Genetic and chemometric properties of Maltese olive oils : developing an analytical profile for protected determination of origin certification

Abstract

The determination of the singularity of the Maltese olive oil provides an opportunity for local producers to purpose a PDO certification. This objective of this study was to identify chemical and genetic parameters which enable the discrimination of Maltese olive oils. Prior to the identification of markers, a preliminary survey of quality was conducted whereby it was shown that monocultivar olive oils grown locally were of sufficiently high quality to be classified as extra virgin olive oils. The minor constituents present in EVOOs were subjected to a more detailed study as chemical markers. Phenolic compounds were extracted and quantified using microtiter assays. The antioxidant activity of these extracts was determined using different redox-based assays including FRAP, ACC and CUPRAC assays whilst the radical scavenging activity towards different radicals, including DPPH, ABTS and NO, was determined. It was shown that EVOOs derived from the indigenous cultivars had a significantly lower TPC and TFC whilst no significant difference was observed in the TdPC when compared to EVOOs from other Mediterranean countries. The low TPC and TFC reflected the significantly lower antioxidant and radical scavenging activity of the indigenous cultivars. Whilst microtiter plate assays showed that the indigenous cultivars had a lower phenolic content, application of HPLC for phenolic profiling revealed that both the indigenous and locally grown foreign cultivars had a significantly higher content of six phenolic compounds, namely p-coumaric acid, tyrosol acetate, 3,4 DHPEA-EDA, p-HPEA-EDA and two unidentified compounds. Elemental characterisation via the application of semi-quantitative XRF analysis enabled discrimination not only between the geographical origin of the EVOOs but also between EVOOs and other refined seed oils. Application of classical statistics and chemometrics on the phenolic profiling showed that geographical discrimination between the different samples was possible. The application of chemometric techniques namely PCA, PLS-DA, PNN, SLC-DA on data derived from different chemical techniques namely fluorescence (SEEF and EEM), FTIR, NMR and direct infusion mass spectrometry not only enabled the complete discrimination of the different EVOOs but enabled the identification of markers which had the highest discriminatory power. In the case of SEEF spectroscopy, compounds having emissions in the 380-480 nm and 650-700 nm ranges were found to have the greatest discriminating power, as confirmed through the use of 3-way chemometric analysis (PARAFAC and N-PLS) on the EEM offering comparable results to those obtained using SEEF. For NMR, 1H chemical shifts in the 3.5-4.5, 6.7-7.0 and 9.1-9.5 ppm had the most discriminate power and were attributed to the presence of glycerides, terpenic, phenolic and carbonyl containing compounds. In the case of DI-MS under positive ESI, it was found that the data obtained was highly redundant, nonetheless minor phenolic compounds identified through their m/z values were found to be the most discriminate. FTIR spectroscopy was found to be one of the most effective chemical fingerprinting methodologies owing to its simplicity, with compounds having functional groups which vibrated at 500-1500 cm-1 being the most discriminate. Application of genetic analysis on the three indigenous cultivars using SSR markers revealed that cultivar discrimination was possible via the use of two most informative markers namely DCA-3 and GAPU 101. Analysis of these markers on genomic DNA showed that the ‘Bidni’ cultivar consisted of a homozygous population whereby the individual trees are clones of each other showing a very similar allelic pattern to olive cultivars cultivated in northern Tunisia. In the case of the ‘Malti’ cultivar, the population was highly heterozygous, showing very similar allelic patterns to Spanish, Greek and Southern Italian cultivars, suggesting a multi-cultivar population coined under the same nomenclature. In the case of ‘Bajda’ at the two loci studied this could not be distinguished from another Leucocarpic cultivar grown in southern Italy baring the name of Morachia, Cannellina, Bianca, and Chiarita. From the results obtained it was concluded that the authenticity of the Maltese EVOO can be defined using both genetics analysis and chemical fingerprinting methods.