Olive Mill Wastewater Extract Research Articles

Olive oil enrichment in phenolic compounds during malaxation in the presence of olive leaves or olive mill wastewater extracts

A combined study was performed to determine the effect of olive leaf aqueous extract (OLE), olive mill wastewater (OMWW) or water addition during the malaxation step on total phenolic content (TPC), antioxidant activity, virgin olive oil composition and quality parameters of olive oil. For these purposes, the Folin‐Ciocalteu assay, 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay, and HPLC‐ESI‐TOF/MS were employed. The experiments were carried out at semi‐industrial scale in a cold extraction olive oil mill using olives of Koroneiki cultivar. Olive oil produced by olive paste which was malaxed without adding water, OMWW, or OLE was employed as control. The use of OMWW and OLE allowed obtaining oils with higher significant TPC compared to that malaxed with water (146.2 and 151.8 vs. 132.8 μg Eq GAE/g oil, respectively). The antioxidant activity followed the same trend. The use of OMWW and OLE enabled an oil enrichment in oleuropein derivatives, especially in 3,4‐DHPEA‐EDA. Despite the fact that acidity and oxidation (K270 and K232 values) were higher in the aforementioned oils compared to the oil extracted with water, all values were under the European Union commission parameters for extra virgin olive oil (EVOO) denomination. However, only the oil extracted using OLE presented slightly higher fruity, bitter, and pungent positive attributes compared to the control and to the oil extracted with water, thus, making OLE a promising by‐product for improving olive oils.Practical applications: This research provides new insight for the improvement of olive oil extraction process in order to obtain high‐phenol EVOO which could be useful for the olive oil, nutraceuticals, and cosmeceuticals industries. Besides, this study could serve as a starting point for improving the olive oil production process, while reducing the impact of its by‐products.The addition of Olive mil wastewater (OMWW) and olive leaf extract during malaxation, allowed obtaining enriched oils with a higher content in total phenolics, and antioxidant activity compared to those obtained adding water. These enriched oils presentes higher quantities of oleuropein derivatives and specifically of 3,4‐DHPEA‐EDA.

Enrichment of Phenolic Compounds from Olive Mill Wastewater and In Vitro Evaluation of Their Antimicrobial Activities

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.

Dietary Administration of Olive Mill Wastewater Extract Reduces Campylobacter spp. Prevalence in Broiler Chickens

Food wastes are sources of compounds that can be used as natural additives in the food and feed industry. The olive oil industry produces two main wastes: aqueous waste (olive mill wastewater) and solid waste (pomace or olive cake). These by-products are rich in phenols, which are antioxidant and antimicrobial compounds able to inhibit or delay the growth of several bacteria in vitro. The dietary effect of both olive mill wastewater polyphenolic extract (OMWPE) and dehydrated olive cake (DOC) on the prevalence of Campylobacter spp. in broiler chickens was investigated. A commercial basal diet was supplemented with either OMWPE- or DOC-enriched maize at two dosages (low: 16%; high: 33%). The prevalence of Campylobacter spp. shedding was evaluated at 21, 35, and 49 days of age. The prevalence of Campylobacter spp. differed among groups only at 49 days of age. Both OMWPE groups showed a lower (p < 0.05) prevalence compared to the control group. The odds ratio evaluation showed that the higher dose of OMWPE reduced the possibility of shedding 11-fold compared to the control group (p < 0.001). These results highlight the potential use of olive by-products against Campylobacter spp. in poultry.

Antimicrobial Activity of Olive Mill Wastewater Extract Against Pseudomonas Fluorescens Isolated from Mozzarella Cheese.

Olive mill wastewater polyphenol extract was tested for antimicrobial activity against 64 strains of Pseudomonas fluorescens responsible for mozzarella discolouration. The extract showed a minimum inhibitory concentration (MIC)50 value of 5 mg/mL and a MIC90 value of 7 mg/mL. The MBC50 and MBC90 values corresponded to 6 and 8 mg/mL, respectively. The MIC concentration (7 mg/mL) was demonstrated to have a bacteriostatic effect while maintaining the bacterial concentration on the levels of the inoculum for 48 hours. The 3/2 MIC concentration was responsible for four logs CFU/mL depletion in colony count after 24 h. As the extract concentration decreased from MIC value, no inhibitory effects were recorded.

Olive Mill Waste Extracts: Polyphenols Content, Antioxidant, and Antimicrobial Activities.

Natural polyphenols extracts have been usually associated with great bioactive properties. In this work, we investigated in vitro antioxidant and antimicrobial potential of the phenolic olive mill wastewater extracts (OWWE) and the olive cake extracts (OCE). Using the Folin Ciocalteux method, OWWE contained higher total phenol content compared to OCE (8.90 ± 0.728 g/L versus 0.95 ± 0.017 mg/g). The phenolic compounds identification was carried out with a performance liquid chromatograph coupled to tandem mass spectrometry equipment (HPLC-ESI-MS). With this method, a list of polyphenols from OWWE and OCE was obtained. The antioxidant activity was measured in aqueous (DPPH) and emulsion (BCBT) systems. Using the DPPH assay, the results show that OWWE was more active than OCE and interestingly the extracts originating from mountainous areas were more active than those produced from plain areas (EC50 = 12.1 ± 5.6 μg/mL; EC50 = 157.7 ± 34.9 μg/mL, resp.). However, when the antioxidant activity was reversed in the BCBT, OCE produced from plain area was more potent than mountainous OCE. Testing by the gel diffusion assay, all the tested extracts have showed significant spectrum antibacterial activity against Staphylococcus aureus, whereas the biophenols extracts showed more limited activity against Escherichia coli and Streptococcus faecalis.

Hydroxytyrosol is not genotoxic in vitro

Hydroxytyrosol (HT) is an olive-derived phenol endowed with several biological activities, some of which demonstrated in humans. Indeed, the European Food Safety Authority (EFSA) allows the health claim that HT (≥5mg/d) “protects LDL particles from oxidative damage”. In terms of safety, HT has been investigated as the predominant part of raw olive mill waste water extracts that have been granted the Generally Recognized as Safe (GRAS) status. Also, a long-term toxicological study of HT proposed a NOAEL of 500mg/kg/d.As several HT-containing supplements and functional foods are entering the market, we sought to investigate the genotoxic and mutagenic potential of HT, using well-established in vitro models, i.e. the chromosomal aberration assay and the Ames test (by using the Salmonella typhimurium TA 100, TA98, TA1535, and TA1537 strains and Escherichia coli WP2(pKM101)), with and without S9-induced metabolic activation). Even though we cannot rule out that prolonged exposure to HT and its metabolites might have untoward effects, the results of this study indicate that HT is non-genotoxic and non-mutagenic at concentrations that far exceed those attainable after intake.

Toxicological evaluation of pure hydroxytyrosol

Of all the phenolic constituents of olives and extra virgin olive oil, hydroxytyrosol is currently being actively exploited as a potential supplement or preservative to be employed in the nutraceutical, cosmeceutical, and food industry. In terms of safety profile, hydroxytyrosol has only been investigated as the predominant part of raw olive mill waste water extracts, due to the previous unavailability of appropriate quantities of the pure compound. We report the toxicological evaluation of hydroxytyrosol and, based on the results, propose a No Observed Adverse Effects Level (NOAEL) of 500mg/kg/d.

New innovation in order to recover the polyphenols of olive mill wastewater extracts for use as a biopesticide against the Euphyllura olivina and Aphis citricola

Results from physicochemical analysis of olive mill wastewater (OMW) have shown that total suspended solids (5.170 to 0.658 g/L), chemical oxygen demand (124.400 ± 25.442 g/L), biochemical oxygen demand (60.593 ± 6.276), total organic carbon (3.317 ± 0.350 g/L), and Kjeldahl nitrogen (0.126 ± 0.044 g/L) far exceed discharge standards. The concentrations of the major constituents of phenolic extracts of OMW can cause mortality of the pests Euphyllura olivina and Aphis citricola. Analysis of variance and use of the Sheffe test showed that exposure for 24 h to olive mill wastewater polyphenol extracts had a significant (P < 0.01) effect on survival of larval stages and adults, depending on concentration. LC50 and CL99 lethal concentrations were 3.642–18.864 g/L and 98.029–225.711 g/L respectively (confidence intervals). Recovery of polyphenols from vegetable waste for use as a biopesticide contributes to protection of citrus and olive trees against diseases, thus solving an environmental problem and avoiding use of synthetic insecticides.

Stability and antioxidant potential of purified olive mill wastewater extracts

A purified olive extract (POE) rich in phenolic and oleosidic compounds was prepared from olive mill wastewaters by adsorption onto an amphoteric polymer resin with the yield of 2.2% (w/v). In addition to complex and simple phenolic compounds found in the POE, the highly hydrophilic elenolic acid and DEDA (dialdehydic form of decarboxymethyl elenolic acid, a non-phenolic secoiridoids) were also present in high concentration (45%, w/w). Among the phenolic constituents, the major compounds are hydroxytyrosol and its glucoside, DEDA linked to hydroxytyrosol (Hy-DEDA), p-coumaric acid and caffeic acid, verbascoside and some flavonoids. The high content (23%, w/w in gallic acid equivalent) and structural diversity of these phenolic compounds confer superior antioxidant potential of POE with an IC50 less than 8.5μg/ml determined by DPPH assay. The stability of bioactive components during storage of the extract and changes in antioxidant properties were studied under different conditions. Determination of kinetics of degradation revealed that air/oxygen is the determinant factor which influences the stability of POE under low temperature storage conditions, while exposure to sunlight did not have significant effect on their stability. Whilst an increase in storage temperature decreased the total content of phenolic compounds (20–24% reduction) and non-phenolic secoiridoid aglycons, a high antioxidant capacity was still observed particularly in the nitrogen-protected POE samples (93–95% of the initial value). This was believed to be due to the transformation of hydroxytyrosol-containing complex phenols, especially the secoiridoid ester Hy-DEDA, into free phenolic monomer. This contributed significantly to the maintenance of a high antioxidant potency of the whole extract during storage. The high antioxidant, radical scavenging activity and stability of the extract suggest potential applications in the food and pharmaceutical industries.

Synergistic Antibacterial Effects of Polyphenolic Compounds from Olive Mill Wastewater

Polyphenols or phenolic compounds are groups of secondary metabolites widely distributed in plants and found in olive mill wastewater (OMW). Phenolic compounds as well as OMW extracts were evaluated in vitro for their antimicrobial activity against Gram-positive (Streptococcus pyogenes and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae). Most of the tested phenols were not effective against the four bacterial strains when tested as single compounds at concentrations of up to 1000 μg mL−1. Hydroxytyrosol at 400 μg mL−1 caused complete growth inhibition of the four strains. Gallic acid was effective at 200, and 400 μg mL−1 against S. aureus, and S. pyogenes, respectively, but not against the gram negative bacteria. An OMW fraction called AntiSolvent was obtained after the addition of ethanol to the crude OMW. HPLC analysis of AntiSolvent fraction revealed that this fraction contains mainly hydroxytyrosol (10.3%), verbascoside (7.4%), and tyrosol (2.6%). The combinations of AntiSolvent/gallic acid were tested using the low minimal inhibitory concentrations which revealed that 50/100–100/100 μg mL−1 caused complete growth inhibition of the four strains. These results suggest that OMW specific fractions augmented with natural phenolic ingredients may be utilized as a source of bioactive compounds to control pathogenic bacteria.

Cytoprotective effects of olive mill wastewater extract and its main constituent hydroxytyrosol in PC12 cells

Evidence from epidemiological studies indicates that adherence to diets rich in secondary plant metabolites, such as polyphenols and phenolic acids, possibly reduces the risk of age-dependent neurodegeneration and subsequent cognitive decline. Olive mill wastewater is particularly rich in the ortho-diphenol hydroxytyrosol (HT) that can be recovered with ad hoc techniques. In a previous study, we showed that hydroxytyrosol-rich olive mill wastewater extract protects dissociated brain cells not only in vitro but also ex vivo after subchronic oral administration of the extract to mice. As plant extracts often contain constituents with potentially confounding activities, our present study aimed to test in vitro whether HT at concentrations present in olive mill wastewater extract is able to protect PC12 cells with similar efficiency. Following a short-term exposure (30 min) to the compounds of interest, cells were subjected to oxidative or nitrosative stress by adding either ferrous iron or sodium nitroprusside to the cell culture medium for 18 h, respectively. Cytotoxicity was assessed by measuring MTT reduction, cellular ATP levels and mitochondrial membrane potential in the absence and presence of HT or HT-rich olive mill wastewater extract. The results we obtained mainly confirm our previous observation of promising cytoprotection of brain cells by HT-rich olive mill wastewater extract in different stressor paradigms. Furthermore, correlation analyses revealed that the observed cytoprotective effects in PC12 cells are likely due to HT present in the extract.

Hydroxytyrosol-Rich Olive Mill Wastewater Extract Protects Brain Cells in Vitro and ex Vivo

Elevated oxidative and nitrosative stress both impair the integrity and functioning of brain tissue, especially in aging. As long-term intake of plant foods rich in antioxidant phenolics, such as extra virgin olive oil, positively modulates surrogate markers of many human pathological alterations, the interest in cheap and abundant sources of such phenolics is rapidly growing. Olive mill wastewater is particularly rich in hydroxytyrosol, an o-diphenol with powerful antioxidant, anti-inflammatory, and antithrombotic activities. Due to the deleterious effect of oxidative stress on brain cell survival, the efficacy of a hydroxytyrosol-rich extract to attenuate Fe2+- and nitric oxide (NO)-induced cytotoxicity in murine-dissociated brain cells was investigated. The addition of either Fe2+ or SNP (an NO donor) caused both a severe loss of cellular ATP and a markedly depolarized mitochondrial membrane potential. Preincubation with hydroxytyrosol significantly attenuated the cytotoxic effect of both stressors, although with different efficiencies. Mice feeding studies were performed to assess the brain bioactivity of hydroxytyrosol ex vivo. Subchronic, but not acute, administration of 100 mg of hydroxytyrosol per kilogram body weight for 12 days enhanced resistance of dissociated brain cells to oxidative stress, as shown by reduced basal and stress-induced lipid peroxidation. Also, basal mitochondrial membrane potential was moderately hyperpolarized (P < 0.05), an effect suggestive of cytoprotection. In synthesis, the ex vivo data provide the first evidence of neuroprotective effects of oral hydroxytyrosol intake. Hydroxytyrosol; olive mill wastewater; dissociated brain cells; oxidative stress; brain; Mediterranean diet.

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

Ethyl acetate extracts of olive mill waste water (OMWW) were prepared, under in optimal conditions, using a continuous counter-current extraction unit. The antiradical and antioxidant activities of the OMWW extract as well as pure phenolic compounds identified in this extract were evaluated. Results showed that pure hydroxytyrosol and 3,4-dihydroxyphenyl acetic acid had the highest radical-scavenging effects on 1,1-diphenyl-2-picrylhydrazyl radical and the highest antioxidant activities using the β-carotene linoleate model system. The effect of addition of individual phenolic compounds and OMWW extract to refined olive and husk oils was compared with that of control, BHA and BHT at 50 °C. Unexpectedly, 3,4-dihydroxyphenyl acetic acid had the highest protective effect against oil oxidation. Oils with added 3,4-dihydroxyphenyl acetic acid had lower PV than oils with added hydroxytyrosol, the most studied powerful antioxidant. Moreover, the addition of OMWW extract, at 500 ppm, resulted in lower PV values than BHA, p-hydroxyphenylacetic acid, tyrosol on the control. The results suggested that 3,4-dihydroxyphenyl acetic acid, hydroxytyrosol and OMWW extract possess useful antioxidant properties and may be used as alternatives in the search for natural replacement of synthetic antioxidant food additives.

Isolation of a yeast, Trichosporon cutaneum, able to use low molecular weight phenolic compounds: application to olive mill waste water treatment

AbstractThis study deals with the degradation of phenolic compounds in olive oil mill waste; a highly polluting material in olive oil‐producing countries because of its abundance and the toxicity of its phenolic compound content. This investigation confirms the ability of an isolated yeast, identified as Trichosporon cutaneum, to degrade phenolic compounds extracted from olive mill waste water (OMW). The yeast was adapted to the OMW by an enrichment culture. The results of this biotransformation were a decrease in the phenolic content and hence a reduction in the phytotoxic effects of the effluent after the yeast treatment. The kinetic growth of the isolated yeast on phenol over a range of concentrations (0.3–3.0 g dm−3) was studied. The ability of the strain to assimilate simple monomeric phenols and alkyl phenols, at a concentration of 1 g dm−3, in a synthetic liquid medium used as the sole carbon source was investigated in a batch culture. The aromatic ring cleavage pathway occurred in the yeast through catechol oxidation. Using various concentrations of ethyl acetate extract from OMW as the sole carbon source, the yeast exhibited growth on the substrate up to 7 g dm−3 equivalent of phenols. A significant reduction of COD after the treatment of the OMW extract by the yeast isolate was noticed. The removal of phenol and COD exceeded 80% of the original loading after 8 days of treatment, for extracts containing initial COD in the range 19 to 72 g dm−3. Copyright © 2004 Society of Chemical Industry

Hydroxytyrosol, as a component of olive mill waste water, is dose- dependently absorbed and increases the antioxidant capacity of rat plasma

Hydroxytyrosol is the most potent phenolic antioxidant of olive oil and olive mill waste water (OMWW) and its biological activities have stimulated research on its potential role in cardiovascular protection. However, evidence of the absorption of OMWW phenolics and on their possible in vivo activity has, until now, never been provided. Three groups male Sprague-Dawley rats were administered 1, 5, or 10 mg/Kg of the OMWW extract, respectively, providing 41.4, 207, and 414 μg/Kg of hydroxytyrosol, respectively. Urine was collected for 24 h and the urinary levels of hydroxytyrosol were quantified by mass spectrometry. Hydroxytyrosol was dose-dependently (R2=0.95) absorbed and excreted in the urines mostly as a glucuronide conjugate. Further, the administration of an hydroxytyrosol-rich OMWW extract (10 mg/kg) to the rats was also associated with an increase of their plasma antioxidant capacity. Future experiments will eventually further clarify its metabolic fate and its in vivo actions.