Virgin olive oil (VOO) quality assessment relies heavily on sensory analysis, specifically the Panel test, which identifies and quantifies positive attributes like fruitiness and negative attributes (defects). Recently, there's been significant interest in linking sensory characteristics to specific volatile compounds. This would improve the Panel test by identifying volatile markers for common defects, creating reference materials for training, and classifying olive oils based on their fruity type. While research focuses on primary defects like "fusty/muddy sediment," "musty/humid," "winey-vinegary/acid-sour," "rancid," and "frostbitten olives," secondary defects such as "grubby" are less studied. The "grubby" defect describes the flavor of oil from olives heavily infested with olive fruit fly (Bactrocera oleae) larvae. This pest is a major problem in the Mediterranean. Larval feeding damages olive tissue, affecting oil quality, with the severity depending on factors such as infestation extent, fruit ripeness, and storage conditions. Previous studies explored the relationship between olive fly infestation and volatile compounds, often noting increased C6 alcohol levels and variations in C6 aldehydes. However, the results concerning C6 aldehydes have been inconsistent. It's also suggested that mechanical damage isn't the sole cause of volatile profile changes, with additional plant-insect interaction mechanisms and increased microbial activity playing a role. Common defects in oils from infested olives are "fusty," "musty," and "rancid," but "grubby" is less frequently reported. The sensory perception of "grubby" is unclear, potentially a combination of taste and odor characteristics that may overlap with oxidation and fermentation-related changes. Sometimes, even with 100% infestation, the sensory defect may be absent, resulting only in a reduction of positive sensory characteristics. This study aimed to investigate the relationship between the intensity of the "grubby" defect and the volatile compound composition of VOO resulting from varying degrees of olive fruit fly infestation. The goal was to identify volatile markers to distinguish defective oils from healthy ones, enhancing our understanding of this defect's causes. Olives were processed immediately after harvest to minimize oxidation and fermentation interferences. This study also assessed the effects of olive fly infestation on fatty acid composition and basic properties of olive pastes.

Several studies have investigated the impact of olive fly infestation on olive oil quality and volatile composition. Angerosa et al. (1992) were among the first to explore the connection between infestation intensity and specific volatile compounds. Subsequent research, including work by Tamendjari et al. (2004), Brkić Bubola et al. (2018), and Notario et al. (2022), examined this topic, often reporting an increase in C6 alcohols levels and inconsistent changes in C6 aldehydes. Alagna et al. (2016) suggested that changes in volatile profiles aren't solely due to mechanical tissue damage but also involve complex plant-insect interactions. The presence of microbial and oxygen activity, due to exocarp damage, leads to fermentative processes increasing ethanol and branched-chain alcohols and ethyl acetate. Commonly reported defects in oils from infested olives include "fusty," "musty," and "rancid." However, reports of "grubby" as a primary defect are rare, often appearing alongside other defects (Tamendjari et al., 2004; Koprivnjak et al., 2010). The sensory characteristics of the "grubby" defect remain not fully defined, possibly representing a combination of taste and odor attributes influenced by oxidation and fermentation.

Olive fruits of the Leccino cultivar were harvested at a ripening index (RI) of 3.0. Three groups were prepared: L-0% (healthy fruits), L-100% (fruits with at least one olive fly puncture or exit hole), and L-50% (a 1:1 mix of L-0% and L-100%). Each group was processed in triplicate within 24 hours of harvesting using an Abencor system. Water and oil content were determined gravimetrically. Quality parameters (free fatty acids (FFA), peroxide value (PV), and spectrophotometric indices) were analyzed according to IOC methods. Fatty acid methyl esters (FAMEs) were determined using gas chromatography. Sensory analysis was conducted by an IOC-approved panel using a 10 cm intensity scale for various attributes, including the "grubby" defect. Volatile compound analysis was performed using headspace solid-phase microextraction with gas chromatography/mass spectrometry (HS-SPME-GC/MS). Sensory and chemical data were analyzed using one-way ANOVA and Tukey's test. Pearson correlation coefficients assessed relationships between infestation, defect intensity, and volatile concentrations. Hierarchical cluster analysis (HCA) and partial least squares discriminant analysis (PLS-DA) were performed using MetaboAnalyst 5.0.

Olive fly infestation didn't significantly affect water and oil content in olive pastes. A slight but significant increase in FFA and PV was observed only in L-100% VOO, indicating minimal oxidation. Fatty acid composition remained largely unaffected by infestation. Sensory analysis revealed that the intensity of "other ripe fruits" increased in oils from infested fruits, while the intensity of "olive fruitiness," "apple," "green leaf/grass," bitterness, and pungency decreased, particularly in L-100% VOO. The "grubby" defect was the most prominent off-flavor in oils from infested fruits, described as cocoa butter-like, fatty, and sweet. Its intensity increased with the degree of infestation. About 20 volatile compounds showed strong positive correlations with both infestation level and "grubby" defect intensity. These included ethyl 2-methylbutyrate, propanoic acid, dimethyl sulfoxide, 2-methylbutanal, 3-methylbutanal, and methyl acetate, often associated with fermentation. Increased concentrations of dimethyl sulfoxide, 2-methylbutanal, and 3-methylbutanal may contribute to the fatty and cocoa butter notes. Compounds associated with oxidation, like 4-methyl-5H-furane-2-one, 6-methyl-5-hepten-2-one, octanal, hexanal, heptanal, E-2-nonen-1-ol, 1-octanol, E-2-heptenal, benzeneacetaldehyde, and E,E-2,4-heptadienal also showed strong positive correlations with infestation and "grubby" defect intensity. Terpenes like β-ocimene and α-farnesene also increased significantly with infestation. HCA successfully clustered VOOs based on infestation level, separating L-100% oils due to higher levels of several volatile compounds. PLS-DA also successfully separated the groups, with ethyl 2-methylbutyrate, 1-octanol, α-farnesene, 4-methyl-5H-furan-2-one, octanal, and β-ocimene being most important for separation. A second PLS-DA comparing L-0% and L-100% highlighted 3-methylbutanal, 2-methylbutanal, β-ocimene, ethyl 2-methylbutyrate, dimethyl sulfoxide, 4-methyl-5H-furan-2-one, α-farnesene, and 6-methyl-5-hepten-2-one as key discriminatory volatile markers.

This study successfully identified a strong correlation between olive fly infestation, the intensity of the "grubby" sensory defect, and specific volatile compounds in Leccino olive oil. The early processing and avoidance of storage minimized interferences from other defects commonly associated with olive fly infestation, allowing a more precise characterization of the "grubby" defect and its chemical origins. The identified volatile markers are likely a result of both oxidative and fermentative processes induced by the fly infestation, along with the release of terpenes as a plant defense response. The combined contribution of these volatile compounds likely shapes the perception of "grubby" as a complex, multi-faceted flavor profile. This research provides significant insight into the chemical basis of this important olive oil defect, offering potential for instrumental support of sensory evaluation. Specifically, these volatile markers could be used to develop rapid and objective methods for detecting and quantifying the "grubby" defect in olive oil samples.

This study demonstrates a clear link between olive fly infestation and the "grubby" defect in olive oil, identifying key volatile markers associated with its occurrence and intensity. The findings refine the sensory description of the defect and suggest a chemical basis involving a combination of oxidative and fermentative degradation products, along with defense-related terpenes. This research contributes to a more comprehensive understanding of olive oil quality and provides valuable tools for instrumental support of sensory analysis in olive oil classification and quality control. Further research on other cultivars is recommended to confirm the generalizability of these findings and expand the database of volatile markers associated with the "grubby" defect.

This study focused on the Leccino cultivar, limiting the generalizability of the findings to other olive cultivars. The specific conditions of infestation (ripening stage, time between harvest and processing) may also have influenced the results. The study did not investigate the specific microbial populations involved in the fermentative processes, and further research to identify these microbes would enhance our understanding. The relatively small sample size could affect the statistical power of the analysis.