Chemical and cellular antioxidant activity of phytochemicals purified from olive mill waste waters

The isolation and identification of a phytocomplex from olive mill waste waters (OMWW) was achieved. The isolated phytocomplex is made up of the following three phenolic compounds: hydroxytyrosol (3,4-DHPEA), tyrosol (p-HPEA) and the dialdehydic form of decarboxymethyl elenolic acid, linked with (3,4-dihydroxyphenyl)ethanol (3,4-DHPEA-EDA). The purification of this phytocomplex was reached by partial dehydration of the OMWW, followed by liquid-liquid extraction with ethyl acetate and middle pressure liquid chromatography (MPLC) on a Sephadex LH-20 column. The phytocomplex accounted for 6% of the total phenolic content of the OMWW. The phytocomplex and individual compounds were tested for antioxidant capacity by the oxygen radical absorbance capacity (ORAC) method. The ORAC phytocomplex produced 10,000 ORAC units/g dry weight, whereas the cellular antioxidant activity, measured by the cellular antioxidant activity in red blood cell (CAA-RBC) method, demonstrated that the phytocomplex and all of the components are able to permeate the cell membrane thus exhibiting antioxidant activity inside the red blood cells. Our phytocomplex could be employed in the formulation of fortified foods and nutraceuticals, with the goal to obtain substantial health protective effects due to the suitable combination of the component molecules.     

Antioxidant and other biological activities of olive mill waste waters.

During olive oil production, large volumes of water are generated and subsequently discarded. Olives contain a variety of bioactive components, and some of them, according to their partition coefficients, end up in the water phase. The current investigation aimed at comparing different methods for the extraction of biologically active components of the olive mill waste waters (OMWW) and evaluating the in vitro antioxidant and anti-inflammatory activities of the resulting extracts. The results indicate that OMWW extracts are able to inhibit human LDL oxidation (a process involved in the pathogenesis of atherosclerosis) and to scavenge superoxide anions and hypochlorous acid at concentrations as low as 20 ppm. Finally, two of the three extracts also inhibited the production of leukotrienes by human neutrophils. The potency of the extracts depended on their degree of refinement: extracts containing only low molecular weight phenols were the most effective.     

Production,characterization, and effects on tomato of humic acid-like polymerin metal derivatives from olive oil mill waste waters

The dark polymeric organic fraction rich in potassium recovered from olive oil mill waste waters (OMWW) and named polymerin and the potassium salified deglycosylated polymerin derivative (K-SDpolymerin) were easily transformed into their metal derivatives by saturation with various metals, including Na, Cu, Zn, Mn, Fe, and Al. Saturated metal polymerins were characterized by diffuse reflectance infrared Fourier transform spectroscopy and atomic absorption spectrometry. Tests on tomato plants of the various polymerins showed that only the soluble polymerin, K-SDpolymerin, and the insoluble Mn-SDpolymerin were significantly toxic. The toxic effects of OMWW on tomato at the original concentration and diluted 1:10 were much stronger than those of any polymerin. The possible exploitation of polymerins as bioamendments and/or metal biointegrators as a functon of their phytotoxic effects, their humic acid-like nature, and their richness in macro- and micronutrient metals is also discussed.     

Bioactivity and analysis of biophenols recovered from olive mill waste

Biophenols have attracted increasing attention during the past few years due to their biological activities and natural abundance and are potential targets for the food and pharmaceutical industries. Olive mill waste (OMW) is rich in biophenols and typically contains 98% of the total phenols in the olive fruit, making value addition to OMW an attractive enterprise. The phenolic profile of OMW is complex, yet this complexity has not been fully exploited in the valorization of the waste. Most work on the bioactivity of OMW has focused on antioxidant and antimicrobial activities. The analytical techniques used to identify and quantify active biophenols are also reviewed.     

Novel secoiridoids with antioxidant activity from Australian olive mill waste

A new biophenolic secoiridoid was identified in Australian Frantoio olive mill waste (OMW) extracts. Isolation, purification, and structure elucidation were performed. Hydroxytyrosyl acyclodihydroelenolate, the first nonaldehydic acyclic secoiridoid, is reported. A second compound was identified as p-coumaroyl-6'-secologanoside (comselogoside), and although it has been identified recently in OMW and leaves, this is the first time it has been identified in both OMW and olive fruits. UV, mass spectral, and NMR data are given for both compounds. The two compounds were quantified by HPLC-DAD, and their antioxidant potential was assessed against the classical olive biophenols, hydroxytyrosol and oleuropein, by the in vitro DPPH radical scavenging assay.     

Investigation of Australian olive mill waste for recovery of biophenols

Olive mill waste is a potential source for the recovery of phytochemicals with a wide array of biological activities. Phytochemical screening of hexane, methanol, and water extracts revealed a diversity of compounds, perhaps overlooked in previous studies through intensive cleanup procedures. Methanol and water extracts contained large amounts of biophenols, and further testing of polar extraction solvents, including ethyl acetate, ethanol, propanol, acetone, acetonitrile, and water/methanol mixtures, highlighted the latter as the solvent of choice for extraction of the widest array of phenolic compounds. Stabilization of the resulting extract was best achieved by addition of 2% (w/w) sodium metabisulfite. Quantitative data are reported for nine biophenols extracted using 60% (v/v) methanol in water with 2% (w/w) sodium metabisulfite. Six compounds had recoveries of greater than 1 g/kg of freeze-dried waste: hydroxytyrosol glucoside, hydroxytyrosol, tyrosol, verbascoside, and a derivative of oleuropein.     

Polyphenolic content in olive oil waste waters and related olive samples

The production of olive oil yields a considerable amount of waste water, which is a powerful pollutant and is currently discarded. Polyphenols and other natural antioxidants, extracted from olives during oil extraction process, partially end up in the waste waters. Experimental and commercial olive oil waste waters from four Mediterranean countries were analyzed for a possible recovering of these biologically interesting constituents. Identification and quantitation of the main polyphenols were carried out by applying HPLC-DAD and HPLC-MS methods. Representative samples of ripe olives were also analyzed at the same time to correlate, if possible, their polyphenolic profiles with those of the corresponding olive oil waste waters. The results demonstrate that Italian commercial olive oil waste waters were the richest in total polyphenolic compounds with amounts between 150 and 400 mg/100 mL of waste waters. These raw, as yet unused, matrices could represent an interesting and alternative source of biologically active polyphenols.     

Availability of triazine herbicides in aged soils amended with olive oil mill waste

Amendments are frequently added to agricultural soils to increase organic matter content. In this study, we examined the influence of alperujo, an olive oil mill waste, on the availability of two triazine herbicides, terbuthylazine and atrazine, in two different sandy soils, one from Sevilla, Spain, and the other from Minnesota. The effect of aging on herbicide sorption and bioavailability was also studied. Soils were amended with alperujo at a rate of 3-5% (w:w) in laboratory studies. Apparent sorption coefficients for the triazine herbicides were calculated as the ratio of the concentrations of each herbicide sequentially extracted with water, followed by aqueous methanol, at each sampling time. These data showed greater sorption of terbuthylazine and atrazine in amended soils as compared to nonamended soils, and an increase in the amount of herbicide sorbed with increasing aging time in nonamended soils. The triazine-mineralizing bacterium Pseudomonas sp. strain ADP was used to characterize triazine bioavailability. Less mineralization of the herbicides by Pseudomonas sp. strain ADP was observed in soils amended with alperujo, as compared to the unamended soils, and, despite the increase in sorption with aging in unamended soils, herbicide mineralization also increased in this case. This has been attributed to Pseudomonas sp. strain ADP first using alperujo as a more readily available source of N as compared to the parent triazines. In summary, addition of alperujo to the soils studied was shown to increase triazine herbicides sorption and hence to reduce its availability and potential to leach.     

Fate of diuron and terbuthylazine in soils amended with two-phase olive oil mill waste

The addition of organic amendments to soil increases soil organic matter content and stimulates soil microbial activity. Thus, processes affecting herbicide fate in the soil should be affected. The objective of this work was to investigate the effect of olive oil production industry organic waste (alperujo) on soil sorption-desorption, degradation, and leaching of diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and terbuthylazine [N2-tert-butyl-6-chloro-N4-ethyl-1,3,5-triazine-2,4-diamine], two herbicides widely used in olive crops. The soils used in this study were a sandy soil and a silty clay soil from two different olive groves. The sandy soil was amended in the laboratory with fresh (uncomposted) alperujo at the rate of 10% w/w, and the silty clay soil was amended in the field with fresh alperujo at the rate of 256 kg per tree during 4 years and in the laboratory with fresh or composted alperujo. Sorption of both herbicides increased in laboratory-amended soils as compared to unamended or field-amended soils, and this process was less reversible in laboratory-amended soils, except for diuron in amended sandy soil. Addition of alperujo to soils increased half-lives of the herbicides in most of the soils. Diuron and terbuthylazine leached through unamended sandy soil, but no herbicide was detected in laboratory-amended soil. Diuron did not leach through amended or unamended silty clay soil, whereas small amounts of terbuthylazine were detected in leachates from unamended soil. Despite their higher sorption capacity, greater amounts of terbuthylazine were found in the leachates from amended silty clay soils. The amounts of dissolved organic matter from alperujo and the degree of humification can affect sorption, degradation, and leaching of these two classes of herbicides in soils. It appears that adding alperujo to soil would not have adverse impacts on the behavior of herbicides in olive production.     

A new process for the management of olive oil mill waste water and recovery of natural antioxidants

The high polyphenol content of the wastewater is the major environmental problem caused by the olive mills. A pilot scale system for the treatment of the olive oil mills wastewater was developed aiming at the recovery of high added value-contained polyphenols and the reduction of the environmental problems. The treatment system consists of three main successive sections: The first one includes successive filtration stages aiming at the gradual reduction of the wastewater suspended solids up to a limit of 25 microm. The second section includes passing of the filtered wastewater through a series of adsorbent resins (XAD16 and XAD7HP) in order to achieve the de-odoring and decolorization of the wastewater and the removal/ recovery of the polyphenol and lactone content. The third section of the procedure includes the thermal evaporation and recovery of the organic solvents mixture, which has been used in the resin regeneration process, and finally the separation of the polyphenols and other organic substance contents using fast centrifuge partition chromatography. The final outcome of the whole procedure is (i) an odorless yellowish wastewater with a 99.99% reduced content in polyphenols and 98% reduced COD, (ii) an extract rich in polyphenols and lactones with high antioxidant activity and high added value, (iii) an extract containing the coloring substances of the olive fruit, and (iv) pure hydroxytyrosol.     

Degradation treatment of waste water from olive processing

The aim of the process under consideration is the degradation of organic pollutants contained in waste water from olive processing. Degradation takes place by processing the polluted water in a tank which contains soil that has certain chemical and biochemical characteristics. The effect of the treatment is an enrichment of the soil with readily-assimilable nutrient substances to the extent that the soil pollutant mixture becomes soil-compost. Neither sludge nor solid residual products are formed in the process, since they too undergo degradation. The treatment of water and sludge is completed on parcels of land underlain with a water-tight base. The watertight floor prevents filtration of polluting substances in the treated water from percolating into the underlying soil.     

Mitigation of olive mill wastewater toxicity

The toxicity of olive mill wastewaters (OMW) is commonly attributed to monomeric phenols. OMW were treated in an aerated, stirred reactor containing agricultural soil, where the oxidative polymerization of phenols took place. In 24 h, OMW monomeric phenols decreased by >90%. This resulted in a corresponding reduction in phytotoxicity, as measured by germination tests with tomato and English cress seeds, and in microbial toxicity, as measured by lag phase duration in Bacillus cereus batch growth. Soil germination capability after irrigation with OMW was assessed in long-term pot experiments. The relative germination percentage of tomato was higher when the soil was irrigated with treated OMW rather than with untreated ones, although it was lower than the control (e.g., soil irrigated with distilled water). At longer incubation times, a complete recovery of the soil germination capability was achieved with treated, but not with untreated, OMW.     

Verbascoside, isoverbascoside, and their derivatives recovered from olive mill wastewater as possible food antioxidants

Olive oil processing industries generate substantial quantities of phenolic-rich byproducts, which could be valuable natural sources of antioxidants. This work is focused on the recovery and structural characterization of antioxidant compounds from olive mill wastewater (OMWW), a polluting byproduct of the olive oil production process. Phenolics were extracted from the waste material using a membrane technology coupled to low-pressure gel filtration chromatography on a Sephadex LH-20. The LH-20 fraction was, in turn, characterized for its phenolic composition by HPLC-DAD-MS/MS analyses. Verbascoside, isoverbascoside, β-hydroxyverbascoside, β-hydroxyisoverbascoside, and various oxidized phenolics were identified. Uptake of verbascoside, purified from the LH-20 fraction, by HT-29 cells, an established model system for studying drug transport properties, was also assayed. Finally, the antioxidant activities of the LH-20 fraction and verbascoside were characterized by two different techniques. Individual verbascoside was more active as a scavenger of reactive oxygen species and as a chemopreventive agent protecting low-density lipoproteins from oxidative damage than the LH-20 fraction.     

Genotoxic potential of waste waters from a leather industry

The genotoxic potential of tannery waste water was evaluated by means of the induction of gene conversion and point mutation in Saccharomyces cerevisiae D7 strain. Neither a toxic nor a genotoxic response was obtained after a 2 hr incubation of raw water samples with the strain. A dose-related increase in the induced gene conversion and mitotic reversion was obtained when dilutions of the raw effluent were incubated for 24 hr. Samples of the waste water were also passed through an XAD2 resin column. The column was sequentially eluted with ethyl ether, chloroform and methanol. The ether and methanol fractions showed a positive genotoxic response.     

Impact of cultivar, harvesting time, and seasonal variation on the content of biophenols in olive mill waste

Olive mill waste (OMW) contains substantial amounts of valuable antioxidant biophenols that can be recovered for possible applications in food, pharmaceutical, and cosmetic industries. However, the impact of cultivar, harvesting time, and seasonal variation on the phenolic composition of OMW has not yet been assessed. Total phenols, antioxidant activity, and phenol profiles of OMW extracts from five different Australian-grown cultivars (Barnea, Correggiola, Manzanillo, Mission, and Paragon) were studied at four different harvesting times in the 2004 season. The impact of seasonal variation was assessed by comparing total phenol content, antioxidant activity, and phenol profile of two cultivars (Correggiola and Mission) harvested in the 2004 and 2005 seasons. The phenol content and antioxidant activity at different harvesting times were mainly a function of the olive cultivar. Harvesting time had a quantitative effect rather than a qualitative effect on the phenol profile. Intercultivar and harvesting time variation accounted for a 2-5-fold change in the total phenol and antioxidant capacity, while levels of individual biophenols experienced up to 50-fold change. The phenol content and antioxidant capacity of OMW significantly changed between seasons with different variation patterns for different cultivars.     

Ultrasonic extraction of phenols from olive mill wastewater: comparison with conventional methods

Recovery of phenols from olive mill wastewater (OMWW) was studied, comparing five sample preparation methods: filtration, solid-phase (SPE), liquid-liquid (LLE) and ultrasonic (US)-assisted extraction of liquid and solid (freeze-dried) OMWW. Results showed that ultrasonication is a good alternative to conventional solvent extractions, providing higher recoveries at both levels of individual and total phenol yields. Sonication of liquid OMWW in organic solvent was more efficient vs its nonassisted counterpart (agitation), but did not provide a representative phenol chromatogram due to ethyl acetate use. By contrast, the US-assisted extraction of freeze-dried OMWW (3 × 20 min) in 100% methanol (1.5 g/25 mL, w/v) offered the highest qualitative-quantitative phenol yields without any US-induced alterations. Moreover, freeze-drying is an excellent preservation of initial liquid OMWW, holding a great potential for delayed analysis. This study is also the first report that Slovenian OMWW may be utilized as a valuable source of phenols, especially hydroxytyrosol and tyrosol.     

Influence of two-phase olive mill waste application to soil on terbuthylazine behaviour and persistence under controlled and field conditions

Purpose  

Terbuthylazine is one of the most common herbicides used to control weeds in olive groves. Application of two-phase olive mill waste (OW) to soils may play a fundamental role in the management of leaching losses of pesticides, especially in Mediterranean areas where soils are characterized by low organic matter levels. We evaluated the impact of OW amendments on the sorption–desorption, degradation, leaching, and persistence of the herbicide terbuthylazine in a representative olive grove soil from Portugal.     

Analysis of metal cations and inorganic anions in olive oil mill waste waters by atomic absorption spectroscopy and ion chromatography. Detection of metals bound mainly to the organic polymeric fraction

Metal cations were quantitatively detected by atomic absorption spectrometry in samples of olive oil mill waste waters obtained by a pressure process (omww(1)) (K, 17.1; Mg, 2.72; Ca, 2.24; Na, 0.40; Fe, 0.123; Zn, 0.0630; Mn, 0.0147; Cu, 0.00860 g L(-)(1)) and a centrifugation process (omww(2)) (K, 9.80; Mg, 1.65; Ca, 1.35; Na, 0. 162; Fe, 0.0330; Zn, 0.0301; Mn, 0.00910; Cu, 0.00980 g L(-)(1)). The inorganic anions, determined in the same samples by ion chromatography, proved to be Cl(-), H(2)PO(4)(-), F(-), SO(4)(2)(-), and NO(3)(-) (1.61, 1.05, 0.66, 0.52, and 0.023 g L(-)(1), respectively, in omww(1) and 0.61, 0.40, 0.25, 0.20, and 0.0090 g L(-)(1), respectively, in omww(2)). Most of the metal cations were revealed to be bound to the omww organic polymeric fraction (opf), composed of polysaccharides, phenol polymers, and proteins. Opf relative molecular weight was substantially estimated in the range between 1000 and 30000 Da for approximately 75% and in the range from 30000 to 100000 Da for approximately 25%. The free residual cations pool proved to be neutralized by the inorganic counteranions. Finally, the possible exploitation of this material in agriculture and in environmental biotechnology processes is also discussed in the light of its chemical and biochemical oxygen demand parameters.     

Production in large quantities of highly purified hydroxytyrosol from liquid-solid waste of two-phase olive oil processing or "Alperujo"

The effect of hydrothermal treatment of two-phase olive waste (alperujo) on the solubilization of hydroxytyrosol was studied. Different conditions of saturated steam were assayed. A high amount of hydroxytyrosol was solubilized and increased with increasing steaming temperature and time, reaching 1.4-1.7 g/100 g of dry alperujo. The effect of acidic (H(2)SO(4)) and basic (NaOH) catalysts was also evaluated. Acid-catalyzed treatment was more effective at milder conditions, whereas the alkali-catalyzed conditions were not very suitable. In the present study, the extracted hydroxytyrosol was purified by means of a new, simple, and inexpensive chromatographic system, under international patent application (PCT/ES02/00058). From 1000 kg of alperujo, with 70% humidity, can be obtained approximately 4.5-5 kg of hydroxytyrosol. After a purification process, at least 3 kg of hydroxytyrosol, at 90-95% purity, would be obtained. The purified compound was identified by HPLC/UV and (1)H and (13)C NMR analyses, and its antioxidant activity was tested on refined olive oil without antioxidants by Rancimat method. The oxidative stability of refined olive oil was increased by a factor of 1.71 in the presence of 100 ppm of hydroxytyrosol.     

Short-term effects of olive mill waste water (OMW) on chemical and biochemical properties of a semiarid Mediterranean soil

Olive mill waste water (OMW), a by-product of the olive mill industry, is produced in large amounts in Mediterranean countries. Olive mill waste water contains a high organic load, substantial amounts of plant nutrients but also several compounds with recognized toxicity towards living organisms. Moreover, OMW may represent a low cost source of water. Thus, the use of OMW for soil fertigation is a valuable option for its disposal, provided that its impact on soil chemical and biochemical properties is established. Investigations were performed on the short-term influence of OMW on several chemical and biochemical properties of a soil from a continental semi-arid Mediterranean region (Morocco). The soil was amended with 0, 18 and 36 ml 100 g⁻¹ soil of OMW (corresponding to a field rate of 0, 40 and 80 m³ ha⁻¹, respectively) and changes in various functionally related properties such as microbial biomass, basal respiration, extractable C and N, and soil hydrolases and oxido-reductases activities were measured over time. The variations of the main physical and chemical properties as well as the residual phytotoxicity of OMW amended and non-amended soils as assessed by tomato seed germination tests were also monitored. Temporary and permanent changes in several chemical and biochemical soil properties occurred following OMW application, thus being these properties varied in sensitivity to the applied disturbance. A sudden increase of total organic C, extractable N and C, available P and extractable Mn and Fe contents were measured. Simultaneously, a rapid increase of soil respiration, dehydrogenase and urease activities and microbial biomass (at 14 day incubation) of OMW amended soils occurred. In contrast, the activities of phosphatase, β-glucosidase, nitrate reductase and diphenol oxidase decreased markedly. The soil became highly phytotoxic after OMW addition (large decline of soil germination capability), mainly at 80 m³ ha⁻¹ OMW. After 42 days' incubation, however, a complete recovery of the soil germination capability and a residual phytotoxicity of about 30% were observed with 40 and 80 m³ ha⁻¹ OMW, respectively. These findings indicate that the impact of OMW on soil properties was the result of opposite effects, depending on the relative amounts of beneficial and toxic organic and inorganic compounds present. The toxic compounds contained in OMW most likely counteracted the beneficial effect of organic substrates provided, which promoted the growth and activity of indigenous microorganisms.