Oxidative stability of sunflower oil enriched with ethyl acetate extracts from olive mill by-products during oxidation tests and frying

In this study, the effects of olive mill wastewater (OMW) and olive pomace (OP) extracts (1 and 2 mg/g), lecithin (L, 1–5 mg/g), and ascorbyl palmitate (AP, 0.2 mg/g) on the oxidative stability of refined sunflower oil (SFO) and its purified triacylglycerols (TAGs) were investigated under accelerated oxidation conditions. Induction period of TAGs increased from 6.84 to 11.27 hr, with OMW methanol extract (1 mg/g), while OP extracts provided less protection (9.79 hr) at 90°C in Rancimat. The K232 values of the SFO with extracts (1 mg/g) and AP (0.2 mg/g) was lower than those of butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and α-tocopherol containing oil samples at 60°C. Binary combinations of extracts with AP (0.2 mg/g) or L (5 mg/g) also effectively decreased the K232 and K270 values of TAGs during oxidation. In conclusion, OMW and OP extracts can be synergistically used with L or AP for stabilization of SFO against oxidation. Practical applications Generally, edible oils contain high amount of unsaturated fatty acids which are susceptible to oxidation during heat required food processes. Natural antioxidants are preferably used for increasing oxidative stability of edible oils. Olive mill byproducts contain valuable phenolic compounds that have high antioxidant capacity. In this study, we studied antioxidant efficiencies of the olive wastewater and pomace extracts in sunflower oil in combinations with lecithin and ascorbyl palmitate. These combinations can be successfully used to increase the oxidative stability of sunflower oil.

In this study, oxidative stability of soybean oil (SBO) enriched with ethyl ecetate extracts of olive by-products was investigated. Total phenolic contents, phenolic profiles and antioxidant activities of olive wastewater (OMWW) and olive pomace (OP) extracts were also determined. Total phenolic contents of extracts obtained from OMWW and OP were 134.45 and 281.43 mg gallic acid equivalent (GAE)/g extract, respectively. While antioxidant activities of OMWW extracts in the linoleic acid emulsion were in the range of 85.79 % and 88.54 %, OP extracts had 83.30 % and 90.09 % at different concentrations (0.5, 1, 2 ve 3 mg/mL) after incubation at 37 °C. β-carotene bleaching activities of the extracts at 50 °C were found as 26.80-66.63% in OMWW extracts and 18.76-53.32% in OP extracts, respectively. 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of OP extracts were higher than those of OMWW extracts and ranged from 30.6% to 87.7% in OP extracts and 16.6% to 54.1% in OMWW extracts at these concentrations. Both the antioxidant and antiradical activities of extracts significantly increased with increased concentration (p

Cardiovascular toxicity in cancer patients receiving chemotherapy remains one of the most undesirable side effects, limiting the choice of the most efficient therapeutic regimen, including combinations of different anticancer agents. Anthracyclines (doxorubicin) and antimetabolites (5-fluorouracil (5-FU), capecitabine) are among the most known agents used in breast cancer and other neoplasms and are associated with cardiotoxic effects. Extra-virgin olive oil (EVOO) is rich in polyphenols endowed with antioxidant cardioprotective activities. Olive mill wastewater (OMWW), a waste product generated by EVOO processing, has been reported to be enriched in polyphenols. In this study, we investigated the activities of polyphenol-rich extract from OMWW, A009, in cooperation with chemotherapy on two breast cancer cell lines, namely, BT459 and MDA-MB-231, in a cardio-oncology perspective. The effects of A009 on cardiac cells were also investigated with and without chemotherapeutic agents. Cell viability was determined on BT459 and MDA-MB-231 (i.e., breast cancer cells) and H9C2 (i.e., rat cardiomyocytes) cells, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A spheroids assay was used as a 3D in vitro model on BT459 and MDA-MB-231 cells. For in vivo studies, the murine sponge assay of angiogenesis was used as a model of breast cancer-associated vascularization. The embryo of Danio rerio (zebrafish) was used to detect the cardioprotective activities of the OMWW. We found that the A009 extract exhibited antiangiogenic activities induced by breast cancer cell supernatants and increased T-cell recruitment in vivo. The combination of the OMWW extracts with doxorubicin or 5-FU limited BT459 and MDA-MB-231 cell viability and the diameter of 3D spheroids, while mitigating their toxic effects on the rat H9C2 cardiomyocytes. Cardioprotective effects were observed by the combination of OMWW extracts with doxorubicin in zebrafish embryos. Finally, in human cardio myocytes, we observed 5-FU-induced upregulation of the inflammatory, senescence-associated cytokine IL6 and p16 genes, which expression was reduced by OMWW treatment. Our study demonstrates that the polyphenol-rich purified OMWW extract A009 combined with cancer chemotherapy could represent a potential candidate for cardiovascular protection in breast cancer patients, while increasing the effects of breast cancer chemotherapy.

The production of olive oil generates massive quantities of by-product called olive mill wastewater (OMWW). The uncontrolled disposal of OMWW poses serious environmental problems. The OMWW effluent is rich in several polyphenolic compounds. Liquid-liquid extraction of OMWW using ethyl acetate solvent was used to enrich phenolic compounds under investigation. Total phenolic and flavonoid content and antioxidant activity of the extract were determined. HPLC coupled to photodiode array (PDA) detector was used to analyze the main three phenolic compounds of OMWW, namely, hydroxytyrosol, tyrosol, and oleuropein. The antimicrobial activity of the extract was also investigated. Additionally, the OMWW extract was used as natural preservative and antioxidants for olive oil. Results showed that OMWW is very rich in phenolic compounds and has strong antioxidant activity. HPLC analysis showed that the extract contains mainly hydroxytyrosol and tyrosol but no oleuropein. The OMWW extract showed also positive activities as antibacterial (gram positive and gram negative) and antifungal as well as activities against yeast. The addition of OMWW extract to olive oil samples has an effect on the stability of olive oil as reflected by its acid value, peroxide value, K232 and K270, and total phenolic content.

The valorization of olive pomace through the extraction of phenolic compounds at an industrial scale is influenced by several factors that can have a significant impact on the feasibility of this approach. These include the types and levels of phenolic compounds that are present, the impact that seasonal variation and cultivar type have on the phenolic compound content in both olive pomace and mill effluents and the technological approach used to process the olive crop. Chemical analysis of phenolic compounds was performed using an HPLC-diode-array detector (DAD)-qTOF system, resulting in the identification of 45 compounds in olive mill wastewater and pomace, where secoiridoids comprised 50–60% of the total phenolic content. This study examined three different factors that could impact the phenolic compound content of these processing streams, including cultivar types typically grown on local farms in Slovenia, the type of downstream processing used and seasonality effects. Olive crop varieties sourced from local farms showed high variability, and the highest phenolic content was associated with the local variety “Istrska Belica”. During processing, the phenolic content was on average approximately 50% higher during two-phase decanting compared to three-phase decanting and the type of compound present significantly different. An investigation into the seasonal effects revealed that the phenolic content was 20% higher during the 2019 growing season compared to 2018. A larger sample size over additional growing seasons is required to fully understand the annual variation in phenolic compound content. The methods and results used in this study provide a basis for further analysis of phenolic compounds present in the European Union’s olive crop processing residues and will inform techno-economic modelling for the development of olive biorefineries in Slovenia.

Efficient production of lignin-modifying enzymes and phenolics removal in submerged fermentation of olive mill by-products by white-rot basidiomycetes

Olive mill wastewater (OMW) was used as a source to extract radical scavenging compounds, in order to study their possible valorization as natural antioxidants. Their antioxidant activity (IC50 0.32 µg/ml) was found to be more potent than that of a commercial antioxidant: Ascorbic acid (AA) (IC50 1.91 µg/ml). The olive mill wastewater extract (OMWE) and AA were separately added to two vegetable oils, sunflower oil and rapeseed oil. These oils were then put under simulated storage conditions (accelerated test at 60°C) for 4 weeks. The physicochemical parameters, analysis of fatty acids, phytosterols, and tocopherols were the key parameters used to monitor the evolution of the oxidation state. The results obtained showed that the oils treated with the OMWE underwent oxidative deterioration slower than that of the control (without additives), and that phytosterols and tocopherols content decreased as a function of storage time following first-order kinetics. The use of the OMWE helped reduce the loss of both phytosterols and tocopherols in 4 weeks by half compared to the control samples, with the exception of total phytosterols in rapeseed oil where the loss reduction was not as important (about 2% loss reduction). Practical application Antioxidants are widely used in the food industry, as they prevent the development of rancid aromas that lead to harmful consequences during storage and cooking. The practical implication of the study is that natural radical scavenging compounds extracted from OMW can be efficiently used to enhance the quality and safety of refined oils stored at high temperature conditions.

Co-composting of olive pomace, olive mill waste water, dairy and poultry manure, and straw was conducted using 15 identical cylindrical 100-L reactors under laboratory conditions at five different C/N ratios. The changes in physical and chemical parameters of composting such as dry matter, organic matter, total phosphorus, ammonium nitrogen, nitrate nitrogen, pH, reduction in volume, and relative heat generation were evaluated based on the initial and final conditions. The results showed that when considering the maximum changes in physical and chemical parameters of the composting process, the ideal initial C/N ratios were determined to be between 25.97 to 33.07 applying regression analysis using the Gaussian equation. Conversely, the results based on ANOVA analysis suggested that the initial C/N ratio of the most ideal mixture (46% olive pomace, 36% separated dairy manure, 12% poultry manure, 5% straw, 1% olive mill wastewater) was 27.5 for this study. Comparing this composting recipe to the other one tested in this study, the use of straw, which is more valuable for agricultural use, is limited to 5%. Consequently, more separated dairy manure and olive pomace could be used in composting.

Introduction: Extra virgin olive oil is a basic component of the Mediterranean diet; it contains several molecules with antioxidant, anti-inflammatory, anti-angiogenic and pro-apoptotic properties against a variety of cancers and influencing cellular metabolism too. Olive mill waste water (OMWW), a liquid residue produced in vast amounts during olive oil extraction, represents an environmental issue but on the other side it is a rich source of phytochemicals with potential health benefits. Particularly, OMWWs are rich in different phenolic compounds, that show promise in preventing the development of several disease disorders, including cancer. We have assessed the anti-angiogenic and anti-tumor properties exerted by a nutraceutical from purified OMWW extract, in in vitro and in vivo models, and evaluated its effect in a human pilot study upon informed consent. Experimental procedures: OMWWs ability to affect cell proliferation and survival on different cancer cell lines was assessed by MTT. OMWWs interference with human umbilical endothelial vein cells (HUVECs) tube formation, migration and invasive capacities was studied by endothelial cell morphogenesis and migration assays. The inhibition of angiogenesis and tumor cell growth was investigated by the matrigel assay and tumour xenograft. To evaluate contribution of potential metabolomic biomarkers, SANIST (surface-activated chemical ionization-electrospray-NIST technology) mass spectrometric data-based platform has been employed on sera from a pilot study (Aiello et al, in preparation). Results: OMWW extract shown a potent inhibitory effect inhibiting cells proliferation of both cancer cell lines and HUVECs.OMWW inhibited several molecular targets expression, including IL8, Angiogenin, mTOR, VEGF, CXCR4, CXCL12 and CXCL8, in tumor cells and in TNF-α-stimulated HUVEC. We nalyzed sera from volunteers, after nutraceutical intervention with Oliphenoliaand we assessed several biological parameters. Endogenous vitamin D was significantly increased, as well as albumin, direct bilirubin, and calcium. We also observed glycemia and potassium decrease after Oliphenolia assumption. A previously unknow form of Vitamin D was also detected by mass spectrometry. Conclusions: OMWW extract has shown a stronger anti-angiogenic and anti-tumor properties, with potential chemo-preventive potentials. Results of human pilot study shows Oliphenolia as a promising and interesting natural dietary supplement, rich in polyphenol compounds, that can exert anti-inflammatory and antioxidant activities. Acknowledgements: This study was supported by a donation from the Fattoria La Vialla di Gianni, Antonio e Bandino Lo Franco - SAS (Arezzo, Italy). We are grateful to Professor Anna Aiello, and Professor Calogero Caruso, Palermo University for the pilot study. Citation Format: Adriana Albini, Luana Calabrone, Francesca Albini, Paola Corradino, Simone Cristoni, Douglas M. Noonan. A nutraceutical supplement derived from olive oil byproducts exhibits potential cancer preventive properties in preclinical studies and increases vitamin D levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7298.

The use of polyphenolic extract, purified hydroxytyrosol and 3,4-dihydroxyphenyl acetic acid from olive mill wastewater for the stabilization of refined oils: a potential alternative to synthetic antioxidants

The present study was conducted to determine and compare the oxidative stability of soybean and sunflower oils using differential scanning calorimetry (DSC). These edible oils were enriched with marjoram (Origanum majorana L.), thyme (Thymus vulgaris L.), and oregano (Origanum vulgare L.) extracts at three different concentrations and synthetic antioxidant (BHA). The fatty acid composition of studied oils was determined by gas chromatography mass spectrometry to evaluate the content of unsaturated fatty acids that are sensitive to oxidation process. Oil samples were heated in the DSC at different heating rates (4.0, 7.5, 10.0, 12.5, and 15.0 °C min−1) and oxidation kinetic parameters (activation energy, pre-exponential factor, and oxidation rate constant) were calculated. The results showed that the oxidative stability of sunflower oil samples enriched with oregano extracts and soybean oil supplemented with thyme extracts was improved compared to samples without the addition of herbal plant extracts and the synthetic antioxidant.

Olive mill wastewater (OMW) is one of the most environmentally concerning food processing effluents due to its phytotoxicity. Recently, several bioactive compounds with potential applications in food, pharmaceutical, and agricultural industries have been identified in OMW. This study aimed to compare, for the first time, the physico-chemical characteristics and biological activity of OMW obtained by two different types of three-phase decanters: a traditional one and a water-saving ARA decanter. DPPH, ABTS, FRAP, and β-carotene bleaching tests were used to investigate the antioxidant effects. The inhibition of key enzymes involved in hyperglycemia and hypolipidemia were also assessed. A high concentration of phenolic compounds was found in OMW obtained by the ARA-controlled system. Hydroxytyrosol resulted as the dominant compound, with a content of 502.3 mg/kg. OMW extract obtained by ARA decanter resulted as the most active in the FRAP test, with value of 67.23 µMFe (II)/g. A moderate inhibitory activity was found against α-amylase, α-glucosidase, and lipase enzymes. Data obtained by this study evidenced that the use of the ARA decanter allows for obtaining OMW extract characterized by a higher content of phytochemicals in comparison to those obtained by the traditional phase decanter, and a consequent higher biological activity. At the same time, the use of this equipment allows for the reduction of environment impact.

The solid fraction of olive mill waste water (OMWW) was separated from OMWW and then the solutes in the solid fraction of OMWW were extracted with ethanol. The detection of polyphenols in the ethanol extract showed the presence of polyphenols in the solid fraction of OMWW. Effects of solvent-to-solid ratio, extraction and agitation time on the extraction of polyphenols from the solid fraction of OMWW were examined and the maximum amount of polyphenol was extracted from the solid fraction of OMWW with a solvent-to-solid ratio of 15 at 70 min of extraction and 10 min of agitation time. Percent yields and purities of the polyphenols extracted from solid fraction of OMWW were higher than those of the polyphenols extracted from olive pomace with ethanol at 70 min of extraction and 10 min of agitation time with solvent-to-solid ratio of 15.

The disposal of olive mill wastewater (OMW) produced during oil extraction in Mediterranean countries creates a significant environmental problem because of the great volumes of effluent produced (10-12 Mm3 year-1), heavy pollution load (40-80 g L‑1 biological oxygen demand (BOD), 50-150 g L‑1 chemical oxygen demand (COD)) and phytotoxic properties (because of phenolic compounds). During the last 30 years, many OMW treatment methods have been proposed and tested, but their application at the olive mill level is limited because of the high investment and/or running costs and technical expertise required. Existing technologies for OMW management have been identified and evaluated with the LIFE+ OLEICO+ project following certain criteria that are currently being used in Portugal, Spain, Italy and Greece. The results show that several viable technologies for OMW treatment do exist (composting, electro-coagulation, hydrolysis-oxidation, phyto-remediation, co-digestion, energy production); however, they require capital investment and maintenance costs that cannot be afforded by a small or medium olive mill plants. On the other hand, application of OMW to olive orchards can be a low-cost alternative method for OMW treatment in regions with small olive oil mill enterprises. Annual rates up to 1500 L tree-1 were applied in five equal doses, at 20-day intervals between November and February. Soil analyses were performed before the onset and after the end of the OMW application period. The response of olive trees to OMW application was monitored by measuring plant nutritional status, photosynthesis and yield. Furthermore, the possibility of groundwater pollution by the application of fresh OMW was investigated with lysimeters. OMW-treated soil had higher K content throughout the experiment. Phenols were decomposed rapidly, and therefore the phenolic content before the onset of a new OMW application period was negligible. The nutritional status, physiology and yield of olive trees were not affected by the application of OMW. The total olive orchard area required for the annual OMW production was 3.6 ha (280 trees ha-1), which is easily available around an olive mill. The cost of application is €0.007 L‑1 OMW, which seems reasonable compared with more sophisticated methods. Detailed study for each case is required in order to determine the application dose according to soil and climatic conditions of the area. The results show that several viable technologies do exist; however, the implementation cost is not negligible.

Addition of antioxidants to oils is essential to retard its oxidation. Nowadays, due to safety concerns, there is a significant demand for replacing synthetic antioxidants with natural ones. Sesame seed coat is a waste product of sesame processing. Ultrasound-assisted extraction of polyphenols from sesame coat was investigated. The highest yield of polyphenols was 7.23 mg gallic acid equivalent (GAE)/g dry nonroasted sesame coat. It was obtained with aqueous ethanol (80%) at a material:solvent ratio of 1:20 (wt/vol) using an ultrasound intensity of 30% of the maximal output power (300 W) for 40 min. High-performance liquid chromatography (HPLC) of the dried extract revealed the presence of sesamin and sesamolin at 65.967 and 38.737 mg/g, respectively. Catechin, p-coumaric acid, and chlorogenic acid were the most abundant polyphenols in the extract. The addition of sesame coat extract enhanced the oxidative stability of sunflower oil (423% increase). Practical applications Oxidative rancidity is a quality problem of refined vegetable oils. The extraction of polyphenols as a natural source of antioxidants from dried sesame coat using ultrasound was studied. Findings indicate that sesame coat extract improved oxidative stability of sunflower oil. Antioxidant efficacy of the extract was greater than that of synthetic antioxidant BHT.