Multivariate analysis of NMR fingerprint of the unsaponifiable fraction of virgin olive oils for authentication purposes

A new approach to the geographical characterisation of virgin olive oils (VOOs) based on the ¹H NMR fingerprint of the unsaponifiable matter is presented. The ¹H NMR spectra of the unsaponifiable fraction of virgin olive oils from Spain, Italy, Greece, Tunisia, Turkey, and Syria were analysed by several pattern recognition techniques (LDA, PLS-DA, SIMCA, and CART). PLS-DA (PLS-1 approach) obtained the best classification results for all classes. Moreover, ¹H NMR spectra of the bulk oil, and its corresponding unsaponifiable fraction, as well as the subfractions of the unsaponifiable fraction (alcohol, sterol, hydrocarbon, and tocopherol fractions) were studied in the search for the markers that multivariate techniques revealed to be related to the geographical origin of olive oils. Additionally, a preliminary study regarding ¹H NMR data of the bulk oil and the corresponding unsaponifiable fraction of VOOs suggested that these spectral data contained complementary information for the geographical characterisation of VOOs.     

UNSAPONIFIABLE MATTER AND OXIDATIVE STABILITY OF COMMERCIALLY PRODUCED INDIAN RICE BRAN OILS

Chemically and physically refined rice bran oils (RBO) were investigated for their content and composition of unsaponifiable matter and oxidative stability. The chemically and physically refined rice bran oils contained 2.6 and 4.5% unsaponifiable matter, respectively. The content and composition of unsaponifiable matter for chemically and physically refined RBO were phytosterols, 0.96%and 1.40%; 4-methyl sterols, 0.51%and 1.12%; triterpene alcohols, 0.52% and 0.86%; and nonpolar components, 0.61% and 1.13%, respectively. The corresponding oxidative stability for the two oils were 2.80 h and 4.37 h, respectively. The content of unsaponifiable matter was influenced by residual amounts of wax and oryzanol in the refined oil as shown by the model experiments. Thus physical refining of rice bran oil requires a thorough dewaxing step to obtain a refined oil without haziness and with a lower amount of unsaponifiable matter.     

Waxy fraction containing long-chain aliphatic aldehydes in virgin olive oils

Long-chain aliphatic aldehydes are natural minor components occurring in the cuticle of numerous plant species and also evidenced in virgin olive oils. The fraction containing these compounds can be isolated from the oil samples by using a solid-phase extraction silica-gel cartridge and then directly analysed by GC on a 5% diphenyl-95% dimethylsiloxane capillary column, using an on column-injection system. The proposed methodology showed that extra virgin olive oils contain long-chain aliphatic aldehydes, with even carbon-atom numbers from C₂₂ to C₃₀. Quantitative results, using the synthesised aldehyde C₂₁ as internal standard, give concentrations of total long-chain aliphatic aldehydes in a variable range below 116 mg kg⁻¹, being hexacosanal (C₂₆-al) the most abundant aldehyde. The different experimental conditions utilised during olive oil extraction processes influence the total aldehydes concentration. Besides contribution to the knowledge of the minor-component composition present in olive oil, their interest and relationship with wax esters, aliphatic alcohols and n-alkanes are discussed.     

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.     

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.     

High performance size-exclusion chromatography analysis of polar compounds applied to refined, mild deodorized, extra virgin olive oils and their blends: An approach to their differentiation

An investigation was carried out to evaluate the use of High Performance Size-Exclusion Chromatography (HPSEC) of polar compounds of refined, mild deodorized, extra virgin olive oils as well as of their blends, in attempting to reveal significant differences in the amounts of the substance classes constituting polar compounds among these oils. Two sets of blends were prepared by mixing an extra virgin olive oil with both refined and mild deodorized olive oils in increasing amounts. The obtained data highlighted that the triacylglycerol oligopolymers were absent or present in traces in the extra virgin olive oil, while their mean amount was equal to 0.04 g/100 g and 0.72 g/100 g in mild deodorized and refined olive oils, respectively. Oxidized triacylglycerols and diacylglycerols were more abundant in mild deodorized oil and refined oil than in extra virgin olive oil. The Factorial Discriminant Analysis of the data showed that the HPSEC analysis could reveal the presence of refined/mild deodorized oils in extra virgin olive oils. In particular, the classification functions obtained allowed designation of mixtures containing at least 30 g/100 g of mild deodorized oil and all those containing refined olive oil as deodorized oil, therefore as oils subjected to at least a mild refining treatment.     

Are virgin olive oils obtained below 27 °C better than those produced at higher temperatures?

Within the European Union, indications of ‘first cold pressing’ and ‘cold extraction’ can only be used for virgin olive oil (VOO) obtained below 27 °C from mechanical processing. Three different malaxing temperatures (25, 35 and 45 °C) are here evaluated for the quality of the VOO obtained in a continuous industrial plant. The oils were stored at room temperature in the dark for 12 months. Initially, oil obtained from a blend of Frantoio/Leccino cultivars (F/L) had higher acidity and peroxide levels and lower phenolic content than a Coratina cultivar (Cor). The oxidative stability of the oils positively correlated with malaxation temperature (F/L, R² = 0.818; Cor, R² = 0.987) as the phenolic content was directly proportional to the temperature (F/L, R² = 0.887; Cor, R² = 0.992). Only oils obtained at 45 °C were rejected because of ‘heated or burnt’ off-flavour. Decarboxymethylation of secoiridoids and further hydrolysis of phenolic esters occurred during storage. The oxidation products of derivatives of tyrosol and hydroxytyrosol were detected after nine months in both the F/L and Cor samples. Thus, VOO obtained at a processing temperature lower than 27 °C does not show higher chemical and sensory qualities than VOO obtained at 35 °C.     

Multivariate characterization of table olives according to their mineral nutrient composition

Different commercial presentations of table olives were characterized by their mineral compositions. Cu, Fe, Mn, Zn, Ca, Mg, Na, K, and P were determined. The processing of table olives affects the mineral content of commercial presentations and significant differences (p < 0.05) were found among green (Spanish style), directly brined, and ripe olives. A predictive discriminate analysis showed that the most discriminating elements were Fe, K, Na, Mn, Cu, and P (among styles) and with Ca (among cultivars). A good classification and cross-validation was observed in the case of elaboration styles but discrimination among cultivars was less conclusive. A further analysis of the confusion matrix, according to cultivars, showed that the lower classification efficiency, in this case, was mainly due to misclassification of samples from Manzanilla and Gordal cultivars. The analysis of the confusion matrix can be useful when the assessment of its results is not obvious.     

Seed lipids of Sesamum indicum and related wild species in Sudan. The sterols

Oil extracted from seeds of different varieties of the cultivated Sesamum indicum L and three related wild species, viz S alatum Thonn, S radiatum Schum & Thonn, and S angustifolium (Oliv) Engl, were analysed for their total unsaponifiables and for the contents and composition of the three sterol fractions (desmethyl, monomethyl and dimethyl sterols). The sterols were analysed after saponification by preparative TLC, capillary GC and GC-MS of their TMS ethers. Oils from the wild species contained more unsaponifiable material (2.3-2.4%) compared with the cultivated species (1.1-1.3%). Considerable differences were observed in the total sterol contents and the relative proportions of the three sterol fractions in the oils from the four species studied. Sitosterol, campesterol, stigmasterol and Δ⁵-avenasterol were the major desmethyl sterols in all four species. The monomethyl sterol fraction consisted primarily of obtusifoliol, gramisterol, cycloeucalenol and citrostadienol. Cycloartenol and 24-methylene cycloartanol were the predominant dimethyl sterols. Variations in the composition of the three sterol fractions were observed between S indicum oils traditionally pressed by the camel-driven expellers and laboratory extracted oils from the same seeds.     

α-Tocopherol and fatty acids contents of some Tunisian table olives (Olea europea L.): Changes in their composition during ripening and processing

An experimental investigation was carried out on Tunisian olive-fruits of Meski, Sayali and Picholine cultivars. α-Tocopherol and fatty acids (FA) contents were analyzed, during both ripening and processing, according to the Spanish style. The relationship between oil, unsaponifiable and α-tocopherol contents was determined only during ripening. A genetic effect on FA composition was observed throughout the sampling periods. The highest oleic acid content was found in Sayali cultivar at green stage (78.5% of total FA). α-Tocopherol was positively correlated with unsaturated FA content (R = 0.71, p < 0.05), and oil amount (R = 0.984; R = 0.976; R = 0.952, p < 0.05 for Picholine, Sayali and Meski, respectively), but it was not correlated with unsaponifiable matter. In processed olive-fruits, the results showed primarily, that processing according to the Spanish style is not restricted to green olive-fruits but can be successfully used in cherry olives with guaranteed quality and nutritional value of processed product (Meski and Picholine) related to FA content. Secondly, both α-tocopherol and FA amounts decreased during processing for all cultivars. This decrease was cultivar dependent. It was more pronounced in the black fruit than in the green one for the same cultivar. During fermentation, pH variation showed the same profile in all cultivars. Final pH values at the end of fermentation depend on the concentration of free FA (acidity) in the brine.     

Changes in the quality of fish oils due to storage temperature and time

The effects of storage temperature on oil quality of various fishes were examined. Fish oils extracted from horse mackerel, shad, garfish and golden mullet were used in oil analyses. Crude fish oils obtained by a solvent extraction method were stored at +4 °C and −18 °C. Chemical quality of the oils was evaluated with various parameters, including iodine, ester, acid, saponification, peroxide, and thio-barbituric acid values and unsaponifiable matter at various time intervals for 150 days of storage. All quality parameters, except iodine and ester values, increased during storage at both of temperatures. Sample oils stored at +4 °C preserved acceptable characteristics for 90 days. Acceptability tolerance was found to be 120 days for shad oil and 150 days for golden mullet, garfish and horse mackerel oils stored at −18 °C. The highest oxidative deterioration was observed in shad oils. Among all samples, garfish oil showed the greatest stability against oxidation.     

Minor compounds in the phenolic fraction of virgin olive oils

The phenolic fraction of 34 virgin olive oils was analysed by gas chromatography–mass spectrometry (GC–MS) with the aim to identify new compounds at low level. Twenty-seven compounds previously described in olive oils were identified; several new minor compounds with phenolic structure were detected in the samples: amongst them, 4-hydroxyphenylacetaldehyde, trans-isoeugenol (trans-2-methoxy-4-(1-propenyl)-phenol), 1,4-dihydroxy-2,6-dimethoxybenzene, 3,4-dihydroxybenzyl alcohol and 3,4-dihydroxyphenylacetic acid were identified by their mass spectra and confirmed using standards. In 34 virgin olive oils (cv. Nocellara del Belice) the mean concentrations for these five substances were always below 0.2 mg kg⁻¹ and only in two samples the level of 3,4-dihydroxyphenylacetic acid reached 1.47 and 1.97 mg kg⁻¹, respectively. These compounds could be used to characterise olive oils; low concentrations of 3,4-dihydroxyphenylacetic acid may show the initial autoxidation processes of olive oils.     

Influence of olive paste preparation conditions on virgin olive oil triterpenic compounds at laboratory-scale

The influence of olive paste preparation conditions on the triterpenic content of virgin olive oils from Arbequina and Picual cultivars was investigated. For this purpose, three sieve diameters of the hammer mill (4, 5, and 6 mm), two malaxation temperatures (20 and 30 °C), and two malaxation times (20 and 40 min) were tested. Results obtained showed that for Arbequina oils, a finer crushing level resulted in higher maslinic acid and erythrodiol content. Increasing malaxing temperature and time lead to a rise in both oleanolic and maslinic acid concentration, whereas erythrodiol content increased only for the longer malaxation time. For Picual oils, higher concentrations of oleanolic acid, maslinic acid, and uvaol were obtained by prolonging the paste malaxation time. A finer crushing level resulted also in an increase of maslinic acid content. These findings suggest that virgin olive oil triterpenic composition can be improved by regulating olive paste preparation conditions.     

A facile NMR method for the quantification of total,free and esterified sterols in virgin olive oil

This study describes a simple analytical technique based on NMR spectroscopy for the determination of free and esterified sterols in olive oil. Free sterols were determined by ³¹P NMR upon derivatization of free sterols with a phosphorous reagent, whereas total (free + esterified) sterols were obtained from ¹H NMR. The present NMR method was validated by performing a comparison study with the reference method of gas chromatography. The agreement between NMR and GC methods was evaluated by using the Bland and Altman statistical analysis. The distribution of the data points in the bias plot showed that ∼96% of the measurements of total, free and esterified sterols in 24 extra virgin olive oil samples from various regions of Greece were within the limits of agreement of the two methods.     

Effect of storage on refined and husk olive oils composition: Stabilization by addition of natural antioxidants from Chemlali olive leaves

Two samples of refined olive and husk oils have been analysed in order to evaluate the influence of storage time on their quality. The following parameters were determined: peroxide values, absorption coefficients K270 and K232, Rancimat induction time, sterols and fatty acid contents. Six months storage at 50 °C in the dark revealed a loss in oil stability. This finding was reflected by the greater increase in peroxide value and a decrease of Rancimat induction time and sterol content. The enrichment of refined olive and husk oils with olive leaves and its hydrolysate extract resulted in an appreciable resistance to oxidative deterioration due to its phenolic antioxidants content. Oleuropein and hydroxytyrosol were the major compounds in Chemlali olive leaves extract and hydrolysate solution, respectively. The antiradical activity of leaves extract as well as its hydrolysate solution was evaluated and compared to that of the BHT. The antioxidant activity of the enriched refined olive and husk oils with leaves and hydrolysate extracts at 400 ppm showed that the latter had the highest protective effect against oil oxidation. Oils with added hydrolysate extract had the lower peroxide value and the higher stability measured with a Rancimat method. After six months of storage the induction time increased from 23.3 to 83.5 h for refined olive oil and from 16.6 to 49 h for husk oil. Furthermore, during oil storage, there was no significant variation in fatty acid composition. However, the total sterol concentration of the oils treated with hydrolysate extract increased. The results suggested that hydrolysate and leaves extracts are excellent antioxidants and can serve as substitutes for synthetic antioxidants.     

Discrimination of olive oil adulterated with vegetable oils using dielectric spectroscopy

The study focused on application of dielectric spectroscopy to identify the adulteration of olive oil. The dielectric properties of binary mixture of oils were investigated in the frequency range of 101 Hz–1 MHz. A partial least squares (PLS) model was developed and used to verify the concentrations of the adulterant. Furthermore, the principal component analysis (PCA) was used to classify olive oil sample as distinct from other adulterants based on their dielectric spectra. The results showed that the dielectric spectra of binary mixture of olive oil spiked with other oils increased with increasing concentration of soy, corn, canola, sesame, and perilla oils from 0% to 100% (w/w) over the measured frequency range. PLS calibration model showed a good prediction capability for the concentrations of the adulterant. For olive oil adulterated with soy oil, the results showed that the RMS was 0.053, sd(RMS), 0.017 and Q² value was 0.967. PCA classification plots for all oil samples showed clear performance in the differentiation for the different concentrations of the adulterant. Each of the oil samples could be easily grouped in different clusters using dielectric spectra. From the results obtained in this research, dielectric spectroscopy could be used to discriminate the olive oil adulterated with the different types of the oils at levels of adulteration below 5%.     

樟树籽油抗氧化能力及物质基础研究

植物油中天然抗氧化成分对油脂的加工、货架期及营养品质具有重要影响。为研究樟树籽油抗氧化活性及物质基础,本文分别对其脂肪酸组成、不皂化物含量、总酚含量和DPPH自由基清除率进行测定,分析总多酚及不皂化物对樟树籽油抗氧化活性的贡献率,明确樟树籽油抗氧化活性物质基础。结果表明,樟树籽油中主要为中链脂肪酸癸酸和月桂酸,二者含量占总脂肪酸的94.80%,不皂化物含量为0.55±0.01% ,总多酚含量为33.07 ±0.63 mg.kg_^-1。樟树籽油对DPPH自由基清除率的IC₅₀值为0.13 g.mL_^-1。相同质量浓度的樟树籽油、不皂化物和总多酚对DPPH自由基清除率分别为98.92±1.58%、38.49±0.66%、49.60±3.78%。因此,不皂化物及总酚对樟树籽油抗氧化贡献率分别为38.91%、50.14%。研究结果表明樟树籽油具有较强的抗氧化能力,其抗氧化活性物质主要为多酚类成分和不皂化物,二者对樟树籽油抗氧化能力的总贡献率高达89.05%。本研究初步明确了樟树籽油的抗氧化活性成分,可为樟树籽油的开发利用奠定基础。     

Fatty Acids,Sterols, Polyphenols,and Chlorophylls of Olive Oils Obtained from Tunisian Wild Olive Trees (Olea europaea L. Var. Sylvestris)

Olive (Olea europaea L.) includes cultivated olive trees (var. europaea) and wild olive trees or oleaster (var. sylvestris) as two botanical varieties. These olive varieties were widely spread in the Mediterranean Region. The aim of this study was to determine fatty acid compositions, sterols, polyphenols, and chlorophylls of oils obtained from 12 wild olive trees from Northern Tunisia. Two dominated oil cultivars in Tunisia (Chétoui and Chemlali) were also used to compare results. The fatty acid methyl ester and the sterol compositions were analyzed using gas-liquid chromatography and thin layer chromatography methods, respectively. The polyphenols and chlorophylls were determined using the calorimetrical method. Results indicated that oils extracted from wild olives displayed good balanced fatty acid compositions, sterols, polyphenols, and chlorophylls. Qualitatively, for wild and cultivated olive oils, the oil has an identical composition, whereas the quantitative variation showed that some wild trees seem to be interesting oil sources as two Tunisian dominated cultivars. The highest oleic acid and polyphenol contents were 71.55% and 537.6 mg/kg of oil found in wild olives (OIch2, OIch1). The β-sitosterol was the major sterolic fraction and ranged from 84.72 to 75.70% according to the wild olives. Consequently, wild olives would be a new future edible olive oil source, as well as commonly cultivated ones.     

The influence of the malaxation temperature on the activity of polyphenoloxidase and peroxidase and on the phenolic composition of virgin olive oil

The effect of the malaxation temperature under sealed conditions on the qualitative and quantitative composition of the phenolic compounds in virgin olive oils produced from four Italian cultivars was assessed for two atmospheric conditions. In both cases, the results show a positive relationship between temperature and the concentration of the derivatives of the secoiridoid aglycones; the effect of the temperature on the oxidoreductases that promote oxidation (polyphenoloxidase and peroxidase) was investigated to determine their optimal temperatures and thermal stability. While olive peroxidase (POD) showed the highest activity at 37 °C and high stability in the temperature range tested, polyphenoloxidase (PPO) exhibited the optimum activity at approximately 50 °C, but showed low stability at 40 °C, with a large variation in stability according to the olive cultivar. These results may contribute to an understanding of the increase in the phenol concentration found in virgin olive oils obtained following higher temperatures of malaxation.     

Pigments profile in monovarietal virgin olive oils from various Italian olive varieties

This paper presents the investigation of the chlorophyll and carotenoid pigments composition in monovarietal virgin olive oils produced from the five main olive varieties (Minuta, Ottobratica, Calabrese, Ogliarola, Baddarica) cultivated in Sicily (southern Italy), from four main olive varieties (DolceAgogia, Moraiolo, Leccino, Frantoio) cultivated in Umbria (central Italy), and from three main olive varieties (Leccino, Oliva Nera di Collecorto, Noccioluta) cultivated in Molise (central Italy). Reversed-phase high performance liquid chromatography using a C-30 column with photodiode array detection was used for pigments analyses. In all, 19 compounds were identified and quantified in 60 olive oils samples. The qualitative pigments pattern was similar among the varieties investigated, whereas quantitative differences were found among the different cultivars; among the varieties investigated in this work, the oils from Umbria showed the highest pigment content (34.19 ppm in average), followed by the oils from Molise (18.61 ppm in average) and the oils from Sicily which showed the lowest pigment contents (13.38 ppm in average). In general, pheophytin a was the major component (range 0.49–19.42 ppm), followed by β-carotene (range 1.27–9.30 ppm) and lutein (range 0.44–5.12 ppm). Those differences may be due to genetic factors and/or geographical differences. Moreover, auroxanthin was detected for the first time in olive oils and was detected only in olive oils from Umbria and Molise regions. The ratio between the two isochromic pigment fractions, namely the ratio between the chlorophyll and carotenoid fractions showed an average value close to unity. The lutein/β-carotene ratio was less than one in the majority of the cases. These parameters, along with other analytical parameters, could be used as indicators of typicality in olive oils. The presence of a specific pigment profile in olive oils could infact be used to guarantee the genuineness of the product, since the quality control of food requires a precise knowledge of the pigments composition of the original products.