The detection of the presence of hazelnut oil in olive oil by free and esterified sterols

Genuine olive and hazelnut oils from diverse geographical origins, as single varieties and blends, were mixed at different percentages and analysed by the method based on the quantification of free and esterified sterols. Two formulas based on three sterols (Campesterol, Δ⁷-stigmastenol and Δ⁷-avenasterol) together with empirical decision rules were able to detect the presence of hazelnut oil in olive oil when the percentage of the former was more than 6–8%, although this figure was much lower in the most of the adulterations. Results of univariate and multivariate statistical procedures based on the analysis of 116 samples are presented in support of the method efficiency.     

Separation and determination of sterols in olive oil by HPLC-MS

This study presents the first liquid chromatographic method for the identification and quantification of seven phytosterols in olive oil. Sterols were identified and quantified by liquid chromatography with mass spectrometry detection in positive APCI (atmospheric pressure chemical ionisation) mode. The samples were saponified by refluxing with 2 N ethanolic KOH, and the non-saponificable fraction was extracted with diethyl ether. This fraction was subjected to thin layer chromatography (TLC) on silica gel plates and then the band of sterols was isolated and extracted with methanol. Sterols were quantified by LC-MS, on a Waters Atlantis 5 μm dC₁₈2,1 × 150 mm column with a gradient of acetonitrile/water (0.01% acetic acid) at a flow of 0.5 mL min⁻¹; column temperature 30 °C. The method presents values between 123 and 677 ng mL⁻¹ for detection limits, with relative standard deviations between 4.0% and 5.4% at a concentration of 5 mg L⁻¹ for each sterol. Sterol contents were determined in a virgin olive oil, a refined olive oil, an olive-pomace oil and a crude olive-pomace oil.     

Phytosterol content of sea buckthorn (Hippophae rhamnoides L.) seed oil: Extraction and identification

Phytosterols in sea buckthorn (Hippophae rhamnoides L.) seed oil extracted by cold pressing, hexane, and supercritical carbon dioxide were identified by GC–MS and FID. Compounds identified were campesterol, clerosterol, lanosterol, sitosterol, β-amyrin, sitostanol, Δ⁵-avenasterol, Δ²⁴⁽²⁸⁾-stigmasta-en-ol, α-amyrin, Δ^5,24(25)-stigmastadienol, lupeol, gramisterol, Δ⁷-sitosterol, cycloartenol, cycloeucalenol, Δ⁷-avenasterol, 28-methylobtusifoliol, 24-methylenecycloartanol, erythrodiol, citrostadienol, uvaol, and oleanol aldehyde. Sitosterol and Δ⁵-avenasterol were, quantitatively, the most important phytosterols. Total sterols and most individual sterols differed significantly (P ? 0.05) among all three extraction methods with supercritical carbon dioxide extracting the highest total sterol levels (1640 mg/100 g oil) and cold pressed the lowest levels (879 mg/100 g oil).     

The effect of citric acid on the phenolic contents of olive oil

Response surface methodology was used to optimise the combined effects of malaxation time (t) and aqueous citric acid solution (C_A) added at the beginning of the malaxation step on total polyphenols (TP) and o-diphenols (OD) content and the antiradical power (ARP) of the olive oil extracted from the Italian olive fruits of Coratina cultivar. Different tests were performed according to a 3² full factorial design, varying t from 30 to 90 min and the C_A from 5 to 15 ml/kg_paste. Overall optimal conditions identified by a numerical optimisation for the three responses were found to be at t = 30 min and C_A = 13.79 ml/kg_paste under which the model predicted TP = 604.52 μgCAE/g_oil, OD = 80.44 μgCAE/g_oil and ARP = 28.73 μgDPPH/μlextract. There were also linear correlations between TP (R² = 0.8176) and OD (R² = 0.8633) values of olive oil and waste water. The results of this study demonstrate that considerably short malaxation time and relatively small amounts of citric acid were required to enhance the quality of olive oil. The outcome of our study will therefore be of great value for the commercial production of olive oil with high level of polyphenols and o-diphenols.     

Gas chromatographic–mass spectrometric characterisation of triterpene alcohols and monomethylsterols in developing Olea europaea L. fruits

Five triterpene alcohols and four 4-monomethylsterols were identified by GC–MS during the ripening of Picholine olive. The quantitative characterisation of these compounds was performed using GC–FID. The results showed that the maximum level of total triterpene alcohols (263.68 mg/100 g oil) was reached at 26th week after the flowering date (WAF) of olive; whilst the highest level of total 4-monomethylsterols (234 mg/100 g oil) was attained at 24th WAF of fruit. The percentage of these two classes represented 20–33% of total phytosterols during olive maturity. 24-Methylene cycloartenol (12–207 mg/100 g oil) and cycloartenol (27–198 mg/100 g oil) were the predominant triterpene alcohols during the ripening of Picholine olive; whereas citrostadienol (30–161 mg/100 g oil) and cycloeucalenol (11–74 mg/100 g oil) were the main 4-monomethylsterol compounds followed by obtusifoliol and gramisterol. β-Amyrin, δ-amyrin and traroxerol were less present in Picholine olive and they accounted for 14% of total triterpene alcohols at complete maturity of fruit. The level of these methylsterols was overwhelmed by the amount of 4-desmethylsterols at each stage of Picholine olive maturity.     

Gas chromatography screening of bioactive phytosterols from mono-cultivar olive oils

Phytosterols (PS) from nine samples of olive oil from Olea europaea L., the Carolea, Cassanese and Coratina mono-cultivars, have been analyzed by gas chromatography. Coratina virgin olive oil (VOO) from the month of November showed highest contents of β-sitosterol (5491 mg kg⁻¹) and Δ⁵ avenasterol (1767 mg kg⁻¹). Olive pomace oil had the lowest total content of all PS, when compared to VOO. These results suggests that, PS can be an important regulatory factor for the functional quality of olive oil along the agro-industry chain from the orchard to nutraceutical.     

Direct determination of phenolic compounds and phospholipids in virgin olive oil by micellar liquid chromatography

A simple HPLC method is reported for fast separation and determination of phenolic compounds (tyrosol, caffeic acid, p-coumaric acid and oleuropeina) and phospholipids (phosphatidylethanolamine and phosphatidylcholine) in virgin olive oil samples. The samples were diluted with 2-propanol and injected into the column directly without previous extraction. Samples with an olive oil content of up to 65% were injected without any problems. The analytes were separated on a C-18 column by a micellar mobile phase containing 0.07 M SDS and 2.5% 2-propanol at pH 3, and were detected at 210 nm. Linear calibration curves [r² > 0.997] were obtained with detection limits ranging from 0.052 to 0.16 μg/g and 1 to 8.6% repeatability for the phenolic compounds. Several virgin olive oil samples were analysed and the recovery values were around 110%.     

Sterols,fatty alcohol and triterpenic alcohol changes during ripe table olive processing

The aim of the work was to study the effects of processing on the unsaponifiable matter, sterols and fatty and triterpenic alcohol in ripe olive fat (Manzanilla and Hojiblanca cultivars) and to disclose the most influential factors using GLM, PCA and DA. There were significant effects of cultivars or ps on unsaponifiable matter, β-sitosterol, Δ⁵-avenasterol, total sterols, 1-docosanol, 1-tetracosanol (ps), erythrodiol and percentage erythrodiol + uvaol. The values of most of these parameters were within the limits established by the EU Directives for olive and pomace oils but classification of the respective oils was not conclusive. Predictive discriminant analysis using these variables permitted 100% success in the classification according to cultivars and ps (68% in the case of cross validation). Results revealed that some influential steps should be re-designed, particularly the storage phase, in order to minimise changes in the studied fat components during ripe olive processing.     

Refining of high oleic safflower oil: Effect on the sterols and tocopherols content

The purpose of this work was to study the influence of the industrial process steps on free fatty acids, peroxide value (PV), p-anisidine value (PAV), trans fatty acids, tocopherols and sterols (free, esterified and total) in high oleic safflower oil. Degumming, bleaching and deodorization steps removed 91.4% of free fatty acids, 96.31% of oxidation primary products (PV), and 54.57% of oxidation secondary products (PAV), from crude high oleic safflower oil. Degumming neither affected the content of sterified sterols nor its proportion with respect to the crude oil. A significant increment (p<0.05) in the content of free sterols was observed during degumming and bleaching due to the acid-catalyzed hydrolysis of steryl esters. A significant reduction (p<0.05) in the content of total sterols during bleaching was observed, which is attributed to a reduction in the sterified sterol fraction. During deodorization, free sterols were distilled from oil, with a gradual reduction in the total sterol content as a function of the deodorization temperature. α- and γ-tocopherols represented 93.3% of the total tocopherols in high oleic safflower crude oil. The refining process removed 28.5% of the tocopherols. Deodorization was the main step which increased the level of trans fatty acids as an effect of temperature and heating time.     

Direct analysis of glucuronidated metabolites of main olive oil phenols in human urine after dietary consumption of virgin olive oil

In the present study we report on a UPLC-MRM validated method for the simultaneous direct analysis of main glucuronidated metabolites of olive oil phenols: tyrosol, hydroxytyrosol and its O-methyl metabolite homovanillyl alcohol in human urine after dietary olive oil ingestion. The developed method was linear within the concentration range 20–2000 ng/mL with adequate recovery of analytes (>86%). Intra- and inter-day precision and accuracy were according to standard requirements for method validation criteria. Using the developed method, urinary concentrations and excretion rates of glucuronides of olive oil phenols were successfully estimated in an intervention study with 11 healthy volunteers supplemented with a dietary dose of virgin olive oil (VOO) (50 mL). Therefore, about 13% of the consumed olive oil polyphenols were recovered in 24-h urine, where 75% of them were in the form of glucuronides (3′- and 4′-O-hydroxytyrosol glucuronides, 4′-O-glucuronides of tyrosol and homovanillyl alcohol) and 25% as free compounds.     

Determination of copper with 5,5-dimethylcyclohexane-1,2,3-trione 1,2-dioxime 3-thiosemicarbazone in olive oils by adsorptive stripping square wave voltammetry

In the present paper a method for the determination of Cu in olive oil samples by adsorptive stripping square wave voltammetry (Ad-SSWV) is presented. It has been proven that Cu reacts with 5,5-dimethylcyclohexane-1,2,3-trione 1,2-dioxime 3-thiosemicarbazone, DCDT, in strongly acid media giving rise to a complex. In Ad-SSWV the complex Cu–DCDT experiments an adsorptive reductive process which promotes the appearance of a peak at −0.570 V. The extraction process of Cu from olive oil is carried out with hot concentrated HCl. Calibration graph has been constructed from 0 to 35 ng mL⁻¹ and the detection limit was 0.49 ng mL⁻¹. The method has been applied to commercial olive oils samples and the amounts of Cu found are very similar to those obtained when samples are analysed by AAS.     

High throughput flow injection bioluminometric method for olive oil antioxidant capacity

This paper describes a rapid flow injection automated method for the determination of olive oil total antioxidant capacity. The chemistry involved is the horseradish peroxidase (HRP) catalysed oxidation of luminol by hydrogen peroxide. Oxidation results in light emission (bioluminescence) that is enhanced using p-iodophenol sensitizer. Olive oil (0.7 mL) is extracted with two 0.7 mL aliquots of 80–20% (v/v) methanol–water solvent. A 17 μL aliquot of the extract containing hydrophilic antioxidants is injected in a phosphate buffer channel that subsequently merges with a luminol–HRP–p-iodophenol reagent stream. Bioluminescence resulting after merging the mixture with a hydrogen peroxide stream is suppressed upon increasing antioxidants’ concentration resulting in negative peaks due to hydrogen peroxide consumption by antioxidants. The method has been optimized on (a) number of manifold channels, (b) flow rates, (c) coil length and (d) HRP, hydrogen peroxide and p-iodophenol concentrations. Detection limit is calculated at 1.5 × 10⁻⁷ M gallic acid, linear range is between 1.0 × 10⁻⁶ and 1 × 10⁻⁴ M and precision is better than 2.8% RSD (n = 4). The fully automated method is achieving a rate of sampling equal 180 probes per hour. The proposed method is applied for the assessment of 50 extra-virgin olive oil samples of different Greek cultivars and regions.     

Alcoholysis of salicornia oil using free and covalently bound lipase onto chitosan beads

Porcine pancreatic lipase was immobilized on chitosan by covalent binding and retention of its activity was examined. The activities of free and immobilized lipase were determined using olive oil as substrate. The free and immobilized enzymes showed pH 9 as optimum and retained 50% of activity after five cycles. When the substrate concentration was kept constant and enzyme concentration was varied, the K_m and V_max were observed to be 4.0 × 10⁻⁷ and 0.32, and 3.32 × 10⁻⁷ and 0.32, respectively, for free and covalently bound enzyme. This indicates that there is no possibility of conformational change during immobilization. Immobilized enzyme showed improved thermal and storage stability. Alcoholysis of salicornia oil, mediated by free and immobilized lipase, was carried out at 25 °C using methanol in hexane and acetone. Free and immobilized enzyme in hexane produced, respectively, 45% and 55% of fatty acid methyl ester after 12 h.     

Stability of avocado oil during heating: Comparative study to olive oil

The stability of the saponifiable and unsaponifiable fractions of avocado oil, under a drastic heating treatment, was studied and compared to that of olive oil. Avocado and olive oil were characterised and compared at time 0 h and after different times of heating process (180 °C). PUFA/SFA (0.61 at t = 0) and ω-6/ω-3 (14.05 at t = 0) were higher in avocado oil than in olive oil during the whole experiment. Avocado oil was richer than olive oil in total phytosterols at time 0 h (339.64; 228.27 mg/100 g) and at 9 h (270.44; 210.30 mg/100 g) of heating. TBARs was higher in olive oil after 3 h, reaching the maximum values in both oils at 6 h of heating treatment. Vitamin E was higher in olive oil (35.52 vs. 24.5 mg/100 g) and it disappeared earlier in avocado oil (at 4 vs. 5 h). The stability of avocado oil was similar to that of olive oil.     

Phenolic acid analysis and antioxidant activity assessment of oil palm (E. guineensis) fruit extracts

Phenolic compounds in oil palm fruit (E. guineensis) were extracted in soluble free (SFP), insoluble-bound (ISBP) and esterified (EFP) forms. The total phenolic content (TPC) of the oil palm fruit extracts was determined using the Folin–Ciocalteu method and found to range from 5.03 to 9.04 g/L per g of dried weight (DW). The antioxidant activities of oil palm phenolic extracts were analysed using free radical scavenging assays and results showed that oil palm phenolic extracts contained antioxidant activities in the order of ISBP > EFP > SFP. Eight different phenolic acids were identified and quantified using a simple reversed-phase high performance liquid chromatography (HPLC) with a diode array detector (DAD) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Ferulic, p-hydroxybenzoic and p-coumaric acid were the dominant phenolic acids found in oil palm fruit extracts and ranged from 55 to 376 μg/g of DW.     

Sterols and triterpene diols in olive oil as indicators of variety and degree of ripening

Sterols and triterpene diols in olive oil as indicators of variety and degree of ripening derived from three olive varieties and produced at three different harvesting periods were studied. In order to test the stability of the proposed indicators, oils obtained were stored for 12 months at three different temperatures. Thirty-six samples in total were subjected to GC analysis and results were processed by multivariate chemometric methods (MANOVA, PCA, and SLDA). Campesterol, β-sitosterol, Δ⁷-campesterol/Δ^5,24-stigmastadienol, clerosterol, uvaol, and campestanol/Δ⁷-avenasterol were established as the indicators of variety of fresh oils, while when stored oils were included in the model, the final three compounds were substituted by 24-methylene-cholesterol/stigmasterol. The most important variables for differentiating fresh oils according to degree of ripening were Δ⁷-campesterol/β-sitosterol, uvaol/stigmasterol, clerosterol/Δ⁵-avenasterol and sitostanol/uvaol, while stored oils were differentiated by campestanol/stigmasterol, erythrodiol, stigmasterol/Δ⁷-campesterol, Δ⁵-avenasterol, 24-methylene-cholesterol/β-sitosterol and 24-methylene-cholesterol. Results demonstrated that sterols and triterpene diols can be used as indicators of variety and degree of ripening among virgin olive oils.     

The effect of sterols on the oxidation of edible oils

The effect of sterols on the oxidation of a triglyceride mixture, similar in composition to olive oil, has been studied at 180°C. Δ⁵-Avenasterol and fucosterol are effective as antioxidants, whilst other sterols, including cholesterol and stigmasterol, are ineffective. The antioxidant effect of Δ⁵-avenasterol increases with concentration in the range 0·01% to 0·1%.An hypothesis is presented to explain the effectiveness of the sterols as antioxidants. It is concluded that lipid free radicals react rapidly with sterols at unhindered allylic carbon atoms. Isomerisation leads to a relatively stable allylic tertiary free radical, which is slow to react further, and this interrupts the autoxidation chain.     

Study on the Chemical Nature of Sterols contained in Bulgarian Sunflower Oil

The free sterols, the sterol esters and the sterol glycosides of the crude sunflower oil as well as those of the technical lecithin, the pitch and the deodorizer distillate of the latter oil were isolated by preparative TLC. The nature of sterols contained in the isolated sterol derivatives was elucidated by GLC and combined GLC-MS. Major sterols of all examined sterol fractions are sitosterol, campesterol and stigmasterol, the amount of sitosterol being prevalent. Unknown sterol with a molecular weight of 4²⁸ is present in sterols of the crude oil and the deodorizer distillate. Sterols of the deodorizer distillate contain an unknown sterol with a molecular weight of 430. Stigmasterol is present in the sterol fraction of the deodorizer distillate in high amounts. It was established that Δ⁷-sterols of the crude oil occur only in esterified form. The hydrolysis of the sterol derivatives in acid medium leads to dehydration products known as steroid dienes and disteroid ethers. Hydrolysis without dehydration was achieved by enzyme preparations. Some sterols of the crude oil were esterified with the same higher fatty acids contained in the glycerides of the sunflower oil.     

Effect of chemical and physical pretreatments on the convective drying of cape gooseberry fruits (Physalis peruviana)

Drying of cape gooseberry fruits is a slow process because of the low permeability to moisture of the fruit’s waxy skin. In this work, the effect of chemical pretreatments (sunflower oil/K₂CO₃ or olive oil/K₂CO₃ at 28 °C, and NaOH/olive oil at 96 °C) and physical pretreatments (blanching) to break down the waxy surface and accelerate moisture diffusion during drying, was assessed. Drying was carried out at 60 °C and 2 m/s air velocity for 10 h. The lowest moisture content (0.27 kg water/kg db), the highest vitamin C content (0.36 mg/g), and the greatest rehydration capacity (1.89) were obtained in fruits pretreated with olive oil (9.48%) and K₂CO₃ (4.74%). However, the greatest changes in color (ΔE^* = 15.05) and chroma (ΔC^* = 9.03) were also associated to fruits pretreated with olive oil and K₂CO₃. The effective diffusivity of water during drying was 7.37 × 10⁻¹¹ m²/s in pretreated samples compared with 6.61×10⁻¹¹ m²/s for untreated samples.     

Analysis of free and esterified sterols in edible oils by online reversed phase liquid chromatography-gas chromatography (RPLC-GC) using the through oven transfer adsorption desorption (TOTAD) interface

An online reversed phase liquid chromatography-gas chromatography (RPLC-GC) method is proposed to quantify free, total and esterified sterols of edible oils. To determine free sterols the diluted oils are injected into the liquid chromatograph, where free sterols are separated from triglycerides and the sterol fraction is automatically transferred to the gas chromatograph to be analysed. To determine total sterols the samples were saponified with potassium hydroxide in ethanolic solution and the unsaponifiable fraction was extracted with diethyl ether. The extract was then analysed by RPLC-GC, avoiding the laborious thin layer chromatography step used in the Official European Union (EU) Method. The relative standard deviations (RSDs) from the absolute peak area varied from 7.6% to 15.8%. Limits of detection (LODs) were less than 8.5 mg/kg. No variability in retention time was observed. The method was applied to the determination of total sterols in edible oil samples and the results were compared with those obtained with the Official EU Method. Good agreement was found between both methods, except in the case of campesterol.