HS-SPME-GC-MS and Chemometrics for the Quality Control and Clustering of Monovarietal Extra Virgin Olive Oil: A 3-Year Study on Terpenes and Pentene Dimers of Italian Cultivars.

Terpenes and pentene dimers are less studied volatile organic compounds (VOCs) but are associated with specific features of extra virgin olive oils (EVOOs). This study aimed to analyze mono- and sesquiterpenes and pentene dimers of Italian monovarietal EVOOs over 3 years (14 cultivars, 225 samples). A head space-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method recently validated was used for terpene and pentene dimer quantitation. The quantitative data collected were used for both the characterization and clustering of the cultivars. Sesquiterpenes were the molecules that most characterized the different cultivars, ranging from 3.908 to 38.215 mg/kg; different groups of cultivars were characterized by different groups of sesquiterpenes. Pentene dimers (1.336 and 3.860 mg/kg) and monoterpenes (0.430 and 1.794 mg/kg) showed much lower contents and variability among cultivars. The application of Kruskal-Wallis test-PCA-LDA-HCA to the experimental data allowed defining 4 clusters of cultivars and building a predictive model to classify the samples (94.3% correct classification). The model was further tested on 33 EVOOs, correctly classifying 91% of them.

Markers of sensory dynamics in phenols-rich virgin olive oils under optimal storage conditions.

Early changes in sensory quality of phenols-rich virgin olive oil (VOO) and their relationship with the chemical changes are less studied in the literature. Therefore, the objective of this study was to propose a predictive model of dynamics of sensory changes based on specific chemical markers. The evolution of the sensory quality of phenol-rich VOOs from Tuscan cultivars stored under optimal storage conditions (i.e., absence of light, no O2 exposure, low temperature) was investigated using a multi-step methodological approach combining sensory (official sensory analysis (so-called Panel Test), Descriptive Analysis and Temporal Dominance of Sensation) and chemical measurements. The sensory map from descriptive data was related to the phenolic and volatile profiles, measured using HPLC-DAD and HS-SPME-GC-MS, respectively. A predictive model of the sensory changes over storage based on chemical compounds was developed. Results showed that very early changes involving phenolic and volatile compounds profiles occur in VOOs stored under optimal storage conditions, which turn in changes in sensory properties evaluated by the official panel test, the descriptive analysis and the temporal dominance of sensation. Furthermore, a chemical marker of sensory dynamics of oils during storage was identified as the ratio between two groups of secoiridoids. The proposed model, supported by the mentioned chemical marker, has the potential of improving the control of sensory changes in phenols-rich virgin olive oils during storage in optimal conditions.

Analysis of Volatile Hydrocarbons (Pentene Dimers and Terpenes) in Extra Virgin Olive Oil: Optimization by Response Surface Methodology and Validation of HS-SPME-GC-MS Method.

A head space-solid phase microextraction-gas chromatography-mass spectrometery (HS-SPME-GC-MS) method for the simultaneous analysis of pentene dimers from lipoxygenase (LOX) pathway, monoterpenes, and sesquiterpenes in extra virgin olive oil (EVOO) was proposed. A Doehlert design was performed; the conditions of the HS-SPME preconcentration step (extraction temperature, extraction time, sample amount, and desorption time) were optimized by response surface methodology, allowing defining the method operable design region. A quantitative method was set up using the multiple internal standard normalization approach: four internal standards were used, and the most suitable one was selected for area normalization of each external standard. The quantitative method was successfully validated and applied to a series of monocultivar EVOOs. This is the first paper in which a quantitative method using commercial standards has been proposed for the analysis of an important class of molecules of EVOO such as pentene dimers. The optimized method is suitable for routine analysis aimed at characterizing high quality EVOOs.

Exploitation of virgin olive oil by-products (Olea europaea L.): phenolic and volatile compounds transformations phenomena in fresh two-phase olive pomace ('alperujo') under different storage conditions.

BACKGROUND: Much effort has recently been spent for re-using virgin olive oil by-products as nutraceutical ingredients for human diet thanks to their richness in bioactive phenols, but their management is not easy for producers. We aimed to provide useful information for a better management of fresh olive pomace before drying, by studying the phenolic and volatile compounds transformations phenomena of fresh olive pomace stored under different conditions planned to simulate controlled and uncontrolled temperature conditions in olive oil mills. RESULTS: The evolution of the phenolic and volatile compounds was studied by high-performance liquid chromatography-diode array detector mass spectrometry (HPLC-DAD-MS) and headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). The phenolic profile varied rapidly during storage: the verbascoside content decreased about 70% after 17 days even at 4 °C, while the content of simple phenols such as hydroxytyrosol and caffeic acid increased over time. The low temperature was able to slow down these phenomena. A total of 94 volatile organic compounds (VOCs) were detected in the fresh olive pomace, with a prevalence of lipoxygenase (LOX) VOCs (78%), mainly aldehydes (19 490.9 µg kg-1 ) despite the higher number of alcohols. A decrease in LOX volatiles and a quick development of the ones linked to off-flavors (carboxylic acids, alcohols, acetates) were observed, in particular after 4 days of storage at room temperature. Only storage at 4 °C allowed these phenomena to be slowed down. CONCLUSION: To preserve the natural phenolic phytocomplex of fresh olive pomace before drying and to avoid off-flavors development, storage in open containers must be avoided and a short storage in cold rooms (7-10 days) is to be preferred. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Volatile Profile of Two-Phase Olive Pomace (Alperujo) by HS-SPME-GC-MS as a Key to Defining Volatile Markers of Sensory Defects Caused by Biological Phenomena in Virgin Olive Oil.

An olive pomace from the two-phase decanter stored in different conditions was used as a model to simulate the detrimental biological phenomena occurring during olive oil processing and storage. A group of EVOO and defective oils were also analyzed. The volatile fraction was studied with HS-SPME-GC-MS; 127 volatiles were identified (55 of which tentatively identified) and evaluated over time. Seven volatiles were tentatively identified for the first time in olive oil; the role of C6 alcohols in detrimental biological phenomena was highlighted. Suitable volatile markers for defects of microbiological origin were defined, particularly the fusty/muddy sediment. They were then applied to olive oils with different quality categories; one of the markers was able to discriminate among EVOOs and all the defective samples, including the borderline ones. The marker was constituted by the sum of concentrations of 10 esters, 4 alcohols, 1 ketone, and 1 α-hydroxy-ketone but no carboxylic acids.

Virgin Olive Oil Volatile Compounds: Composition, Sensory Characteristics, Analytical Approaches, Quality Control, and Authentication.

Volatile organic compounds strongly contribute to both the positive and negative sensory attributes of virgin olive oil, and more and more studies have been published in recent years focusing on several aspects regarding these molecules. This Review is aimed at giving an overview on the state of the art about the virgin olive oil volatile compounds. Particular emphasis was given to the composition of the volatile fraction, the analytical issues and approaches for analysis, the sensory characteristics and interaction with phenolic compounds, and the approaches for supporting the Panel Test in virgin olive oil classification and in authentication of the botanical and geographic origin based on volatile compounds. A pair of detailed tables with a total of approximately 700 volatiles identified or tentatively identified to date and tables dealing with analytical procedures, sensory characteristics of volatiles, and specific chemometric approaches for quality assessment are also provided.

New Volatile Molecular Markers of Rancidity in Virgin Olive Oils under Nonaccelerated Oxidative Storage Conditions.

Evolution of the volatile profile of two extra-virgin olive oils with very different fatty acid composition (monounsaturated fatty acid/polyunsaturated fatty acid ratio) stored in several nonaccelerated oxidative conditions was studied by a validated headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method. The role of C8 volatile compounds in oxidative processes was highlighted, and controversial aspects regarding the origin of some volatiles were clarified. Specific volatile markers for rancidity were proposed: sum of pentanal, hexanal, nonanal, E-2-heptenal, propanoic acid, and hexanoic acid for oils stored in the dark; sum of pentanal, heptanal, nonanal, decanal, E-2-heptenal, E-2-decenal, E,E-hepta-2,4-dienal, and E,E-deca-2,4-dienal, octane for oils stored under light exposure; sum of pentanal, nonanal, decanal, E-2-heptenal, E-2-decenal, E,E-hepta-2,4-dienal, nonan-1-ol, propanoic acid, octane, 6-methylhept-5-en-2-one, and oct-1-en-3-ol for oils stored under light exposure with oxygen in headspace. A simplified marker (sum of pentanal, nonanal and E-2-heptenal) suitable for all conditions was also proposed.

Headspace Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry Quantification of the Volatile Profile of More than 1200 Virgin Olive Oils for Supporting the Panel Test in Their Classification: Comparison of Different Chemometric Approaches.

A reliable and robust tool for supporting the panel test in virgin olive oil classification is still required. We propose four chemometric approaches based on t test, principal component analysis (PCA) and linear discriminant analysis (LDA), applied for combining sensorial data, and chemical measurements. The former was from the panel test, and the latter was from headspace solid-phase microextraction-gas chromatography-mass spectrometry quantitation of 73 volatile organic compounds (VOCs) of 1223 typical commercial virgin olive oils, with most of them recognized as difficult to classify with accuracy by the panel test. The approaches were developed and validated, and the best results, with 83.5% correct classification, were using the PCA-LDA approach. Among the other methods, developed for proposing simplified procedures based on a smaller number of VOCs, the best method gave 80.1% correct classification only using 10 VOCs. All of the approaches suggested that octane, heptanal, pent-1-en-3-ol, Z-3-hexenal, nonanal, and 4-ethylphenol should be considered as a basis of volatiles for classification of olive oil samples.

An effective HPLC-based approach for the evaluation of the content of total phenolic compounds transferred from olives to virgin olive oil during the olive milling process.

BACKGROUND: Several studies demonstrate a strong interest in learning more about phenolic transfer during oil extraction, with the main goal of increasing the phenolic concentration in olive oils. We aimed to propose and apply a new methodological approach for evaluating phenolic transfer from olives into oil during milling, based on the quantification of phenolic content in whole lyophilized fruits and the corresponding oils and considering the oil extraction yields. RESULTS: We investigated the phenols transferred into the oil during olive milling in continuous extraction systems in Tuscany. In 2012, oils were extracted from cultivar Frantoio by a two-phase extraction system; in 2016, oils were extracted from cultivars Leccio del Corno and Arbequina by a three-phase extraction system. Results highlighted very low percentages of extracted phenols: up to 0.40% by the two-phase system and up to 0.19% by the three-phase system (0.08% for cultivar Arbequina and 0.19% for cultivar Leccio del Corno). CONCLUSION: The usefulness of a simple and effective methodological approach for evaluating the extracted phenols was highlighted. Values of extracted phenols were up to 25 times lower than previous literature data. The proposed approach is applicable in all types of milling processes. © 2017 Society of Chemical Industry.

Multiple internal standard normalization for improving HS-SPME-GC-MS quantitation in virgin olive oil volatile organic compounds (VOO-VOCs) profile.

The commercial value of virgin olive oils (VOOs) strongly depends on their classification, also based on the aroma of the oils, usually evaluated by a panel test. Nowadays, a reliable analytical method is still needed to evaluate the volatile organic compounds (VOCs) and support the standard panel test method. To date, the use of HS-SPME sampling coupled to GC-MS is generally accepted for the analysis of VOCs in VOOs. However, VOO is a challenging matrix due to the simultaneous presence of: i) compounds at ppm and ppb concentrations; ii) molecules belonging to different chemical classes and iii) analytes with a wide range of molecular mass. Therefore, HS-SPME-GC-MS quantitation based upon the use of external standard method or of only a single internal standard (ISTD) for data normalization in an internal standard method, may be troublesome. In this work a multiple internal standard normalization is proposed to overcome these problems and improving quantitation of VOO-VOCs. As many as 11 ISTDs were used for quantitation of 71 VOCs. For each of them the most suitable ISTD was selected and a good linearity in a wide range of calibration was obtained. Except for E-2-hexenal, without ISTD or with an unsuitable ISTD, the linear range of calibration was narrower with respect to that obtained by a suitable ISTD, confirming the usefulness of multiple internal standard normalization for the correct quantitation of VOCs profile in VOOs. The method was validated for 71 VOCs, and then applied to a series of lampante virgin olive oils and extra virgin olive oils. In light of our results, we propose the application of this analytical approach for routine quantitative analyses and to support sensorial analysis for the evaluation of positive and negative VOOs attributes.

New isobaric lignans from Refined Olive Oils as quality markers for Virgin Olive Oils.

Herein we describe the influence of olive oil refining processes on the lignan profile. The detection of new isobaric lignans is suggested to reveal frauds in commercial extra-Virgin Olive Oils. We analyzed five commercial olive oils by HPLC-DAD-TOF/MS to evaluate their lignan content and detected, for the first time, some isobaric forms of natural (+)-pinoresinol and (+)-1-acetoxypinoresinol. Then we analyzed partially and fully-refined oils from Italy, Tunisia and Spain. The isobaric forms occur only during the bleaching step of the refining process and remain unaltered after the final deodorizing step. Molecular dynamic simulation helped to identify the most probable chemical structures corresponding to these new isobars with data in agreement with the chromatographic findings. The total lignan amounts in commercial olive oils was close to 2mg/L. Detection of these new lignans can be used as marker of undeclared refining procedures in commercial extra-virgin and/or Virgin Olive Oils.

Whole Lyophilized Olives as Sources of Unexpectedly High Amounts of Secoiridoids: The Case of Three Tuscan Cultivars.

The phenolic profiles of three typical Tuscan olive cultivars, Frantoio, Moraiolo, and Leccino, stored in different conditions (fresh, frozen, and whole lyophilized fruits), have been compared during the ripening period. Our main goals were to evaluate the phenolic content of whole freeze-dried fruits and to test the stability of the corresponding cake in oxidative-stress conditions. The comparison of fresh and whole freeze-dried fruits from the 2012 season gave unexpected results; e.g., oleuropein in lyophilized fruits was up to 20 times higher than in fresh olives with values up to 80.3 g/kg. Over time we noted that the olive pastes obtained from lyophilized olives contained highly stable phenolic compounds, even under strong oxidative stress conditions. Finally, it was also observed that the cake/powder obtained from unripe freeze-dried olives was very poor in oil content and therefore quite suitable for use in nutritional supplements rich in phenolic compounds, such as secoiridoids, which are not widely present in the human diet.

Nutraceutical properties of chestnut flours: beneficial effects on skeletal muscle atrophy.

Plants contain a wide range of non-nutritive phytochemicals, many of which have protective or preventive properties for human diseases. The aim of the present work has been to investigate the nutraceutical properties of sweet chestnut flour extracts obtained from fruits collected from 7 geographic areas of Tuscany (Italy), and their ability in modulating skeletal muscle atrophy. We found that the cultivars from different geographic areas are characterized by the composition and quantity of various nutrients and specific bioactive components, such as tocopherols, polyphenols and sphingolipids. The nutraceutical properties of chestnut sweet flours have been evaluated in C2C12 myotubes induced to atrophy by serum deprivation or dexamethasone. We found that the pretreatment with both total extracts of tocopherols and sphingolipids is able to counterbalance cell atrophy, reducing the decrease in myotube size and myonuclei number, and attenuating protein degradation and the increase in expression of MAFbx/atrogin-1 (a muscle-specific atrophy marker). By contrast, polyphenol extracts were not able to prevent atrophy. Since we also found that γ-tocopherol is the major form of tocopherol in sweet flour and its content differs depending on the procedure of sweet flour preparation, the mechanisms by which γ-tocopherol as well as sphingolipids affect skeletal muscle cell atrophy have been also investigated. This is the first evidence that chestnut sweet flour is a natural source of specific bioactive components with a relevant role in the prevention of cell degeneration and maintenance of skeletal muscle mass, opening important implications in designing appropriate nutritional therapeutic approaches to skeletal muscle atrophy.