Development of Analytical Strategies for the Determination of Olive Fruit Bioactive Compounds Using UPLC-HRMS and HPLC-DAD. Chemical Characterization of Kolovi Lesvos Variety as a Case Study.

In this study, an overall survey regarding the determination of several bioactive compounds in olive fruit is presented. Two methodologies were developed, one UPLC-Q-TOF-MS method for the determination of olive fruit phenolic compounds and one HPLC-DAD methodology targeting the determination of pigments (chlorophylls and carotenoids), tocopherols (α-, ß, -γ, δ-) and squalene. Target and suspect screening workflows were developed for the thorough fingerprinting of the phenolic fraction of olives. Both methods were validated, presenting excellent performance characteristics, and can be used as reliable tools for the monitoring of bioactive compounds in olive fruit samples. The developed methodologies were utilized to chemical characterize the fruits of the Kolovi olive variety, originating from the island of Lesvos, North Aegean Region, Greece. Twenty-five phenolic compounds were identified and quantified in Kolovi olives with verbascoside, hydroxytyrosol, oleacein and oleomissional found in significantly high concentrations. Moreover, 12 new bioactive compounds were identified in the samples using an in-house suspect database. The results of pigments analysis suggested that Kolovi variety should be characterized as low pigmentation, while the tocopherol and squalene content was relatively high compared to other olive varieties. The characterization of Kolovi olive bioactive content highlighted the high nutritional and possible economic value of the Kolovi olive fruit.

Antioxidant quantification in different portions obtained during olive oil extraction process in an olive oil press mill.

BACKGROUND: Different antioxidant compounds are generally transferred from olives to olive oil during the production process. This work characterized the principal total bioactive compounds (tocopherols and phenols) in olives, olive oil and by-products of four cultivars grown in Calabrian areas (southern Italy), considering the effect of harvesting period. Antioxidant capacity, total and individual phenolic compounds were also analysed. RESULTS: Drupes, olive paste, pomace and olive wastewater showed similar phenolic compounds, while olive oil possessed a different composition, suggesting that phenols are not only transferred from drupe to oil, but also they change during oil production. Tocopherols varied among cultivars and harvesting period: generally, they were more abundant in samples produced in the first harvesting period. Qualitative and quantitative differences in phenolic composition and antioxidant activity were significantly found among cultivars in all the matrices. CONCLUSION: The highest amount of total phenolic antioxidants ended up in olive wastewater with variability due to the olive cultivar, while only a small part of them finished in the oil. This work shows evidence that the availability of bioactive compounds in different portions from the olive oil extraction belong to different varietal origins. In particular, new information was acquired on Ottobratica Calipa, a new olive clone, that produced an olive oil with an interesting antioxidant amount. © 2020 Society of Chemical Industry.

Changes in volatile organic composition of olive oil extracted from cv. 'Leccino' fruit subjected to ethylene treatments at different ripening stages.

BACKGROUND: Olive, as a non-climacteric fruit, is presumed to be ethylene independent with regard to ripening triggering/coordination. Nevertheless, studies have demonstrated that postharvest ethylene treatments induce changes in composition and properties also of non-climacteric fruits, including aroma profiles, a key quality parameter of extra virgin olive oils. Olive fruit of cv. 'Leccino' harvested at two distinct ripening stages (less advanced ripening, LAR; and more advanced ripening, MAR, with Jaén index of 4.58 and 5.10, respectively) were subjected to ethylene (1000 ppm in air) treatment for 24 h before oil extraction. RESULTS: Based on multivariate analysis of volatile organic compound (VOCs), the effect of ethylene treatment appeared to be more pronounced in MAR samples. However, differences in organoleptic analysis were also detected in ethylene-treated LAR olive oils. Ethylene seems to selectively affect linolenic/linoleic acid metabolism, particularly concerning the C5 pathway, and reduce specific defect-associated compounds. CONCLUSION: Exogenous ethylene applied to cv. 'Leccino' olives before processing was effective in inducing specific changes in the VOC profiles of the resulting oil. The effect was different depending on the ripening stage of the harvested olives. The lipoxygenase pathway (including the production of C5 compounds) and fermentative-related compounds appeared to be affected by the treatment. © 2020 Society of Chemical Industry.

Utilization of New Technologies in the Production of Pharmaceutical Olive Oil.

Olive oil is a key ingredient in the Mediterranean diet and offers many health benefits. However, many factors affect the quality and quantity of olive oil such as olive tree diseases and olive-related pests. Unfortunately, the procedure of identifying pests or the outbreak of a disease is time-consuming, and it depends heavily on the size of the olive grove. Through the use of ICT, remote monitoring of the olive grove can be achieved, by collecting environment-related data and having an overview of the olive grove's overall health. In this paper we propose a low-cost dense network of sensors that collects daily data regarding the olive grove, thus, providing the possibility to prevent infestation of olive fruit fly and/or the outbreak of olive tree-related disease.

Isotopic Traceability (13C and 18O) of Greek Olive Oil.

In recent years, isotopic analysis has been proven a valuable tool for the determination of the origin of various materials. In this article, we studied the 18O and 13C isotopic values of 210 olive oil samples that were originated from different regions in Greece in order to verify how these values are affected by the climate regime. We observed that the δ18O isotopic values range from 19.2 ‱ to 25.2 ‱ and the δ13C values range from -32.7 ‱ to -28.3 ‱. These differences between the olive oils' isotopic values depended on the regional temperature, the meteoric water, and the distance from the sea. Furthermore, we studied the 13C isotopic values of biophenolic extracts, and we observed that they have same capability to differentiate the geographic origin. Finally, we compared the isotopic values of Greek olive oils with samples from Italy, and we concluded that there is a great dependence of oxygen isotopes on the climatic characteristics of the different geographical areas.

Biogas Potential of the Side Streams Obtained in a Novel Phenolic Extraction System from Olive Mill Solid Waste.

The olive oil production is an important industrial sector in many Mediterranean areas, but it is currently struggled by the necessity of a proper valorisation of the olive mill solid waste or alperujo. The alperujo is the main by-product generated during the two-phase olive oil extraction, accounting for up to 80% of the initial olive mass. The alperujo is a source of valuable compounds, such as the pomace olive oil or highly interesting phenolic compounds. In the present research, a novel biorefinery approach has been used for phenolic compounds recovery. However, the extraction of these valuables compounds generates different exhausted phases with high organic matter content that are required to be managed. This study consists of the evaluation of the anaerobic biodegradability of the different fractions obtained in a novel biorefinery approach for the integral valorisation of alperujo. The results show that the different phases obtained during the biorefinery of the alperujo can be effectively subjected to anaerobic digestion and no inhibition processes were detected. The highest methane yield coefficients were obtained for the phases obtained after a two-months storages, i.e., suspended solids and liquid phase free of suspended solids, which generated 366 ± 7 mL CH4/g VS and 358 ± 6 mL CH4/g VS, respectively. The phenol extraction process reduced the methane yield coefficient around 25% due to the retention of biodegradable compounds during the extraction process. Regardless of this drop, the anaerobic digestion is a suitable technology for the stabilization of the different generated residual phases, whereas the high market price of the extracted phenols can largely compensate the slight decrease in the methane generation.

Volatile and Sensory Profiles of Algerian Extra-Virgin Olive Oil from Souidi and Zeletni Cultivars.

In this study, the volatile and sensory profiles of monovarietal extra-virgin olive oil (EVOO) from two less widespread Algerian autochthonous cultivars (Souidi and Zeletni) were obtained using headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry and a panel test, respectively. A total of 14 and 10 volatile compounds belonging to different chemical classes were identified and quantified in the Souidi and Zeletni EVOOs, respectively. Zeletni EVOO contains 2.07 times more (E)-2-hexenal than Souidi EVOO. In addition, the amounts of C6 compounds from LA and LnA, as well as the total amount of the compounds of the LOX pathway were higher in Zeletni than in the Souidi EVOO. Another important finding was the predominance of sesquiterpene ß-ocimene in the composition of the volatile fraction of Souidi EVOO. The sensory profiles of the EVOOs analyzed were characterized by fruity, bitter and pungent sensory positive attributes, perceived at medium intensity in both oils studied.

Cold-Pressing Olive Oil in the Presence of Cryomacerated Leaves of Olea or Citrus: Nutraceutical and Sensorial Features.

The nutraceutical properties of extra-virgin olive oil (EVOO) can be further improved by the addition of olive leaves during olive pressing. However, while Citrus leaves are rich sources of bioactive substances, no data are available in the literature about the effect of Citrus leaf addition on the nutraceutical and sensorial profiles of olive oil. This study aimed at comparing the chemical and sensorial qualities of olive oils obtained from ripe olives pressed together with either Olea or Citrus spp. (lemon or orange) cryomacerated leaves. General composition parameters as well as major antioxidants and antioxidant activity were measured. A panel test evaluation, as well as headspace volatile characterization (headspace solid phase microextraction, HS-SPME), were also performed. All data were compared with an EVOO extracted from the same olive batch used as control. It was possible to obtain Leaf Olive Oils (LOOs) characterized by a higher (p < 0.05) content of antioxidants, compared to the control sample, and the highest oleuropein concentration was detected in the olive oil extracted in presence of olive leaf (+50% in comparison with the control). All the LOOs showed a higher smell complexity and the scent of ripe fruit was generally mitigated. Lemon and olive LOOs showed the best smell profile.

Innovative technologies in virgin olive oil extraction process: influence on volatile compounds and organoleptic characteristics.

BACKGROUND: Innovative technologies are experimentally applied to the virgin olive oil extraction process in order to make it continuous and more efficient. Most of the efforts aim at overcoming the limitations of the traditional malaxation step, which, however, is essential for the development of virgin olive oil sensory notes. RESULTS: Compared to the traditional process, innovative technologies based on the heat exchanger led generally to a decrement in volatile lipoxygenase (LOX) alcohols linked to alcohol dehydrogenase activity and, conversely, to a slightly increase in volatile LOX esters. Aldehydes from the same pathway were not significantly affected. However, an industrial combined plant constructed from a heat exchanger, low-frequency ultrasound device and microwave apparatus determined the highest 'fruity' intensity perceived by panellists, in accordance with the highest value of total volatiles, with values significantly higher than heat exchanger alone, which, instead, had the lowest levels of hexanal and LOX alcohols. The pungent taste showed the same trend observed for 'fruity' intensity, whereas bitter taste did not show significant differences among trials. CONCLUSION: The introduction of ultrasound, coupled with heat exchanger and microwave, seemed not to modify the behaviour of enzymes of the LOX pathway, and the obtained virgin olive oils showed volatiles and organoleptic characteristics not significantly different from those obtained by the traditional olive oil extraction process. These findings provided the first insights into the effect of the combination of innovative technologies in the olive oil extraction process on virgin olive oil volatiles and sensory characteristics. © 2019 Society of Chemical Industry.

Influence of the feed pipe position of an industrial scale two-phase decanter on extraction efficiency and chemical-sensory characteristics of virgin olive oil.

BACKGROUND: Nowadays, olive oil extraction is basically achieved by means of two-phase decanters, which allow a reduction of water consumption and the leaching of phenolic compounds. Despite this, most of the working settings derive from studies carried out on three-phase decanters. Hence, the aim of the present study was to assess the influence of two-phase decanter feed pipe position (FP) on the extraction efficiency and chemical-sensory characteristics of virgin olive oil. Three different positions were considered: at 825 mm (FP1), 610 mm (FP2) and 520 mm (FP3) from the outlet of the oily phase. RESULTS: Position FP3 allowed the highest oil recovery (up to 10%), the lowest percentage of oil in the olive pomace and, in general, a regular trend in terms of oil extraction efficiency. However, the oily must that came out of the decanter was not completely clean in terms of residual content of solid sediment and water. The feeding position partially affected the profile of antioxidant compounds. CONCLUSION: In two-phase decanters, loading the olive paste close to the outlet of the oily phase is recommended to increase the extraction efficiency without jeopardizing the chemical-sensory characteristics of virgin olive oil. © 2018 Society of Chemical Industry.

Concentrates of triterpenic acids obtained from crude olive pomace oils: characterization and evaluation of their potential antioxidant activity.

BACKGROUND: Pentacyclic triterpenic acids (TA) are phytochemicals of increasing nutritional interest owing to their bioactive properties, such as anti-inflammatory, antitumoral, antihyperglycemic and hepatoprotective. Crude olive pomace oils constitute a non-exploited significant source of these compounds. In the present study, concentrates of TA were extracted and characterized from crude olive pomace oils that were obtained by centrifugation and subsequent solvent extraction, respectively. Specifically, the concentrates were obtained from the byproduct generated in the filtration of the oils. The solids were subjected to Soxhlet extractions with hexane to remove the residual oil and then with ethanol for the TA extraction. RESULTS: Concentrates containing 850-980 g kg-1 TA were isolated from the oils obtained by centrifugation, whereas those isolated from oils obtained by hexane extraction presented levels of TA that ranged from 510 to 900 g kg-1 . Oleanolic (OA) and maslinic (MA) acids were the TA found in the concentrates. The relative contents of OA and MA were, respectively, 30:70 (w/w) and 77:23 (w/w). All concentrates also presented phenolic compounds at levels of g kg-1 and displayed slight antioxidant properties. CONCLUSION: Concentrates of TA, containing MA and OA, can be readily obtained from a byproduct generated by filtration of crude olive pomace oils. Concentrates isolated from oils obtained by centrifugation were rich in MA, whereas those from oils extracted with hexane were rich in OA. The concentrates showed slight antioxidant properties that can be mainly attributed to the presence of phenolic compounds and not to TA. © 2018 Society of Chemical Industry.

Isolation and characterization of phenol degrading bacterium strain Bacillus thuringiensis J20 from olive waste in Palestine.

This study aimed at isolation of phenol degrading bacteria from olive mill wastes in Palestine. The efficiency of phenol removal and factors affecting phenol degradation were investigated. A bacterial strain (J20) was isolated from solid olive mill waste and identified as Bacillus thuringiensis based on standard morphological, biochemical characteristics and 16SrRNA sequence analysis. The strain was able to grow in a phenol concentration of 700 mg/L as the sole carbon and energy source. The culture conditions showed a significant impact on the ability of these cells to remove phenol. This strain exhibited optimum phenol degradation performance at pH 6.57 and 30 °C . Under the optimized conditions, this strain could degrade 88.6% of phenol (700 mg/L) within 96 h when the initial cell density was OD600 0.2. However, the degradation efficiency could be improved from about 88% to nearly 99% by increasing the cell density. Immobilization of J20 was carried out using 4% sodium alginate. Phenol degradation efficiency of the immobilized cells of J20 was higher than that of the free cells, 100% versus 88.6% of 700 mg/L of phenol in 120 h, indicating the improved tolerance of the immobilized cells toward phenol toxicity. The J20 was used in detoxifying crude OMWW, phenolic compounds levels were reduced by 61% compared to untreated OMWW after five days of treatment. Hence, B. thuringiensis-J20 can be effectively used for bioremediation of phenol-contaminated sites in Palestine. These findings may lead to new biotechnological applications for the degradation of phenol, related to olive oil production.

State of the Art on Functional Virgin Olive Oils Enriched with Bioactive Compounds and Their Properties.

Virgin olive oil, the main fat of the Mediterranean diet, is per se considered as a functional food-as stated by the European Food Safety Authority (EFSA)-due to its content in healthy compounds. The daily intake of endogenous bioactive phenolics from virgin olive oil is variable due to the influence of multiple agronomic and technological factors. Thus, a good strategy to ensure an optimal intake of polyphenols through habitual diet would be to produce enriched virgin olive oil with well-known bioactive polyphenols. Different sources of natural biological active substances can be potentially used to enrich virgin olive oil (e.g., raw materials derived from the same olive tree, mainly olive leaves and pomaces, and/or other compounds from plants and vegetables, mainly herbs and spices). The development of these functional olive oils may help in prevention of chronic diseases (such as cardiovascular diseases, immune frailty, ageing disorders and degenerative diseases) and improving the quality of life for many consumers reducing health care costs. In the present review, the most relevant scientific information related to the development of enriched virgin olive oil and their positive human health effects has been collected and discussed.

Specification of a new de-stoner machine: evaluation of machining effects on olive paste's rheology and olive oil yield and quality.

BACKGROUND: An industrial prototype of a partial de-stoner machine was specified, built and implemented in an industrial olive oil extraction plant. The partial de-stoner machine was compared to the traditional mechanical crusher to assess its quantitative and qualitative performance. The extraction efficiency of the olive oil extraction plant, olive oil quality, sensory evaluation and rheological aspects were investigated. RESULTS: The results indicate that by using the partial de-stoner machine the extraction plant did not show statistical differences with respect to the traditional mechanical crushing. Moreover, the partial de-stoner machine allowed recovery of 60% of olive pits and the oils obtained were characterised by more marked green fruitiness, flavour and aroma than the oils produced using the traditional processing systems. CONCLUSION: The partial de-stoner machine removes the limitations of the traditional total de-stoner machine, opening new frontiers for the recovery of pits to be used as biomass. Moreover, the partial de-stoner machine permitted a significant reduction in the viscosity of the olive paste. © 2016 Society of Chemical Industry.

How 'ground-picked' olive fruits affect virgin olive oil ethanol content, ethyl esters and quality.

BACKGROUND: Olives dropped on the ground naturally sometimes are not separated from those fresh and healthy collected from the tree for harvest and processing. In this work we compared the quality, ethanol content and bioactive components of virgin olive oils from ground-picked olives, tree-picked fruits and their mixture. RESULTS: Ground-picked olives produced 'Lampante' virgin olive oils; these are of a lower quality category, because of important alterations in chemical and sensory characteristics. Ethyl esters showed the highest values, although under the regulated limit. The mixture of ground and tree-picked olives gave oils classified as 'virgin' because of sensory defects, although the quality parameters did not exceed the limits for the 'extra' category. Ethanol content showed a significant increase in the oils from ground- picked olives and their mixture with respect to those from tree-picked fruits. Furthermore, bioactive compounds showed a significant decrease as fruit quality was poorer. CONCLUSION: Ground-picked olives must be harvested and processed separately since they produce low-quality virgin olive oils with sensory defects and lower concentrations of bioactive compounds. The higher acidity and ethanol concentration observed in oils from ground-picked fruits or their mixture may help ethyl ester synthesis during storage. © 2015 Society of Chemical Industry.

Comparison Between Different Flavored Olive Oil Production Techniques: Healthy Value and Process Efficiency.

Three different flavoring methods of olive oil were tested employing two different herbs, thyme and oregano. The traditional method consist in the infusion of herbs into the oil. A second scarcely diffused method is based on the addition of herbs to the crushed olives before the malaxation step during the extraction process. The third innovative method is the implementation of the ultrasound before the olive paste malaxation. The objective of the study is to verify the effect of the treatments on the quality of the product, assessed by means of the chemical characteristics, the phenol composition and the radical scavenging activity of the resulting oils. The less favorable method was the addition of herbs directly to the oil. A positive effect was achieved by the addition of herbs to the olive paste and other advantages were attained by the employment of ultrasound. These last two methods allow to produce oils "ready to sell", instead the infused oils need to be filtered. Moreover, the flavoring methods applied during the extraction process determine a significant increment of phenolic content and radical scavenging activity of olive oils. The increments were higher when oregano is used instead of thyme. Ultrasound inhibited the olive polyphenoloxidase, the endogenous enzyme responsible for olive oil phenol oxidation. This treatment of olive paste mixed with herbs before malaxation was revealed as the most favorable method due to the best efficiency, reduced time consumption and minor labor, enhancing the product quality of flavored olive oil.

Effect of Extra Virgin Olive Oil on Biomarkers of Inflammation in HIV-Infected Patients: A Randomized, Crossover, Controlled Clinical Trial.

BACKGROUND: Premature atherosclerosis in HIV-infected patients is associated with chronic infection by itself and adverse effects of antiretroviral treatment (ART). Extra virgin olive oil (EVOO) has a beneficial effect on the cardiovascular system because of its anti-inflammatory properties. The objective of this study was to determine whether the consumption of EVOO improves inflammation and atherosclerosis biomarkers in HIV-infected patients receiving ART. MATERIAL AND METHODS: This randomized, crossover, controlled trial included 39 HIV-positive male participants who consumed 50 mL of EVOO or refined olive oil (ROO) daily. Four participants dropped out of the study. Leukocyte count, erythrocyte sedimentation rate (ESR), high-sensitivity C-reactive protein (hsCRP), interleukin-6, fibrinogen, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, malondialdehyde, glutathione-peroxidase, superoxide dismutase, oxidized LDL and von Willebrand factor were determined before the first and after each of the 2 intervention periods. Intervention and washout periods lasted for 20 and 14 days, respectively. RESULTS: In participants with >90% compliance (N=30), hsCRP concentrations were lower after EVOO intervention (geometric mean [GM], 1.70 mg/L; 95% confidence interval [CI], 1.15-2.52) compared to ROO administration (GM, 2.92 mg/L; 95% CI, 1.95-4.37) (p=0.035). In participants using lopinavir/ritonavir, ESR and hsCRP concentrations decreased 62% and 151%, respectively, after EVOO administration. In the whole study population (N=35) we found no difference in analyzed biomarkers after EVOO administration. CONCLUSIONS: Our exploratory study suggests that EVOO consumption could lower hsCRP in patients on ART.

Chemical and Biological Investigation of Olive Mill Waste Water - OMWW Secoiridoid Lactones.

Olive mill waste water is the major byproduct of the olive oil industry containing a range of compounds related to Olea europaea and olive oil constituents. Olive mill waste water comprises an important environmental problem in olive oil producing countries, but it is also a valuable material for the isolation of high added value compounds. In this study, an attempt to investigate the secoiridoid content of olive mill waste water is described with the aid of ultrahigh-performance liquid chromatography-electrospray ionization (±)-high-resolution mass spectrometry and centrifugal partition chromatography methods. In total, seven secoiridoid lactones were isolated, four of which are new natural products. This is the first time that a conjugate of hydroxytyrosol and a secoiridoid lactone has been isolated from olive mill waste water and structurally characterized. Furthermore, the range of isolated compounds allowed for the proposal of a hypothesis for the biotransformation of olive secoiridoids during the production of olive mill waste water. Finally, the ability of the representative compounds to reduce the intracellular reactive oxygen species was assessed with the dichlorofluorescein assay in conjunction with the known antioxidant agent hydroxytyrosol.

Assessment of the olive oil extraction plant layout implementing a high-power ultrasound machine.

The objective of this study is to assess the effects of installation and operation of a high-power ultrasound machine (HPU) for the treatment of olive paste by using ultrasound technology in order to evaluate the best way installation and the best definition of the operating conditions of the machine. The study was conducted installing in an industrial olive oil mill a continuous processing ultrasound machine, which used a frequency of 20 kHz able to work at 3200 kg h-1 as feed capacity. Checking of performance has been carried out by the assessment of the different operating and process conditions, assessing in particular the impact of the ultrasound treatment before and after the malaxation phase on performance indicators of the continuous olive oil plant (plant extractability, olive paste rheological characteristic) and on selected chemical properties of the olive oil extracted (quality parameters, antioxidant content, and volatile profiles). In the tested conditions, high-power ultrasound treatment did not produce significant effect on the legal parameters (free acidity, peroxide index and spectrophotometric indexes), while a significant increase in the content of phenolic compounds was generally observed; higher enhancements were more evident when the high-power ultrasound treatment was carried out before the malaxation phase.

Application of response surface methodology (RSM) for the optimization of supercritical CO2 extraction of oil from patè olive cake: Yield, content of bioactive molecules and biological effects in vivo.

The two-phase technology for olive oil extraction generates large amounts of patè olive cake (POC), a by-product that is rich in bioactive health-promoting compounds. Here, response surface methodology (RSM) was used to maximize supercritical-CO2 oil extraction from POC, while minimizing operative temperature, pressure and time. Under the optimal parameters (40.2 °C, 43.8 MPa and time 30 min), the oil yield was 14.5 g·100 g-1 dw (~65% of the total oil content of the freeze-dried POC matrix), as predicted by RSM. Compared with freeze-dried POC, the oil contained more phytosterols (13-fold), tocopherols (6-fold) and squalene (8-fold) and was a good source of pentacyclic triterpenes. When the biological effects of POC oil intake (20-40 µL·die-1) were evaluated in the livers of BALB/c mice, no significant influence on redox homeostasis was observed. Notably, a decline in liver triglycerides alongside increased activities of NAD(P)H:Quinone Oxidoreductase 1, Carnitine Palmitoyl-CoA Transferase and mitochondrial respiratory complexes suggested a potential beneficial effect on liver fatty acid oxidation.