Evolution of monovarietal virgin olive oils as a function of chemical composition and oxidation status

Abstract Virgin Olive Oil (VOO) shelf life is determined by the varietal-specific chemical composition and principally by the of phenolic composition. The aim of this study was to investigate the changes in fatty acid profile, phenolic composition, and quality parameters of nine Italian monovarietal VOOs obtained under the same pedoclimatic, agronomic and technological conditions and stored for 12 months at 15 °C in the dark. The varieties with medium-high concentrations of secoiridoids and balanced values between the individual molecules were those with the highest stability. Orthogonal Projections to Latent Structures (OPLS) regression revealed that oleuropein derivatives and phenolic alcohols had the highest antioxidant activity. OPLS discriminant analysis separated well fresh and stored oils. PV, K270, tyrosol, hydroxytyrosol, and oxidated oleacein were the most effective indicators of VOO ageing. Oleacein and oleocanthal decreased after storage, phenolic alcohols, oleacein and ligstroside aglycon increased. Graphical Abstract


Introduction
Virgin Olive Oil (VOO) consumption contributes to lowering the risk of cardiovascular and neurodegenerative diseases, atherosclerosis, several types of cancer, and diabetes, such health benefits have been attributed principally to the presence of hydrophilic phenols, specifically oleacein and oleocanthal (Foscolou et al. 2018). Phenols protect also VOO lipids from oxidation by free radical scavenging and quenching singlet oxygen (Castellani et al. 2008;Krichene et al. 2015). Autoxidation is a natural process of edible oils containing fatty acids (FA) with double bonds, and the higher is the number of double bonds, the higher is the oxidation rate (Choe and Min 2006). Lipids' hydroperoxides and conjugated forms are the first oxidation products, which in turn may form short-chain fatty acids and volatile compounds, responsible of several VOO off-flavours indicators of oil ageing (Choe and Min 2006;Esposto et al. 2020;Caipo et al. 2021).
Light and oxygen exposure, high temperatures, and the presence of enzymatic activity in dispersed water promote oxidation process (Salvo et al. 2017;Esposto et al. 2020;Caipo et al. 2021;Mousavi et al. 2021). Storage temperatures below 20 C, absence of light and oxygen are the conditions that better contribute to extend VOO shelf life (Krichene et al. 2015;Mousavi et al. 2021), which, thanks to the high oleic acid content and to the presence of antioxidants like phenolic compounds, tocopherols, and pigments could last up to 24 months (Esposto et al. 2020;Alonso-Salces et al. 2021;Mousavi et al. 2021). However, remains ambiguous the role of antioxidants in determining VOO stability. In general, secoiridoids and phenolic alcohols had the strongest antioxidant activity (Esposto et al, 2020), but contrasting results have been reported about the role of individual molecules (Krichene et al. 2015;Miho et al, 2020;Alonso-Salces et al, 2021;Caipo et al 2021). The discrepancies observed in literature could be presumably attributed to the difference of experimental conditions (storage containers and headspace, temperatures, and light exposure) and to the specific chemical composition of the VOO samples analysed. Indeed, it seems that the varietalspecific ratio between antioxidants, rather than the individual concentration of antioxidant molecules, plays the principal role in determining VOO evolution during time.
For this reason, this study aims to (1) deepen the knowledge on the storage dynamics of VOOs obtained from 3 Italian (Coratina, Leccino and Frantoio) and 6 local Sardinian (Bosana, Nera di Oliena, Pizz 'e Carroga, Semidana, Sivigliana da Olio, and Tonda di Villacidro) varieties; (2) understand better the relationship between chemical composition and VOO oxidation status determined by the VOO quality parameters. We determined the changes in fatty acid (FA) and phenolic composition, and quality parameters (free acidity, peroxide value (PV), K232, and K270), of the 9 monovarietal VOOs obtained under the same pedoclimatic, agronomic and technological conditions, stored for 12 months at 15 C in the dark. The relationship between quality parameters variation and VOO chemical composition was evaluated applying both Orthogonal Projections to Latent Structures (OPLS) and OPLS Discriminant Analysis (OPLS-DA). Such multivariate approaches can provide a clear and comprehensive view about the role of both major and minor VOO components on quality parameters (OPLS) and able to identify those molecules most affected by storage (OPLS-DA). Recent studies have already applied similar statistical approach (PLS) on this field, but only focused on the identification of shelf-life and aging predictors, such as oxidation products of volatile compounds (Esposto et al. 2020;Alonso-Salces et al. 2021;Caipo et al. 2021).

Results and discussion
The variability on the quality parameters was highly related to the varietal factor and less to storage (SM2). Free acidity was similar between all samples and always below 0.45% (SM3). After storage the increase of acidity and K232 were significant but of limited relevance (23% and 15%, respectively). K270 and PV showed stronger increases (44% and 128%, respectively) than K232. In terms of quality parameters variations, Leccino, Nera di Oliena, Pizz 'e Carroga, Semidana, and Tonda di Villacidro VOOs exhibited the highest stability. Oleic acid ratio ranged between 68.16% (Semidana) and 81.68% (Coratina), whereas linoleic acid between 6.49% (Coratina) and 13.84% (Semidana). Our findings in FA composition (SM3) were in general close to the range values reported previously in literature for the same varieties (Deiana et al. 2021). According to literature (G omez-Alonso et al. 2007;Di Stefano and Melilli 2020), FA composition underwent limited changes during the storage period, only polyunsaturated fatty acids (PUFA) decreased significantly. In general, VOOs samples with initial high oleic acid and secoiridoids levels reported the lowest FA variations. Fifteen phenolic compounds were identified and quantified by HPLC-DAD (SM4). Oleacein (20-765 mg kg À1 ), oleocanthal (18-768 mg kg À1 ), oleuropein and ligstroside aglycones (27-583 mg kg À1 and 16-195 mg kg À1 , respectively) were the most representative. The varietal-specific phenolic profiles observed were in line with those reported in literature (Deiana et al. 2019). The different concentration levels should be ascribed to technological (Boselli et al. 2009) or environmental factors (Deiana et al. 2021). Only phenolic acids and vanillin were not significantly affected by storage (SM2). Apigenin also showed little losses (-10%), whilst the other flavonoid luteolin decreased by above 60%. The phenolic acids stability and the different flavonoids' behaviour agrees with literature (Boselli et al. 2009;Su arez et al. 2011). 1-acetoxypinoresinol (3-46 mg kg À1 ) suffered significant losses, on average À36%. A strong decrease of oleacein, oleocanthal, and the dialdehydic forms of oleuropein and ligstroside aglycon was observed. The oxidated form of oleacein, present only in few fresh samples, was quantified in all the stored samples. In line with literature (Krichene et al. 2015;Esposto et al. 2020;Alonso-Salces et al. 2021), oleuropein derivatives depletion was stronger than ligstroside ones. Conversely, oleuropein and ligstroside aglycon increased after the storage period. Increasing trend, or alternate bearing, of aglycon forms was already observed by other authors and the main differences were related to the varietal factor (Boselli et al. 2009;Su arez et al. 2011;Lozano-S anchez et al. 2013;Di Stefano and Melilli 2020;Esposto et al. 2020). Su arez et al. (2011 attributed the aglycon increase to the presence of respective dimers which could had been released the molecules during storage. The presence of dimer structures was also notified in our previous work where the same extraction and bottling protocol were adopted (Deiana et al. 2019). Finally, hydroxytyrosol and tyrosol increased after the storage period (145% and 60%, respectively).
To understand better the role of the phenolic and FA composition in determining the VOO stability during ageing processes, OPLS-DA (SM5) and OPLS (SM6) were performed. OPLS-DA discriminated well between fresh and stored samples (SM5a), showing good reliability and predictive ability (SM7 and SM8). Oxidized oleacein, hydroxytyrosol, tyrosol, ligstroside aglycon, PV, K270, luteolin, the dialdehydic form of oleuropein aglycon, and 1-acetoxypinoresinol were the variables mostly involved in oil aging (SM5b). Similar results were obtained also by Esposto et al. (2020) and Caipo et al. (2021) performing PLS models setting storage time as dependent variable.
Throughout OPLS regression models, the initial quality parameter values, and their variation after 12-months storage (expressed as the difference between the final and initial values, D) were put in relation with the initial values of quality parameters (when not set as dependent variable), phenolic compounds, and fatty acids (SM6). Higher secoiridoids concentration, mainly oleuropein derivatives, and low PUFA content contributed to limit PV in fresh oils (SM6a). PV variation (DPV) was promoted first by higher PUFA and PV initial values (SM6b), indeed, the presence of free fatty acids and hydroperoxides enhance the peroxidation process (Choe and Min 2006). Conversely to PV models, the higher oleacein and oleocanthal levels the higher K232 initial values and DK232 (SM6c, SM6d). Only phenolic acids and phenolic alcohols seemed to constrain the formation of conjugated dienes (K232). FA composition played a marginal role in K232 models. High levels of secoiridoids were also positively correlated with K270 initial values (SM6e), only oleacein seemed to limit K270 increase (SM6f). OPLS regression analyses and the changes observed in monovarietal VOOs, revealed that oils with medium-high concentrations of secoiridoids and well-balanced ratio between the individual antioxidant molecules (e. g., Leccino, Nera di Oliena, Pizz 'e Carroga, and Semidana) held the strongest stability. This suggests that the VOO oxidative stability depends first on the synergic effect of the antioxidant molecules, rather than the presence at high concentrations of a single molecule with a proven high antioxidant power. The results obtained contributed to understand the role of the varietal specific VOO components on its shelf life and provide useful indications on varietal choice to producers, which should look at a product with well-balanced chemical composition, also re-considering minor varieties of Italian olive germplasm.

Experimental
See Supplementary materials for materials and methods (plant material, meteoclimatic conditions (SM1), VOO extraction and storage, determination of quality parameters, FA, and phenolic compounds, analysis of data), tables and figures.