A highly automated method for simultaneous determination of sterols, erythrodiol and uvaol in olive oils

Gu, Qiang; Yi, Xin; Shen, Jinling; Shi, Jing; Liu, Yijun; Shao, Jiahui

doi:10.1039/c6ay00357e

Cited by 8 publications

(1 citation statement)

References 30 publications

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“…(Meziane & Kadi, 2008), σπόρους του φυτού Jatropha curcas L. (Santos et al, 2015), κελύφη καρύδας (Sulaiman et al, 2013), σπόρους του φυτού Ρίκινου (Amarante et al, 2014), σόγια (Toda et al, 2016, Dagostin et al, 2015, λιναρόσπορο (Kostić et al, 2013), και βαμβακόσπορο (Saxena, Sharma, & Sambi, 2011 (Meziane & Kadi, 2008, Amarante et al, 2014, Amin, Hawash, Diwani, & Rafei, 2010, Rodrigues, Pinto, & Fernandes, 2008 (Kostić et al, 2013, Meziane & Kadi, 2008. (Boskou, 2011, Gu et al, 2016, Yuan, Ju, Jin, Ren, & Liu, 2015. Η κινητική μελέτη διεξήχθη χωριστά για τις επιμέρους κύριες στερόλες (βσιτοστερόλη, καμπεστερόλη, στιγμαστερόλη) και για το σκουαλένιο του πυρηνέλαιου.…”

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Εναλλακτικές Μέθοδοι Εκχύλισης Φυτικών Λιπαρών Και Βιοδραστικών Συστατικών Τους

Chanioti¹,

Χανιώτη²

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Olive pomace is the solid by-product resulting from the olive oil production by the continuous centrifugation process, performed through a three-phase or a two-phase system. It is a low-cost material produced in large quantities and the common practice for its valorization is the recovery of its oil (olive-pomace oil) in small-scale industrial plants. The olive pomace oil displays almost similar composition to olive oil, however olive pomace oil has higher unsaponifiable matter content than olive oil, and it is particularly richer in certain sterols (such as β-sitosterol) and in squalene; these bioactive compounds have a remarkable biological activity. Besides being a source of oil, olive pomace is also an important source of other bioactive compounds, such as phenolic compounds. In recent years, the recovery of these bioactive compounds has been proposed, which can be used as antioxidants and antimicrobial agents in food, pharmaceutical and cosmetic systems.The aim of this PhD dissertation was to evaluate innovative alternative extraction methods against conventional (by organic solvent) to obtain high-quality olive-pomace oil enriched in bioactive compounds such as sterols and squalene, as well as to obtain phenolic compounds directly from the olive pomace with improved properties (bioactivity) that can be incorporated into food systems (to produce functional products) providing benefits for human health.The extraction process using organic solvent was studied as the most established method in the food industry in order to recover oil from oilseeds. Olive pomace with initial moisture content of 45% w/w, d.w. was used and it was dried until moisture content 4.5% w/w, d.w. and ground to a particle size of 1 mm. A kinetic study and modeling of the extraction process was carried out to obtain both the olive pomace oil and its unsaponifiable matter (estimated as USM% of oil). The kinetic study extraction experiments were performed on a scale of 25 g of dried olive pomace and using hexane as a solvent to examine the main parameters affecting the process, such as temperature (T=40-60 °C), solid:liquid ratio (S:L=1:4-1:12 g/mL), agitation speed (AS=100-800 rpm) and extraction time (t=1-60 min), and the evolution of the process affecting its cost and to investigate its sustainability on an industrial scale. Based on the results, the mechanism of the mass transfer of the oil and its unsaponifiable compounds in the solvent was revealed and it was found that the fastest transfer of soluble components to the solvent was carried out at the beginning step of the process (washing step), followed by a transfer rate decrease step (diffusion step) until it reached equilibrium, corresponding to the maximum extraction yield. The experimental data for the oil and its unsaponifiable matter (USM% of oil) were used to estimate the variables of two mathematical models: the model of So & Macdonald, (1986) and the model of Sulaiman et al. (2013). The experimental data showed a very good fit to the So & Macdonald model (1986) providing high determination coefficient values (R2>0.996) and low values of relative deviations (0.40<ARD<3.05). However, the model of Sulaiman et al. (2013) provided lower determination coefficient (R2>0.981) and higher values of relative deviations (3.75<ARD<5.73). From the designed parameters for both models, higher temperatures (60 °C), solid: liquid ratio (1:12 g/mL) and agitation speed (800 rpm) led to increased oil yield (5.83-7.11% olive pomace oil) and favored the extraction of unsaponifiable matter in the extracted oil (USM%=3.40-4.23% of oil), while the mass transfer coefficients were increased (kw, kd for the model of So & Macdonald and k·α for the model of Sulaiman et al.) during the extraction. The So & Macdonald (1986) kinetic model variables for extracting both the oil and its unsaponifiable matter under different conditions (temperature-T, solid: liquid ratio-S:L and agitation speed-AS) proved again the predominance of the washing stage in relation to the diffusion stage, since the washing coefficients (1.05 <kw<2.27) were higher than the diffusion coefficients (0.11<kd<0.21). The values of enthalpy change (ΔH) and entropy change (ΔS) were positive, while the values of Gibbs free energy change (ΔG) were negative, indicating that the process was endothermic, irreversible and spontaneous. Under equilibrium conditions, the oil yield increases by a factor of about 1.096 and 1.054 for the models of So & Macdonald and Sulaiman et al., respectively, for every 10 °C rise in temperature.The kinetic model proposed by So & Macdonald (1986) was used to correlate experimental data for selected bioactive compounds of the unsaponifiable matter of olive pomace oil, such as squalene and major sterols (β-sitosterol, campesterol, stigmasterol) (mg/g oil), using the same extraction parameters (T=40-60 °C, S: L=1:4-1:12 g/mL, AS=100-800 rpm, and t=1-60 min). Similarly to the extraction of the oil and its unsaponifiable matter, the kinetics of these individual components are characterized by a rapid washing step and a subsequent slower diffusion step. Regarding the kinetic extraction model variables for squalene, β-sitosterol, stigmasterol and campesterol under different conditions (T, S:L, AS), the washing step proved again the prevalent step in relation to the diffusion step, since the washing coefficients (kw) were higher than the diffusion coefficients (kd). Among the tested values, higher temperature (60 °C), solid: liquid ratio (1:12 g/mL) and agitation speed (800 rpm) resulted in an increased concentration of β-sitosterol (0.81-1.18 mg/g of oil), campesterol (0.16-0.20 mg/g oil), stigmasterol (0.10-0.14 mg/g oil) and squalene (3.95-4.20 mg/g oil) of the extracting oils.Furthermore, extraction using a co-solvent was examined to recover components of different polarity. Isopropyl alcohol was used as co-solvent, and various mixtures of hexane (H):isopropanol (I) in various ratios (H:I=3:1, H:I=3:2, H:I=4:1, H:I=9: 1) were evaluated in terms of the oil yield, the unsaponifiable matter (USM%) as well as the major sterols and squalene content in the extracted oil. The extraction experiments were carried out on a Soxhlet apparatus on a scale of 25 g of dried olive pomace with a particle size (PS) of 1 mm and a solid/liquid ratio (S:L) of 1:8 g/mL for 8 h. Among the solvent mixtures tested, the H:I=3:2 mixture exhibited the highest oil recovery (13.70% olive pomace), the largest unsaponifiable matter in the extracted oil (USM=4.92% of oil) and high sterols and squalene concentration (total sterols=1,706 mg/g oil, squalene=5,217 mg/g oil).Ultrasonic assisted - extraction (UAE), an "eco-friendly" extraction method that provides efficient and selective oil extraction while protecting the bioactive components from degradation, was applied to extract both the olive pomace oil and to recover its bioactive compounds that constitute its unsaponifiable matter such as sterols and squalene. RSM and Box-Behnken experimental design identified polynomial equations correlating the oil yield, as well as the unsaponifiable matter (USM%), the total content of phenolic compounds (TPC) and the antioxidant activity (DPPH) of the extracted olive pomace oil with the process parameters (T=40-60 °C, S:L=1:4-1:12 g/mL and PS=0.5-2 mm). The extraction experiments were carried out on a scale of 25 g of dried olive pomace and a predetermined volume of hexane in an ultrasonic bath for 1 h. The maximum oil yield (11.22% olive pomace) was achieved with T=60 °C, S:L=1:12 g/mL and PS=0.5 mm. The maximum value of USM% of oil was 4.50% and was achieved at optimal conditions T=55 °C, S:L=1:12 g/mL and PS=0.5 mm. The maximum TPC value of the olive pomace oil was 0.261 mg GA/g of oil and was obtained by extraction of the olive pomace in conditions: T=50 °C, S:L=1:8 g/mL and PS=0.9 mm. The maximum antioxidant activity (DPPH) of the olive pomace oil was found to be 0.211 mg Trolox/g oil and was achieved at T= 55 °C, S:L=1:8 g/mL and PS=0.9 mm. Through the model, the optimization of the recovery of the major sterols (β-sitosterol, campesterol and stigmasterol) and squalene, that are the main components of the unsaponifiable fraction of the oil, and the effect of the main extraction parameters (T=40-60 °C and S:L=1:4-1:12 g/mL) were examined. The maximum β-sitosterol concentration was found to be 1.399 mg/g at T=60 °C and S:L=1:12 g/mL. The maximum concentration of campesterol was 0.220 mg/g and was achieved at T=55 °C for S:L=1:10 g/mL. The maximum concentration of stigmasterol was found to be 0.105 mg/g, by applying T=50 °C and S:L=1:12 g/mL. Furthermore, the maximum concentration of squalene was found to be 4.575 mg/g at T= 60 °C and S:L=1:12 g/mL.In addition, the combination of H:I=3:2 mixture and ultrasound extraction under the optimal conditions (T=50-60 °C, S:L=1:12 g/mL, t=1 h) was examined and showed similar results to those obtained by applying UAE and using hexane under the same conditions in terms of oil yield, unsaponifiable matter (USM%) and the individual sterols of the olive pomace oil. The optimal conditions of ultrasonic assisted extraction (UAE) were compared with the conventional Soxhlet extraction (SE) (extraction conditions: S:L=1:8 g/mL, T≈78 °C, t=8h, PS=1 mm , solvent=hexane and H:I=3:2 mixture) with respect to the extraction yield of olive-pomace oil, USM%, individual major sterols, squalene, TPC and antioxidant activity (DPPH) of recovered oil. It was found that the olive pomace oil under optimal UAE conditions showed higher yield (11%) and better quality compared to its unsaponifiable matter (USM=4.51% of oil), the total content of phenolic compounds (TPC=0.255 mg GA/g of oil) and the antioxidant activity of the extracted oil (DPPH=0.225 mg Trolox/g oil) from the corresponding extracted oil obtained by Soxhlet (SE) with hexane as solvent (USM=3.68% of oil, TPC=0.207 mg GA/g oil and DPPH=0.189 mg Trolox/g oil, respectively). It should be noted that olive pomace oil obtained by UAE reached about 82% of the corresponding oil yield and 92% of its total unsaponifiable matter (USM%) and showed higher TPC (5.5%) and antioxidant activity (DPPH) (by 8%) than that obtained by the SE method and the H:I=3:2 mixture as solvent. As far as the individual sterols and squalene are concerned, the β-sitosterol concentration obtained under optimal UAE conditions was higher (1.349 mg/g of oil) than that obtained by the SE method and using hexane (0.911 mg/g oil) or mixture H:I=3:2 (1.121 mg/g oil). The extracted oil by the SE method and the H:I=3:2 mixture had a similar concentration of campesterol (0.228 mg/g oil) to oil obtained by UAE (0.208 mg/g oil) and lower stigmasterol concentration 0.101 mg/g oil) compared to the one obtained by UAE (0.115 mg/g oil). The concentration of squalene in the extracted oils was found to decrease in the following order: SE-H:I=3:2>UAE-hexane≈UAE-H:I=3:2>SE-hexane.Furthermore, the extraction of phenolic compounds directly from the olive pomace was investigated. The effect of the type of organic solvent and its concentration in water (% v/v) was examined by conventional extraction in terms of the total content of phenolic components (TPC), the antioxidant activity (DPPH) of the extracts as well as the main phenolic components of the extracts. Organic solvents such as ethanol, methanol and acetone in different ratios in water (50, 70 and 90% v/v) were examined, and the conventional extraction was optimized by two-level Box-Behnken design (T=60 °C, S:L=1:12.5 g/mL, PS of dried olive pomace=1 mm, t=4 h). According to the results of the study, ethanol 70% v/v was chosen as the extraction solvent providing extracts with the highest total content of phenolic compounds (TPC=23,061 mg GA/g olive pomace d.w.), the maximum antioxidant activity (DPPH=18,156 g of olive pomace d.w./g DPPH), and with the highest concentration of oleuropein (OL), hydroxytyrosol (HY), rutin (RU) and the total determined phenolic components of the extracts (SUM) (OL=0.834 mg/g olive pomace, HY=1.04 mg/g olive pomace, RU=0.493 mg/g olive pomace and SUM=2.41 mg/g olive pomace).In addition, the effect of the enzymes on the yield of phenolic compounds from the olive pomace was investigated. The enzymes selected were a mixture of pectinase and polygalacturonase (Novozym 33095) and when the optimal concentration of the enzyme solution (1% v/v) was determined, the effect of temperature (T=40 and 60 °C) and time (t=1 and 4 h) on aqueous extraction (CEW), aqueous enzymatic extraction (CEE) and extraction using 70% ethanol (CEEth) for the recovery of its phenolic compounds (S:L=5 g/mL, PS of dried olive pomace=1 mm) was evaluated. The aqueous extraction was performed using buffer pH=4,5. The use of ethanol 70% resulted in the recovery of extracts with the maximum total content of phenolic compounds (TPC=23.88 mg GA/g olive pomace d.w.) and the maximum antioxidant activity (DPPH=18.36 g olive pomace d.w./g DPPH). However, by applying the aqueous enzymatic extraction at 60 °C, more phenolic-enriched extracts with higher antioxidant activity compared to the aqueous extraction were obtained (TPC=11.41 mg GA/g olive pomace d.w. for the CEE and TPC=9.92 mg GA/ g olive pomace d.w. for CEW, respectively). The highest concentration of OL was obtained with ethanol (OL=0.868 mg/g olive pomace), followed by aqueous enzymatic extraction (OL=0.549 mg/g olive pomace) for 4 h at 60 °C. Also, ethanol 70% was the most effective solvent for HY extraction, however the use of enzymes provided extracts with a concentration of HY that reached 88% of the corresponding of extracts obtained by ethanol as solvent.In addition, the application of ultrasound (UAE) was investigated for the extraction of phenolic compounds from olive-pomace. Process parameters that influence the effectiveness of the method (solvent type=ethanol, methanol and acetone, concentration=50, 70 and 90% v/v, T=40, 60, 80 °C) were tested and the UAE optimized in terms of total content of phenolic compounds (TPC), antioxidant activity (DPPH) and the main phenolic components of the extracts (S:L=1:12.5 g/mL, PS of dried olive pomace=1 mm, t=30 min, frequency=60 kHz). The maximum TPC was found to be 21.77 mg GA/g of olive pomace d.w. by applying T= 60 °C and ethanol at 70% concentration. The maximum value of the antioxidant activity was 20.95 g of olive pomace d.w./g of DPPH by applying T=60 °C and 70% ethanol. OL, HY and SUM exhibited the maximum concentration (OL=0.674 mg/g of olive pomace, HY=1.161 mg/g of olive pomace and SUM=2.32 mg/g of olive pomace) in the extracts obtained by applying T=60 °C and ethanol at a concentration of 70%, while the maximum concentration of RU was found at 0.421 mg/g of olive pomace by applying T=70 °C and 70% ethanol. Comparing the results with those obtained by the conventional method, it was found that the TPC of the extracts obtained under optimal conditions using UAE reached 93% of that obtained by the CE method and also the ultrasound extract showed 13% less antioxidant activity (DPPH) compared to that obtained by the CE. The extract obtained under optimal UAE conditions showed higher HY concentration of 11% and the SUM was similar (98%) to that of the conventional method (CE). However, it should be noted that the optimum result from UAE was obtained at reduced extraction time (t=30 min), whereas the corresponding result with the CE method was obtained for t=4 h.Microwave assisted - extraction (MAE) was investigated as another alternative extraction method for the recovery of phenolic compounds from olive pomace and the effect of temperature (T=40 and 60 °C) and time (t=5 and 30 min) on aqueous extraction (MAEW), aqueous enzymatic extraction (MAEE), and extraction with ethanol 70% (MAEEth) (S:L=1:12.5 g/mL, PS of dried olive pomace=1 mm, microwave power=200 W) was evaluated. The use of 70% ethanol and microwave (MAEEth) resulted in the recovery of extracts with maximum TPC [≥73% of TPC and ≥5% less antioxidant activity than the one obtained by conventional extraction method (CEEth) at 60 °C for 4 h, respectively, which has been extracted in a shorter time (t=30 min)]. It is also worth noting that by applying the microwave aqueous enzymatic extraction (MAEE) at 60 °C for 30 min, the extracts showed ⁓ 21% more phenolic content (TPC) and better antioxidant activity of ⁓9% compared to the corresponding extracts obtained by the aqueous enzymatic extraction by the conventional method (CEE) at 60 °C for 4 h (TPC=14.37 mg GA/g olive pomace d.w. and DPPH=20.23 g olive pomace d.w./g DPPH for MAEE, respectively). By using ethanol 70% and MAE (MAEEth), OL was efficiently extracted [≥83% more OL compared to the conventional method (CEEth)]. The application of MAE and the use of enzymes (MAEE) for 30 min at 60 °C gave extracts of a similar OL concentration (OL=0.549 mg/g olive pomace) than those obtained using enzyme and conventional extraction (CEE) at 60 °C for 4h. The SUM of the MAE extracts appeared to be generally higher, and specifically for 30 min at 60 °C, it was 64%, 27% and 28% higher using 70% ethanol (SUM=7.040 mg/g olive pomace for MAEEth), aqueous enzymatic extraction (SUM=2,463 mg/g olive pomace for MAEE) and aqueous extraction (SUM=1,975 mg/g olive pomace for MAEW) respectively, compared to that of the corresponding extracts obtained by conventional extraction.The homogenate assisted extraction (HAE) was evaluated as an innovative extraction method for the recovery of phenolic compounds from the olive pomace and the effect of parameters such as temperature (T=40 and 60 °C) and agitation speed (AS=4000 and 12000 rpm) on aqueous extraction (HAEW), aqueous enzymatic extraction (HEEE) and ethanol 70% extraction (HEEEEth) (S:L=1:12.5 g/mL, t=30 min) was evaluated. In addition, the enzyme pretreatment of the olive pomace (1% v/v enzyme solution at 40 °C for 1 h) was examined before extraction with a high speed homogenizer. The use of ethanol 70% and high speed homogenizer (HAEEth) led to the recovery of extracts with the maximum content of phenolic compounds (TPC=26.37 mg GA/g olive pomace d.w.), which is 12% higher than the corresponding content of the extracts by conventional extraction method (CEEth) and the maximum antioxidant activity (DPPH=17.62 g olive pomace d.w./g DPPH), about 5% more active than those obtained by conventional extraction (CEEth) at 60 °C for 4 h and obtained in less time (t=30 min). By applying aqueous enzymatic extraction and using a high-speed homogenizer (HAEE) at 60 °C at 12000 rpm for 30 min, extracts showed ≥37% more total phenolic content (TPC=18.38 mg GA/g olive pomace d.w.) compared to the corresponding extracts obtained by the aqueous enzymatic extraction by the conventional method (CEE) at 60 °C for 4 h and 20% more total phenolic content than the corresponding extracts obtained by the aqueous enzymatic extraction using microwaves ( MAEE) at 60 °C for 30 min. Comparing the results for the enzyme pretreatment of the olive pomace, it was proved that they were not significantly different from those of the aqueous enzymatic extraction without pretreatment, thus the activity of the enzyme mixture is sufficient during the application of HAE. Ethanol 70% and high speed homogenizer (HAEEth) extracted effectively about 86% more OL (OL=6.34 mg/g olive pomace) compared to the conventional method (CEEth). The use of high speed homogenizer and enzymes (HAEE) gave extracts of similar HY concentration compared to those obtained using 70% ethanol (HAEEth) (HY=0.32 mg/g olive pomace). The concentration of RU in ethanolic extracts was found to be higher (0.66 mg/g olive pomace) compared to the corresponding aqueous enzyme extraction (RU=0.33 mg/g olive pomace). The SUM of the extracts using 70% ethanol (HAEEth) appeared to be generally higher; in particular, for 30 min at 60 °C and 12000 rpm, it was 67% greater than that of the corresponding extracts obtained by conventional extraction method (CEEth). In general, the combination of enzymes and high-speed homogenizer (HAEE) contributed to extracts with more enriched phenolic profiles and higher concentration of individual phenolic compounds compared to conventional extraction (CEW and CEE) and microwave extraction (MAEW and MAEE).The application of HHPAE as an innovative extraction method for the recovery of phenolic compounds from olive pomace was investigated and the effect of pressure (P=300 and 600 MPa) and time (t=5 and 10 min) on aqueous extraction (HHPAEW), aqueous enzymatic extraction (HHPAEE) and ethanol 70% extraction (HHPEAEEth) (S:L=1:12.5 g/mL, PS of dried olive pomace=1 mm, T=25 °C). The use of ethanol 70% (HHPAEEth) and aqueous enzymatic extraction (HHPAEE) combined with high hydrostatic pressure led to the recovery of extracts with the maximum content of phenolic compounds. The ethanolic extracts (TPC=16.52 mg GA/g olive pomace d.w.) consisted of ⁓71% of the corresponding extracts by conventional extraction method (CEEth) and were obtained in less time (t=10 min). Furthermore, by applying aqueous enzymatic extraction and using high hydrostatic pressure (HHPAEE) at 600 MPa for 10 min, extracts were obtained by ≥23% more total phenolic content (TPC=14.84 mg GA/g olive pomace d.w.) compared to the corresponding extracts obtained by the aqueous enzymatic extraction and the conventional method (CEE) at 60 °C for 4 h and 36% more total phenolic content, compared to the corresponding extracts obtained by the aqueous extraction and the use of high hydrostatic pressure (HHPAEW) at 600MPa for 10 min (TPC=9.25 mg GA/g olive pomace d.w.). The extracts with a maximum antioxidant activity were obtained by applying HHPAEE at 600MPa for 10 min (DPPH=20.32 g olive pomace d.w/g DPPH) and were about 90% as effective as those resulting from the application of conventional (CEE) extraction at 60 °C for 4 h as well as 73% and 32%, respectively more active compared to those obtained by aqueous extraction and extraction with 70% ethanol using HHP (DPPH=35.21 g olive pomace d.w./g DPPH for HHPAEW and DPPH=26.85 g olive pomace d.w./g DPPH for HHPAEEth, respectively). Ethanol 70% and HHP extracted effectively ⁓84% more OL (OL=4.42 mg/g olive pomace) compared to the conventional method (CEEth). The use of HHP and enzymes (HHPAEE) gave extracts of similar HY concentration (HY=0.38 mg/g olive pomace) and RU (RU=0.22 mg/g olive pomace) than those obtained using ethanol 70 % (HHPAEEth). The SUM of the extracts using 70% ethanol was generally higher and in particular at 600 MPa for 10 min (SUM=5.13 mg/g olive pomace) was 52% higher than that of the corresponding extracts obtained by conventional method extraction (CEEth) (at 60 °C for 4 h).New green eutectic solvents (NADES) have been studied in combination with alternative innovative extraction methods, such as the use of high-speed homogenizer (HAE), microwaves (MAE), ultrasounds (UAE) and high hydrostatic pressure (HHPAE) to recover phenolic compounds from olive pomace. Four different eutectic solvent systems (NADES) consisting of a mixture of choline chloride with maltose (DES-MA), glycerol (DES-GLY), citric acid (DES-CA) and lactic acid (DES-LA) and different process parameters, depending on the extraction method (S:L=1:12.5 g/mL, PS of dried olive pomace=1 mm, t=30 min). The DES-CA showed the best extraction yield with respect to TPC and the antioxidant activity (DPPH) of the extracts obtained by applying HAE (TPC=34.08 mg GA/g olive pomace d.w., DPPH=5.11 g olive pomace d.w./g DPPH) and UAE (TPC=20.14 mg GA/g olive pomace d.w., DPPH=20.69 g olive pomace d.w./g DPPH) and DES-LA by applying MAE (TPC=29.57 mg GA/g olive pomace d.w., DPPH=17.51 g olive pomace d.w./g DPPH) and HHPAE (TPC=25.96 mg GA/g olive pomace d.w., DPPH=15.67 g olive pomace d.w./g DPPH), respectively. HAE has been shown to be the most effective method achieving extracts with the highest yields, strongest antioxidant activity and highest concentration of OL (OL=12,86 mg/g olive pomace), RU (RU=1,71 mg/g olive pomace) as well as SUM (SUM=18.30 mg/g olive pomace) compared to MAE, UAE and HHPAE. HPLC analysis of the extracts showed that almost all of the extracts obtained by eutectic solvents (NADES) in all of tested methods were more enriched in phenolic compounds than those with conventional solvents [ethanol 70%, water (buffer pH=4.5)].Moreover, applications in food systems were examined in order to evaluate antioxidant activity of the extracts of the phenolic compounds from olive pomace. In particular, the antioxidant activity of olive pomace extract and its fractions (hexane, chloroform, ethyl acetate, and water) was studied in a standard unsaturated fatty acid such as linoleic acid and in a vegetable oil such as sunflower oil (at a concentration of 200 ppm). The ethanolic extract and the ethyl acetate fraction had a stronger antioxidant activity (2.32 g extract/g DPPH for the ethanolic extract and 1.20 g extract/g DPPH for the ethyl acetate fraction) than the hexane fraction (247, 2 g extract/g DPPH), the chloroform fraction (5.41 g extract/g DPPH) and the water fraction (3.71 g extract/g DPPH) against linoleic acid oxidation (at 40 °C for 140 h). In addition, the higher antioxidant activity in thermal oxidation of sunflower oil was observed by the fraction of ethyl acetate having the highest concentration in total content of phenolic compounds (TPC= 08.41 mg GA/g extract). The antioxidant activity of the ethanolic extract and its fractions on thermal oxidation of sunflower oil (for 60 h at 80 °C) based on the peroxide values was: BHT>ethyl acetate fraction>ethanol extract>chloroform fraction>water fraction>hexane fraction>α-tocopherol>control.Furthermore, the incorporation of the phenolic compounds of the olive pomace as they are and/or encapsulated in emulsions based on olive oil (enriched olive oil) has been applied and their antioxidant activity and oxidative stability in enriched olive oils have been investigated. Enriched olive oils (with the addition of phenolic compounds TPC≈0,115 mg GA/g of oil) were studied under accelerated oxidation conditions (storage at 80 °C). The peroxide value, the p-anisidine value, and the absorption coefficients K232 and K270 of all the enriched olive oils were significantly lower than those of the original olive oil during the 15-day storage, indicating that the olive-pomace is also an important source of phenolic compounds that are capable of improving the oxidative stability of the oils. In addition, the results of the change of the oxidative stability of the original olive oil and the enriched olive oil under accelerated conditions with the Rapid Oxitest® method showed that the original olive oil was oxidized more rapidly [induction period-IP=22.13 h], while the phenolic compounds from the olive-pomace were able to prolong the oxidative stability of the enriched olive oils, showing higher IP values (IP=23.59 h-27.99 h) compared to the original olive oil. Therefore, the extracts from olive pomace could be used as natural additives in the food industry as they enhance the oxidative stability of the final products.Therefore, the study reveals that there are interesting alternatives to conventional extraction in order to obtain high-quality olive-pomace oil enriched in bioactive compounds (sterols and squalene) as well as to obtain phenolic compounds (oleuropein, hydroxytyrosol, rutin etc.) directly from the olive pomace with improved properties that can be incorporated into food systems, leading to the production of new/innovative products.

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