Effect of steam treatment of alperujo on the composition, enzymatic saccharification, and in vitro digestibility of alperujo

The solid waste from two-phase olive oil extraction or "alperujo" was submitted to steam treatment at high pressure or temperature, 200 degrees C for 5 min, in the presence and absence of mild acid catalyst. This treatment made easier the separation of the solid and liquid fractions. Besides the recovery of certain valuable components from the liquid fraction (the antioxidant hydroxytyrosol, low molecular weight oligosaccharides, glucose, mannitol, etc.), the major components of the solid residue could be also exploited. In this study, changes in composition of alperujo due to steam treatment were determined. The process reduced appreciably the hemicellulose concentrations (75-88%), removed a substantial portion of Klason lignin and protein (50%), and led to an extensive solubilization of alperujo (55-67%). Cellulose was very resistant to autohydrolysis and acid-catalyzed hydrolysis, so the solid residue was enriched in fat (13-18 g/100 g of dry steam-treated alperujo) and cellulose (15-25 g/100 g of dry and defatted steam-treated alperujo). The steam-treated material can be efficiently saccharified with commercial cellulase. The best hydrolysis yields were attained, up to 80%, when the treated material was post-treated with NaOH. The possibility of using this steam-treated alperujo in animal feeding was evaluated by an in vitro digestibility test, using the pepsin-cellulase method. The treatment affected positively the nutritional characteristics of alperujo with an increase in its in vitro (dry and organic matter) digestibility (8-10% higher than untreated material). In vitro digestibility and cellulose accessibility to enzymatic hydrolysis were improved by the alkali post-treatment.     

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.     

Total recovery of the waste of two-phase olive oil processing: isolation of added-value compounds

A process for the value addition of solid waste from two-phase olive oil extraction or "alperujo" that includes a hydrothermal treatment has been suggested. In this treatment an autohydrolysis process occurs and the solid olive byproduct is partially solubilized. From this water-soluble fraction can be obtained besides the antioxidant hydroxytyrosol several other compounds of high added value. In this paper three different samples of alperujo were characterized and subjected to a hydrothermal treatment with and without acid catalyst. The main soluble compounds after the hydrolysis were represented by monosaccharides xylose, arabinose, and glucose; oligosaccharides, mannitol and products of sugar destruction. Oligosaccharides were separated by size exclusion chromatography. It was possible to get highly purified mannitol by applying a simple purification method.     

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.     

Phenolic compounds in virgin olive oil. 2. Reappraisal of the extraction, HPLC separation, and quantification procedures

The extraction procedures (solid/liquid SPE and liquid/liquid LLE) and HPLC separation and quantification methods of polyphenolic compounds have been checked in virgin olive oils in order to explain the differences in content reported in the literature. The work has been carried out on oils prepared from one cultivar and produced under the same protocol. The extraction methods are practically equivalent, but the SPE technique is more favorable because it is faster and simpler. It has been proved that the chromatographic features and the method of chemical expression of the concentrations may greatly affect the final values. Thus, under the same analytical method, the total concentration values of polyphenols of the same oil show variations from 18% to 80%, according to the formality of expression as gallic acid, caffeic acid, or tyrosol equivalents. The role of the nature and spectrophotometric features of the phenols and of the internal standard is also discussed, and it was found to be an important source of reported variation. A gradient separation with an eluent mixture acetonitrile-sulfuric acid (0.1 mol/L), detection at 225 nm, and quantitative calculation of polyphenolic compounds in oils (expressed as tyrosol equivalents, THY(eq)) is proposed.     

Antioxidant effect of phenolic compounds, alpha-tocopherol, and other minor components in virgin olive oil

The effect of acidity, squalene, hydroxytyrosol, aldehydic form of oleuropein aglycon, hydroxytyrosyl acetate, tyrosol, homovanillic acid, luteolin, apigenin, alpha-tocopherol, and the mixtures hydroxytyrosol/hydroxytyrosyl acetate, hydroxytyrosol/tyrosol, and hydroxytyrosol/alpha-tocopherol on the oxidative stability of an olive oil matrix was evaluated. A purified olive oil was spiked with several concentrations of these compounds and, then, subjected to an accelerated oxidation in a Rancimat apparatus at 100 degrees C. Acidity, squalene, homovanillic acid, and apigenin showed negligible effect. At the same millimolar concentrations, the different o-diphenolic compounds yielded similar and significant increases of the induction time, alpha-tocopherol a lesser increase, and tyrosol a scarce one. At low concentrations of o-diphenols and alpha-tocopherol, a linear relationship between induction time and concentration was found, but at high concentrations the induction time tended toward constant values. To explain this behavior, a kinetic model was applied. The effect of the mixtures hydroxytyrosol/hydroxytyrosyl acetate was similar to that of a single o-diphenol at millimolar concentration equal to the sum of millimolar concentrations of both compounds. Concentrations of tyrosol >0.3 mmol/kg increase the induction time by 3 h. The mixtures hydroxytyrosol/alpha-tocopherol showed opposite effects depending on the relative concentrations of both antioxidants; so, at hydroxytyrosol concentrations <0.2 mmol/kg, the addition of alpha-tocopherol increased the induction time, whereas at higher hydroxytyrosol concentrations, the alpha-tocopherol diminished the stability.     

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.     

Small branches of olive tree: a source of biophenols complementary to olive leaves

The extraction of biophenols (BPs) from small branches (fibrous softwood) of olive tree accelerated by microwave assistance is proposed for the first time. Under optimal working conditions, no further extraction of the target analytes was achieved after 10 min, so complete removal of them within this interval was assumed (amounts ca. to 19000, 1000, 2000, 900, and 700 mg/kg of oleuropein, verbascoside, tyrosol, alpha-taxifolin, and hydroxytyrosol, respectively; the three last BPs are absent in branch-free olive leaves). The extracts required no cleanup or concentration prior to injection into a chromatograph-photodiode array detector assembly for individual separation-quantification. Extraction from this raw material was also implemented in continuous and discontinuous-continuous extractors using ultrasound assistance and superheated liquids, respectively, as auxiliary energies, and the results were compared with those obtained by microwave-assisted extraction. The simultaneous extraction of small branches and leaves from olive tree provided extracts with a higher variety of BPs, but either extracts richer in oleuropein and verbascoside without tyrosol, alpha-taxifolin, and hydroxytyrosol or rich in these three BPs can be obtained by separate extraction of leaves and branches, respectively.     

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.     

Mild photochemical synthesis of the antioxidant hydroxytyrosol via conversion of tyrosol

Hydroxytyrosol, a naturally occurred orthodiphenolic antioxidant molecule found in olive oil and olive mill wastewaters, was obtained from the wet hydrogen peroxide photocatalytic oxidation of its monophenolic precursor tyrosol. The liquid-phase oxidation of tyrosol to hydroxytyrosol was performed by use of an iron-containing heterogeneous catalyst (Al-Fe)PILC with the assistance of UV irradiation at 254 nm and at room temperature. The spectroscopic and HPLC data of the synthesized compound proved to coincide fully with those of a pure sample obtained by continuous countercurrent extraction. This reaction was found to be light-induced. The hydroxytyrosol synthesis reaction reached its maximum yield of 64.36% under the optimized operating conditions of 3.6 mM tyrosol, 0.5 g L(-1) catalyst, and 10(-2) M H2O2 with the assistance of UV light. Increasing the initial hydrogen peroxide concentration more than 10(-2) M has a diminishing return on the reaction efficiency. Catalyst can be recuperated by means of filtration and then reused in a next run after regeneration since its activity did not significantly decrease (<10%). The reaction synthesis is operationally simple and could find application for industrial purposes.     

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.     

Development of a sensitive and specific solid phase extraction--gas chromatography-tandem mass spectrometry method for the determination of elenolic acid, hydroxytyrosol, and tyrosol in rat urine

A novel gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed, using an ion trap mass spectrometer, for the simultaneous determination of olive oil bioactive components, elenolic acid, hydroxytyrosol, and tyrosol, in rat urine. Samples were analyzed by GC-MS/MS prior to and after enzymatic treatment. A solid phase extraction sample pretreatment step with greater than 80% analytical recoveries for all compounds was performed followed by a derivatization reaction prior to GC-MS/MS analysis. The calibration curves were linear for all compounds studied for a dynamic range between 1 and 500 ng. The limit of detection was in the mid picogram level for tyrosol and elenolic acid (300 pg) and in the low picogram level for hydroxytyrosol (2.5 pg). The method was applied to the analysis of rat urine samples after sustained oral intake of oleuropein or extra virgin olive oil as a diet supplement.     

Supercritical fluid extraction and high-performance liquid chromatographic determination of phloroglucinols in St. John's Wort (Hypericum perforatum L.)

A small-scale supercritical fluid extraction (SFE) method was developed for the selective extraction of phloroglucinols from St. John's wort (SJW) leaf/flower mixtures using supercritical carbon dioxide (CO(2)). The extraction efficiency was investigated as influenced by pressure, temperature, time, and modifier. The optimized condition of SFE was carried out at 3.80 x 10(4) kpa (5500 psi) and 50 degrees C. Samples were held in static extraction for 10 min, followed by a dynamic extraction for 90 min at the flow rate of 1 mL/min. A simple and sensitive HPLC method was developed for the analysis of hyperforin and adhyperforin, the major phloroglucinols, in the SFE extract of SJW.     

Engineering of a tyrosol-producing pathway, utilizing simple sugar and the central metabolic tyrosine, in Escherichia coli

Metabolic engineering was applied to the development of Escherichia coli capable of synthesizing tyrosol (2-(4-hydroxyphenyl)ethanol), an attractive phenolic compound with great industrial value, from glucose, a renewable carbon source. In this strain, tyrosine, which was supplied not only from the culture medium but also from the central metabolism, was converted into tyrosol via three steps: decarboxylation, amine oxidation, and reduction. The engineered strain synthesized both tyrosol and 4-hydroxyphenylacetate (4HPA), but disruption of the endogenous phenylacetaldehyde dehydrogenase gene shut off 4HPA production and improved the production of tyrosol as a sole product. The engineered mutant strain was capable of producing 0.5 mM tyrosol from 1% (w/v) glucose during a 48 h shake flask cultivation.     

Tagetolone and tagetenolone: two phytotoxic polyketides from Alternaria tagetica

Two new phytotoxic polyketides, tagetolone (1) and tagetenolone (2), in addition to tyrosol and p-hydroxybenzoic acid, have been isolated from the organic crude extract of culture filtrates from the fungal pathogen Alternaria tagetica. Complete characterization of all structures was carried out following a careful analysis of their spectroscopic data (IR, MS, (1)H and (13)C NMR, and 2D NMR experiments).     

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.     

Effect of a new thermal treatment in combination with saprobic fungal incubation on the phytotoxicity level of alperujo

Byproducts generated from food industries, such as olive oil mills, have been studied to decrease harmful pollution and their environmental consequences. In this work, a new thermal pretreatment and saprobic fungal incubation to detoxify alperujo (two-phase olive mill waste) have been evaluated in view of its use as fertilizer in agriculture. The sequential use of both methods simplifies the thermal conditions and incubation times of the fungal treatment. Optimization of the thermal treatment from 150 to 170 °C for 45 and 15 min, respectively, reduced the incubation time with Coriolpsis rigida from 20 to 10 weeks needed to reduce phytotoxic effects on tomato plants. Therefore, the combination of thermal and biological treatments will allow the development of the potential benefits of alperujo to improve nutrients in agricultural soil.     

Selective recovery of tagatose from mixtures with galactose by direct extraction with supercritical CO2 and different cosolvents

A selective fractionation method of carbohydrate mixtures of galactose/tagatose, using supercritical CO(2) and isopropanol as cosolvent, has been evaluated. Optimization was carried out using a central composite face design and considering as factors the extraction pressure (from 100 to 300 bar), the extraction temperature (from 60 to 100 degrees C), and the modifier flow rate (from 0.2 to 0.4 mL/min, which corresponded to a total cosolvent percentage ranging from 4 to 18% vol). The responses evaluated were the amount (milligrams) of tagatose and galactose extracted and their recoveries (percent). The statistical analysis of the results provided mathematical models for each response variable. The corresponding parameters were estimated by multiple linear regression, and high determination coefficients (>0.96) were obtained. The optimum conditions of the extraction process to get the maximum recovery of tagatose (37%) were 300 bar, 60 degrees C, and 0.4 mL/min of cosolvent. The predicted value was 24.37 mg of tagatose, whereas the experimental value was 26.34 mg, which is a 7% error from the predicted value. Cosolvent polarity effects on tagatose extraction from mixtures of galactose/tagatose were also studied using different alcohols and their mixtures with water. Although a remarkable increase of the amount of total carbohydrate extracted with polarity was found, selective extraction of tagatose decreased with increase of polarity of assayed cosolvents. To improve the recovery of extracted tagatose, additional experiments outside the experimental domain were carried out (300 bar, 80 degrees C, and 0.6 mL/min of isopropanol); recoveries >75% of tagatose with purity >90% were obtained.     

Akt/FOXO3a/SIRT1-mediated cardioprotection by n-tyrosol against ischemic stress in rat in vivo model of myocardial infarction: switching gears toward survival and longevity

Moderate consumption of wine has been associated with decreased risk of cardiovascular events. Recently we have shown that white wine is equally as cardioprotective as red wine. However, unlike resveratrol (polyphenol in red wine), the white wine component, n-tyrosol [2-(4-hydroxyphenyl)ethanol] has not been explored for its cardioprotective effect and mechanism of action. Therefore, the present study was designed to evaluate the effect of tyrosol treatment (5 mg/kg/day for 30 days) on myocardial ischemic stress in a rat in vivo model of Myocardial Infarction (MI) and to identify key molecular targets involved in this mechanism. MI was induced by Left Anterior Descending (LAD) coronary artery ligation. Reduced infarct size (32.42 vs 48.03%) and cardiomyocyte apoptosis (171 vs 256 counts/100 HPF) were observed along with improvement in the myocardial functional parameters such as LVIDs (5.89 vs 6.58 mm), ejection fraction (51.91 vs 45.09%), and fractional shortening (28.46 vs 23.52%) as assessed by echocardiography in the tyrosol-treated animals when compared to the nontreated controls. We have also observed significant increase in the phosphorylation of Akt (1.4-fold), eNOS (3-fold) and FOXO3a (2.6-fold). In addition, tyrosol induced the expression of longevity protein SIRT1 (3.2-fold) in the MI group as compared to the non-treated MI control. Therefore tyrosol's SIRT1, Akt and eNOS activating power adds another dimension to the white wine research, because it adds a great link to the French paradox. In conclusion these findings suggest that tyrosol induces myocardial protection against ischemia related stress by inducing survival and longevity proteins that may be considered as anti-aging therapy for the heart. However, human intervention studies would be necessary before establishing any recommendations about dietary habits for tyrosol intake or administration of dietary supplements containing tyrosol.     

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.