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.     

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.     

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.     

Antimicrobial activity of olive solutions from stored Alpeorujo against plant pathogenic microorganisms

The aim of this work was to assess the in vitro antimicrobial effects that wastewaters from alpeorujo oil extraction have against phytopathogenic bacteria and fungi. Alpeorujo was stored for 6 months and then processed to extract its oil, pomace, and a new liquid waste (OWSA), which was characterized by its content in phenolic compounds. OWSA at 20% decreased bu >4 log the population of Erwinia spp., Pseudomonas spp., and Clavibacter spp. viable cells in test tubes, whereas OWSA at 50% in agar medium was necessary to inhibit mycelial growth of most fungi. It was found that the bactericidal effect was due to the joint action of low molecular mass phenolic compounds, although neither hydroxytyrosol, its glucosides, hydroxytyrosol glycol, nor a glutaraldehyde-like compound individually explained this bioactivity. Hence, OWSA constitutes a promising natural solution to fight plant phytopathogenic bacteria and fungi.     

Oxidative transformation of natural and synthetic phenolic mixtures by Trametes versicolor laccase

The efficiency of Trametes versicolor laccase in the transformation of phenols (caffeic acid, catechol, hydroxytyrosol, methylcatechol, protocatechuic acid, syringic acid, m-tyrosol, 3-hydroxybenzoic acid, 3-hydroxyphenylacetic acid, 2,6-dihydroxybenzoic acid, 4-hydroxybenzaldehyde) usually present in waste water, such as that derived from an olive oil factory, was investigated. According to their response to 24 h laccase action the 11 phenolic compounds were classified in three groups: reactive (88-100% transformation), intermediate reactive (transformation lower than 50%), and recalcitrant (not transformed at all). The enzyme was able to transform the 11 substrates even when they were present in a mixture and also toward a phenolic extract from a Moroccan olive oil mill waste water (OMW) sample. The disappearance of protocatechuic, 3-hydroxyphenylacetic, and 2,6-dihydroxybenzoic acids, and 4-hydroxybenzaldehyde was enhanced whereas that of caffeic acid and m-tyrosol was depressed when the phenols were present in the mixture. A reduction of enzyme activity occurred in single and/or complex phenolic mixtures after enzymatic oxidation. No correspondence between phenol transformation and disappearance of enzymatic activity was, however, observed. The overall results suggest that laccases are effective in the transformation of simple and complex phenolic mixtures.     

Optimization of microwave-assisted extraction for the characterization of olive leaf phenolic compounds by using HPLC-ESI-TOF-MS/IT-MS(2)

In the present work, a simple and rapid method for the extraction of phenolic compounds from olive leaves, using microwave-assisted extraction (MAE) technique, has been developed. The experimental variables that affect the MAE process, such as the solvent type and composition, microwave temperature, and extraction time, were optimized using a univariate method. The obtained extracts were analyzed by using high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap tandem mass spectrometry (ESI-IT-MS(2)) to prove the MAE extraction efficiency. The optimal MAE conditions were methanol:water (80:20, v/v) as extracting solvent, at a temperature equal to 80 °C for 6 min. Under these conditions, several phenolic compounds could be characterized by HPLC-ESI-MS/MS(2). As compared to the conventional method, MAE can be used as an alternative extraction method for the characterization of phenolic compounds from olive leaves due to its efficiency and speed.     

Antioxidant phenols in barley (Hordeum vulgare L.) flour: comparative spectrophotometric study among extraction methods of free and bound phenolic compounds

Phenolic compounds are found in both free and bound forms in cereals. The majority is in the insoluble bound form, that is, bound to cell wall material, such as ferulic acid and its derivatives. The antioxidant properties of the phenolic compounds in grains are associated with the health benefits of grains and grain products. The extraction capacity of several solvent mixtures, for extracting free phenols from barley flours, and the possibility of employing a rapid automated solvent extraction method were studied. The extraction yield of each method was evaluated by correlating several spectrophotometric indices (absorption at 280, 320, and 370 nm and total phenolic compounds by the Folin-Ciocalteu method) with the antioxidant activities of the barley extracts (scavenging activity by the 2,2-diphenyl-1-picrylhydrazyl method). Interesting results were obtained when ethanol and acetone-based extraction mixtures were employed to extract free phenols. A comparison was made between alkaline and acid hydrolysis. The extraction yield of bound phenolic compounds increased when the digestion time for alkaline hydrolysis was prolonged.     

Analysis of total contents of hydroxytyrosol and tyrosol in olive oils

The most abundant phenolic compounds in olive oils are the phenethyl alcohols hydroxytyrosol and tyrosol. An optimized method to quantify the total concentration of these substances in olive oils has been described. It consists of the acid hydrolysis of the aglycons and the extraction of phenethyl alcohols with a 2 M HCl solution. Recovery of the phenethyl alcohols from oils was very high (<1% remained in the extracted oils), and the limits of quantification (LOQ) were 0.8 and 1.4 mg/kg for hydroxytyrosol and tyrosol, respectively. Precision values, both intraday and interday, remained below 3% for both compounds. The final optimized method allowed for the analysis of several types of commercial olive oils to evaluate their hydroxytyrosol and tyrosol contents. The results show that this method is simple, robust, and reliable for a routine analysis of the total concentration of these substances in olive oils.     

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.     

Effects of extraction solvent mixtures on antioxidant activity evaluation and their extraction capacity and selectivity for free phenolic compounds in barley (Hordeum vulgare L.)

Four kinds of solvent extracts from three Chinese barley varieties (Ken-3, KA4B, and Gan-3) were used to examine the effects of extraction solvent mixtures on antioxidant activity evaluation and their extraction capacity and selectivity for free phenolic compounds in barley through free radical scavenging activity, reducing power and metal chelating activity, and individual and total phenolic contents. Results showed that extraction solvent mixtures had significant impacts on antioxidant activity estimation, as well as different extraction capacity and selectivity for free phenolic compounds in barley. The highest DPPH* and ABTS*+ scavenging activities and reducing power were found in 80% acetone extracts, whereas the strongest *OH scavenging activity, O2*- scavenging activity, and metal chelating activity were found in 80% ethanol, 80% methanol, and water extracts, respectively. Additionally, 80% acetone showed the highest extraction capacity for (+)-catechin and ferulic, caffeic, vanillic, and p-coumaric acids, 80% methanol for (-)-epicatechin and syringic acid, and water for protocatechuic and gallic acids. Furthermore, correlations analysis revealed that TPC, reducing power, DPPH* and ABTS*+ scavenging activities were well positively correlated with each other (p < 0.01). Thus, for routine screening of barley varieties with higher antioxidant activity, 80% acetone was recommended to extract free phenolic compounds from barley. DPPH* scavenging activity and ABTS*+ scavenging activity or reducing power could be used to assess barley antioxidant activity.     

Model study on the effect of 15 phenolic olive mill wastewater constituents on seed germination and Vibrio fischeri metabolism

Olive mill wastewaters (OMW) can be a severe problem when disposed of as untreated because of their high organic load, elevated concentration of polyphenols, and moderately low biodegradability. In the present study, the acute toxicity of 15 compounds with low molecular weight (<350 Da), catechol, four benzoic acids, three phenylacetic acids, three phenylethanols, and four cinnamic acids, already isolated from the reverse osmosis in the fractionation of OMW, was assessed on the marine bacterium Vibrio fischeri and on the seeds of two dicotyledonous species Cucumis sativus and Lepidium sativum, and on one monocotyledon Sorghum bicolor. Results of phytotoxicity showed that the most toxic compounds were catechol (EC50s ranging from 0.40 mmol/L for S. bicolor to 1.09 for C. sativus) and hydroxytyrosol, (EC50s ranging from 0.47 mmol/L for S. bicolor to 1.55 for C. sativus) while the toxic potential on bacteria was particularly elevated with EC50 values 1 or 2 orders less than phytotoxicity. These results suggested that the risk of OMW disposal may be more elevated for the water compartment than for the soil.     

Separation and identification of phenolic compounds in olive oil by coupling high-performance liquid chromatography with postcolumn solid-phase extraction to nuclear magnetic resonance spectroscopy (LC-SPE-NMR)

This study reports the first application of the hyphenated LC-SPE-NMR technique using postcolumn solid-phase extraction to the direct analysis of phenolic compounds in the polar part of olive oil. Apart from the identification and structure elucidation of simple phenols (hydroxytyrosol, tyrosol, vanillic acid, vanillin, p-coumaric acid, hydroxytyrosol, and tyrosol acetates), lignans (pinoresinol and 1-acetoxypinoresinol), flavonoids (apigenin and luteolin), and a large number of secoiridoid derivatives, this technique enables the identification of several new phenolic components, which had not been reported previously as constituents in the polar part of olive oil.     

Interactions of ferric ions with olive oil phenolic compounds

The ferric complexing capacity of four phenolic compounds, occurring in olives and virgin olive oil, namely, oleuropein, hydroxytyrosol, 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA), and 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (3,4-DHPEA-EDA), and their stability in the presence of ferric ions were studied. At pH 3.5, all compounds formed a reversible 1:1 complex with ferric ions, but hydroxytyrosol could also form complexes containing >1 ferric ion per phenol molecule. At pH 5.5, the complexes between ferric ions and 3,4-DHPEA-EA or 3,4-DHPEA-EDA were relatively stable, indicating that the antioxidant activity of 3,4-DHPEA-EA or 3,4-DHPEA-EDA at pH 5.5 is partly due to their metal-chelating activity. At pH 7.4, a complex containing >1 ferric ion per phenol molecule was formed with hydroxytyrosol. Oleuropein, 3,4-DHPEA-EA, and 3,4-DHPEA-EDA also formed insoluble complexes at this pH. There was no evidence for chelation of Fe(II) by hydroxytyrosol or its derivatives. At all pH values tested, hydroxytyrosol was the most stable compound in the absence of Fe(III) but the most sensitive to the presence of Fe(III).     

Changes in phenolic composition and antioxidant activity of virgin olive oil during frying

The concentration of hydroxytyrosol (3,4-DHPEA) and its secoiridoid derivatives (3,4-DHPEA-EDA and 3,4-DHPEA-EA) in virgin olive oil decreased rapidly when the oil was repeatedly used for preparing french fries in deep-fat frying operations. At the end of the first frying process (10 min at 180 degrees C), the concentration of the dihydroxyphenol components was reduced to 50-60% of the original value, and after six frying operations only about 10% of the initial components remained. However, tyrosol (p-HPEA) and its derivatives (p-HPEA-EDA and p-HPEA-EA) in the oil were much more stable during 12 frying operations. The reduction in their original concentration was much smaller than that for hydroxytyrosol and its derivatives and showed a roughly linear relationship with the number of frying operations. The antioxidant activity of the phenolic extract measured using the DPPH test rapidly diminished during the first six frying processes, from a total antioxidant activity higher than 740 micromol of Trolox/kg down to less than 250 micromol/kg. On the other hand, the concentration of polar compounds, oxidized triacylglycerol monomers (oxTGs), dimeric TGs, and polymerized TGs rapidly increased from the sixth frying operation onward, when the antioxidant activity of the phenolic extract was very low, and as a consequence the oil was much more susceptible to oxidation. The loss of antioxidant activity in the phenolic fraction due to deep-fat frying was confirmed by the storage oil and oil-in-water emulsions containing added extracts from olive oil used for 12 frying operations.     

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.     

Effect of solvent, temperature, and solvent-to-solid ratio on the total phenolic content and antiradical activity of extracts from different components of grape pomace

Grape byproducts were subjected to an extraction process under various different experimental conditions (namely, solvent type, temperature, solvent-to-solid ratio, time contact, and raw material) in order to study the effect of these conditions on the yield of phenolic compounds and the corresponding antiradical activity of extracts. Although the order of decreasing capacity to extract soluble materials was ethanol > methanol > water, methanol was the most selective for extracting phenolic compounds. Temperature and solvent-to-solid ratio were found to have a critical role in extraction efficiency; values of 50 degrees C (between 25 and 50 degrees C) and 1:1 (between 1:1 and 5:1) maximized the antiradical activity of phenolic extracts. In addition, extracts from grape samples previously subjected to distillation reached higher antiradical values in comparison to those coming directly from pressing; in both cases, seed extracts showed better results than those of stem when ethanol or water was employed, whereas the opposite occurred in the case of methanol. These differences were attributed to the different phenolic compositions of the considered fractions.     

Comparison of different extraction solvent mixtures for characterization of phenolic compounds in strawberries

Eight different solvent mixtures containing acetone or methanol pure or combined with an acid (acetic, formic, hydrochloric) were tested for their efficiency for extraction of phenolic compounds from strawberries belonging to five groups of polyphenols: anthocyanins, flavonols, flavan-3-ols, hydroxycinnamic acid derivatives and conjugated forms of ellagic acid. Twenty-eight compounds from these five groups have been detected and quantified using HPLC-DAD-ESI-MS(n). The yield of each phenolic compound and group was evaluated with regard to the extraction solvent composition. Acetone containing extraction mixtures were superior to the ones containing methanol for extraction yield of total phenolic compounds, which was especially pronounced for the groups of flavan-3-ols and conjugated forms of ellagic acid. The mixture acetone/acetic acid (99:1, v/v) gave the best results for the qualitative and quantitative assay of the polyphenols present in strawberries since all 28 compounds were detected only in these extracts in quantities higher or comparable to the other extraction solvents tested.     

Inhibition of hemoglobin- and iron-promoted oxidation in fish microsomes by natural phenolics

Natural phenolic antioxidants have been tested in hake (Merluccious merluccious) microsomes as inhibitors of lipid oxidation promoted by fish muscle prooxidants: hemoglobin (Hb), enzymatic NADH-iron and nonenzymatic ascorbate-iron. The phenolics selected were as follows: (a) a grape phenolic extract (OW), (b) a fraction (IV) with isolated grape procyanidins with a medium-low degree of polymerization and galloylation percentage, (c) hydroxytyrosol obtained from olive oil byproducts, and (d) a synthetic phenolic antioxidant, propyl gallate. All compounds delayed lipid oxidation activated by Hb, enzymatic NADH-iron, and nonenzymatic ascorbate-iron, excluding hydroxytyrosol that was not an effective antioxidant on oxidation promoted by nonenzymatic iron. The relative antioxidant efficiency was independent of the prooxidant system, IV > propyl gallate > OW > hydroxytyrosol, and showed a positive correlation with their incorporation into microsomes (p < 0.05). The reducing capacity or ability for donating electrons and the chelating properties may also contribute to the antioxidant activity of phenolics, although these factors were less decisive than their affinity for incorporating into the microsomes. Conversely, the inhibition of Hb oxidation by phenolics and their polarity did not seem to play an important role on antioxidant mechanism. These results stressed the importance of incorporating the exogenous antioxidants into the membranes where are located key substances for fish lipid oxidation (highly unsaturated phospholipids, iron-reducing enzymes, and endogenous alpha-tocopherol).     

Lettuce and chicory byproducts as a source of antioxidant phenolic extracts

A process to obtain enriched antioxidant phenolic extracts from lettuce (baby, romaine, and iceberg cultivars) and chichory byproducts as a way to valorize these byproducts was developed. Two extraction protocols using water and methanol as solvent were used. Amberlite XAD-2 nonionic polymeric resin was used to purify the extracts. The extraction yield, phenolic content, and phenolic yield were evaluated as well as the antioxidant capacity of the extracts (DPPH, ABTS, and FRAP assays). Baby and romaine lettuce byproducts showed the highest water extract yields [27 and 26 g of freeze-dried extracts/kg of byproduct fresh weight (fw), respectively], whereas baby and iceberg lettuce showed highest methanol extract yields (31 and 23 g of freeze-dried extracts/kg of byproduct fw, respectively). Methanol extraction yielded a raw extract with a high phenolic content, the baby and chicory extracts being the richest with approximately 50 mg of phenolics/g of freeze-dried extract. Regarding the purified extracts, water extraction yielded a higher phenolic content, baby and chicory being also the highest with mean values of approximately 190 and 300 mg of phenolics/g of freeze-dried extract, respectively. Both raw and purified extracts from baby and chicory showed the higher antioxidant contents (DPPH, ABTS, and FRAP assays). The antioxidant capacity was linearly correlated with the phenolic content. The results obtained indicate that lettuce byproducts could be, from the industrial point of view, an interesting and cheap source of antioxidant phenolic extracts to funcionalize foodstuffs.     

Influence of cultivar and concentration of selected phenolic constituents on the in vitro chemiopreventive potential of olive oil extracts

One of the main olive oil phenolic compounds, hydroxytyrosol (3,4-DHPEA), exerts in vitro chemopreventive activities (antiproliferative and pro-apoptotic) on tumor cells through the accumulation of H(2)O(2) in the culture medium. However, the phenol composition of virgin olive oil is complex, and 3,4-DHPEA is present at low concentrations when compared to other secoiridoids. In this study, the in vitro chemopreventive activities of complex virgin olive oil phenolic extracts (VOO-PE, derived from the four Italian cultivars Nocellara del Belice, Coratina, Ogliarola, and Taggiasca) were compared to each other and related to the amount of the single phenolic constituents. A great chemopreventive potential among the different VOO-PE was found following this order: Ogliarola > Coratina > Nocellara > Taggiasca. The antiproliferative and pro-apoptotic activities of VOO-PE were positively correlated to the secoiridoid content and negatively correlated to the concentration of both phenyl alcohols and lignans. All extracts induced H(2)O(2) accumulation in the culture medium, but this phenomenon was not responsible for their pro-apoptotic activity. When tested in a complex mixture, the olive oil phenols exerted a more potent chemopreventive effect compared to the isolated compounds, and this effect could be due either to a synergistic action of components or to any other unidentified extract constituent.