Combining different biomarkers to distinguish Chemlali virgin olive oils from different geographical areas of Tunisia.

BACKGROUND: Despite their high potential, Tunisian virgin olive oils (VOOs) are mainly exported in bulk or blended with VOOs of other origin, hindering their international market placement. To face this situation, their valorization is needed by highlighting their unique features and by developing tools to guarantee their geographical authenticity. Compositional features of Chemlali VOOs produced in three Tunisian regions were assessed to identify suitable authenticity markers. RESULTS: Quality indices ensured the quality of the VOOs studied. Volatile compounds, total phenols, fatty acid (FA) and chlorophylls are significantly influenced by the region of origin, which was justified by the differences found in soil and climatic conditions of the three geographical regions. To explore the capabilities of these markers for the geographical authentication of Tunisian Chemlali VOOs, classification models based on partial least squares-discriminant analysis (PLS-DA) were developed by grouping the minimum number of variables allowing the highest discrimination power, minimizing in this way the analytical procedure. The PLS-DA authentication model based on combining volatile compounds with FA or with total phenols achieved a correct classification of 95.7% of the VOOs according to their origin, as assessed by 10%-out cross-validation. Sidi Bouzid Chemlali VOOs achieved 100% of correct classification, while the misclassification between Sfax and Enfidha ones did not exceed 10%. CONCLUSIONS: These results allowed to establish the most promising and affordable combination of markers for the geographical authentication of Tunisian Chemlali VOOs from distinct production regions and provide the basis to further develop authentication models based on wider datasets. © 2023 Society of Chemical Industry.

Revealing adulterated olive oils by triacylglycerol screening methods: Beyond the official method.

Official control methods to detect olive oil (OO) adulteration fail to provide satisfactory consumer protection. Thus, faster and more sensitive screening tools are needed to increase their effectiveness. Here, the official method for adulterant detection in OO was compared with three untargeted screening methods based on triacylglycerol analysis using high-throughput (FIA-HESI-HRMS; HT-GC-MS; HPLC-RID) and pattern recognition techniques (PLS-DA). They were assayed on a set of genuine and adulterated samples with a high natural variability (n = 143). The sensitivity of the official method was 1 for high linoleic (HL) blends at ≥2 % but only 0.39 for high oleic (HO) blends at ≥5 %, while specificity was 0.96. The sensitivity of the screening methods in external validation was 0.90-0.99 for the detection of HL and 0.82-0.88 for HO blends. Among them, HT-GC-MS offered the highest sensitivity (0.94) and specificity (0.76), proving to be the most suitable screening tool for OO authentication.

Using fluorescence excitation-emission matrices to predict bitterness and pungency of virgin olive oil: A feasibility study.

Unlike other food products, virgin olive oil must undergo an organoleptic assessment that is currently based on a trained human panel, which presents drawbacks that might affect the efficiency and robustness. Therefore, disposing of instrumental methods that could serve as screening tools to support sensory panels is of paramount importance. The present work aimed to explore excitation-emission fluorescence spectroscopy (EEFS) to predict bitterness and pungency, since both attributes are related with fluorophore compounds, such as polar phenols. Bitterness and pungency intensities of 250 samples were provided by an official sensory panel and used to build and compare partial least squares regressions (PLSR) with the excitation-emission matrix. Both PARAFAC scores and two-way unfolded data led to successful PLSR. The most relevant PARAFAC scores agreed with virgin olive oil phenolic spectra, evidencing that EEFS would be the fit-for-purpose screening tool to support the sensory panel.

Geographical authentication of virgin olive oil by GC-MS sesquiterpene hydrocarbon fingerprint: Verifying EU and single country label-declaration.

According to the last report from the European Union (EU) Food Fraud Network, olive oil tops the list of the most notified products. Current EU regulation states geographical origin as mandatory for virgin olive oils, even though an official analytical method is still lacking. Verifying the compliance of label-declared EU oils should be addressed with the highest priority level. Hence, the present work tackles this issue by developing a classification model (PLS-DA) based on the sesquiterpene hydrocarbon fingerprint of 400 samples obtained by HS-SPME-GC-MS to discriminate between EU and non-EU olive oils, obtaining an 89.6% of correct classification for the external validation (three iterations), with a sensitivity of 0.81 and a specificity of 0.95. Subsequently, multi-class discrimination models for EU and non-EU countries were developed and externally validated (with three different validation sets) with successful results (average of 92.2% of correct classification for EU and 96.0% for non-EU countries).

Stepwise strategy based on 1H-NMR fingerprinting in combination with chemometrics to determine the content of vegetable oils in olive oil mixtures.

1H NMR fingerprinting of edible oils and a set of multivariate classification and regression models organised in a decision tree is proposed as a stepwise strategy to assure the authenticity and traceability of olive oils and their declared blends with other vegetable oils (VOs). Oils of the 'virgin olive oil' and 'olive oil' categories and their mixtures with the most common VOs, i.e. sunflower, high oleic sunflower, hazelnut, avocado, soybean, corn, refined palm olein and desterolized high oleic sunflower oils, were studied. Partial least squares (PLS) discriminant analysis provided stable and robust binary classification models to identify the olive oil type and the VO in the blend. PLS regression afforded models with excellent precisions and acceptable accuracies to determine the percentage of VO in the mixture. The satisfactory performance of this approach, tested with blind samples, confirm its potential to support regulations and control bodies.

Methods to determine the quality of acid oils and fatty acid distillates used in animal feeding.

Acid oils and fatty acid distillates are by-products from the refining of edible oils and fats. They are used as feed ingredients, but their highly variable composition sometimes affects the productive parameters of the animals. Thus, their quality control and standardization are necessary. The official methods recommended for crude and refined fats and oils must be modified to give reliable results when applied to acid oils and fatty acid distillates. This article summarizes the drawbacks that were encountered during the setup of the analytical methods and how were they overcome by adapting the methods to these type of fat samples. Some methods such as the determinations of fatty acid composition, tocopherol and tocotrienol content, unsaponifiable matter, acidity and peroxide value had to be minimally adapted. However, others such as the determinations of moisture and volatile matter, insoluble impurities, lipid classes and p-anisidine value showed important drawbacks that required a more significant adaptation.•All the analytical methods have been successfully applied to acid oils and fatty acid distillates.•A detailed description of the sample preparation for analysis and applied analytical methods is provided as a compendium of methods in the supplementary material.•These methods will be extremely useful to improve the quality control of these heterogeneous feed ingredients.

Rapid direct analysis to discriminate geographic origin of extra virgin olive oils by flash gas chromatography electronic nose and chemometrics.

At present, the geographical origin of extra virgin olive oils can be ensured by documented traceability, although chemical analysis may add information that is useful for possible confirmation. This preliminary study investigated the effectiveness of flash gas chromatography electronic nose and multivariate data analysis to perform rapid screening of commercial extra virgin olive oils characterized by a different geographical origin declared in the label. A comparison with solid phase micro extraction coupled to gas chromatography mass spectrometry was also performed. The new method is suitable to verify the geographic origin of extra virgin olive oils based on principal components analysis and discriminant analysis applied to the volatile profile of the headspace as a fingerprint. The selected variables were suitable in discriminating between "100% Italian" and "non-100% Italian" oils. Partial least squares discriminant analysis also allowed prediction of the degree of membership of unknown samples to the classes examined.

Determination of volatile thiols in virgin olive oil by derivatisation and LC-HRMS, and relation with sensory attributes.

Volatile thiols were determined by derivatisation followed by ESI-LC-HRMS in twenty-five virgin olive oils (VOOs) pertaining to different commercial classes. 4-Methoxy-2-methyl-2-butanethiol (4MMB) was quantified, and 3-methyl-2-butenethiol and methanethiol were identified for the first time in VOO by non-target analysis. In order to evaluate the influence of volatile thiols on VOO aroma, the sensory notes, defects and secondary attributes were determined in samples according to European regulation by an Official VOO Panel. A significant correlation was found between the concentration of 4MMB in VOO samples and the 'fig tree leaf' sensory attribute. To confirm these results, 4MMB odour detection threshold and odour recognition were investigated.

Determination of volatile thiols in lipid matrix by simultaneous derivatization/extraction and liquid chromatography-high resolution mass spectrometric analysis. Application to virgin olive oil.

A simple, reliable, selective and sensitive method for the determination of volatile thiols at trace levels in lipid matrices was developed. The method consisted of a single-step derivatization/extraction procedure followed by electrospray ionization-high performance liquid chromatography-high resolution mass spectrometry (ESI-HPLC-HRMS) analysis using ebselen as a derivatization agent. The analytical conditions were optimized in a lipid model system (triolein) and virgin olive oil (VOO), both spiked with seven representative volatile thiols. The method was validated and the sensitivity, precision, accuracy and selectivity were evaluated. The experimental limit of quantification (LOQ) of the tested thiols in the VOO matrix ranged from 0.05 to 0.5ng/kg. Moreover, the intra-day relative standard deviation (RSD) was in general lower than 10%, except for esters, which reached RSD values of around 13%. The inter-day RDS ranged between 9.6% and 36.6%. The recovery in VOO ranged from 79% to 20% for derivatives with the highest and lowest polarity, respectively. The methodology was applied to the analysis of some VOO samples. Within this first probe sample, some of the VOO analyzed presented concentrations of 4-methoxy-2-methyl-2-butanethiol above the LOQ, ranging between 0.2 and 1.9ng/kg. The advantage of using full-scan acquisition mode when working with HRMS is that it allows the use of a non-target approach based on the fragmentation of thiol derivatives yielding a main product ion at m/z 275.9922 [C13H10ONSe](+) and that corresponded to the ebselen moiety of the derivatives. Any positive finding satisfied the identification and confirmation criteria established during the validation: retention time drift <3-fold the standard deviation of the method, mass accuracy <2ppm with real resolution ≥20,000 (full width at half maximum) at the mass range of interest, and the presence of isotope ions containing (80)Se and (78)Se, respectively.

Insight into virgin olive oil secoiridoids characterization by high-resolution mass spectrometry and accurate mass measurements.

With the aim to enhance characterization of virgin olive oil (VOO), high resolution mass spectrometry (HRMS) and high resolution tandem mass spectrometry (HRMS/MS), in positive and negative electrospray ionization (ESI) modes, coupled to fused-core reverse phase chromatography, were applied to distinct VOO phenolic extracts after the optimization of chromatographic conditions, ESI and fragmentation parameters. HRMS, but also HRMS/MS resulted fundamental to progress in secoiridoids structural elucidation. The former revealed that the secoiridoid composition of VOO was far more complicated than previously reported, while the latter helped clarify product ion elemental composition allowing new fragmentations, in addition to those reported in the literature, to be put forward. In particular, for the first time, different product ions with the same nominal mass were unequivocally identified in the spectra of secoiridoid compounds, confirming the greater capacity of HRMS/MS to clarify structure than low-resolution MS. Furthermore, and differing from previous studies, the multiple isomers of the main VOO secoiridoids could be differentiated on the basis of their HR product ion spectra in positive mode.

Ultrahigh resolution mass spectrometry and accurate mass measurements for high-throughput food lipids profiling.

In the present study, accurate mass measurements by ultrahigh resolution mass spectrometry with Orbitrap-Exactive working at resolving power R: 100,000 (m/z 200, full width at half maximum) with an accuracy better than 2 ppm in all the mass range (m/z 200 to 2000) were used to show a detailed molecular composition of diverse edible oils and fats. Flow injection was used to introduce samples into the mass spectrometer, obtaining a complete analysis of each sample in less than 10 min, including blanks. Meticulous choice of organic solvents and optimization of the ion source and Orbitrap mass analyzer parameters were carried out, in order to achieve reproducible mass spectra giving reliable elemental compositions of the lipid samples and to prevent carry over. More than 200 elemental compositions attributable to diacylglycerols, triacylglycerols (TAGs), and their oxidation products have been found in the spectra of food lipids from different origin. Several compounds with very close molecular mass could only be resolved through ultrahigh resolution, allowing detailed and robust TAG profiling with a high characterization potential.

The activity of healthy olive microbiota during virgin olive oil extraction influences oil chemical composition.

The activity of olive microbiota during the oil extraction process could be a critical point for virgin olive oil quality. With the aim to evaluate the role of microbiological activity during the virgin olive oil extraction process, just before oil extraction freshly collected healthy olive fruits were immersed in contaminated water from an olive mill washing tank. The oils extracted were then compared with control samples from the same batch of hand-picked olives. The presence of lactic and enteric bacteria, fungi and Pseudomonas on the surface of olives was proved to be much higher in washed than in control olives, with increments in cfu/g between 2 and 3 orders of magnitude. The biogenesis of volatile compounds and the extraction of olive polyphenols and pigments were significantly influenced by the microbiological profile of olives even without any previous storage. In most cases the effect of olive microbiota on oil characteristics was greater than the effect exerted by malaxation time and temperature. Oils from microbiologically contaminated olives showed lower amounts of C5 volatiles and higher levels of C6 volatiles from the lipoxygenase pathway and some fermentation products. On the other hand, a decrease of chlorophylls, pheophytins, xanthophylls and the ratio chlorophyll/pheophytin was observed in these oils. Likewise, the microbiological activity during oil extraction led to significantly lower amounts of polyphenols, in particular of oleuropein derivatives. These differences in olive oil chemical composition were reflected in oil sensory characteristics by the decrease of the green and bitter attributes and by the modification of the oil color chromatic ordinates.

Evolution of sesquiterpene hydrocarbons in virgin olive oil during fruit ripening.

Despite the potential of sesquiterpene hydrocarbons in olive oil authentication, their metabolism in Olea europaea is poorly understood, and little is known about their biochemical regulation in olives as a function of ripening. To ascertain some metabolic relationships between sesquiterpene hydrocarbons and olive ripening, the content of sesquiterpene hydrocarbons was assessed in virgin olive oils from two olive varieties grown in the same geographical area and produced at different harvesting periods. During the ripening, the accumulation of sesquiterpenes in the olive itself, and thus in the oil, differed according to their molecular structure: bicyclic sesquiterpenes, showed decreasing concentrations the later the harvest, while acyclic farnesene-like compounds progressively increased through the olive ripening process. This is first evidence that the accumulation of sesquiterpene hydrocarbons in olive, and hence in olive oil, is modulated during ripening. Therefore, the degree of ripening of olives should be taken into consideration when considering the sesquiterpenic profile of virgin olive oil for their authentication.

Influence of olives' storage conditions on the formation of volatile phenols and their role in off-odor formation in the oil.

Volatile phenols are spoilage compounds of many foods, which have also been detected in the volatile fraction of defective virgin olive oils (VOOs). However, their formation in olive fruits or VOOs, as well as the factors affecting their production, has yet to be elucidated. In the present study, the evolution of volatile phenols was monitored for the first time in VOOs obtained from olives stored during different periods under two different conditions of limited aerobiosis. Moreover, their odor activity values (OAVs) in VOO samples were calculated as a first assessment of their sensory importance, and the microbiological profile of the olives' surface was evaluated at each sampling point in order to clarify the possible causes of volatile phenols formation. Although volatile phenols seem to acquire their sensory significance at advanced stages of olives' alteration, they were significantly correlated to the time of olives' storage and in accordance with sensory evaluation, indicating that they could be considered as analytical indices of olive fruits' degradation during storage, likely reflecting the microbiological activity.

Assessment of some diterpenoids in commercial distilled gin.

In the present study the qualitative and quantitative determination of diterpenoids in commercial distilled gin was carried out. This widely consumed juniper-based spirit is aromatized using Juniper (Juniperus communis) berries. Although juniper reportedly contains several diterpenic compounds, no studies have addressed the diterpenic composition of juniper-based spirits or beverages. With this objective, here we followed a multilevel factorial experimental design to optimize a direct immersion-solid phase microextraction (DI-SPME) method coupled to gas chromatography/mass spectrometry and analyzed eight commercial brands of gin. With total concentrations ranging from 10 to 190 microg L(-1), manool, manoyl oxide and trans-totarol were the most abundant diterpenoids of the 10 identified or tentatively identified at variable but not negligible concentrations in the distilled gin samples. The diterpenic composition allowed the brands to be differentiated. This indicates that these compounds contribute to the sensory characteristics of the distinct commercial brands, thus guaranteeing the authenticity and consequently the quality of the product.

Determination of volatile phenols in virgin olive oils and their sensory significance.

Volatile phenols are strong odorants produced by microbial activity and reported in several foods, but very scarce information is available on their presence in virgin olive oils (VOOs) and on their relation with VOO chemical and sensory quality. In the present paper, a factorial experimental design was applied for the development of a suitable solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) analytical method for the analysis of volatile phenols in olive oil. The memory effects demonstrated by SPME fibres required the optimization of desorption conditions to minimize experimental errors. A series of nine volatile phenols were identified and quantified for the first time in VOOs by analyzing samples with distinct off-flavours. Their limits of detection and quantification (microg/kg) were largely below the odour detection thresholds (ODTs) calculated in this study (mg/kg), confirming the capacity of the technique to assess the target compounds at early stages of the oil sensory alteration. The odour activity values (OAVs) of volatile phenols were calculated in VOOs facilitating a first assessment of their potential importance in the aroma of the product.

Monoterpene and sesquiterpene hydrocarbons of virgin olive oil by headspace solid-phase microextraction coupled to gas chromatography/mass spectrometry.

In the present article, a headspace solid-phase microextraction method coupled to GC/MS was developed and applied for the simultaneous determination of mono- and sesquiterpenic hydrocarbons in virgin olive oils of different olive variety and geographical origin. Analysis of various oils resulted in the simultaneous detection of 15 monoterpenes and 30 sesquiterpenes. Some of these hydrocarbons were previously reported to be constituents of virgin olive oil terpenoid fraction, although we also detected some terpenic hydrocarbons that have not previously been documented as present in virgin olive oil. Significant differences were detected in the proportion of terpenic compounds in oils obtained from different olive varieties grown in different geographical areas. The monoterpene, and particularly the sesquiterpene composition of olive oil may be used to distinguish samples from different cultivar and geographical areas.

Simultaneous determination of volatile and semi-volatile aromatic hydrocarbons in virgin olive oil by headspace solid-phase microextraction coupled to gas chromatography/mass spectrometry.

A reliable, simple and relatively fast method for the simultaneous determination of volatile and semi-volatile aromatic hydrocarbons in virgin olive oil was developed, based on headspace solid-phase microextraction (HS-SPME). The investigation regarded eco-contaminants such as alkylated monoaromatic hydrocarbons from C1- to C4-benzenes and light polyaromatic hydrocarbons up to four aromatic rings. Sampling and chromatographic conditions were optimized by using standard solutions in deodorized olive oil and the analytical performances of the method were determined. The proposed method was then applied to real samples of virgin olive oil were the target hydrocarbons could be identified and quantified. Several of them had not been previously quantified in virgin olive oil. Moreover, by the analysis of olive oil samples an additional number of C4-benzenes could be tentatively identified.

Solid-phase microextraction in the analysis of virgin olive oil volatile fraction: modifications induced by oxidation and suitable markers of oxidative status.

Modifications of virgin olive oil subjected to accelerated storage were evaluated by HS-SPME analysis. To find a suitable marker of oxidative degradation, the volatile compounds showing variable concentration during the oxidative process have been identified and quantified by SPME coupled to GC-MS and GC-FID, respectively. The SPME analysis results were then compared with the parameters usually applied to assess the oxidative status of lipids, such as peroxide value, spectrophotometric absorbance, and loss of unsaturated fatty acids. Finally, the assessment of nonanal has been suggested as a marker of oxidative degradation. This rapid, inexpensive, and reliable method may allow screening of oils prior to testing by a panel of assessors.

Solid-phase microextraction in the analysis of virgin olive oil volatile fraction: characterization of virgin olive oils from two distinct geographical areas of northern Italy.

SPME was employed to characterize the volatile profile of virgin olive oils produced in two geographical areas of northern Italy: the region of the Gulf of Trieste and the area near Lake Garda. There are as yet no data on the headspace composition of virgin olive oils from these regions, characterized by particular conditions of growth for Olea europaea. Using the SPME technique coupled to GC-MS and GC-FID, the volatile components of 42 industrially produced virgin olive oil samples were identified and the principal compounds quantitatively analyzed. Significant differences in the proportion of volatile constituents from oils of different varieties and geographical origins were detected. The results suggest that besides the genetic factor, environmental conditions influence the volatile formation.