A simple method for preparing chlorophyll free phenols from olive leaves and efficiently enriching it in refined olive oil

A simple method for preparing chlorophyll free phenols from olive leaves and efficiently enriching it in refined olive oil | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A simple method for preparing chlorophyll free phenols from olive leaves and efficiently enriching it in refined olive oil Yunfei Huang, Wenqing He, Ruifeng Wang, Yangyang Jia, Lu Li, Yawei Xu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4020617/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract The refining process almost completely removes phenols from refined olive oil (ROO). Enriching ROO with olive phenols can significantly enhance its quality and health benefits. However, current enrichment methods are inefficient and overlook the negative impact of chlorophyll present in the phenol extract on the oil. In this study, we developed a straightforward two-step dissolve and resaturate process to prepare chlorophyll-free phenols from olive leaves and efficiently enrich ROO using the ultrasonic pulsed probe method. Under optimal conditions, the total phenol content in ROO increased by 424 mg/kg, including increases of 158.5 mg/kg (37.4%) for oleacein, 29.5 mg/kg (7.0%) for hydroxytyrosol, and 17.9 mg/kg (4.2%) for oleuropein aglycone. This also significantly enhanced the antioxidant activity, oxidative stability, and some flavor characteristics of ROO. Our study offers a straightforward, practical, and effective strategy for the valuable use of olive leaves and for improving the quality of ROO. refined olive oil olive leaves olive phenols phenol-enriched olive oil oleacein antioxidant activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Extra virgin olive oil (EVOO) is considered one of the healthiest vegetable oils and a crucial component of the Mediterranean diet, thanks to its high levels of oleic acid, phytosterols, tocopherols, and phenols (Sánchez de Medina et al. 2017). The phenolic composition is considered the main reason for EVOO's health benefits (Gorzynik-Debicka et al. 2018 ). The European Food Safety Authority (EFSA) has issued a health declaration stating that every 1000 grams of olive oil must contain at least 250 mg of total phenolic compounds, such as hydroxytyrosol and its derivatives (EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) 2011 ). However, refined olive oil (ROO) almost completely lost phenols due to the refining process, which includes filtering, neutralization, distillation, degumming, bleaching, and high-heat deodorization, significantly falling short of the healthy concentrations required by EFSA health claims. Therefore, enriching refined olive oil with olive polyphenols is an effective strategy to enhance its quality and health benefits. Hydroxytyrosol, oleacein and oleuropein aglycone are representative phenols in olive oil. They have been reported to contribute to olive oil's health benefits, including anticancer, anti-atherosclerotic, anti-obesity, anti-inflammatory, and antioxidant activities (Y. Huang et al. 2024 ). Coincidentally, olive leaves, rich in phenolic compounds like hydroxytyrosol, oleuropein, luteolin-7-O-glucoside, and oleacein, which are also desirable in olive oil, suggest that olive leaves can serve as an economical raw material for the phenolic enrichment of ROO. Enriching ROO with olive leaf phenols could improve their quality and health benefits and also valorize olive leaves, typically used as animal forage or burned as waste (Özcan and Matthäus 2017 ). Currently, two methods have been reported for enriching oil with phenols using olive leaves as raw materials. The first method involves macerating crushed olive leaves in the oil for a week and then filtering out the leaf fragments (Baccouri et al. 2022 ; Japón-Luján et al. 2008 ; Nenadis et al. 2010 ). This method is simple but time-consuming and offers low enrichment, increasing the total phenol content by only 20 mg/kg (Sevim 2013 ). The second method involves extracting target phenols from the leaves with alcohol solutions, then mixing the phenolic extract with the oil, and removing the alcohol from the two-phase system using a rotary evaporator (Sánchez de Medina et al. 2011). Japón-Luján and Luque De Castro (2008) extracted phenols from olive leaves using ethanol, mixed the alcoholic extract with the oil, and then stirred and centrifuged it to obtain oil enriched mainly with oleuropein, apigenin-7-glucoside, luteolin-7-glucoside, and verbascoside. Although this technique is simple and improves phenol enrichment to 80mg/kg, it still falls far short of meeting public health needs. Moreover, it is time-consuming and inefficient, increases the oil's viscosity, and leaves residual alcohol, resulting in poor oil flavor and characteristics. Additionally, previous studies overlooked the negative impact of chlorophyll co-existing in the olive leaf phenol extract on the oil, which not only darkened its color but also increased photosensitivity and significantly reduced its oxidative stability. Therefore, developing a method to prepare chlorophyll-free olive leaf phenol extracts is urgently needed. This study aims to develop a simple and effective method for preparing chlorophyll-free phenol extracts from olive leaves and efficiently enriching them in ROO. The quality and sensory characteristics of the ROO before and after enrichment were comprehensively evaluated. We believe our data will offer practical and effective strategies for the high-value utilization of olive leaves and improving the quality of ROO. 2. Materials and methods 2.1. Samples, Standards, and Reagents Tender olive leaves for this research were collected in October 2022 from ‘Picual’ in Xichang City, Sichuan Province, China. Subsequently, they were freeze-dried at -40°C for 48 hours, ground to a uniform particle size (diameter ≤ 0.4 mm) using an ultra-micro-mill, and stored at 4℃ until use. ROO was sourced from Monini, Italy, and the two extra-virgin olive oils (EVOO1, EVOO2) were purchased from Mueloliva, Spain, and Olivoila, China. Methanol and acetonitrile reagents for high-performance liquid chromatography (HPLC) were sourced from Fisher, USA. Deionized water in the mobile phase was obtained from Millipore Milli-Q water purification system, USA. Standards for tyrosol, oleuropein, apigenin, luteolin-7-glucoside, luteolin, and quercetin were obtained from Yuan Ye, China; oleacein standards were from Phytolab, Germany. Each standard (10 mg) was dissolved in 10 mL of methanol or acetonitrile, then diluted to various concentrations. The peak area for each concentration was measured by HPLC, using the peak area as the y-axis and concentration as the x-axis to create a standard curve. Solutions were stored in the dark at -20℃ until needed. 2.2. Enrichment of ROO with Phenols Extracts from Olive Leaves 2.2.1. Treatment of Chlorophyll Olive Oil (COO) Olive leaf powder was mixed with an 80% ethanol solution at a 1:15 mass ratio and extracted using ultrasound at 490 W for 30 minutes at 30°C. Phenolic extracts were obtained through filtration. This extraction process was repeated three times. The phenolic extract was rotary evaporated to dryness and then freeze-dried for 30 hours to yield a green solid powder for enrichment. The enriched powder was mixed with ROO in a 1:5 mass ratio, and ultrasonic pulse treatment was performed with a water bath set at 35℃. Ultrasonic power was set at 300 W, treatment time at 20 minutes, and frequency at 300 Hz. Clarified enriched olive oil, termed Chlorophyll Olive Oil (COO), was obtained by centrifuging at 10,000 rpm for 7 minutes and filtering. 2.2.2. Treatment of Phenolic-Enriched Olive Oil (PEOO) Phenols were extracted from olive leaf powder using 80% ethanol in water as previously described. The phenolic extract was then rotary evaporated to dryness. The extract was fully solubilized in a 40% ethanol solution, ultrasonicated for 5 minutes at 490W and 30°C, then transferred to a 50ml centrifuge tube and centrifuged at 8000 rpm for 5 minutes. Chlorophyll was precipitated, and the supernatant was retained. The extract was freeze-dried for 30 hours to yield a yellow solid powder for enrichment. The enriched powder was mixed with ROO in mass ratios of 1:3, 1:5, 1:10, 1:15, and 1:20. Ultrasonic power settings were 150W, 188W, 225W, 263W, 300W. Ultrasonic treatment times were 0, 5, 10, 15, and 20 minutes. Ultrasonic pulse treatment was conducted with a water bath maintained at 35℃. Ultrasonic frequency was set at 300 Hz. Clarified, enriched olive oil, termed Treatment of Phenolic-Enriched Olive Oil (PEOO), was obtained. 2.2.3. Treatment of Ultrasonic Pulsed Probe Olive Oil (UPOO) The extraction process was as previously described. The enriched powder was mixed with ROO at a 1:10 mass ratio. Ultrasonic power was set at 225W. Ultrasonic treatment time was 5 minutes. Ultrasonic pulse treatment was conducted with a water bath at 35℃. Ultrasonic frequency was set at 300 Hz. Clarified, enriched olive oil, termed Ultrasonic Pulsed Probe Olive Oil (UPOO), was obtained. 2.2.4. Treatment of Vibratory Olive Oil (VOO) Prepared the yellow solid powder used for enrichment according to the method of FEOO. The enriched powder was mixed with ROO at a 1:5 mass ratio and shaken in the dark for 120 minutes at 190 rpm in a water bath at 35°C. Clarified, enriched olive oil, termed Vibratory Olive Oil (VOO), was obtained by centrifuging at 10,000 rpm for 7 minutes and filtering. 2.2.5. Treatment of Ultrasonic-Bath Olive Oil (UBOO). The initial process aligns with the above description. The enriched powder was mixed with ROO at a 1:5 mass ratio and ultrasonicated in a water bath at 35°C for 20 minutes using an ultrasonic cleaner. Ultrasonication frequency was set at 300 Hz. Clarified Ultrasonic-Bath Olive Oil (UBOO) was obtained by centrifuging at 10,000 rpm for 7 minutes. 2.3. Determination of Phenol Content in ROO and Enriched Olis by HPLC-DAD. Total phenolic content was determined using the method of (Sánchez de Medina et al. 2011) expressed as gallic acid equivalents per gram. A model 1220 series high-performance liquid chromatograph (Agilent, USA), equipped with a TC-C18 column (250 mm × 4.6 mm), was utilized. Mobile phase A consisted of 0.2% phosphoric acid in water, and mobile phase B was a 1:1 (v/v) mixture of methanol and acetonitrile. The flow rate was set at 1.0 mL/min. The column temperature was maintained at 30°C, with a sample injection volume of 10 µL. The gradient profile was as follows: 0 min at 4% B, progressing to 50% B at 40 min, 60% B at 45 min, 100% B from 60 to 70 min, and returning to 4% B at 72 min, maintained until 82 min. Chromatograms were obtained at 280 nm. Standard curves were created using the concentrations of tyrosol, oleuropein, apigenin, luteolin-7-glucoside, luteolin, quercetin, oleacein, and their corresponding peak areas in the chromatograms. Quantitative analysis was performed by using the chromatographic peak areas. Quantitative analysis for hydroxytyrosol and oleuropein aglycone utilized the internal standard method with syringic acid, expressing results in tyrosol equivalents per gram. A 10 µL injection of an external calibration standard solution (0.030 mg/mL tyrosol versus 0.015 mg/mL syringic acid) was administered, with chromatograms recorded at 280 nm. The response factor ratio of syringic acid to tyrosol, recorded as RRFsyr/tyr, was calculated. The content of hydroxytyrosol and oleuropein aglycone was calculated by comparing their peak areas in the chromatograms to that of syringic acid. The calculation formula is as follows: Three independent determinations were conducted on the same sample. Results were reported in milligrams of compounds per kilogram of oil. $$（mg/kg）=\frac{A\times 1000\times {RRF}_{syr/tyr}\times Ws}{As\times W}$$ A: is the peak areas of the hydroxytyrosol or oleuropein aglycone recorded at 280 nm; As: is the area of the syringic acid internal standard recorded at 280 nm; W: is the weight of the oil used in g; Ws: is the weight of the syringic acid used as internal standard in 1 mL of solution added to the sample, in mg. 2.4. Characterization of Olive Phenols by LC-TOF/MS. The phenolic composition of olive oil and leaves was determined using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-TOF/MS). For phenolic composition analysis, a Waters Vion IMS QTOF (USA) equipped with an ESI ion source in negative ion mode was used. Settings included a source voltage of 3 kV, source temperature of 150°C, m/z range of 115–1000, collision energy of 10–25 eV, column temperature of 40°C, and a flow rate of 0.25 mL/min. The elution gradient followed this sequence: starting at 4% B at 0 min, increasing to 50% B by 16 min, 60% B by 18 min, reaching 100% B at 24 min, maintained through 28 min, then returning to 4% B at 28.8 min and maintained until 32.8 min. An injection volume of 1 µL was used, with all samples being filtered through 0.22 µm nylon filters prior to analysis. 2.5. Determination of Color Parameters 2.5.1. L*, a*, b*, Y, x, y The Ultrascan VIS (HunterLab) system was employed to measure the colorimetric values of olive oil samples. Initially, calibration was performed using a standard black and white board. Subsequently, the olive oil was positioned in a vessel under a light source to record the values of L*, a*, b*, Y, x, and y. L* and Y indicate luminance, with higher values signifying brighter colors. a* and x represented the degree of red-green, where positive values indicate red and negative values green. b* and y indicated the degree of yellow-blue, with positive values signifying yellow and negative values blue. Each sample is measured three times in parallel. 2.5.2. C*, h* The chromaticity (C*) and hue angle (h*) of the olive oil were calculated using a* and b*. C* represents color saturation, with larger values indicating higher purity. h* represents the color angle, indicating hue within a 0-360 range, with 0 for red, 180 for green, and 360 for blue. The calculation formulas are as follows: $${C}^{\text{*}}=\sqrt{{a}^{\text{*}2}+{b}^{\text{*}2}}$$ $${h}^{\text{*}}={\text{tan}}^{-1}(\frac{{b}^{\text{*}}}{{a}^{\text{*}}}\left)\right({a}^{\text{*}}>0,{b}^{\text{*}}>0)$$ $${h}^{\text{*}}={\text{tan}}^{-1}(\frac{{b}^{\text{*}}}{{a}^{\text{*}}})+180({a}^{\text{*}}0)$$ 2.5.3. Chlorophyll and Carotenoids Following the method of (Tarchoune et al. 2019 ), 2 mL of olive oil was diluted tenfold with hexane. The absorbance of the solution was measured using a spectrophotometer. The chlorophyll content's maximum absorption wavelength was at 670 nm, while that of carotenoids was at 470 nm. The specific extinction coefficients are 613 for chlorophyll and 2000 for carotenoids, respectively. The pigment concentration calculation formula is as follows: Chlorophyll (mg/kg) = (A 670 × 10 6 ) / (613 × 100 × d) Carotenoids (mg/kg) = (A 470 × 10 6 ) / (2000 × 100 × d) Where A is the absorbance and d is the spectrophotometer cell thickness (1cm). 2.6. Free Acidity, Peroxide Value and Oxidative Stability 2.6.1. Free Acidity Free acidity, indicating the free fatty acid content in oils and fats, is a crucial quality parameter. A 10g sample of olive oil was diluted to 50mL with an ether-ethanol solution (1:1, v/v). A 0.1M potassium hydroxide ethanol solution served as the titrant, with phenolphthalein as the indicator, until the pink color persisted for at least 10 seconds. Free acidity in the oil sample was expressed as oleic acid percentage (g oleic acid/kg oil). The calculation formula is as follows: $$Acid Value=V\times c\times \frac{M}{1000}\times \frac{100}{m}=\frac{V\times C\times M}{10\times m}$$ V: volume of potassium hydroxide ethanol solution (mL); c: molar concentration of potassium hydroxide ethanol solution (mol/L); M: molar mass conversion factor for oleic acid (= 282); M: weight of sample oil (g); 2.6.2. Peroxide Value The peroxide value measures substances in the sample that oxidize potassium iodide under specified conditions. 2.0 g of olive oil sample was weighed, 25 mL of chloroform-acetic acid solution (4:6, v/v) added, stirred to dissolve, covered, shaken for 1 minute, and left in the dark at room temperature for 5 minutes. Approximately 75 mL of distilled water and 1.0 g of 10 g/L starch solution were added as an indicator to titrate the released iodine with 0.01 mol/L sodium thiosulfate solution. The titration volume was recorded, and the peroxide value was expressed in milliequivalents of reactive oxygen species per kilogram of oil (meq O 2 /kg oil). Three measurements were taken for the test material, along with a blank test. The formula is as follows: $$PV=\frac{V\times T\times 1000}{m}$$ V: volume of standard sodium thiosulfate solution (mL); T: molar concentration of standard sodium thiosulfate solution (mol/L); M: weight of sample oil (g); 2.6.3. Oxidative Stability The oxidative stability of COO was tested using UV irradiation at 30°C, with peroxide values determined every 24 hours. The oxidative stability of the oils was assessed with a 65℃ oven oxidation test. Each oil sample, weighing 15.0 g, was stored in a constant temperature oven at 65℃. Peroxide values were determined bi-daily at a set time to analyze the degree of fat and oil oxidation over 2 weeks. 2.7. Evaluation of Antioxidant Activity Antioxidant activity in olive oil's polar fractions was assessed using DPPH, ABTS, and FRAP methods. Polar fraction extraction followed Nakbi et al. ( 2010 )'s method, with optimizations. 2.5 g of olive oil was weighed, mixed with 4.5 mL of methanol, and shaken in the dark at 2000 r/min for 20 minutes. The sample stood in the dark until fully separated; the supernatant was then transferred to a 10 mL brown volumetric flask. This process was repeated twice to obtain the polar fractions. Following Nakbi et al. ( 2010 )'s method. 200 µL of the polar extract was diluted in 5 mL of methanol; 2 mL of this solution was mixed with 2 mL of DPPH-methanol and reacted in the dark at room temperature for 2 hours. Absorbance was measured at 517 nm, adjusted to zero using a methanol blank. A standard curve for Trolox concentration versus DPPH scavenging rate (20–400 µmol/L) was plotted as y = 0.1444x-0.3851, R²=0.9935. Trolox equivalent antioxidant activity was calculated and expressed as µmol TE/100g of oil; the scavenging rate was denoted as R. R (%) =[1-(A2-A3)/A1] × 100% A1:2.0mL DPPH-methanol solution + 2.0mL methanol solution; A2:2.0mL DPPH-methanol solution + 2.0mL oil sample methanol dilution; A3:2.0mL oil sample methanol dilution + 2.0mL methanol solution. Following and optimizing Re et al. ( 1999 )'s method. The ABTS free radical solution was left to stand in the dark at room temperature for 12–16 hours. Before measurement, the solution was diluted with methanol to an absorbance (OD) of 0.700 ± 0.020 to prepare the ABTS solution. 200µL of the polar extraction solution was diluted in 5mL methanol, mixed with 2mL ABTS solution in a brown bottle, and left in the dark at room temperature for 20 minutes. Absorbance was measured at 734nm, zeroed with a methanol blank. A standard curve of Trolox concentration versus scavenging rate (20–400 µmol/L) was established as y = 0.0875x + 66.608, R²=0.9588. Trolox equivalent antioxidant activity was calculated, with results expressed as µmol TE/100g of oil and the scavenging rate denoted as R. R (%) = (A2-A1)/A2×100% Al: ABTS solution + methanol dilution of the polar extraction solution of the oil sample; A2: ABTS solution. The Fe³⁺ reducing antioxidant capacity (FRAP) was assessed using Benzie and Strain ( 1996 )'s method, with minor modifications. The FRAP solution was made from 300 mmol/L acetate buffer (pH 6.6), 10 mmol/L TPTZ, and 20 mmol/L FeCl 3 , in a 10:1:1 ratio (v:v:v). 0.5 mL of the sample dilution was mixed with 1.5 mL of preheated FRAP solution at 37℃, reacted for 10 minutes at the same temperature, and absorbance was measured at 593 nm using ethanol as the blank. A standard curve was plotted with Trolox (20–200 µmol/L) as the reference: y = 0.0089x + 0.0701, R²=0.9998. The antioxidant activity of the samples, measured by FRAP, was quantified as the amount of Trolox equivalent required to achieve the same absorbance value (µmol TE/100g). 2.8. Sensory Attribute of the Enriched-Olive Oils A panel of 25 untrained members, randomly chosen from the students and staff of Huazhong Agricultural University's College of Food Science and Technology, tested selected samples for sensory evaluation and consumer preference. Red illumination was used to mask color differences in the oil samples. Sensory assessments took place in quiet, individual compartments. In the consumer acceptability test, panelists rated the samples on a nine-point scale, from non-detectable (0) to very strong (9), for both smell and taste according to the intensity of odor and flavor. In the consumer preference test, panelists ranked the samples according to preference and rated each on a nine-point scale, from very likeable (1) to very dislikeable (9). Each oil's specific flavor was evaluated, with several flavors receiving individual scores. Flavors were rated on a nine-point scale, from undetectable (0) to very strong (9), based on their strength. 2.9. Statistical Analysis All experiments reported mean ± SD from three measurements (n = 3). Multivariate and one-way ANOVA analyses were conducted using SPSS software (version 22.0, USA), with Tukey post hoc tests applied at p < 0.05. Figure 1 . Effect of chlorophyll from olive leaf extracts to olive oil enriched with phenolics. (A) the appearance of refined olive oil (ROO), chlorophyll olive oil (COO) and two extra virgin olive oil (EVOO1 and EVOO2). (B) changes in peroxide values of ROO, phenolic-enriched olive oil (PEOO), and COO with light. Table 1 Color parameters of seven groups of oil. ROO VOO UBOO COO PEOO EVOO1 EVOO2 L* 93.23 ± 0.01 92.98 ± 0.18 90.19 ± 0.10 23.48 ± 0.32 88.24 ± 0.16 86.97 ± 0.04 83.98 ± 0.08 a* -3.05 ± 0.02 -3.36 ± 0.05 -3.64 ± 0.16 -7.24 ± 0.06 -5.32 ± 0.05 -2.69 ± 0.01 -0.05 ± 0.03 b* 15.39 ± 0.16 16.09 ± 0.62 16.72 ± 0.10 19.66 ± 0.30 27.06 ± 0.26 88.96 ± 1.02 97.22 ± 1.56 Y 83.50 ± 0.02 82.58 ± 0.49 81.74 ± 0.09 3.92 ± 0.10 72.56 ± 0.34 69.94 ± 0.07 64.03 ± 0.15 x 0.34 ± 0.00 0.34 ± 0.00 0.34 ± 0.00 0.37 ± 0.00 0.36 ± 0.00 0.45 ± 0.00 0.47 ± 0.00 y 0.36 ± 0.00 0.37 ± 0.00 0.37 ± 0.00 0.45 ± 0.00 0.39 ± 0.00 0.48 ± 0.00 0.49 ± 0.00 C* 15.69 ± 0.16 16.43 ± 0.61 17.11 ± 0.10 20.95 ± 0.27 27.58 ± 0.26 89.00 ± 1.02 97.22 ± 1.56 H* 178.63 ± 0.00 178.64 ± 0.00 178.64 ± 0.01 178.78 ± 0.01 178.62 ± 0.00 178.46 ± 0.00 178.43 ± 0.00 Chlorophyll (mg/kg) 2.50 ± 0.25 3.05 ± 0.34 4.08 ± 0.65 192.55 ± 3.22 11.96 ± 0.25 9.03 ± 0.34 12.72 ± 0.59 Carotenoids(mg/kg) 1.62 ± 0.08 1.80 ± 0.10 2.22 ± 0.25 49.79 ± 1.18 5.82 ± 0.06 7.73 ± 0.08 8.68 ± 0.104 3. Results and discussion 3.1. Removal of chlorophyll from phenol extract of olive leaves is essential for the quality of phenolic-enriched oil. While olive leaves are excellent for phenolic enrichment of olive oil due to their high phenolic content, alcohol extraction also inevitably removes chlorophyll. Following previous methods (Sánchez de Medina et al. 2011), we first extracted phenols from olive leaves with alcohol and then enriched the olive oil with this extract. As depicted in Fig. 1 A, chlorophyll present in olive leaf phenolic extracts colored the olive oil dark green, significantly detracting from its visual appeal. After enriching with dried phenolic extracts in an 80% ethanol solution, the chlorophyll content in ROO rose from 2.50 ± 0.25 mg/kg to 192.55 ± 3.22 mg/kg. Besides affecting ROO's appearance, photosynthetic pigments like chlorophyll can also cause unwanted photosensitized oxidation. Figure 1 B illustrates that PEOO and COO underwent identical extraction and enrichment processes, except COO did not undergo chlorophyll removal. Following enrichment, the peroxide values of both PEOO and COO slightly increased but remained similar. When the three groups of oils were exposed to UV light continuously, the peroxide value of COO increased by 25.82 meq O 2 /kg after 24 hours, which was much higher than that of ROO (3.31 meq O 2 /kg) and PEOO (5.07 meq O 2 /kg). The peroxide value of COO increased much higher than ROO and PEOO after 48 and 72 hours of UV irradiation. Clearly, chlorophyll significantly affects not just the appearance but also the oxidative stability of olive oil. However, previous studies on olive oil enrichment from olive leaves overlooked chlorophyll's impact (Japón-Luján and Luque De Castro 2008; Malheiro et al. 2013 ; Sánchez de Medina et al. 2011; Vidal et al. 2022 ). For example, Japón-Luján and Luque De Castro (2008) extracted olive leaf phenols using ethanol, and mixed and shaking it with olive oil to obtain a phenol-rich oil, without mentioning the effect of chlorophyll in it. Therefore, developing an effective method to remove chlorophyll from olive leaf phenolic extracts while preserving the original phenolic compounds is essential. 3.2. Developing a practical strategy for preparing chlorophyll free olive phenols extract from olive Leaves. Preliminary tests indicate that the primary phenols in olive leaves, akin to those in olive oil, include hydroxytyrosol, luteolin-7-O-glucoside, oleacein, and oleuropein aglycone. Given health and safety considerations, solvents like ethanol and water are commonly used for extracting phenols from olive leaves. Phenols from water extraction, due to their high polarity, are insoluble in olive oil; adding ethanol facilitates the dissolution of characteristic olive phenols. We examined how varying ethanol-water ratios affect the phenolic content from ultrasonic extraction of olive leaves, with results presented in Fig. 2 A. The four representative phenols were most efficiently extracted using an 80% ethanol-water solution. However, chlorophyll also dissolved efficiently when the ethanol concentration exceeded 80%. The presence of chlorophyll in extracts negatively affects oil quality and oxidative stability, necessitating a simple method to separate chlorophyll from olive leaf phenol extracts. Given the logP values of hydroxytyrosol, luteolin-7-O-glucoside, oleacein, quercetin, luteolin, and oleuropein aglycone (0.02, -0.09, 2.16, 1.92, 2.40, and 1.01, respectively), which are significantly lower than those of chlorophyll a (10.23) and b (10.36), separation of characteristic olive phenols and chlorophyll based on solubility differences in a specific solvent combination appears feasible. As shown in Fig. 2 A, chlorophyll extraction is minimal when ethanol concentration is below 40%. We employed a two-step dissolve and resaturate process for separation. The extract, obtained with 80% ethanol, was evaporated, dried, and then redissolved in an ethanol solution below 80%. Mixing and centrifuging followed, retaining phenols in the solution and precipitating chlorophyll. Figure 2 B shows that as the ethanol concentration for redissolution decreased gradually from 80–40%, the phenol content slightly changed, while chlorophyll content significantly decreased. Further decreasing the ethanol concentration resulted in minimal changes to the chlorophyll content. Using 40% ethanol for redissolution maximized the retention of the four phenols and achieved the highest phenol-to-chlorophyll ratio (Fig. 2 C), indicating it as the optimal redissolution solvent. The retention rates for hydroxytyrosol, luteolin-7-O-glucoside, oleacein, and oleuropein aglycone were 106.65%, 96.44%, 88.01%, and 84.40% respectively, with 95.59% of chlorophyll removed. The increase in hydroxytyrosol may result from the degradation of oleuropein in the extract, producing more hydroxytyrosol (Fernández-Mar et al. 2012 ; Xie 2019 ). In summary, we established a simple and practical strategy for preparing virtually chlorophyll-free olive phenol extracts from olive leaves using a two-step dissolve and resaturate process. Figure 4 . HPLC chromatogram for ROO, VOO, UBOO, COO, PEOO, EVOO1 and EVOO2 at 280nm. Peaks correspond to (1) hydroxytyrosol; (2) tyrosol; (3) syringic acid (internal standard); (4) luteolin-7-glucoside; (5) oleacein; (6) oleuropein; (7) quercetin; (8) luteolin; (9) apigenin; (10) oleuropein aglycone. Table 2 Phenolic composition and content of seven groups of oil by HPLC. Compound ROO Vibratory Olive Oil (VOO) Ultrasonic-Bath Olive Oil (UBOO) Chlorophyll Olive Oil (PEOO) Phenolic-Enriched Olive Oil (PEOO) EVOO1 EVOO2 Total Polyphenol 27.78 ± 4.25 62.21 ± 3.29 78.14 ± 5.33 407.15 ± 7.19 451.75 ± 12.03 334.60 ± 25.72 245.73 ± 19.42 Hydroxytyrosol 1.83 ± 0.19 5.74 ± 0.27 10.52 ± 0.37 11.59 ± 1.41 31.29 ± 2.07 26.01 ± 2.71 14.22 ± 2.53 Tyrosol 1.68 ± 0.32 3.07 ± 0.25 4.11 ± 0.12 10.26 ± 0.67 6.47 ± 0.72 14.24 ± 1.58 10.64 ± 0.44 Luteolin-7-O-glucoside 0.90 ± 0.19 1.71 ± 0.11 2.13 ± 0.07 15.97 ± 0.74 33.21 ± 1.98 4.77 ± 0.58 4.58 ± 1.32 Oleacein 3.93 ± 0.06 12.92 ± 0.71 27.76 ± 0.54 99.33 ± 4.75 162.43 ± 13.82 69.02 ± 5.19 52.28 ± 4.05 Oleuropein 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 14.42 ± 0.17 12.02 ± 1.58 5.44 ± 3.48 12.26 ± 0.40 Quercetin 2.43 ± 0.05 2.48 ± 0.06 2.52 ± 0.11 17.37 ± 0.90 13.77 ± 1.56 5.33 ± 0.83 7.25 ± 1.86 Luteolin 0.80 ± 0.10 0.66 ± 0.05 0.00 ± 0.00 10.86 ± 0.48 6.18 ± 0.72 1.07 ± 0.23 2.30 ± 0.22 Apigenin 0.40 ± 0.20 0.71 ± 0.03 0.88 ± 0.06 4.01 ± 0.17 3.51 ± 0.36 0.00 ± 0.00 0.00 ± 0.00 Oleuropein Aglycone 2.86 ± 0.59 1.63 ± 0.06 0.39 ± 0.68 43.00 ± 1.64 20.78 ± 0.88 128.49 ± 6.68 78.58 ± 7.27 3.3. Establishing a simple method for efficiently enriching olive leaves phenol extracts in refined olive oils. Two existing methods enrich phenols in olive oil from olive leaves. The first method involves soaking crushed olive leaves in oil for an extended period before filtering out leaf fragments. The second method extracts target phenols from olive leaves using alcohol, then mixes this extract with olive oil, and finally removes the alcohol to obtain the enriched oil. Both methods were unsatisfactorily effective, increasing total phenol content to below 100 mg/kg, insufficient for refined olive oil to meet the EFSA's health claim requirements for phenolic content. Therefore, there is a need for an efficient enrichment method. We first compared three common enrichment methods: water bath oscillation, ultrasonic water bath, and ultrasonic pulse probe. The solid-oil ratio and temperature were consistent across all three treatments, with a 120-minute treatment time for the oscillating water bath and 20 minutes for the other two methods. The results of the phenolic enrichment treatment of ROO by each method are shown in Fig. 3 A. After enrichment, the total phenol content was 62.21 ± 3.29 mg/kg for VOO, 78.14 ± 5.33 mg/kg for UBOO, and 269.15 ± 14.79 mg/kg for UPOO, all exceeding the blank group's 27.78 ± 4.25 mg/kg. Among the three devices, only the ultrasonic pulsed probe-treated ROO exceeded the EFSA's specified 250 mg/kg phenolic content. After selecting the ultrasonic pulsed probe method, we explored its parameters further, with results presented in Figs. 3 B-D. Figure 3 B demonstrates the significance of the enriched solids-to-oil ratio for increasing total phenol content in the oil. Although a 1:3 ratio yielded the highest increase in total phenols, the increase was only 13.0 mg/kg more than the 1:5 ratio, making 1:5 the economically favorable choice. Figure 3 C identifies 300W as the optimal ultrasonic power for phenolic enrichment. Figure 3 D indicates that total phenol content in the oil first increased then decreased with prolonged ultrasound time, likely due to oil oxidation degrading the phenolics; the optimal ultrasound duration was 10 minutes. Using optimal conditions, we produced PEOO with a phenolic content of 451.75 ± 12.03 mg/kg, a 67.8% increase over UPOO (269.15 ± 14.79 mg/kg), which hadn't undergone process optimization. Additionally, our enrichment effect was 5 to 20 times greater than reported in previous studies. We then analyzed and compared the phenolic composition of PEOO with that of ROO, VOO, UBOO, and two EVOOs. Figure 4 displays HPLC plots at 280 nm for the enriched oils after different enrichment treatments, visualizing the enrichment effects on ROO. The enrichment resulted in a phenolic composition of ROO similar to that of EVOO. The most significant increases in ROO phenol content were in oleacein, luteolin-7-O-glucoside, and hydroxytyrosol, with increases of 158.50 mg/kg, 32.31 mg/kg, and 29.46 mg/kg, respectively. These phenols contributed to 37.4%, 7.6%, and 6.9% of the total phenol increase, respectively. Oleacein offers various health benefits, including obesity regulation, anticancer, and anti-inflammatory effects (Y. Huang et al. 2024 ), and adds a pleasant bitter and spicy flavor to olive oil (Genovese et al. 2021 ). Following our enrichment process, the oleacein content in PEOO was 2.4 to 3.1 times higher than in the two EVOOs. The enriched oleacein content was 3.69 times higher than previously reported methods (42.95 mg/kg) (Sánchez de Medina et al. 2011). To our knowledge, no reports exist on enriching ROO with such large amounts of oleacein. Luteolin-7-O-glucoside, abundant in olive leaves but scarce in olive oil, has significant anti-inflammatory and antioxidant activities. Increasing its content benefits olive oil's health properties (Park and Song 2019 ). Following enrichment, luteolin-7-O-glucoside content in PEOO was nearly 8 times that of EVOO, exceeding VOO and UBOO, and significantly higher than the 1.60–1.42 µg/mL reported by others (Japón-Luján et al. 2008 ). Hydroxytyrosol, a key phenol in olive leaves, is also present in olive oil. Hydroxytyrosol, found in high-quality olive oil, has strong antioxidant activity (Kiritsakis 1998 ; Mkaouar et al. 2018 ). The enriched PEOO had higher hydroxytyrosol content than both EVOO groups, with a 7-fold and 3.5-fold increase over VOO and UBOO, respectively, and significantly surpassing previous research findings (Sánchez de Medina et al. 2011). Oleuropein aglycone, produced from oleuropein by enzymes like β-glucanase, is unstable. Oleuropein aglycone offers pharmacological benefits, including anti-Alzheimer's, anti-breast cancer, anti-inflammatory, and hypoglycemic effects (Xu et al. 2018 ). PEOO's enrichment of oleuropein aglycone fell short of expectations. Post-enrichment, the content rose from 2.86 ± 0.59 mg/kg to 20.78 ± 0.88 mg/kg, lower than in both EVOOs. Interestingly, oleuropein aglycone content decreased in VOO and UBOO, likely due to degradation during enrichment. Our method increased the oleuropein content in PEOO by 12.02 mg/kg, achieving a concentration comparable to EVOO and surpassing values reported in literature (Japón-Luján et al. 2008 ). Oleuropein, abundant in olive leaf extract but scarce in olive oil, is attributed to its high polarity. Higher oleuropein levels enhance the bitter flavor. The enrichment method raised the levels of quercetin, luteolin, tyrosol, and apigenin in ROO by 11.34, 5.38, 4.79, and 3.11 mg/kg, respectively, surpassing results documented in literature (Sánchez de Medina et al. 2011; Sanmartin et al. 2019 ). The enriched PEOO had quercetin and luteolin contents 2.6 and 1.9 times, and 5.8 and 2.7 times higher than those in the two EVOOs, respectively. Even after enrichment, the tyrosol content remained lower than that of EVOO. This may stem from two factors. The first is tyrosol's high polarity; the second, the solubility of phenols, varies with the fatty acid composition of the raw oil (Paulo and Santos 2020 ; Sánchez de Medina et al. 2011). Apigenin, undetected in EVOO, is a flavonoid known for its anti-inflammatory, antitumor, antioxidant, and cardiovascular protective effects (Li et al. 2023 ). Our enrichment method introduced this compound into olive oil. In summary, enriching ROO with olive leaf phenolic solid extracts using ultrasonic pulsed probes is effective. Optimal conditions include a 1:5 ratio of enriched solids to oil, 300 W ultrasonic power, and 10 min of ultrasonic time. The enriched PEOO's total phenol content reached 451.75 ± 12.03 mg/kg by the optimal process, 12.3 and 8.4 times higher than VOO and UBOO, respectively. Our method enriched ROO with oleacein, hydroxytyrosol, and luteolin-7-O-glucoside at 162.43 ± 13.82, 31.29 ± 2.07, and 33.21 ± 1.98 mg/kg, respectively, 3.1, 2.2, and 7.3 times higher than in EVOO. Notably, oleacein's increase, representing 37.4% of the total phenolic boost, is unprecedented in other studies. We have developed a straightforward and efficient method to enrich ROO with olive leaf phenol extracts. Figure 5 . Quality Test of ROO, PEOO, EVOO1, EVOO2. (A) relative peroxide values at 14 days. (B) odor level and flavor intensity, consumer preference and sensory positive attributes. (C) sensory defects. Table 3 Free acidity, peroxide value, antioxidant activity of COO, PEOO, EVOO1 and EVOO2. ROO PEOO EVOO1 EVOO2 Free Acidity(Oleic acid %) 0.13 ± 0.01 0.18 ± 0.01 0.35 ± 0.02 0.25 ± 0.01 Peroxide Value(meq O 2 /kg) 4.40 ± 0.13 4.83 ± 0.14 9.63 ± 0.47 18.45 ± 0.32 DPPH 21.78 ± 2.04 154.75 ± 4.72 81.18 ± 0.63 74.88 ± 0.82 ABTS 12.31 ± 0.79 189.58 ± 0.90 136.73 ± 3.43 139.72 ± 4.25 FRAP 22.48 ± 0.19 95.64 ± 0.24 44.02 ± 0.48 38.66 ± 0.26 3.4. Quality evaluation of the oil before and after phenols enrichment. The work described above developed a simple and efficient method for phenol enrichment of ROO. We determined and compared the acid value, peroxide value, oxidative stability, antioxidant capacity, and sensory characteristics of optimally processed PEOO with control oils (ROO, EVOO1, EVOO2) to assess the phenolic enrichment method's impact. 3.4.1. Free Acidity Free acidity, indicating free fatty acid content (often as a percentage of oleic acid), inversely correlates with oil quality. According to the IOC (“IOC COI/T.15/NC No 3/Rev. 18 Trade standard applying to olive oils and olive pomace olis” 2022), EVOO's free acidity must not exceed 0.80%, and ROO's, 0.30%. Table 3 shows that PEOO's free acidity rose by 0.05 units post-enrichment, yet remained below both ROO's standard and that of the two EVOOs. The phenol enrichment method minimally impacted the oil's free fatty acid content. 3.4.2. Peroxide Value The peroxide value measures the oxidation level of fats and fatty acids. It represents the quantity of oxidation products in a 1 kg sample, in millimoles of peroxide. Table 3 indicates PEOO's peroxide value rose post-enrichment yet remained under the IOC's threshold of 5.0 for ROO and significantly below EVOO's 20.0 standard. The olive oil's fatty acids experienced minor oxidation following phenol enrichment but retained their high quality. 3.4.3 Oxidative Stability This experiment dynamically monitored the oil's peroxide value under extreme temperature and ventilation to assess its oxidative stability. Oil samples were maintained at 65°C with continuous ventilation, and their peroxide value measured every 2 days. Figure 4 A displays the relative peroxide values of the four oils; lower change values indicate better oxidative stability. ROO, with the lowest phenol content, exhibited the greatest peroxide value change, while enriched PEOO showed the least, at 0.6 times that of ROO. The peroxide value changes in the four oils negatively correlated with their phenol contents, aligning with findings from another study (Rubió et al. 2012 ). Phenols significantly enhanced the oxidative stability of olive oil. Interestingly, the peroxide change curves for PEOO, EVOO1, and EVOO2, high in phenol content, initially steepened then flattened, while ROO's curve, with minimal phenol, remained linear. The oxidative protection of the oil by phenols was particularly evident in the later stages of the oxidation reaction. In summary, the phenolic enrichment of ROO in this study significantly increased its oxidative stability, surpassing that of commercial EVOO. 3.4.4. Antioxidant Activity This study utilized DPPH, ABTS, and FRAP assays to analyze the antioxidant capacity of enriched oils. A single method is insufficient for a comprehensive assessment of antioxidant properties in food products, as different methods yield a broader range of results (D. Huang et al. 2005 ). Table 3 shows that PEOO's four radical scavenging abilities were significantly stronger than ROO's, consistent with the oxidative stability results. Higher values in DPPH, ABTS, and FRAP assays indicate greater free radical scavenging capacity of the oil. After phenol enrichment, PEOO's DPPH value was 7.1 times that of ROO, and 1.9 and 2.1 times higher than EVOO1 and EVOO2, respectively. PEOO's ABTS value was 15.4 times that of ROO and 1.4 times higher than EVOO1 and EVOO2. PEOO's FRAP value was 4.3 times that of ROO, and 2.2 and 2.5 times higher than EVOO1 and EVOO2, respectively. A comprehensive comparison revealed that the four methods complemented each other, showing that phenolic-enriched PEOO significantly enhanced antioxidant capacity over non-phenolic-enriched ROO, surpassing the commercial EVOOs. Therefore, this study's method significantly increased ROO's antioxidant activity and improved its quality. Table 4 Sensory Evaluation of COO, PEOO, EVOO1 and EVOO2. ROO PEOO EVOO1 EVOO2 Odor level and flavor intensity 3.04 ± 0.79 7.96 ± 0.98 6.24 ± 1.48 5.20 ± 0.66 Consumer preference 6.16 ± 0.57 3.32 ± 0.34 6.52 ± 0.59 5.76 ± 0.30 Positive attributes Fruity 1.04 ± 0.59 1.60 ± 0.46 4.52 ± 0.52 2.64 ± 0.33 Sweet 0.64 ± 1.41 0.76 ± 1.40 1.72 ± 1.51 1.28 ± 1.51 Bitter 0.80 ± 0.41 5.00 ± 0.43 1.64 ± 0.47 2.32 ± 0.15 Pungent 0.52 ± 0.32 3.56 ± 0.44 2.00 ± 0.20 2.40 ± 0.34 Defects Fusty/muddy sediment 0.32 ± 0.31 0.68 ± 0.36 0.16 ± 0.37 0.72 ± 0.17 Musty-humid-earthy 0.32 ± 0.12 1.24 ± 0.87 0.44 ± 0.33 0.76 ± 0.43 Winey-vinegary-acid-sour 0.16 ± 0.07 0.80 ± 1.04 2.08 ± 0.64 1.04 ± 0.52 Metallic 0.50 ± 0.22 1.66 ± 0.45 0.74 ± 0.21 0.98 ± 0.12 Greasy 0.88 ± 0.67 1.20 ± 1.19 1.24 ± 0.73 1.88 ± 0.88 3.4.5. Sensory Evaluation The sensory characteristics of PEOO, ROO, EVOO1, and EVOO2, including odor, flavor intensity, and consumer preference, were evaluated and compared. Figure 4 B and Table 4 show that after enrichment, PEOO's odor intensity, fruity flavor, sweetness, bitterness, and pungency increased by 162%, 54%, 19%, 525%, and 585% respectively, compared to ROO, while consumer preference decreased by 46%. PEOO exhibited stronger odor intensity, bitterness, and pungency, but lower fruitiness, sweetness, and consumer preference than both EVOO groups. The decrease in consumer preference could be attributed to the unfamiliarity of the Chinese with oils having bitter and spicy flavors. Conversely, Spanish consumers preferred olive oils with high bitterness, pungency, and a "fruity" flavor (Zamuz et al. 2020 ). Consumer preferences for olive oil's taste and flavor vary between traditional and non-traditional producing countries (Latino et al. 2022 ). The increase in PEOO's bitterness was mainly due to higher levels of oleacein, oleuropein, and oleuropein aglycone (Marx et al. 2017 , 2022 ), while the increased pungency was likely due to higher levels of ligstroside derivatives (Andrewes et al. 2003 ). Figure 4 C compares the defect sensations of PEOO with those of ROO, EVOO1, and EVOO2. All five defective sensations in PEOO increased compared to ROO, particularly the metallic sensation (232% increase), possibly due to contact with the ultrasound pulse probe's metal. PEOO's fusty/muddy sediment, winey-vinegary-acid-sour, and greasy sensations remained similar to those in EVOO. Overall, this study's methodology endowed ROO with positive attributes, enhancing its characteristic flavor, though the flaws also impacted its organoleptic quality. Fortunately, these flaws are within acceptable limits. 4. Conclusion This study established a simple method to prepare chlorophyll-free phenols from olive leaves and an efficient method to enrich them in refined olive oils. Under optimal conditions, the total phenol content in ROO increased to 424 mg/kg, exceeding the EFSA health claim threshold of 250 mg/kg and surpassing the total phenolic content in EVOO. The phenolic composition of PEOO, treated with the enrichment method, resembled that of EVOO. Oleacein, hydroxytyrosol, and luteolin-7-O-glucoside levels reached 3.1, 2.2, and 7.3 times those in EVOO, respectively. The antioxidant capacity and oxidative stability of PEOO were significantly improved, along with enhancement of certain flavors. Our study offers a simple, practical, and efficient strategy for high-value utilization of olive leaves and improving ROO's quality. Declarations Author Contribution Yunfei Huang and Wenqing He wrote the main original draft. Ruifeng Wang, Yangyang Jia, Lu Li, and Yawei Xu prepared Figures. 1-5. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4020617","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":277892530,"identity":"1d515491-fbf6-4ed3-a41b-133328e1fbf4","order_by":0,"name":"Yunfei Huang","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yunfei","middleName":"","lastName":"Huang","suffix":""},{"id":277892531,"identity":"4ddf8b76-5fd1-444a-9d6f-6ca77e3a057b","order_by":1,"name":"Wenqing He","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wenqing","middleName":"","lastName":"He","suffix":""},{"id":277892532,"identity":"6bac5c32-f858-461e-9769-da8063743a7b","order_by":2,"name":"Ruifeng Wang","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ruifeng","middleName":"","lastName":"Wang","suffix":""},{"id":277892533,"identity":"40b15f66-96e0-4270-86c3-62f181cd8b77","order_by":3,"name":"Yangyang Jia","email":"","orcid":"","institution":"Henan Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yangyang","middleName":"","lastName":"Jia","suffix":""},{"id":277892534,"identity":"808906da-6ca7-46e6-b1d4-46ec3d9f9836","order_by":4,"name":"Lu Li","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Li","suffix":""},{"id":277892535,"identity":"4443451d-484c-4a82-97db-4cdf32e7ee56","order_by":5,"name":"Yawei Xu","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yawei","middleName":"","lastName":"Xu","suffix":""},{"id":277892536,"identity":"12ef97cb-7130-455f-ad4f-4334bc4fee33","order_by":6,"name":"Yuhan Lu","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuhan","middleName":"","lastName":"Lu","suffix":""},{"id":277892537,"identity":"580f4433-51e6-4335-a4d5-f264c2b0178e","order_by":7,"name":"Xiaoxiao Zhang","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxiao","middleName":"","lastName":"Zhang","suffix":""},{"id":277892538,"identity":"b2b07db3-98e4-49f6-a4cf-089fd3a324d8","order_by":8,"name":"Feixue Wu","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Feixue","middleName":"","lastName":"Wu","suffix":""},{"id":277892539,"identity":"2f76dec6-b7c3-478f-a11b-ab2f52da2300","order_by":9,"name":"Chunmei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYBACPmYGNiBlIcfAwNh4gLEBIiqBTwsbRIuEMVBLA5FaGCBaEkGKidTCzvzs0c02ifS17YeBtuyokzc4wHzwNg+DXR5uh7GZG+e2SeRuO5MI1HKGzXDDAbZkax6G5GLcWnjYpMFaDoC0tPEwbjjAYybNw3AgsYGAlnSz8w9BWiTsNxzg/0aUlgSzG2BbDBKBtrAR0MJmJp1zTsJw2w2gLYltCckzD7MZW84xSMaphZ//8DPpnDIbebPz6Q8ffGyrs+073vzwxpsKO5xawICRDcpIABHMIMIAn3oQ+ENIwSgYBaNgFIxoAAD4CVIjebWQjAAAAABJRU5ErkJggg==","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Chunmei","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-03-06 11:19:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4020617/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4020617/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52455234,"identity":"f05f77eb-7666-4a35-a86f-83247daed133","added_by":"auto","created_at":"2024-03-11 19:54:56","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":189697,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of chlorophyll from olive leaf extracts to olive oil enriched with phenolics. (A) the appearance of refined olive oil (ROO), chlorophyll olive oil (COO) and two extra virgin olive oil (EVOO1 and EVOO2). (B) changes in peroxide values of ROO, phenolic-enriched olive oil (PEOO), and COO with light.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4020617/v1/8c62c67749f323c8b82accec.jpeg"},{"id":52455083,"identity":"323ad218-7e22-43f6-b16d-4db831116119","added_by":"auto","created_at":"2024-03-11 19:46:54","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":264682,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of extraction solvents on phenolics and chlorophyll of olive leaves. (A) ethanol-water ratio. (B) conversion of the ratio of ethanol to water. (C) conversion of the ratio of ethanol to water on the ratio of phenolics to chlorophyll.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4020617/v1/2a2b05ca5529e50343879e0c.jpeg"},{"id":52455085,"identity":"e0caff5a-ea49-465e-9d90-724c91139647","added_by":"auto","created_at":"2024-03-11 19:46:54","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":190275,"visible":true,"origin":"","legend":"\u003cp\u003eParameter optimization of phenolic enrichment process for ROO. (A) oils obtained by different enrichment process. (B) enriched solid to oil (g/mL). (C) ultrasonic power (W). (D) ultrasonic time (min) (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4020617/v1/3f9f93e012c32f75b9042a0a.jpeg"},{"id":52455088,"identity":"bdfef7b4-d90e-485e-96dc-fa2afb9d5d34","added_by":"auto","created_at":"2024-03-11 19:46:56","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":133166,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC chromatogram for ROO, VOO, UBOO, COO, PEOO, EVOO1 and EVOO2 at 280nm. Peaks correspond to (1) hydroxytyrosol; (2) tyrosol; (3) syringic acid (internal standard); (4) luteolin-7-glucoside; (5) oleacein; (6) oleuropein; (7) quercetin; (8) luteolin; (9) apigenin; (10) oleuropein aglycone.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4020617/v1/56105297942c6e861ebcf03a.jpeg"},{"id":52455084,"identity":"fd9d8d5d-8945-4cfb-935e-3715d405031a","added_by":"auto","created_at":"2024-03-11 19:46:54","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":207388,"visible":true,"origin":"","legend":"\u003cp\u003eQuality Test of ROO, PEOO, EVOO1, EVOO2. (A) relative peroxide values at 14 days. (B) odor level and flavor intensity, consumer preference and sensory positive attributes. (C) sensory defects.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4020617/v1/e281e7cee542b986439513d5.jpeg"},{"id":52456018,"identity":"941e3efc-bffb-4832-85ed-2e8dcc627441","added_by":"auto","created_at":"2024-03-11 20:03:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1039573,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4020617/v1/179eb4af-e035-4d4d-8170-05189bff4727.pdf"},{"id":52455087,"identity":"c365c0cb-01ad-48ec-85d9-10b9e73f55bd","added_by":"auto","created_at":"2024-03-11 19:46:56","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":80194,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4020617/v1/27e20cf45e9481d378ebd68c.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"A simple method for preparing chlorophyll free phenols from olive leaves and efficiently enriching it in refined olive oil","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eExtra virgin olive oil (EVOO) is considered one of the healthiest vegetable oils and a crucial component of the Mediterranean diet, thanks to its high levels of oleic acid, phytosterols, tocopherols, and phenols (S\u0026aacute;nchez de Medina et al. 2017). The phenolic composition is considered the main reason for EVOO's health benefits (Gorzynik-Debicka et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The European Food Safety Authority (EFSA) has issued a health declaration stating that every 1000 grams of olive oil must contain at least 250 mg of total phenolic compounds, such as hydroxytyrosol and its derivatives (EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, refined olive oil (ROO) almost completely lost phenols due to the refining process, which includes filtering, neutralization, distillation, degumming, bleaching, and high-heat deodorization, significantly falling short of the healthy concentrations required by EFSA health claims. Therefore, enriching refined olive oil with olive polyphenols is an effective strategy to enhance its quality and health benefits.\u003c/p\u003e \u003cp\u003eHydroxytyrosol, oleacein and oleuropein aglycone are representative phenols in olive oil. They have been reported to contribute to olive oil's health benefits, including anticancer, anti-atherosclerotic, anti-obesity, anti-inflammatory, and antioxidant activities (Y. Huang et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Coincidentally, olive leaves, rich in phenolic compounds like hydroxytyrosol, oleuropein, luteolin-7-O-glucoside, and oleacein, which are also desirable in olive oil, suggest that olive leaves can serve as an economical raw material for the phenolic enrichment of ROO. Enriching ROO with olive leaf phenols could improve their quality and health benefits and also valorize olive leaves, typically used as animal forage or burned as waste (\u0026Ouml;zcan and Matth\u0026auml;us \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrently, two methods have been reported for enriching oil with phenols using olive leaves as raw materials. The first method involves macerating crushed olive leaves in the oil for a week and then filtering out the leaf fragments (Baccouri et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jap\u0026oacute;n-Luj\u0026aacute;n et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Nenadis et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This method is simple but time-consuming and offers low enrichment, increasing the total phenol content by only 20 mg/kg (Sevim \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The second method involves extracting target phenols from the leaves with alcohol solutions, then mixing the phenolic extract with the oil, and removing the alcohol from the two-phase system using a rotary evaporator (S\u0026aacute;nchez de Medina et al. 2011). Jap\u0026oacute;n-Luj\u0026aacute;n and Luque De Castro (2008) extracted phenols from olive leaves using ethanol, mixed the alcoholic extract with the oil, and then stirred and centrifuged it to obtain oil enriched mainly with oleuropein, apigenin-7-glucoside, luteolin-7-glucoside, and verbascoside. Although this technique is simple and improves phenol enrichment to 80mg/kg, it still falls far short of meeting public health needs. Moreover, it is time-consuming and inefficient, increases the oil's viscosity, and leaves residual alcohol, resulting in poor oil flavor and characteristics. Additionally, previous studies overlooked the negative impact of chlorophyll co-existing in the olive leaf phenol extract on the oil, which not only darkened its color but also increased photosensitivity and significantly reduced its oxidative stability. Therefore, developing a method to prepare chlorophyll-free olive leaf phenol extracts is urgently needed.\u003c/p\u003e \u003cp\u003eThis study aims to develop a simple and effective method for preparing chlorophyll-free phenol extracts from olive leaves and efficiently enriching them in ROO. The quality and sensory characteristics of the ROO before and after enrichment were comprehensively evaluated. We believe our data will offer practical and effective strategies for the high-value utilization of olive leaves and improving the quality of ROO.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Samples, Standards, and Reagents\u003c/h2\u003e \u003cp\u003eTender olive leaves for this research were collected in October 2022 from \u0026lsquo;Picual\u0026rsquo; in Xichang City, Sichuan Province, China. Subsequently, they were freeze-dried at -40\u0026deg;C for 48 hours, ground to a uniform particle size (diameter\u0026thinsp;\u0026le;\u0026thinsp;0.4 mm) using an ultra-micro-mill, and stored at 4℃ until use. ROO was sourced from Monini, Italy, and the two extra-virgin olive oils (EVOO1, EVOO2) were purchased from Mueloliva, Spain, and Olivoila, China.\u003c/p\u003e \u003cp\u003eMethanol and acetonitrile reagents for high-performance liquid chromatography (HPLC) were sourced from Fisher, USA. Deionized water in the mobile phase was obtained from Millipore Milli-Q water purification system, USA. Standards for tyrosol, oleuropein, apigenin, luteolin-7-glucoside, luteolin, and quercetin were obtained from Yuan Ye, China; oleacein standards were from Phytolab, Germany. Each standard (10 mg) was dissolved in 10 mL of methanol or acetonitrile, then diluted to various concentrations. The peak area for each concentration was measured by HPLC, using the peak area as the y-axis and concentration as the x-axis to create a standard curve. Solutions were stored in the dark at -20℃ until needed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Enrichment of ROO with Phenols Extracts from Olive Leaves\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Treatment of Chlorophyll Olive Oil (COO)\u003c/h2\u003e \u003cp\u003eOlive leaf powder was mixed with an 80% ethanol solution at a 1:15 mass ratio and extracted using ultrasound at 490 W for 30 minutes at 30\u0026deg;C. Phenolic extracts were obtained through filtration. This extraction process was repeated three times. The phenolic extract was rotary evaporated to dryness and then freeze-dried for 30 hours to yield a green solid powder for enrichment. The enriched powder was mixed with ROO in a 1:5 mass ratio, and ultrasonic pulse treatment was performed with a water bath set at 35℃. Ultrasonic power was set at 300 W, treatment time at 20 minutes, and frequency at 300 Hz. Clarified enriched olive oil, termed Chlorophyll Olive Oil (COO), was obtained by centrifuging at 10,000 rpm for 7 minutes and filtering.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Treatment of Phenolic-Enriched Olive Oil (PEOO)\u003c/h2\u003e \u003cp\u003ePhenols were extracted from olive leaf powder using 80% ethanol in water as previously described. The phenolic extract was then rotary evaporated to dryness. The extract was fully solubilized in a 40% ethanol solution, ultrasonicated for 5 minutes at 490W and 30\u0026deg;C, then transferred to a 50ml centrifuge tube and centrifuged at 8000 rpm for 5 minutes. Chlorophyll was precipitated, and the supernatant was retained. The extract was freeze-dried for 30 hours to yield a yellow solid powder for enrichment. The enriched powder was mixed with ROO in mass ratios of 1:3, 1:5, 1:10, 1:15, and 1:20. Ultrasonic power settings were 150W, 188W, 225W, 263W, 300W. Ultrasonic treatment times were 0, 5, 10, 15, and 20 minutes. Ultrasonic pulse treatment was conducted with a water bath maintained at 35℃. Ultrasonic frequency was set at 300 Hz. Clarified, enriched olive oil, termed Treatment of Phenolic-Enriched Olive Oil (PEOO), was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Treatment of Ultrasonic Pulsed Probe Olive Oil (UPOO)\u003c/h2\u003e \u003cp\u003eThe extraction process was as previously described. The enriched powder was mixed with ROO at a 1:10 mass ratio. Ultrasonic power was set at 225W. Ultrasonic treatment time was 5 minutes. Ultrasonic pulse treatment was conducted with a water bath at 35℃. Ultrasonic frequency was set at 300 Hz. Clarified, enriched olive oil, termed Ultrasonic Pulsed Probe Olive Oil (UPOO), was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4. Treatment of Vibratory Olive Oil (VOO)\u003c/h2\u003e \u003cp\u003ePrepared the yellow solid powder used for enrichment according to the method of FEOO. The enriched powder was mixed with ROO at a 1:5 mass ratio and shaken in the dark for 120 minutes at 190 rpm in a water bath at 35\u0026deg;C. Clarified, enriched olive oil, termed Vibratory Olive Oil (VOO), was obtained by centrifuging at 10,000 rpm for 7 minutes and filtering.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5. Treatment of Ultrasonic-Bath Olive Oil (UBOO).\u003c/h2\u003e \u003cp\u003eThe initial process aligns with the above description. The enriched powder was mixed with ROO at a 1:5 mass ratio and ultrasonicated in a water bath at 35\u0026deg;C for 20 minutes using an ultrasonic cleaner. Ultrasonication frequency was set at 300 Hz. Clarified Ultrasonic-Bath Olive Oil (UBOO) was obtained by centrifuging at 10,000 rpm for 7 minutes.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Determination of Phenol Content in ROO and Enriched Olis by HPLC-DAD.\u003c/h2\u003e \u003cp\u003eTotal phenolic content was determined using the method of (S\u0026aacute;nchez de Medina et al. 2011) expressed as gallic acid equivalents per gram.\u003c/p\u003e \u003cp\u003eA model 1220 series high-performance liquid chromatograph (Agilent, USA), equipped with a TC-C18 column (250 mm \u0026times; 4.6 mm), was utilized. Mobile phase A consisted of 0.2% phosphoric acid in water, and mobile phase B was a 1:1 (v/v) mixture of methanol and acetonitrile. The flow rate was set at 1.0 mL/min. The column temperature was maintained at 30\u0026deg;C, with a sample injection volume of 10 \u0026micro;L. The gradient profile was as follows: 0 min at 4% B, progressing to 50% B at 40 min, 60% B at 45 min, 100% B from 60 to 70 min, and returning to 4% B at 72 min, maintained until 82 min. Chromatograms were obtained at 280 nm. Standard curves were created using the concentrations of tyrosol, oleuropein, apigenin, luteolin-7-glucoside, luteolin, quercetin, oleacein, and their corresponding peak areas in the chromatograms. Quantitative analysis was performed by using the chromatographic peak areas. Quantitative analysis for hydroxytyrosol and oleuropein aglycone utilized the internal standard method with syringic acid, expressing results in tyrosol equivalents per gram. A 10 \u0026micro;L injection of an external calibration standard solution (0.030 mg/mL tyrosol versus 0.015 mg/mL syringic acid) was administered, with chromatograms recorded at 280 nm. The response factor ratio of syringic acid to tyrosol, recorded as RRFsyr/tyr, was calculated. The content of hydroxytyrosol and oleuropein aglycone was calculated by comparing their peak areas in the chromatograms to that of syringic acid. The calculation formula is as follows: Three independent determinations were conducted on the same sample. Results were reported in milligrams of compounds per kilogram of oil.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$（mg/kg）=\\frac{A\\times 1000\\times {RRF}_{syr/tyr}\\times Ws}{As\\times W}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eA: is the peak areas of the hydroxytyrosol or oleuropein aglycone recorded at 280 nm;\u003c/p\u003e \u003cp\u003eAs: is the area of the syringic acid internal standard recorded at 280 nm;\u003c/p\u003e \u003cp\u003eW: is the weight of the oil used in g;\u003c/p\u003e \u003cp\u003eWs: is the weight of the syringic acid used as internal standard in 1 mL of solution added to the sample, in mg.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Characterization of Olive Phenols by LC-TOF/MS.\u003c/h2\u003e \u003cp\u003eThe phenolic composition of olive oil and leaves was determined using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-TOF/MS). For phenolic composition analysis, a Waters Vion IMS QTOF (USA) equipped with an ESI ion source in negative ion mode was used. Settings included a source voltage of 3 kV, source temperature of 150\u0026deg;C, m/z range of 115\u0026ndash;1000, collision energy of 10\u0026ndash;25 eV, column temperature of 40\u0026deg;C, and a flow rate of 0.25 mL/min. The elution gradient followed this sequence: starting at 4% B at 0 min, increasing to 50% B by 16 min, 60% B by 18 min, reaching 100% B at 24 min, maintained through 28 min, then returning to 4% B at 28.8 min and maintained until 32.8 min. An injection volume of 1 \u0026micro;L was used, with all samples being filtered through 0.22 \u0026micro;m nylon filters prior to analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Determination of Color Parameters\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. L*, a*, b*, Y, x, y\u003c/h2\u003e \u003cp\u003eThe Ultrascan VIS (HunterLab) system was employed to measure the colorimetric values of olive oil samples. Initially, calibration was performed using a standard black and white board. Subsequently, the olive oil was positioned in a vessel under a light source to record the values of L*, a*, b*, Y, x, and y. L* and Y indicate luminance, with higher values signifying brighter colors. a* and x represented the degree of red-green, where positive values indicate red and negative values green. b* and y indicated the degree of yellow-blue, with positive values signifying yellow and negative values blue. Each sample is measured three times in parallel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. C*, h*\u003c/h2\u003e \u003cp\u003eThe chromaticity (C*) and hue angle (h*) of the olive oil were calculated using a* and b*. C* represents color saturation, with larger values indicating higher purity. h* represents the color angle, indicating hue within a 0-360 range, with 0 for red, 180 for green, and 360 for blue. The calculation formulas are as follows:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$${C}^{\\text{*}}=\\sqrt{{a}^{\\text{*}2}+{b}^{\\text{*}2}}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$${h}^{\\text{*}}={\\text{tan}}^{-1}(\\frac{{b}^{\\text{*}}}{{a}^{\\text{*}}}\\left)\\right({a}^{\\text{*}}\u0026gt;0,{b}^{\\text{*}}\u0026gt;0)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$${h}^{\\text{*}}={\\text{tan}}^{-1}(\\frac{{b}^{\\text{*}}}{{a}^{\\text{*}}})+180({a}^{\\text{*}}\u0026lt;0,{b}^{\\text{*}}\u0026gt;0)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. Chlorophyll and Carotenoids\u003c/h2\u003e \u003cp\u003eFollowing the method of (Tarchoune et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), 2 mL of olive oil was diluted tenfold with hexane. The absorbance of the solution was measured using a spectrophotometer. The chlorophyll content's maximum absorption wavelength was at 670 nm, while that of carotenoids was at 470 nm. The specific extinction coefficients are 613 for chlorophyll and 2000 for carotenoids, respectively. The pigment concentration calculation formula is as follows:\u003c/p\u003e \u003cp\u003eChlorophyll (mg/kg) = (A\u003csub\u003e670\u003c/sub\u003e \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) / (613 \u0026times; 100 \u0026times; d)\u003c/p\u003e \u003cp\u003eCarotenoids (mg/kg) = (A\u003csub\u003e470\u003c/sub\u003e \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) / (2000 \u0026times; 100 \u0026times; d)\u003c/p\u003e \u003cp\u003eWhere A is the absorbance and d is the spectrophotometer cell thickness (1cm).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Free Acidity, Peroxide Value and Oxidative Stability\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Free Acidity\u003c/h2\u003e \u003cp\u003eFree acidity, indicating the free fatty acid content in oils and fats, is a crucial quality parameter. A 10g sample of olive oil was diluted to 50mL with an ether-ethanol solution (1:1, v/v). A 0.1M potassium hydroxide ethanol solution served as the titrant, with phenolphthalein as the indicator, until the pink color persisted for at least 10 seconds. Free acidity in the oil sample was expressed as oleic acid percentage (g oleic acid/kg oil). The calculation formula is as follows:\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\n$$Acid Value=V\\times c\\times \\frac{M}{1000}\\times \\frac{100}{m}=\\frac{V\\times C\\times M}{10\\times m}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eV: volume of potassium hydroxide ethanol solution (mL);\u003c/p\u003e \u003cp\u003ec: molar concentration of potassium hydroxide ethanol solution (mol/L);\u003c/p\u003e \u003cp\u003eM: molar mass conversion factor for oleic acid (=\u0026thinsp;282);\u003c/p\u003e \u003cp\u003eM: weight of sample oil (g);\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Peroxide Value\u003c/h2\u003e \u003cp\u003eThe peroxide value measures substances in the sample that oxidize potassium iodide under specified conditions. 2.0 g of olive oil sample was weighed, 25 mL of chloroform-acetic acid solution (4:6, v/v) added, stirred to dissolve, covered, shaken for 1 minute, and left in the dark at room temperature for 5 minutes. Approximately 75 mL of distilled water and 1.0 g of 10 g/L starch solution were added as an indicator to titrate the released iodine with 0.01 mol/L sodium thiosulfate solution. The titration volume was recorded, and the peroxide value was expressed in milliequivalents of reactive oxygen species per kilogram of oil (meq O\u003csub\u003e2\u003c/sub\u003e/kg oil). Three measurements were taken for the test material, along with a blank test. The formula is as follows:\u003cdiv id=\"Equf\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equf\" name=\"EquationSource\"\u003e\n$$PV=\\frac{V\\times T\\times 1000}{m}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eV: volume of standard sodium thiosulfate solution (mL);\u003c/p\u003e \u003cp\u003eT: molar concentration of standard sodium thiosulfate solution (mol/L);\u003c/p\u003e \u003cp\u003eM: weight of sample oil (g);\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3. Oxidative Stability\u003c/h2\u003e \u003cp\u003eThe oxidative stability of COO was tested using UV irradiation at 30\u0026deg;C, with peroxide values determined every 24 hours.\u003c/p\u003e \u003cp\u003eThe oxidative stability of the oils was assessed with a 65℃ oven oxidation test. Each oil sample, weighing 15.0 g, was stored in a constant temperature oven at 65℃. Peroxide values were determined bi-daily at a set time to analyze the degree of fat and oil oxidation over 2 weeks.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Evaluation of Antioxidant Activity\u003c/h2\u003e \u003cp\u003eAntioxidant activity in olive oil's polar fractions was assessed using DPPH, ABTS, and FRAP methods. Polar fraction extraction followed Nakbi et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)'s method, with optimizations. 2.5 g of olive oil was weighed, mixed with 4.5 mL of methanol, and shaken in the dark at 2000 r/min for 20 minutes. The sample stood in the dark until fully separated; the supernatant was then transferred to a 10 mL brown volumetric flask. This process was repeated twice to obtain the polar fractions.\u003c/p\u003e \u003cp\u003eFollowing Nakbi et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)'s method. 200 \u0026micro;L of the polar extract was diluted in 5 mL of methanol; 2 mL of this solution was mixed with 2 mL of DPPH-methanol and reacted in the dark at room temperature for 2 hours. Absorbance was measured at 517 nm, adjusted to zero using a methanol blank. A standard curve for Trolox concentration versus DPPH scavenging rate (20\u0026ndash;400 \u0026micro;mol/L) was plotted as y\u0026thinsp;=\u0026thinsp;0.1444x-0.3851, R\u0026sup2;=0.9935. Trolox equivalent antioxidant activity was calculated and expressed as \u0026micro;mol TE/100g of oil; the scavenging rate was denoted as R.\u003c/p\u003e \u003cp\u003eR (%) =[1-(A2-A3)/A1] \u0026times; 100%\u003c/p\u003e \u003cp\u003eA1:2.0mL DPPH-methanol solution\u0026thinsp;+\u0026thinsp;2.0mL methanol solution;\u003c/p\u003e \u003cp\u003eA2:2.0mL DPPH-methanol solution\u0026thinsp;+\u0026thinsp;2.0mL oil sample methanol dilution;\u003c/p\u003e \u003cp\u003eA3:2.0mL oil sample methanol dilution\u0026thinsp;+\u0026thinsp;2.0mL methanol solution.\u003c/p\u003e \u003cp\u003eFollowing and optimizing Re et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1999\u003c/span\u003e)'s method. The ABTS free radical solution was left to stand in the dark at room temperature for 12\u0026ndash;16 hours. Before measurement, the solution was diluted with methanol to an absorbance (OD) of 0.700\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020 to prepare the ABTS solution. 200\u0026micro;L of the polar extraction solution was diluted in 5mL methanol, mixed with 2mL ABTS solution in a brown bottle, and left in the dark at room temperature for 20 minutes. Absorbance was measured at 734nm, zeroed with a methanol blank. A standard curve of Trolox concentration versus scavenging rate (20\u0026ndash;400 \u0026micro;mol/L) was established as y\u0026thinsp;=\u0026thinsp;0.0875x\u0026thinsp;+\u0026thinsp;66.608, R\u0026sup2;=0.9588. Trolox equivalent antioxidant activity was calculated, with results expressed as \u0026micro;mol TE/100g of oil and the scavenging rate denoted as R.\u003c/p\u003e \u003cp\u003eR (%) = (A2-A1)/A2\u0026times;100%\u003c/p\u003e \u003cp\u003eAl: ABTS solution\u0026thinsp;+\u0026thinsp;methanol dilution of the polar extraction solution of the oil sample;\u003c/p\u003e \u003cp\u003eA2: ABTS solution.\u003c/p\u003e \u003cp\u003eThe Fe\u0026sup3;⁺ reducing antioxidant capacity (FRAP) was assessed using Benzie and Strain (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1996\u003c/span\u003e)'s method, with minor modifications. The FRAP solution was made from 300 mmol/L acetate buffer (pH 6.6), 10 mmol/L TPTZ, and 20 mmol/L FeCl\u003csub\u003e3\u003c/sub\u003e, in a 10:1:1 ratio (v:v:v). 0.5 mL of the sample dilution was mixed with 1.5 mL of preheated FRAP solution at 37℃, reacted for 10 minutes at the same temperature, and absorbance was measured at 593 nm using ethanol as the blank. A standard curve was plotted with Trolox (20\u0026ndash;200 \u0026micro;mol/L) as the reference: y\u0026thinsp;=\u0026thinsp;0.0089x\u0026thinsp;+\u0026thinsp;0.0701, R\u0026sup2;=0.9998. The antioxidant activity of the samples, measured by FRAP, was quantified as the amount of Trolox equivalent required to achieve the same absorbance value (\u0026micro;mol TE/100g).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Sensory Attribute of the Enriched-Olive Oils\u003c/h2\u003e \u003cp\u003eA panel of 25 untrained members, randomly chosen from the students and staff of Huazhong Agricultural University's College of Food Science and Technology, tested selected samples for sensory evaluation and consumer preference. Red illumination was used to mask color differences in the oil samples. Sensory assessments took place in quiet, individual compartments.\u003c/p\u003e \u003cp\u003eIn the consumer acceptability test, panelists rated the samples on a nine-point scale, from non-detectable (0) to very strong (9), for both smell and taste according to the intensity of odor and flavor.\u003c/p\u003e \u003cp\u003eIn the consumer preference test, panelists ranked the samples according to preference and rated each on a nine-point scale, from very likeable (1) to very dislikeable (9).\u003c/p\u003e \u003cp\u003eEach oil's specific flavor was evaluated, with several flavors receiving individual scores. Flavors were rated on a nine-point scale, from undetectable (0) to very strong (9), based on their strength.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical Analysis\u003c/h2\u003e \u003cp\u003e All experiments reported mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three measurements (n\u0026thinsp;=\u0026thinsp;3). Multivariate and one-way ANOVA analyses were conducted using SPSS software (version 22.0, USA), with Tukey post hoc tests applied at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Effect of chlorophyll from olive leaf extracts to olive oil enriched with phenolics. (A) the appearance of refined olive oil (ROO), chlorophyll olive oil (COO) and two extra virgin olive oil (EVOO1 and EVOO2). (B) changes in peroxide values of ROO, phenolic-enriched olive oil (PEOO), and COO with light.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eColor parameters of seven groups of oil.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eROO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVOO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUBOO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCOO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePEOO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEVOO1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEVOO2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e93.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e92.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e90.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e23.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e88.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e86.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e83.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ea*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-3.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-3.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-3.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e-7.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e-5.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e-2.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e-0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eb*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e16.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e19.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e27.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e88.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e97.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e83.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e82.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e81.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e72.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e69.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e64.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ey\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e16.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e17.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e20.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e27.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e89.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e97.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e178.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e178.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e178.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e178.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e178.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e178.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e178.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChlorophyll (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e192.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e11.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e9.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e12.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarotenoids(mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e49.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e7.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e8.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.104\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e \u003cem\u003e3.1. Removal of chlorophyll from phenol extract of olive leaves is essential for the quality of phenolic-enriched oil.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWhile olive leaves are excellent for phenolic enrichment of olive oil due to their high phenolic content, alcohol extraction also inevitably removes chlorophyll. Following previous methods (S\u0026aacute;nchez de Medina et al. 2011), we first extracted phenols from olive leaves with alcohol and then enriched the olive oil with this extract. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, chlorophyll present in olive leaf phenolic extracts colored the olive oil dark green, significantly detracting from its visual appeal. After enriching with dried phenolic extracts in an 80% ethanol solution, the chlorophyll content in ROO rose from 2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mg/kg to 192.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22 mg/kg. Besides affecting ROO's appearance, photosynthetic pigments like chlorophyll can also cause unwanted photosensitized oxidation. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB illustrates that PEOO and COO underwent identical extraction and enrichment processes, except COO did not undergo chlorophyll removal. Following enrichment, the peroxide values of both PEOO and COO slightly increased but remained similar. When the three groups of oils were exposed to UV light continuously, the peroxide value of COO increased by 25.82 meq O\u003csub\u003e2\u003c/sub\u003e/kg after 24 hours, which was much higher than that of ROO (3.31 meq O\u003csub\u003e2\u003c/sub\u003e/kg) and PEOO (5.07 meq O\u003csub\u003e2\u003c/sub\u003e/kg). The peroxide value of COO increased much higher than ROO and PEOO after 48 and 72 hours of UV irradiation.\u003c/p\u003e \u003cp\u003eClearly, chlorophyll significantly affects not just the appearance but also the oxidative stability of olive oil. However, previous studies on olive oil enrichment from olive leaves overlooked chlorophyll's impact (Jap\u0026oacute;n-Luj\u0026aacute;n and Luque De Castro 2008; Malheiro et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; S\u0026aacute;nchez de Medina et al. 2011; Vidal et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For example, Jap\u0026oacute;n-Luj\u0026aacute;n and Luque De Castro (2008) extracted olive leaf phenols using ethanol, and mixed and shaking it with olive oil to obtain a phenol-rich oil, without mentioning the effect of chlorophyll in it. Therefore, developing an effective method to remove chlorophyll from olive leaf phenolic extracts while preserving the original phenolic compounds is essential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Developing a practical strategy for preparing chlorophyll free olive phenols extract from olive Leaves.\u003c/h2\u003e \u003cp\u003ePreliminary tests indicate that the primary phenols in olive leaves, akin to those in olive oil, include hydroxytyrosol, luteolin-7-O-glucoside, oleacein, and oleuropein aglycone. Given health and safety considerations, solvents like ethanol and water are commonly used for extracting phenols from olive leaves. Phenols from water extraction, due to their high polarity, are insoluble in olive oil; adding ethanol facilitates the dissolution of characteristic olive phenols. We examined how varying ethanol-water ratios affect the phenolic content from ultrasonic extraction of olive leaves, with results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The four representative phenols were most efficiently extracted using an 80% ethanol-water solution. However, chlorophyll also dissolved efficiently when the ethanol concentration exceeded 80%. The presence of chlorophyll in extracts negatively affects oil quality and oxidative stability, necessitating a simple method to separate chlorophyll from olive leaf phenol extracts.\u003c/p\u003e \u003cp\u003eGiven the logP values of hydroxytyrosol, luteolin-7-O-glucoside, oleacein, quercetin, luteolin, and oleuropein aglycone (0.02, -0.09, 2.16, 1.92, 2.40, and 1.01, respectively), which are significantly lower than those of chlorophyll a (10.23) and b (10.36), separation of characteristic olive phenols and chlorophyll based on solubility differences in a specific solvent combination appears feasible. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, chlorophyll extraction is minimal when ethanol concentration is below 40%. We employed a two-step dissolve and resaturate process for separation. The extract, obtained with 80% ethanol, was evaporated, dried, and then redissolved in an ethanol solution below 80%. Mixing and centrifuging followed, retaining phenols in the solution and precipitating chlorophyll. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows that as the ethanol concentration for redissolution decreased gradually from 80\u0026ndash;40%, the phenol content slightly changed, while chlorophyll content significantly decreased. Further decreasing the ethanol concentration resulted in minimal changes to the chlorophyll content. Using 40% ethanol for redissolution maximized the retention of the four phenols and achieved the highest phenol-to-chlorophyll ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating it as the optimal redissolution solvent. The retention rates for hydroxytyrosol, luteolin-7-O-glucoside, oleacein, and oleuropein aglycone were 106.65%, 96.44%, 88.01%, and 84.40% respectively, with 95.59% of chlorophyll removed. The increase in hydroxytyrosol may result from the degradation of oleuropein in the extract, producing more hydroxytyrosol (Fern\u0026aacute;ndez-Mar et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Xie \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In summary, we established a simple and practical strategy for preparing virtually chlorophyll-free olive phenol extracts from olive leaves using a two-step dissolve and resaturate process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. HPLC chromatogram for ROO, VOO, UBOO, COO, PEOO, EVOO1 and EVOO2 at 280nm. Peaks correspond to (1) hydroxytyrosol; (2) tyrosol; (3) syringic acid (internal standard); (4) luteolin-7-glucoside; (5) oleacein; (6) oleuropein; (7) quercetin; (8) luteolin; (9) apigenin; (10) oleuropein aglycone.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhenolic composition and content of seven groups of oil by HPLC.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eROO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVibratory Olive Oil (VOO)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUltrasonic-Bath Olive Oil (UBOO)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChlorophyll Olive Oil (PEOO)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePhenolic-Enriched Olive Oil (PEOO)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEVOO1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEVOO2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Polyphenol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e27.78\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e62.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e78.14\u0026thinsp;\u0026plusmn;\u0026thinsp;5.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e407.15\u0026thinsp;\u0026plusmn;\u0026thinsp;7.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e451.75\u0026thinsp;\u0026plusmn;\u0026thinsp;12.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e334.60\u0026thinsp;\u0026plusmn;\u0026thinsp;25.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e245.73\u0026thinsp;\u0026plusmn;\u0026thinsp;19.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydroxytyrosol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e31.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e26.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e14.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTyrosol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e10.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e14.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e10.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuteolin-7-O-glucoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e15.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e33.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e4.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e4.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOleacein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e12.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e27.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e99.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e162.43\u0026thinsp;\u0026plusmn;\u0026thinsp;13.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e69.02\u0026thinsp;\u0026plusmn;\u0026thinsp;5.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e52.28\u0026thinsp;\u0026plusmn;\u0026thinsp;4.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOleuropein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e12.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e5.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e12.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e13.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e5.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e7.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuteolin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e10.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApigenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOleuropein Aglycone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e43.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e20.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e128.49\u0026thinsp;\u0026plusmn;\u0026thinsp;6.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e78.58\u0026thinsp;\u0026plusmn;\u0026thinsp;7.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Establishing a simple method for efficiently enriching olive leaves phenol extracts in refined olive oils.\u003c/h2\u003e \u003cp\u003eTwo existing methods enrich phenols in olive oil from olive leaves. The first method involves soaking crushed olive leaves in oil for an extended period before filtering out leaf fragments. The second method extracts target phenols from olive leaves using alcohol, then mixes this extract with olive oil, and finally removes the alcohol to obtain the enriched oil. Both methods were unsatisfactorily effective, increasing total phenol content to below 100 mg/kg, insufficient for refined olive oil to meet the EFSA's health claim requirements for phenolic content. Therefore, there is a need for an efficient enrichment method.\u003c/p\u003e \u003cp\u003eWe first compared three common enrichment methods: water bath oscillation, ultrasonic water bath, and ultrasonic pulse probe. The solid-oil ratio and temperature were consistent across all three treatments, with a 120-minute treatment time for the oscillating water bath and 20 minutes for the other two methods. The results of the phenolic enrichment treatment of ROO by each method are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. After enrichment, the total phenol content was 62.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.29 mg/kg for VOO, 78.14\u0026thinsp;\u0026plusmn;\u0026thinsp;5.33 mg/kg for UBOO, and 269.15\u0026thinsp;\u0026plusmn;\u0026thinsp;14.79 mg/kg for UPOO, all exceeding the blank group's 27.78\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25 mg/kg. Among the three devices, only the ultrasonic pulsed probe-treated ROO exceeded the EFSA's specified 250 mg/kg phenolic content.\u003c/p\u003e \u003cp\u003eAfter selecting the ultrasonic pulsed probe method, we explored its parameters further, with results presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB demonstrates the significance of the enriched solids-to-oil ratio for increasing total phenol content in the oil. Although a 1:3 ratio yielded the highest increase in total phenols, the increase was only 13.0 mg/kg more than the 1:5 ratio, making 1:5 the economically favorable choice. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC identifies 300W as the optimal ultrasonic power for phenolic enrichment. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD indicates that total phenol content in the oil first increased then decreased with prolonged ultrasound time, likely due to oil oxidation degrading the phenolics; the optimal ultrasound duration was 10 minutes.\u003c/p\u003e \u003cp\u003eUsing optimal conditions, we produced PEOO with a phenolic content of 451.75\u0026thinsp;\u0026plusmn;\u0026thinsp;12.03 mg/kg, a 67.8% increase over UPOO (269.15\u0026thinsp;\u0026plusmn;\u0026thinsp;14.79 mg/kg), which hadn't undergone process optimization. Additionally, our enrichment effect was 5 to 20 times greater than reported in previous studies.\u003c/p\u003e \u003cp\u003eWe then analyzed and compared the phenolic composition of PEOO with that of ROO, VOO, UBOO, and two EVOOs. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e displays HPLC plots at 280 nm for the enriched oils after different enrichment treatments, visualizing the enrichment effects on ROO. The enrichment resulted in a phenolic composition of ROO similar to that of EVOO.\u003c/p\u003e \u003cp\u003eThe most significant increases in ROO phenol content were in oleacein, luteolin-7-O-glucoside, and hydroxytyrosol, with increases of 158.50 mg/kg, 32.31 mg/kg, and 29.46 mg/kg, respectively. These phenols contributed to 37.4%, 7.6%, and 6.9% of the total phenol increase, respectively. Oleacein offers various health benefits, including obesity regulation, anticancer, and anti-inflammatory effects (Y. Huang et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and adds a pleasant bitter and spicy flavor to olive oil (Genovese et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Following our enrichment process, the oleacein content in PEOO was 2.4 to 3.1 times higher than in the two EVOOs. The enriched oleacein content was 3.69 times higher than previously reported methods (42.95 mg/kg) (S\u0026aacute;nchez de Medina et al. 2011). To our knowledge, no reports exist on enriching ROO with such large amounts of oleacein. Luteolin-7-O-glucoside, abundant in olive leaves but scarce in olive oil, has significant anti-inflammatory and antioxidant activities. Increasing its content benefits olive oil's health properties (Park and Song \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Following enrichment, luteolin-7-O-glucoside content in PEOO was nearly 8 times that of EVOO, exceeding VOO and UBOO, and significantly higher than the 1.60\u0026ndash;1.42 \u0026micro;g/mL reported by others (Jap\u0026oacute;n-Luj\u0026aacute;n et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Hydroxytyrosol, a key phenol in olive leaves, is also present in olive oil. Hydroxytyrosol, found in high-quality olive oil, has strong antioxidant activity (Kiritsakis \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Mkaouar et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The enriched PEOO had higher hydroxytyrosol content than both EVOO groups, with a 7-fold and 3.5-fold increase over VOO and UBOO, respectively, and significantly surpassing previous research findings (S\u0026aacute;nchez de Medina et al. 2011).\u003c/p\u003e \u003cp\u003eOleuropein aglycone, produced from oleuropein by enzymes like β-glucanase, is unstable. Oleuropein aglycone offers pharmacological benefits, including anti-Alzheimer's, anti-breast cancer, anti-inflammatory, and hypoglycemic effects (Xu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). PEOO's enrichment of oleuropein aglycone fell short of expectations. Post-enrichment, the content rose from 2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59 mg/kg to 20.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 mg/kg, lower than in both EVOOs. Interestingly, oleuropein aglycone content decreased in VOO and UBOO, likely due to degradation during enrichment. Our method increased the oleuropein content in PEOO by 12.02 mg/kg, achieving a concentration comparable to EVOO and surpassing values reported in literature (Jap\u0026oacute;n-Luj\u0026aacute;n et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Oleuropein, abundant in olive leaf extract but scarce in olive oil, is attributed to its high polarity. Higher oleuropein levels enhance the bitter flavor.\u003c/p\u003e \u003cp\u003eThe enrichment method raised the levels of quercetin, luteolin, tyrosol, and apigenin in ROO by 11.34, 5.38, 4.79, and 3.11 mg/kg, respectively, surpassing results documented in literature (S\u0026aacute;nchez de Medina et al. 2011; Sanmartin et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The enriched PEOO had quercetin and luteolin contents 2.6 and 1.9 times, and 5.8 and 2.7 times higher than those in the two EVOOs, respectively. Even after enrichment, the tyrosol content remained lower than that of EVOO. This may stem from two factors. The first is tyrosol's high polarity; the second, the solubility of phenols, varies with the fatty acid composition of the raw oil (Paulo and Santos \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; S\u0026aacute;nchez de Medina et al. 2011). Apigenin, undetected in EVOO, is a flavonoid known for its anti-inflammatory, antitumor, antioxidant, and cardiovascular protective effects (Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our enrichment method introduced this compound into olive oil.\u003c/p\u003e \u003cp\u003eIn summary, enriching ROO with olive leaf phenolic solid extracts using ultrasonic pulsed probes is effective. Optimal conditions include a 1:5 ratio of enriched solids to oil, 300 W ultrasonic power, and 10 min of ultrasonic time. The enriched PEOO's total phenol content reached 451.75\u0026thinsp;\u0026plusmn;\u0026thinsp;12.03 mg/kg by the optimal process, 12.3 and 8.4 times higher than VOO and UBOO, respectively. Our method enriched ROO with oleacein, hydroxytyrosol, and luteolin-7-O-glucoside at 162.43\u0026thinsp;\u0026plusmn;\u0026thinsp;13.82, 31.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07, and 33.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98 mg/kg, respectively, 3.1, 2.2, and 7.3 times higher than in EVOO. Notably, oleacein's increase, representing 37.4% of the total phenolic boost, is unprecedented in other studies. We have developed a straightforward and efficient method to enrich ROO with olive leaf phenol extracts.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Quality Test of ROO, PEOO, EVOO1, EVOO2. (A) relative peroxide values at 14 days. (B) odor level and flavor intensity, consumer preference and sensory positive attributes. (C) sensory defects.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFree acidity, peroxide value, antioxidant activity of COO, PEOO, EVOO1 and EVOO2.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eROO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePEOO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEVOO1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEVOO2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFree Acidity(Oleic acid %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeroxide Value(meq O\u003csub\u003e2\u003c/sub\u003e/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e18.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDPPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e154.75\u0026thinsp;\u0026plusmn;\u0026thinsp;4.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e81.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e74.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eABTS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e189.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e136.73\u0026thinsp;\u0026plusmn;\u0026thinsp;3.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e139.72\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFRAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e22.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e95.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e44.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e38.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Quality evaluation of the oil before and after phenols enrichment.\u003c/h2\u003e \u003cp\u003eThe work described above developed a simple and efficient method for phenol enrichment of ROO. We determined and compared the acid value, peroxide value, oxidative stability, antioxidant capacity, and sensory characteristics of optimally processed PEOO with control oils (ROO, EVOO1, EVOO2) to assess the phenolic enrichment method's impact.\u003c/p\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1. Free Acidity\u003c/h2\u003e \u003cp\u003eFree acidity, indicating free fatty acid content (often as a percentage of oleic acid), inversely correlates with oil quality. According to the IOC (\u0026ldquo;IOC COI/T.15/NC No 3/Rev. 18 Trade standard applying to olive oils and olive pomace olis\u0026rdquo; 2022), EVOO's free acidity must not exceed 0.80%, and ROO's, 0.30%. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that PEOO's free acidity rose by 0.05 units post-enrichment, yet remained below both ROO's standard and that of the two EVOOs. The phenol enrichment method minimally impacted the oil's free fatty acid content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2. Peroxide Value\u003c/h2\u003e \u003cp\u003eThe peroxide value measures the oxidation level of fats and fatty acids. It represents the quantity of oxidation products in a 1 kg sample, in millimoles of peroxide. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e indicates PEOO's peroxide value rose post-enrichment yet remained under the IOC's threshold of 5.0 for ROO and significantly below EVOO's 20.0 standard. The olive oil's fatty acids experienced minor oxidation following phenol enrichment but retained their high quality.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.4.3 Oxidative Stability\u003c/h2\u003e \u003cp\u003eThis experiment dynamically monitored the oil's peroxide value under extreme temperature and ventilation to assess its oxidative stability. Oil samples were maintained at 65\u0026deg;C with continuous ventilation, and their peroxide value measured every 2 days. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA displays the relative peroxide values of the four oils; lower change values indicate better oxidative stability. ROO, with the lowest phenol content, exhibited the greatest peroxide value change, while enriched PEOO showed the least, at 0.6 times that of ROO. The peroxide value changes in the four oils negatively correlated with their phenol contents, aligning with findings from another study (Rubi\u0026oacute; et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Phenols significantly enhanced the oxidative stability of olive oil. Interestingly, the peroxide change curves for PEOO, EVOO1, and EVOO2, high in phenol content, initially steepened then flattened, while ROO's curve, with minimal phenol, remained linear. The oxidative protection of the oil by phenols was particularly evident in the later stages of the oxidation reaction. In summary, the phenolic enrichment of ROO in this study significantly increased its oxidative stability, surpassing that of commercial EVOO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.4.4. Antioxidant Activity\u003c/h2\u003e \u003cp\u003eThis study utilized DPPH, ABTS, and FRAP assays to analyze the antioxidant capacity of enriched oils. A single method is insufficient for a comprehensive assessment of antioxidant properties in food products, as different methods yield a broader range of results (D. Huang et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that PEOO's four radical scavenging abilities were significantly stronger than ROO's, consistent with the oxidative stability results. Higher values in DPPH, ABTS, and FRAP assays indicate greater free radical scavenging capacity of the oil. After phenol enrichment, PEOO's DPPH value was 7.1 times that of ROO, and 1.9 and 2.1 times higher than EVOO1 and EVOO2, respectively. PEOO's ABTS value was 15.4 times that of ROO and 1.4 times higher than EVOO1 and EVOO2. PEOO's FRAP value was 4.3 times that of ROO, and 2.2 and 2.5 times higher than EVOO1 and EVOO2, respectively. A comprehensive comparison revealed that the four methods complemented each other, showing that phenolic-enriched PEOO significantly enhanced antioxidant capacity over non-phenolic-enriched ROO, surpassing the commercial EVOOs. Therefore, this study's method significantly increased ROO's antioxidant activity and improved its quality.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSensory Evaluation of COO, PEOO, EVOO1 and EVOO2.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eROO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePEOO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEVOO1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEVOO2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOdor level and flavor intensity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConsumer preference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive attributes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFruity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSweet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBitter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePungent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDefects\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFusty/muddy sediment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMusty-humid-earthy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWiney-vinegary-acid-sour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetallic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreasy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e \u003ch2\u003e3.4.5. Sensory Evaluation\u003c/h2\u003e \u003cp\u003eThe sensory characteristics of PEOO, ROO, EVOO1, and EVOO2, including odor, flavor intensity, and consumer preference, were evaluated and compared. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e show that after enrichment, PEOO's odor intensity, fruity flavor, sweetness, bitterness, and pungency increased by 162%, 54%, 19%, 525%, and 585% respectively, compared to ROO, while consumer preference decreased by 46%. PEOO exhibited stronger odor intensity, bitterness, and pungency, but lower fruitiness, sweetness, and consumer preference than both EVOO groups. The decrease in consumer preference could be attributed to the unfamiliarity of the Chinese with oils having bitter and spicy flavors. Conversely, Spanish consumers preferred olive oils with high bitterness, pungency, and a \"fruity\" flavor (Zamuz et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Consumer preferences for olive oil's taste and flavor vary between traditional and non-traditional producing countries (Latino et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The increase in PEOO's bitterness was mainly due to higher levels of oleacein, oleuropein, and oleuropein aglycone (Marx et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), while the increased pungency was likely due to higher levels of ligstroside derivatives (Andrewes et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC compares the defect sensations of PEOO with those of ROO, EVOO1, and EVOO2. All five defective sensations in PEOO increased compared to ROO, particularly the metallic sensation (232% increase), possibly due to contact with the ultrasound pulse probe's metal. PEOO's fusty/muddy sediment, winey-vinegary-acid-sour, and greasy sensations remained similar to those in EVOO. Overall, this study's methodology endowed ROO with positive attributes, enhancing its characteristic flavor, though the flaws also impacted its organoleptic quality. Fortunately, these flaws are within acceptable limits.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study established a simple method to prepare chlorophyll-free phenols from olive leaves and an efficient method to enrich them in refined olive oils. Under optimal conditions, the total phenol content in ROO increased to 424 mg/kg, exceeding the EFSA health claim threshold of 250 mg/kg and surpassing the total phenolic content in EVOO. The phenolic composition of PEOO, treated with the enrichment method, resembled that of EVOO. Oleacein, hydroxytyrosol, and luteolin-7-O-glucoside levels reached 3.1, 2.2, and 7.3 times those in EVOO, respectively. The antioxidant capacity and oxidative stability of PEOO were significantly improved, along with enhancement of certain flavors. Our study offers a simple, practical, and efficient strategy for high-value utilization of olive leaves and improving ROO's quality.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e Yunfei Huang and Wenqing He wrote the main original draft. Ruifeng Wang, Yangyang Jia, Lu Li, and Yawei Xu prepared Figures. 1-5. Yuhan Lu, Xiaoxiao Zhang and Feixue Wu prepared Tables. 1-4. Chunmei Li is project consultant who checked the manuscript text. All the authors approved the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This work was supported by the National Natural Science Foundation of China, 32172201.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e The data that support the finding of this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAndrewes, P., Busch, J. L. H. C., De Joode, T., Groenewegen, A., \u0026amp; Alexandre, H. (2003). Sensory Properties of Virgin Olive Oil Polyphenols: Identification of Deacetoxy-ligstroside Aglycon as a Key Contributor to Pungency. \u003cem\u003eJournal of Agricultural and Food Chemistry\u003c/em\u003e, \u003cem\u003e51\u003c/em\u003e(5), 1415\u0026ndash;1420. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf026042j\u003c/span\u003e\u003cspan address=\"10.1021/jf026042j\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaccouri, B., Rajhi, I., Theresa, S., Najjar, Y., Mohamed, S. N., \u0026amp; Willenberg, I. (2022). The potential of wild olive leaves (Olea europaea L. subsp. oleaster) addition as a functional additive in olive oil production: the effects on bioactive and nutraceutical compounds using LC\u0026ndash;ESI\u0026ndash;QTOF/MS. \u003cem\u003eEuropean Food Research and Technology\u003c/em\u003e, \u003cem\u003e248\u003c/em\u003e(11), 2809\u0026ndash;2823. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00217-022-04091-y\u003c/span\u003e\u003cspan address=\"10.1007/s00217-022-04091-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenzie, I. F. F., \u0026amp; Strain, J. J. (1996). 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Bioactive compounds in wine: Resveratrol, hydroxytyrosol and melatonin: A review. \u003cem\u003eFood Chemistry\u003c/em\u003e, \u003cem\u003e130\u003c/em\u003e(4), 797\u0026ndash;813. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2011.08.023\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2011.08.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGenovese, A., Caporaso, N., \u0026amp; Sacchi, R. (2021). 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Consumer Acceptance and Quality Parameters of the Commercial Olive Oils Manufactured with Cultivars Grown in Galicia (NW Spain). \u003cem\u003eFoods\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(4), 427. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods9040427\u003c/span\u003e\u003cspan address=\"10.3390/foods9040427\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"refined olive oil, olive leaves, olive phenols, phenol-enriched olive oil, oleacein, antioxidant activity","lastPublishedDoi":"10.21203/rs.3.rs-4020617/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4020617/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe refining process almost completely removes phenols from refined olive oil (ROO). Enriching ROO with olive phenols can significantly enhance its quality and health benefits. However, current enrichment methods are inefficient and overlook the negative impact of chlorophyll present in the phenol extract on the oil. In this study, we developed a straightforward two-step dissolve and resaturate process to prepare chlorophyll-free phenols from olive leaves and efficiently enrich ROO using the ultrasonic pulsed probe method. Under optimal conditions, the total phenol content in ROO increased by 424 mg/kg, including increases of 158.5 mg/kg (37.4%) for oleacein, 29.5 mg/kg (7.0%) for hydroxytyrosol, and 17.9 mg/kg (4.2%) for oleuropein aglycone. This also significantly enhanced the antioxidant activity, oxidative stability, and some flavor characteristics of ROO. Our study offers a straightforward, practical, and effective strategy for the valuable use of olive leaves and for improving the quality of ROO.\u003c/p\u003e","manuscriptTitle":"A simple method for preparing chlorophyll free phenols from olive leaves and efficiently enriching it in refined olive oil","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-11 19:46:45","doi":"10.21203/rs.3.rs-4020617/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-13T03:52:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-12T19:14:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-11T10:22:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72438916985748622250696046459800144769","date":"2024-06-03T03:00:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315291750867833951820236165442559838340","date":"2024-06-01T09:27:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84347711101696339537238581265860991895","date":"2024-05-06T11:46:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8c6458be-6498-48a4-9037-189aeb6385ff","date":"2024-03-12T14:51:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-10T12:01:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-07T08:43:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-07T08:05:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2024-03-06T09:23:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d2725dde-1b38-4d33-ac8f-2326b37d7593","owner":[],"postedDate":"March 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-05T23:09:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-11 19:46:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4020617","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4020617","identity":"rs-4020617","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}