitate chlorophyll, filter to obtain extract D;

[0018] S5, mix the extract D with 40% ethanol aqueous solution, let stand to separate the layers, and collect the ethanol aqueous phase extract E;

[0019] S6. The aqueous ethanol extract E is distilled to obtain an ethanol-free product, which is then freeze-dried at low temperature to obtain olive leaf extract powder.

[0020] Preferably, in step S1, the olive leaf raw material is selected from the tender leaves of olive trees in late spring of four varieties: Piguar, Corati, Belate, and Godard.

[0021] Preferably, in step S2, material A is mixed with water at a ratio of 1g:30mL to 1g:70mL, acid is added to adjust the pH to 5.5 to 6.5, and the mixture is stirred at 35 to 45°C and 45 to 50% humidity in the dark for 20 to 24 hours. Then, an alkaline solution is added to adjust the pH to 6.5 to 7.0, and the mixture is allowed to stand in the dark for another 20 to 24 hours. Finally, the mixture is vacuum dried at 35 to 45°C and pulverized to obtain reactant material B.

[0022] Preferably, in step S3, the extraction solvent is an aqueous solution containing choline chloride and malic acid in a mass ratio of 1:2 to 1:4. The mass ratio of reactant B to the extraction solvent is 1:10 to 1:14, and the microwave-assisted extraction process conditions are: microwave power 600 to 700 W, microwave treatment time 15 to 30 min, and treatment temperature 40 to 50 °C.

[0023] Preferably, in step S4, the low-temperature standing conditions are: -20 to -5°C for 60 to 120 minutes.

[0024] Preferably, in step S5, the ethanol aqueous solution is a 20% to 55% ethanol aqueous solution, and the volume ratio of extract D to ethanol aqueous solution is 1:1 to 1:4.

[0025] Preferably, in step S6, the distillation process parameters are: steam pressure 0.2–2.0 MPa, distillation until no ethanol remains in the solution and the anhydrous ethanol obtained from distillation is collected for recycling, while the aqueous solution and the precipitated precipitate are stored together at -20–-5°C for 8–12 hours. The low-temperature freeze-drying process parameters are: temperature -40–-30°C, vacuum degree 20–100 Pa, and processing time 20–24 hours.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention provides a method for preparing olive leaf polyphenol extract and polyphenol-refined olive oil containing olive leaf polyphenol extract. The prepared olive leaf polyphenol extract is enriched into refined olive oil, thereby increasing the content of polyphenols to no less than 250 mg / kg, while significantly reducing the chlorophyll content and overcoming the problem of reduced oil yield. Attached Figure Description

[0028] Figure 1 The HPLC chromatograms of the four main polyphenolic substances in the polyphenol-refined olive oil prepared in Example 4 are shown.

[0029] Figure 2 HPLC chromatograms of polyphenol-refined olive oil prepared for different experimental examples.

[0030] Figure 3 The images show a comparison of the appearance and color of the polyphenol-refined olive oil prepared in Examples 2 and 4 with commercially available olive oil (referred to as "refined oil" and "commercially available extra virgin olive oil," respectively). Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. The following embodiments are only used to illustrate the present invention, and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0032] Explanation of the sources of some key experimental materials:

[0033] Refined olive oil: The refined olive oil is from the Italian brand Monini and was purchased online.

[0034] Olive leaf raw materials: Collect 1.0 kg each of four varieties of olive leaves in late spring (March), including Piguar, Corrati, Bellate, and Godard. Mix them evenly in equal proportions, sift out the dark green old leaves, and retain the lighter-colored young leaves. The time interval between picking the olive leaves from the tree should not exceed 48 hours to ensure that the endogenous enzymes in the olive leaves still maintain a high level of enzyme activity.

[0035] Example 1

[0036] This embodiment provides a method for preparing polyphenol-refined olive oil, including the following steps:

[0037] S1, Olive leaf selection and cleaning:

[0038] Using a blower and vibrating screen, small twigs mixed in with the olive leaf raw material are separated and sieved out. The olive leaf raw material mixed in the same proportion is washed multiple times to remove dust and insect eggs adhering to the leaves, resulting in clean olive leaves A. Note: The time interval between olive leaves A and being picked from the tree should not exceed 48 hours to ensure that the endogenous enzymes in the olive leaves still maintain a high level of enzyme activity.

[0039] S2, Dark static preprocessing:

[0040] Fresh, clean olive leaves (A) were mixed with pure water and placed in a high-speed homogenizer at a ratio of 1:30 (g / mL). The mixture was stirred at 50 rpm, and a small amount of acetic acid was added to adjust the pH to 6.0. The temperature was controlled at 40°C, and the ambient humidity was 50%. The mixture was stirred in the dark for 24 hours. The homogenizer was then turned off, and an appropriate amount of sodium bicarbonate was added to adjust the pH to 7.0. The solution was then allowed to stand in the dark for another 24 hours. The solution was dried at 40°C using a vacuum dryer. All dried solids were collected, pulverized using a grinder, and passed through a 100-mesh sieve to obtain olive leaf powder (B) (referred to as "reactant B").

[0041] S3, Microwave extraction of olive leaf polyphenols using a eutectic solvent coupled with a micro-extraction method:

[0042] Preparation of the eutectic solvent (extraction reagent): Choline chloride and malic acid were mixed at a mass ratio of 1:2 and stirred rapidly at 200 rpm in an 80°C water bath for 5–10 minutes to form a viscous, clear liquid. 1.5 times the volume of water was added and mixed thoroughly to obtain the eutectic solvent required for extraction.

[0043] Olive leaf powder B was mixed with a pre-prepared eutectic solvent at a mass ratio of 1:10, and microwave-assisted extraction was performed: microwave power 700W, microwave treatment time 15min, and treatment temperature 50℃. After treatment, olive leaf polyphenol DES extract A (also referred to as "extract C") was obtained through a self-cleaning filter.

[0044] S4, Dechlorosis treatment with olive leaf polyphenol extract:

[0045] Add glacial acetic acid to olive leaf polyphenol DES extract A to adjust the pH to 3.0, mix thoroughly, then slowly add 5% (w / v) sodium bicarbonate solution to adjust the pH to approximately 9.5. Let stand at -20℃ for 2 hours. Chlorophyll precipitates under the control of pH and temperature, while olive leaf polyphenols remain in the DES extract. Take the filtered DES extract (referred to as "extract D") and mix it with 40% ethanol solution at a 1:1 volume ratio, stir thoroughly, let stand to separate the layers, and take the upper 40% ethanol aqueous phase to obtain clear olive leaf polyphenol extract B (referred to as "extract E"), free of chlorophyll.

[0046] S5, freeze-dried into powder:

[0047] Olive leaf polyphenol extract B was distilled to separate ethanol and water using a distillation apparatus at a steam pressure of 0.2 MPa until no ethanol remained in the solution. The anhydrous ethanol obtained from distillation was collected for recycling, while the aqueous solution and the precipitated material were stored together at -20°C for 12 hours. The frozen aqueous solution and the precipitated material were then freeze-dried to obtain dried olive leaf enriched powder A.

[0048] The freeze-drying process conditions were -40℃, 20Pa vacuum, and 24h processing time. Olive leaf enriched powder A was further pulverized using a vibrating ultrafine pulverizer, and then filtered through a vibrating screen with a mesh size of 400 to obtain olive leaf enriched powder B.

[0049] S6, Ultrasonic pulse enrichment processing:

[0050] Olive leaf enrichment powder B was mixed evenly with refined olive oil (commercially available, also referred to as "refined oil") at a mass ratio of 1:10. The enrichment powder and oil were then processed using an ultrasonic pulse crusher. The process parameters were: ultrasonic power of 600W, ultrasonic treatment time of 3 minutes (ultrasonic pulse interval: 5 seconds on, 5 seconds off), ultrasonic frequency of 300Hz, and water bath temperature of 35℃. The resulting enriched refined olive oil A was obtained after ultrasonic crushing.

[0051] S7, Separation Filtration:

[0052] The enriched refined olive oil A was separated into solid particles and clarified olive oil B using a disc centrifuge (centrifugation acceleration 3500g, separation time 10min). Clarified olive oil B was in the upper layer, and the solid particles settled in the lower layer. The clarified olive oil B was left to stand in the dark for 24 hours and then filtered using an industrial-grade oil filter to obtain the final clear and impurity-free polyphenol-enriched olive oil C.

[0053] Example 2

[0054] This embodiment provides a method for preparing polyphenol-refined olive oil, including the following steps:

[0055] The steps for preparing olive leaf polyphenol DES extract A (also referred to as "extract C") in S1 to S3 are the same as those in Example 1.

[0056] S4, Extraction of olive leaf polyphenols (without dechlorosis treatment):

[0057] Olive leaf polyphenol DES extract A (hereinafter referred to as "extract C") was directly mixed with 40% ethanol solution at a volume ratio of 1:1, stirred thoroughly, allowed to stand and separate into layers, and the upper 40% ethanol aqueous phase was taken to obtain clear olive leaf polyphenol extract B (hereinafter referred to as "extract E").

[0058] S5 is freeze-dried into powder.

[0059] The operation steps are the same as those in Example 1.

[0060] S6, Ultra-high voltage coupled ultrasonic pulse enrichment processing:

[0061] Olive leaf enrichment powder B was mixed evenly with refined olive oil at a mass ratio of 1:10. Ultra-high pressure technology was used to dissolve the active ingredients in the olive leaf enrichment powder B into the oil at a pressure of 200 MPa for 3 minutes. The enrichment powder and oil were then processed using an ultrasonic pulse crusher with the following parameters: ultrasonic power of 600 W, ultrasonic processing time of 3 minutes (ultrasonic pulse interval: 5 seconds on, 5 seconds off), ultrasonic frequency of 300 Hz, and water bath temperature of 35℃. The resulting enriched refined olive oil A was obtained after ultrasonic crushing.

[0062] S7, Separation Filtration:

[0063] The enriched refined olive oil A was separated into solid particles and clarified olive oil B using a disc centrifuge (centrifugation acceleration 3500g, separation time 10min). Clarified olive oil B was in the upper layer, and the solid particles settled in the lower layer. The clarified olive oil B was left to stand in the dark for 24 hours and then filtered using an industrial-grade oil filter to obtain the final clear and impurity-free polyphenol-enriched olive oil C.

[0064] Example 3

[0065] This embodiment provides a method for preparing polyphenol-refined olive oil, including the following steps:

[0066] S1, Olive leaf selection and cleaning:

[0067] The operation steps are the same as those in Example 1.

[0068] S2, Dark static preprocessing:

[0069] The operation steps are the same as those in Example 1.

[0070] S3, Microwave extraction of olive leaf polyphenols:

[0071] Olive leaf powder B, which had been pretreated by standing in the dark, was mixed with 80% ethanol aqueous solution (the extraction reagent did not contain choline chloride or malic acid) at a mass ratio of 1:10. Microwave-assisted extraction was performed at a power of 700W for 15 minutes at a temperature of 50℃. After treatment, olive leaf polyphenol ethanol extract A was obtained by using a self-cleaning filter.

[0072] S4, Dechlorosis treatment with olive leaf polyphenol extract:

[0073] The operation steps are the same as those in Example 1.

[0074] S5, freeze-dried into powder:

[0075] The operation steps are the same as those in Example 1.

[0076] S6, Ultra-high voltage coupled ultrasonic pulse enrichment processing:

[0077] The operation steps are the same as those in Example 2.

[0078] S7, Separation Filtration:

[0079] The operation steps are the same as those in Example 2.

[0080] Example 4

[0081] This embodiment provides a method for preparing polyphenol-refined olive oil, including the following steps:

[0082] S1, Olive leaf selection and cleaning:

[0083] The olive leaf raw materials, mixed in equal proportions, are washed multiple times to remove dust, insect eggs, and other contaminants adhering to the leaves, resulting in clean olive leaves A. Note: The time interval between olive leaves A and their removal from the tree should not exceed 48 hours to ensure that the endogenous enzymes in the olive leaves maintain a high level of enzyme activity.

[0084] S2, Dark static preprocessing:

[0085] Fresh, clean olive leaves (A) were mixed with pure water and placed in a high-speed homogenizer at a ratio of 1:30 (g / mL). The mixture was stirred at 50 rpm, and a small amount of acetic acid was added to adjust the pH to 6.0. The temperature was controlled at 40°C, and the ambient humidity was 50%. The mixture was stirred in the dark for 24 hours. The homogenizer was then turned off, and an appropriate amount of sodium bicarbonate was added to adjust the pH to 7.0. The solution was then allowed to stand in the dark for another 24 hours. The solution was dried at 40°C using a vacuum dryer. All dried solids were collected, pulverized using a grinder, and passed through a 100-mesh sieve to obtain olive leaf powder (B) (referred to as "reactant B").

[0086] S3, Microwave extraction of olive leaf polyphenols using a eutectic solvent coupled with a micro-extraction method:

[0087] Preparation of the eutectic solvent (extraction reagent): Choline chloride and malic acid were mixed at a mass ratio of 1:2 and stirred rapidly at 200 rpm in an 80°C water bath for 5–10 minutes to form a viscous, clear liquid. 1.5 times the volume of water was added and mixed thoroughly to obtain the eutectic solvent required for extraction.

[0088] Olive leaf powder B was mixed with a pre-prepared eutectic solvent at a mass ratio of 1:10, and microwave-assisted extraction was performed: microwave power 700W, microwave treatment time 15min, and treatment temperature 50℃. After treatment, olive leaf polyphenol DES extract A (also referred to as "extract C") was obtained through a self-cleaning filter.

[0089] S4, Dechlorosis treatment with olive leaf polyphenol extract:

[0090] Add glacial acetic acid to olive leaf polyphenol DES extract A to adjust the pH to 3.0, mix thoroughly, then slowly add 5% (w / v) sodium bicarbonate solution to adjust the pH to approximately 9.5. Let stand at -20℃ for 2 hours. Chlorophyll precipitates under the control of pH and temperature, while olive leaf polyphenols remain in the DES extract. Take the filtered DES extract (referred to as "extract D") and mix it with 40% ethanol solution at a 1:1 volume ratio, stir thoroughly, let stand to separate the layers, and take the upper 40% ethanol aqueous phase to obtain clear olive leaf polyphenol extract B (referred to as "extract E"), free of chlorophyll.

[0091] S5, freeze-dried into powder:

[0092] Olive leaf polyphenol extract B was distilled to separate ethanol and water using a distillation apparatus at a steam pressure of 0.2 MPa until no ethanol remained in the solution. The anhydrous ethanol obtained from distillation was collected for recycling, while the aqueous solution and the precipitated material were stored together at -20°C for 12 hours. The frozen aqueous solution and the precipitated material were then freeze-dried to obtain dried olive leaf enriched powder A.

[0093] The freeze-drying process conditions were -40℃, 20Pa vacuum, and 24h processing time. Olive leaf enriched powder A was further pulverized using a vibrating ultrafine pulverizer, and then filtered through a vibrating screen with a mesh size of 400 to obtain olive leaf enriched powder B.

[0094] S6, Ultra-high voltage coupled ultrasonic pulse enrichment processing:

[0095] Olive leaf enrichment powder B was mixed evenly with refined olive oil at a mass ratio of 1:10. Ultra-high pressure technology was used to dissolve the active ingredients in the olive leaf enrichment powder B into the oil at a pressure of 200 MPa for 3 minutes. The enrichment powder and oil were then processed using an ultrasonic pulse crusher with the following parameters: ultrasonic power of 600 W, ultrasonic processing time of 3 minutes (ultrasonic pulse interval: 5 seconds on, 5 seconds off), ultrasonic frequency of 300 Hz, and water bath temperature of 35℃. The resulting enriched refined olive oil A was obtained after ultrasonic crushing.

[0096] S7, Separation Filtration:

[0097] The enriched refined olive oil A was separated into solid particles and clarified olive oil B using a disc centrifuge (centrifugation acceleration 3500g, separation time 10min). Clarified olive oil B was in the upper layer, and the solid particles settled in the lower layer. The clarified olive oil B was left to stand in the dark for 24 hours and then filtered using an industrial-grade oil filter to obtain the final clear and impurity-free polyphenol-enriched olive oil C.

[0098] Example 5

[0099] This embodiment provides a method for preparing polyphenol-refined olive oil, including the following steps:

[0100] S1, Olive leaf selection and cleaning:

[0101] The olive leaf raw materials, mixed in equal proportions, are washed multiple times to remove dust, insect eggs, and other contaminants adhering to the leaves, resulting in clean olive leaf A. Note: The time interval between olive leaf A and its removal from the tree should not exceed 48 hours to ensure that the endogenous enzymes in the olive leaves maintain a high level of enzyme activity.

[0102] S2, Pulverization treatment (instead of the dark settling pretreatment in Example 4):

[0103] Fresh, clean olive leaves A are crushed using a pulverizer and passed through a 100-mesh sieve to obtain olive leaf powder B (referred to as "reactant B").

[0104] S3, Microwave extraction of olive leaf polyphenols using a eutectic solvent coupled with a micro-extraction method:

[0105] Preparation of the eutectic solvent (extraction reagent): Choline chloride and malic acid were mixed at a mass ratio of 1:2 and stirred rapidly at 200 rpm in an 80°C water bath for 5–10 minutes to form a viscous, clear liquid. 1.5 times the volume of water was added and mixed thoroughly to obtain the eutectic solvent required for extraction.

[0106] Olive leaf powder B was mixed with a pre-prepared eutectic solvent at a mass ratio of 1:10, and microwave-assisted extraction was performed: microwave power 700W, microwave treatment time 15min, and treatment temperature 50℃. After treatment, olive leaf polyphenol DES extract A (also referred to as "extract C") was obtained through a self-cleaning filter.

[0107] S4, Dechlorosis treatment with olive leaf polyphenol extract:

[0108] Add glacial acetic acid to olive leaf polyphenol DES extract A to adjust the pH to 3.0, mix thoroughly, then slowly add 5% (w / v) sodium bicarbonate solution to adjust the pH to approximately 9.5. Let stand at -20℃ for 2 hours. Chlorophyll precipitates under the control of pH and temperature, while olive leaf polyphenols remain in the DES extract. Take the filtered DES extract (referred to as "extract D") and mix it with 40% ethanol solution at a 1:1 volume ratio, stir thoroughly, let stand to separate the layers, and take the upper 40% ethanol aqueous phase to obtain clear olive leaf polyphenol extract B (referred to as "extract E"), free of chlorophyll.

[0109] S5, freeze-dried into powder:

[0110] Olive leaf polyphenol extract B was distilled to separate ethanol and water using a distillation apparatus at a steam pressure of 0.2 MPa until no ethanol remained in the solution. The anhydrous ethanol obtained from distillation was collected for recycling, while the aqueous solution and the precipitated material were stored together at -20°C for 12 hours. The frozen aqueous solution and the precipitated material were then freeze-dried to obtain dried olive leaf enriched powder A.

[0111] The freeze-drying process conditions were -40℃, 20Pa vacuum, and 24h processing time. Olive leaf enriched powder A was further pulverized using a vibrating ultrafine pulverizer, and then filtered through a vibrating screen with a mesh size of 400 to obtain olive leaf enriched powder B.

[0112] S6, Ultra-high voltage coupled ultrasonic pulse enrichment processing:

[0113] Olive leaf enrichment powder B was mixed evenly with refined olive oil at a mass ratio of 1:10. Ultra-high pressure technology was used to dissolve the active ingredients in the olive leaf enrichment powder B into the oil at a pressure of 200 MPa for 3 minutes. The enrichment powder and oil were then processed using an ultrasonic pulse crusher with the following parameters: ultrasonic power of 600 W, ultrasonic processing time of 3 minutes (ultrasonic pulse interval: 5 seconds on, 5 seconds off), ultrasonic frequency of 300 Hz, and water bath temperature of 35℃. The resulting enriched refined olive oil A was obtained after ultrasonic crushing.

[0114] S7, Separation Filtration:

[0115] The enriched refined olive oil A was separated into solid particles and clarified olive oil B using a disc centrifuge (centrifugation acceleration 3500g, separation time 10min). Clarified olive oil B was in the upper layer, and the solid particles settled in the lower layer. The clarified olive oil B was left to stand in the dark for 24 hours and then filtered using an industrial-grade oil filter to obtain the final clear and impurity-free polyphenol-enriched olive oil C.

[0116] Using commercially available refined olive oil and commercially available extra virgin olive oil as controls, the components of polyphenol-enriched olive oil C prepared in the above experimental examples were tested. The test indicators and methods included:

[0117] 1) Determination of total polyphenol content in olive oil:

[0118] Accurately weigh 2.0 g of olive oil into a 10 mL screw-cap test tube. Add 1.0 mL of internal standard solution (0.015 mg / mL syringic acid). Seal with the screw cap and shake for 30 seconds. Add 5 mL of methanol / water (8:2, v / v) extraction solution and shake for 1 minute. Extract in a 500 W ultrasonic bath at room temperature for 15 minutes. Centrifuge at 5000 rpm for 25 minutes. Collect the supernatant and filter through a 0.45 μm filter.

[0119] A Model 1220 series high-performance liquid chromatograph (Agilent, USA) equipped with an Agilent TC-C18 column was used. Mobile phase A was 0.2% phosphoric acid aqueous solution, and mobile phase B was methanol and acetonitrile (1:1, v / v). The elution flow rate was 1 mL / min. The oven temperature was set to 30 °C, and the sample injection volume was 10 μL. The UV detector provided a signal at 280 nm.

[0120] Elution gradient: 0 min, 4% B; 40 min, 50% B; 45 min, 60% B; 60 min, 100% B; 70 min, 100% B; 72 min, 4% B; 82 min, 4% B.

[0121] Inject 10 μL of external calibration standard solution (0.030 mg / mL tyrosol solution and 0.015 mg / mL syringic acid solution) and record the chromatogram at 280 nm. Calculate and record the response factor ratio of syringic acid to tyrosol, referred to as RRFsyr / tyr.

[0122] 10 μL of the sample solution was injected into the HPLC system, and the chromatogram was recorded at 280 nm. The total polyphenol content of olive oil was calculated by comparing the sum of the peak areas of the main biophenolic compounds (hydroxytyrosol, tyrosol, oleuropein, and privetin and their respective derivatives) in the chromatogram with the peak area of ​​the internal standard compound syringic acid. Three independent determinations were performed on the same sample. The results are expressed as milligrams of compounds per kilogram of oil.

[0123] The calculation formula is as follows:

[0124] RF1μg (Syringic Acid) = Area of ​​Syringic Acid / Number of μg of Syringic Acid Injected

[0125] RF1μg (tyrosol) = Area of ​​tyrosol / Number of μg of tyrosol injected

[0126] RRFsyr / tyr = RF1μg (syringic acid) / RF1μg (tyrosol)

[0127]

[0128] ∑A = the sum of the peak areas of biophenols (hydroxytyrosol, tyrosol, natural and oxidized oleuropein and privetin derivatives, flavonoids and phenolic acids) recorded at 280 nm;

[0129] As = the area of ​​the syringic acid internal standard recorded at 280 nm;

[0130] W = Weight of oil used, in grams;

[0131] Ws = The weight, in mg, of eugenol added to 1 mL of the solution in the sample as an internal standard.

[0132] 2) Determination of the content of single polyphenol compounds in olive oil:

[0133] Weigh 10.0 mg each of the standards for hydroxytyrosol, luteolin, oleacein, and quercetin, and dilute to 10 mL in a brown volumetric flask to obtain a 1.0 mg / mL standard solution. Dilute with methanol to concentrations of 1.0 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, 0.2 mg / mL, and 0.1 mg / mL, respectively. Record the peak area of ​​each compound at 280 nm using the HPLC method described above. Plot a standard curve of peak area versus concentration gradient for each compound. Substitute the peak area corresponding to the sample solution's chromatogram into the calculation to obtain the content.

[0134] 3) Determination of chlorophyll content in olive oil:

[0135] Take 2 mL of olive oil sample and dilute it 10-fold with n-hexane. Measure the absorbance of the solution using a spectrophotometer, and determine the chlorophyll content of the olive oil by colorimetry. The maximum absorption wavelength of chlorophyll is 670 nm. 613 is the specific extinction coefficient of chlorophyll. The pigment concentration is calculated as follows:

[0136] Chlorophyll (mg / kg) = (A 670 ×10 6 ) / (613×100×d)

[0137] Where A is absorbance and d is the thickness of the spectrophotometer cell (1 cm).

[0138] 4) The antioxidant activity of olive oil was tested using the DPPD method and the ABTS method, respectively.

[0139] The test statistics are shown in the table below. Figures 1 to 3 :

[0140]

[0141]

[0142] As can be seen from the table above, the total phenol content in the polyphenol-refined olive oils prepared in Examples 1 and 4 is much higher than 250 mg / kg, while the chlorophyll content is less than 15 mg / kg. In particular, the polyphenol-refined olive oil prepared in Example 4 has a total phenol content much higher than 250 mg / kg, and also contains hydroxytyrosol, luteolin, quercetin, and oleacein. The antioxidant activity of the olive oil is greatly enhanced, resulting in significant health benefits.

[0143] Additionally, it is worth noting that:

[0144] 1) The preparation process of olive leaf polyphenol extract relies on a combination of dark settling treatment, microwave-assisted extraction, and dechlorosis treatment to obtain olive leaf polyphenol extract with high polyphenol content and chlorophyll removal, which significantly increases the polyphenol content.

[0145] 2) Polyphenol-refined olive oil is enriched into conventional refined olive oil by utilizing olive leaf polyphenol extracts, with ultra-high pressure coupled ultrasonic pulse enrichment technology being preferred for better results.

[0146] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An olive leaf polyphenol extract, characterized in that, The preparation method of the olive leaf polyphenol extract includes the following steps: S1, Obtain olive leaf raw material, wash it, and obtain material A; S2, mix material A with water, add acid to adjust the pH to 5.5~6.5, carry out the reaction in the dark under stirring conditions, then add alkaline solution to adjust the pH to 6.5~7.0, let the reaction stand in the dark, vacuum dry, and pulverize to obtain reactant B; S3, Mix reactant B with extraction solvent, wherein the extraction solvent is selected from an aqueous solution containing choline chloride and malic acid, and perform microwave-assisted extraction to obtain extract C; S4, add acid to the extract C to adjust the pH to 2.5~3.5, mix well, then add alkaline solution to adjust the pH to 9.5~10.0, let stand at low temperature to precipitate chlorophyll, filter to obtain extract D; S5, mix the extract D with a 20%~55% ethanol aqueous solution, let it stand to separate into layers, and collect the ethanol aqueous phase extract E; S6. Distill the aqueous ethanol extract E to obtain the ethanol-free product, dry it, and obtain olive leaf extract powder. The temperature for both the dark-avoidance reaction and the dark-restoration reaction was 35~45℃, the ambient air humidity was 45%~50%, and the reaction time was 20~24h. The extraction solvent is selected from an aqueous solution containing choline chloride and malic acid in a mass ratio of 1:2 to 1:

4. The mass ratio of reactant B to extraction solvent is 1:10 to 1:

14. The microwave-assisted extraction process conditions are: microwave power 600 to 700W, microwave treatment time 15 to 30 minutes, and treatment temperature 40 to 50℃.

2. The olive leaf polyphenol extract according to claim 1, characterized in that, In steps S2 and S4, the acid used is glacial acetic acid, and the alkaline solution used is an aqueous solution of sodium bicarbonate.

3. The olive leaf polyphenol extract according to claim 1 or 2, characterized in that, The volume ratio of extract D to aqueous ethanol solution is 1:1 to 1:

4.

4. The olive leaf polyphenol extract according to claim 1 or 2, characterized in that, The distillation process parameters are: steam pressure 0.2~2.0 MPa.

5. A method for preparing polyphenol-refined olive oil, characterized in that, Includes the following steps: Obtain the olive leaf polyphenol extract according to any one of claims 1 to 4; The olive leaf polyphenol extract is mixed with refined olive oil at a mass ratio of 1:15 to 1:40, and then subjected to high-pressure solubilization and / or pulsed ultrasonic solubilization and enrichment treatment. After centrifugation and filtration, refined olive oil with high polyphenol content is obtained.

6. The method for preparing polyphenol-refined olive oil according to claim 5, characterized in that, The process parameters for the high-pressure catalytic melting treatment are: pressure 150~250MPa, treatment time 3~10min.

7. The method for preparing polyphenol-refined olive oil according to claim 5, characterized in that, The process parameters for the pulsed ultrasonic dissolution treatment are as follows: ultrasonic power is 450~650W, total ultrasonic treatment time is 2~10min, ultrasonic pulse interval is 10s working and 5s stopping, ultrasonic frequency is 250~350Hz, and water bath temperature is 30~45℃.

8. A polyphenol-refined olive oil prepared by the preparation method according to any one of claims 5 to 7.

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