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.     

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.     

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.     

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.     

Olive mill waste and glyphosate‐based herbicide addition to olive grove soils: effects on microbial activities and their responses to drying–rewetting cycles

The objective of this experimental study was to determine the effect of agronomic practices usually implemented in olive groves (addition of olive mill waste and herbicides) on soil microbial communities and to test whether drought enhanced such effects. For that purpose, mesocosms containing soil cores from olive groves were incubated for 5 months under either of the three treatments: (i) addition of olive mill waste (OMW), (ii) addition of glyphosate‐based herbicide (Gly treatment) and (iii) both treatments. Half of the mesocosms were subjected or not (controls) to drying–rewetting cycles (D/Rw) for 1 month (1 D/Rw) or 3 months (3 D/Rw). In the controls, 2 months after the Gly treatment, higher lipase activities were observed compared with no practice treatment as well as a significant change in catabolic profiles of cultivable microbial communities. Three months later, lipase activities significantly decreased under the Gly treatment. Addition of OMW together with Gly treatment counteracted the negative effect of the herbicide on lipase activities. After three D/Rw cycles, Gly treatment modified catabolic profiles and induced a decrease in functional diversity. Overall, the combination of glyphosate‐based herbicide with OMW was a conservative practice that maintained soil functioning and led to a better response to D/Rw cycles.     

Effect of processing conditions, prestorage treatment, and storage conditions on the phenol content and antioxidant activity of olive mill waste

The impact of two- and three-phase processing systems and malaxation conditions on phenol content (both total and individual phenols) and antioxidant capacity of laboratory-generated olive mill waste (OMW) was assessed. Two-phase olive processing generated a waste with higher phenol content and antioxidant capacity. Using the two-phase system, both malaxation time and temperature affected the phenol content and antioxidant capacity. The effects of different prestorage drying treatments on phenol content and antioxidant capacity were also compared. Air drying and drying at 60 degrees C resulted in a substantial decrease in the phenol content and antioxidant capacity. Drying at 105 degrees C and freeze-drying produced less degradation. The phenol content and antioxidant capacity of OMW stored at 4 degrees C and of OMW preserved by 40% w/w ethanol and 1% w/w acetic acid and stored at 4 degrees C were monitored for 30 days and compared with those of OMW stored at room temperature. None of these storage conditions could prevent the rapid decrease in phenolic concentrations and antioxidant capacity, which happened within the first 24 h.     

Treated Olive Mill Wastewater Effects on Soil Properties and Plant Growth

Olive-oil production has a vital impact on the socioeconomic development in most Mediterranean countries, where 97.5 % of the world oil is produced. However, the olive-oil extraction process generates considerable quantities of an agro-industrial effluent, olive mill wastewater (OMW), which has negative impact on the environment and biological life. The objective of this study was to evaluate the potential use of OMW treated by different technologies in irrigation and determine its effect on the plant growth and soil quality parameters. Different technologies were used to treat the OMW, the resultant treated OMW was used to irrigate the maize planted in the pot experiment. The results indicated that UOMW increased soil salinity and reduced plant growth, while the treated OMW by different technologies improved plant growth and resulted in lower soil pH. The impact on other soil properties varied depending on the techniques used for treatments. Although treated OMW enhanced plant growth compared with the untreated, the plant growth remained lower than that obtained using the potable water with fertilizers, indicating lack of some essential plant nutrients.     

Hypocholesterolemic effects of phenolic extracts and purified hydroxytyrosol recovered from olive mill wastewater in rats fed a cholesterol-rich diet

In our previous studies, a phenolic-rich extract of olive mill wastewaters (OMW) was prepared under optimal conditions, using a continuous countercurrent extraction unit, and hydroxytyrosol was purified from the obtained OMW extract. The antioxidant activity of OMW extract and hydroxytyrosol was determined by a series of models in vitro. In this study, the hypocholesterolemic effects of hydroxytyrosol and OMW extract in rats fed a cholesterol-rich diet were tested. Wistar rats, fed a standard laboratory diet or a cholesterol-rich diet for 16 weeks, were used. Serum lipid levels, as well as thiobarbituric acid reactive substances (TBARS) and superoxide dismutase and catalase activities in liver were examined. Cholesterol-rich diet-induced hypercholesterolemia was manifested in the elevation of serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C). Administration of a low-dose (2.5 mg/kg of body weight) of hydroxytyrosol and a high-dose (10 mg/kg of body weight) of OMW extract significantly lowered the serum levels of TC and LDL-C while increasing the serum levels of high-density lipoprotein cholesterol (HDL-C). Furthermore, the TBARS contents in liver, heart, kidney, and aorta decreased significantly after oral administration of hydroxytyrosol and OMW extract as compared with those of rats fed a cholesterol-rich diet. In addition, OMW phenolics increased CAT and SOD activities in liver. These results suggested that the hypocholesterolemic effect of hydroxytyrosol and OMW extract might be due to their abilities to lower serum TC and LDL-C levels as well as slowing the lipid peroxidation process and enhancing antioxidant enzyme activity.     

Olive mill wastewater effects on the microbial communities as studied in the field of olive trees by analysis of fatty acid signatures

The aim of this work was to study the effects of spreading olive mill wastewater (OMW) on the soil surface of an olive grove on the soil microbial communities. Analyses of ester-linked fatty acid methyl esters (EL-FAME) were used to assess variations in the soil microbial community structure following land spreading of OMW. Our data provide evidence that agronomic application of OMW has important effects on soil microbial community. Bacteria were relatively more reduced by these treatments than fungi and actinomycetes as revealed by an increased index of fungal/bacterial FAME and actinomycetes/bacterial FAME. Specific FAME markers indicated a significant reduction in the Gram-positive bacteria. However, the relative proportion of the Gram-negative bacteria was not significantly different after agronomic application of OMW. The ratios of cyclopropyl/monoenoic precursors decreased and the total monounsaturated/total saturated fatty acids increased in the OMW amended soils, suggesting that the microbes inhabiting the control soil are more carbon limited than the OMW amended soils. The changes in the FAME pattern of the soil organisms possibly were related (i) to an altered substrate quantity, that is the availability of substrates after the treatments, (ii) the complex nature of OMW which also contains high molecular-mass recalcitrant polyphenols.     

Short-term effects in soil microbial community following agronomic application of olive mill wastewaters in a field of olive trees

Olive mill wastewater (OMW) creates a disposal problem. The large amounts generated, combined with the high phenol and chemical oxygen demand concentrations, are the main difficulties in finding a solution for the management of these wastewaters. We investigated the short-term effect of spreading OMW on the soil surface of an olive grove on the soil microbial communities. Analyse of ester-linked fatty acid methyl ester (EL-FAME) were used to assess variation in soil microbial community structure after agronomic application of OMW. EL-FAME analysis showed significant shifts of specific groups of fatty acids 30 days after application of OMW to a field of olive trees at rates of 0 (control soil), 30, 60, 100, and 150 m³ ha⁻¹ of OMW. In particular, the branched saturated fatty acids indicative of Gram-positive bacteria decreased and the unsaturated fatty acids commonly found in Gram-negative bacteria and fungi increased. The fungal/bacterial ratio measured increased significantly with increasing OMW. Lower cy19/18:1ω7c and cy17/16:1ω7c ratios were found in the amended soil than the control soil, and we interpret that as an indication that nutrient availability may be more limiting in the control soil. Similarly, the relative abundances of monounsaturated fatty acids increased with added OMW, and this is consistent with the presence of high substrate availability in OMW-treated soil. Principal Components Analysis of the FAME profiles showed discrimination between the control soil and OMW amended soil. Differences in fatty acid profiles between OMW-treated soil and control soil suggests that amendment of soil with OMW favors specific groups of organisms. To our knowledge, this is the first report of alterations in the FAME profile in soils due to agronomic application of OMW.     

Long-Term Spreading of Olive Mill Wastewater on Olive Orchard: Effects on Olive Production,Oil Quality,and Soil Properties

In Italy, the law no 574 of 1996 permits and regulates the disposal of olive mill wastewater (OMW), the liquid by-product obtained in oil mill when olive fruits are processed to extract virgin olive oil, by its controlled spreading on cultivated soil. With the aim to verify the long-term effects of the practice on olive production, oil quality, and physical–chemical and microbiological characteristics of soil, different amounts of OMW were spread in February, for 9 years consecutively, on soil cultivated with olive trees. The results obtained confirmed that the controlled spreading of OMW is not harmful for the plant production. On the contrary, OMW spread on olive grove is useful for the fertility of soil and the growth of microflora for the important supply of nitrogen (N), phosphorus (P), potassium (K), and, above all, of organic matter. The evaluated biological properties indicated that OMW spreading stimulates the growth of soil fungal and fungus-like communities and aerobic N₂ fix micro-organisms. With regard to the oil quality, the data indicate that no significant differences were ascertained on the analyzed parameters.     

Solid olive residues: insight into their phenolic composition

Solid olive residues (SOR) are byproducts of the olive-milling process, but they have an increasing importance in the pharmaceutical industry due to their rich content of biophenols. Such compounds are studied widely for their antioxidant and antimicrobial activities, but there is a lack of information about their quantitative recovery. This research highlighted the key role played both by the selection of the cultivar and by the degree of olive fruit ripening on the phenolic content on the SOR. The extraction methods were selected to reach the best quantitative results mainly using a safe food solvent. In light of the results the Soxhlet extraction with ethanol could be proposed as preferential for a higher recovery of verbascoside and its analogues.     

Toward a high yield recovery of antioxidants and purified hydroxytyrosol from olive mill wastewaters

We investigated to develop effective procedures to recover the potentially high-added-value phenolic compounds contained in the discontinuous three-phase olive processing wastewaters (OMW). Particular emphasis was made to extract and purify hydroxytyrosol, one of the major compounds occurring in OMW. Batch optimization experiments showed that ethyl acetate is the most efficient solvent for the recovery of phenolic monomers from OMW. The latter was used with an optimal pH equal to 2. Furthermore, the percentage of each monomer, and particularly hydroxytyrosol, in the extract was maximum for a solvent ratio and a theoretical extraction stage number equal to 2 and 3, respectively. High yield (85.46%) recovery of hydroxytyrosol was achieved from OMW using a three-staged continuous counter-current liquid-liquid extraction unit. Hydroxytyrosol (1.225 g) were extracted per liter of OMW. One gram of hydroxytyrosol per liter of OMW was then purified by means of a chromatographic system which could be adapted to a large scale production process.     

Repeated application of diluted olive mill wastewater induces changes in the structure of the soil microbial community

Repeated application of diluted olive mill wastewater (OMW) is a common disposal method which allows the application of large amounts of OMW and improves the organic matter and nutrient status of Mediterranean soils. However, there is lack of information regarding the effects of this practice on the soil microbial community. A study was carried out to investigate these effects on the structure of the bacterial and fungal community of a loamy sand (LS) and a sandy loam (SL) soil, using denaturating gradient gel electrophoresis (DGGE) fingerprinting. OMW was daily applied as aqueous solutions of 0, 2 and 4%, in the presence or absence of nitrogen fertilization, for a three-month period. Multivariate analysis of the DGGE profiles showed that OMW applications resulted in marked changes in the fungal community in both soils, while nitrogen fertilization diminished these effects. Small effects were evident for the bacteria only in the LS soil and this was attributed to the higher availability of OMW-derived phenolics in this soil which resulted in a direct impact on bacteria. Nitrogen fertilization alleviated the effects of OMW on the bacterial community. We suggest that the impact of OMW on the structure of the soil microbial communities was mostly a result of its indirect effect on the soil nutritional status, which becomes enriched in organic substrates and poor in available nitrogen.     

Changes in the content of bioactive polyphenolic compounds of olive mill wastewater by the action of exogenous enzymes

The aim behind the present research is to develop an enzymatic treatment for olive mill wastewater (OMW) to release high amounts of simple phenolics having high antioxidant value. OMW was hydrolyzed by a mixed enzyme preparation rich in β-glucosidase produced by Aspergillus niger . This research shows that A. niger β-glucosidase played a major role in the release of simple phenolic compounds from OMW. These compounds were recovered by ethyl acetate extraction and identified by HPLC and LC-MS. The main identified phenolic compound is hydroxytyrosol. The results of enzymatic hydrolysis of OMW under optimum conditions indicated a maximum hydroxytyrosol concentration of 2.9 g L(-1) compared to 0.015 g L(-1) contained in the control (test without added enzyme). The above results prove that OMW is a potential substrate for producing hydroxytyrosol through enzymatic hydrolysis of its glycosides.     

Effects of soil application of olive mill wastewaters on the structure and function of the community of arbuscular mycorrhizal fungi

Recycling of olive mill wastewaters (OMW) into agricultural soils is a controversial issue since benefits to soil fertility should counterbalance potential short-term toxicity effects. We investigated the short-term effects of OMW on the soil-plant system, regarding the diversity, structure and root colonization capacity of arbuscular mycorrhizal (AM) fungi and the respective growth response of Vicia faba L, commonly used as green manure in olive-tree plantations. A compartmentalized pot system was used that allowed the establishment of an AM fungal community in one compartment (feeder) and the application of three OMW dose levels in an adjacent second compartment (receiver). At 0, 10, and 30 days after OMW treatment (DAT), V. faba pre-germinated seeds were seeded in the receiver compartment. At harvest, shoot and root dry weights, AM fungal root colonization, soil hyphal length and P availability were recorded in the receiver compartment. In addition, OMW effects on AM fungal diversity in plant roots were studied by DGGE. A transient effect of OMW application was observed; plant growth and AM fungal colonization were initially inhibited, whereas soil hyphal length was stimulated, but in most cases differences were absent when seeding was performed 30 DAT. Similarly, changes induced in the structure of the root AM fungal community were of transient nature. Cloning and sequencing of all the major DGGE bands showed that roots were colonized by Glomus spp. The transient effects of OMW on the structure and function of AM fungi could be attributed to OMW-derived phytoxicity to V. faba plants or to an indirect effect via alteration of soil nutritional status. The high OMW dose significantly increased soil P availability in the presence of AM fungi, suggesting efficient involvement of AM fungi in organic-P minerilization. Overall our results indicate that soil application of OMW would cause transient changes in the AM fungal colonization of V. faba plants, which, would not impair their long-term plant growth promoting ability.     

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.     

Change in soil properties and the soil microbial community following land spreading of olive mill wastewater affects olive trees key physiological parameters and the abundance of arbuscular mycorrhizal fungi

Olive mill wastewater (OMW) constitutes a major environmental problem for Mediterranean countries, where most of the world olive oil production takes place. The recycling of the OMW and its use as water for irrigation in agriculture, provided that its impact on soil and plant is established, is an attractive possibility for the Mediterranean countries. Investigations were performed on the influence of agronomic application of OMW (amount applied: 30, 60, 100 and 150 m³ ha^?1) in a field of olive trees on trees characters (photosynthesis, root-soluble carbohydrate and root colonisation), soil properties, and soil microbial community structure. Specific attention was paid to arbuscular mycorrhizal (AM) fungi. The soil fatty acid methyl ester (FAME) 16:1ω5 was used to quantify biomass of AM fungi and the root FAME 16:1ω5 analysis was used as index for the development of colonisation in the olive trees roots. A significant increase in organic C, C/N ratio, extractable phosphorus and exchangeable potassium was found after one year of agronomic application of OMW. The development of saprophytic fungi was significantly higher in the OMW amended soils, whereas the abundance of the soil FAME 16:1ω5, root FAME 16:1ω5, photosynthetic rates and the amount of the total root-soluble carbohydrate were decreased significantly after agronomic application of OMW. A principal component analysis (PCA) of the trees characteristics profiles showed discrimination between the nonirrigated and the OMW irrigated olive trees. These findings suggest that the altering functioning of arbuscular mycorrhizas should be considered as potential factors mediating olive trees responses to agronomic application of OMW when the OMW dose applied is higher than 30 m³ ha^?1. To our knowledge, this is the first report of alterations in the soil FAME 16:1ω5 and root FAME 16:1ω5 due to land spreading of OMW.     

Olive mill wastewater spreading and AMF inoculation effects in a low-input semi-arid Mediterranean crop succession

ABSTRACT

The study aimed to evaluate, in a marginal semi-arid Mediterranean agro-ecosystem (Sicily-Italy), the effects of arbuscular mycorrhizal fungi (AMF) inoculation and raw olive mill wastewater (OMW) (40 and 80 m³ ha^?1) on forage (durum wheat-snail medick intercropping) yield, and grain production of broad bean and chickpea. AMF inoculation significantly increased (+13.6%) forage dry biomass and durum wheat nitrogen (+22.8%) and phosphorus (+32.5%) uptake. AMF inoculation, significantly promoted broad bean phosphorus uptake (+11.5%) and root nodule number (+13.9%) in the absence of OMW. OMW spreading reduced weeds in the forage (?31.3%), root nodule number (?29.7%) and dry weight (?22.7%) in broad bean. OMW also significantly increased snail medick dry biomass (+19.3%) as compared to control treatments (0, 40 and 80 m³ H₂O ha^?1, average production 361 g m^?2), and broad bean grain yield with a production of 2.46 ± 0.12 and 1.94 ± 0.09 Mg ha^?1 with and without OMW, respectively. During the experiment AMF colonization was not affected by OMW volumes. The results obtained showed that in a marginal Mediterranean agro-ecosystem: 1) OMW, notwithstanding spreading volumes, is a valuable amendment to maximize legume yield while 2) AMF inoculation is a valuable practice to improve biomass production and N and P uptake in wheat.     

DETERMINATION OF OLIVE MILL WASTEWATER TOXIC EFFECTS ON THREE MINT SPECIES GROWN IN HYDROPONIC CULTURE

The effects of two olive mill wastewater (OMW) dilutions upon physiological function and metabolic components of three mint species grown in hydroponic culture were studied. Visible symptoms, chlorosis and stunted growth, were pronounced in peppermint and spearmint; toxicity symptoms in menthe douce were few. Peppermint and spearmint displayed loss of calcium (Ca), magnesium (Mg), and potassium (K); menthe douce revealed accumulation of Ca, sodium (Na), and iron (Fe), under OMW. The loss of Ca negatively affected the development and stability of cell wall and membrane; the Mg deficiency negatively affected the photosynthetic apparatus and carbon fixation. Fv′/ Fm′ was slightly affected under OMW, whereas, Φ_PSII, q _P and Rfd displayed significant reductions. No clear effect of OMW on phenols and carbohydrates among the species were found. Ascorbic acid content in peppermint dropped dramatically; the reduction was lower for spearmint and menthe douce. The OMW resistant species, menthe douce, developed a mechanism to control the physiological and the biochemical status.