On the Squalene Content of CV Chondrolia Chalkidikis and Chalkidiki (Greece) Virgin Olive Oil.

This work is a continuation of efforts to establish the nutritional profile of virgin olive oil (VOO) from cv. Chondrolia Chalkidikis and Chalkidiki and to strengthen its positioning in the global VOO landscape. VOOs produced at an industrial scale in different olive mills of the Chalkidiki (Greece) regional unit as well as VOOs obtained at the laboratory scale from drupes of different maturity stages for four consecutive harvesting years were examined for their squalene (SQ) content using both HPLC and GC procedures. The mean values of SQ were found to be 4228 (HPLC) and 4865 (GC) mg/kg oil (n = 15) and were of the same magnitude as that in VOOs from cv Koroneiki (4134 mg/kg, n = 23). Storage of VOOs in the dark at room temperature for 18 months indicated an insignificant mean SQ content loss (~2%) in comparison to a mean loss of 26% for alpha-tocopherol content. This finding strengthens our view that SQ does not act as a radical scavenger that donates hydrogen atoms to the latter. The four consecutive harvest years studied indicated a clear declining trend in VOO SQ concentration upon olive ripening. To our knowledge, this is the first systematic work concerning the SQ content of Chondrolia Chalkidikis and Chalkidiki VOOs.

The Potential of Virgin Olive Oil from cv. Chondrolia Chalkidikis and Chalkidiki (Greece) to Bear Health Claims according to the European Legislation.

The European food legislation authorizes the use of certain health claims based on a scientific basis. This study aimed to evaluate the fatty acid, tocopherol, and polar phenol composition of virgin olive oil (VOO) from cv. Chondrolia Chalkidikis and Chalkidiki regarding the fulfillment of official requirements for the health claims of 'oleic acid', 'vitamin E', and 'olive oil polyphenols'. The examination of representative industrial VOOs from 15 olive mills of the Chalkidiki regional unit showed that the two cultivars yield oils contained the necessary concentrations of the responsible bioactive compounds. This evidence was further substantiated by a four harvest study whereby olives from different maturity stages were sampled from three olive groves. Oils were extracted at a laboratory scale and examined for their content in the above-mentioned three categories of constituents. Oils produced at industrial scale from olives harvested on the 'technological optimum' stage according to the olive grove proprietor were also analyzed. Extra virgin olive oil of the studied cultivars can safely bear the generic claims for 'oleic acid' and 'vitamin E'. The cultivars present great potential regarding the total hydroxytyrosol and tyrosol content of the extracted oil required to attain the third health claim that may be influenced negatively by manufacturing practices.

Quality aspects of European virgin olive oils with registered geographical indications: Emphasis on nutrient and non-nutrient bioactives.

European virgin olive oil with geographical indications are strictly regulated and are of importance for the producing member states, Spain, Italy, Greece, Portugal, France, Slovenia and Croatia. These products are consumed locally, or within the European Union but are also exported worldwide. The chapter stresses on the importance of combining origin indications with other certifications or opportunities raising from European legislation in the agri-food sector so that to tighten consumer loyalty for this category of products. Emphasis is given to the richness of virgin olive oil in bioactive compounds that are already covered by nutritional and health claims (oleic acid, vitamin E, "polyphenols") and to those compounds that can be exploited in the future toward the same direction (squalene, oleanolic and maslinic acids).

Toward a Harmonized and Standardized Protocol for the Determination of Total Hydroxytyrosol and Tyrosol Content in Virgin Olive Oil (VOO). The Pros of a Fit for the Purpose Ultra High Performance Liquid Chromatography (UHPLC) Procedure.

Τoward a harmonized and standardized procedure for the determination of total hydroxytyrosol and tyrosol content in virgin olive oil (VOO), the pros of a recently published in house validated ultra high performance liquid chromatography (UHPLC) protocol are discussed comparatively with those of other procedures that determine directly or indirectly the compounds hosted under the health claim on "olive oil polyphenols" (EC regulation 432/2012). Authentic VOOs were analyzed with five different liquid chromatographic separation protocols and 1H-NMR one in five different laboratories with expertise in VOO phenol analysis within three months. Data comparison indicated differences in absolute values. Method comparison using appropriate tools (Passing-Bablok regression and Bland Altman analyses) for all protocols vs. the UHPLC one indicated slight or statistically significant differences. The results were also discussed in terms of cost effectiveness, detection means, standard requirements and ways to calculate the total hydroxytyrosol and tyrosol content. Findings point out that the in-house validated fit for the purpose UHPLC protocol presents certain pros that should be exploited by the interested parties. These are the simplicity of sample preparation, fast elution time that increase the number of samples analyzed per day and integration of well-resolved peaks with the aid of only two commercially available external standards. Importance of correction factors in the calculations is stressed.

In House Validated UHPLC Protocol for the Determination of the Total Hydroxytyrosol and Tyrosol Content in Virgin Olive Oil Fit for the Purpose of the Health Claim Introduced by the EC Regulation 432/2012 for "Olive Oil Polyphenols".

An ongoing challenge in olive oil analytics is the development of a reliable procedure that can draw the consensus of all interested parties regarding the quantification of concentrations above the required minimum value of 5 mg of bioactive "olive oil polyphenols" per 20 g of the oil, to fulfill the health claim introduced by the European Commission (EC) Regulation 432/2012. An in-house validated ultra-high performance liquid chromatography (UHPLC) protocol fit for this purpose is proposed. It relies on quantification of the total hydroxytyrsol (Htyr) and tyrosol (Tyr) content in the virgin olive oil (VOO) polar fraction (PF) before and after acidic hydrolysis of their bound forms. PF extraction and hydrolysis conditions were as previously reported. The chromatographic run lasts ~1/3 of the time needed under high performance liquid chromatography (HPLC) conditions, this was also examined. Eluent consumption for the same piece of information was 6-fold less. Apart from being cost effective, a larger number of samples can be analyzed daily with less environmental impact. Two external curves, detection at 280 nm and correction factors for molecular weight difference are proposed. The method, which is fit for purpose, is selective, robust with satisfactory precision (percentage relative standard deviation (%RSD) values < 11%) and recoveries higher than 87.6% for the target analytes (Htyr, Tyr). Standard operational procedures are easy to apply in the olive oil sector.

Addressing analytical requirements to support health claims on "olive oil polyphenols" (EC Regulation 432/2012).

A health claim on "olive oil polyphenols", namely, hydroxytyrosol and its derivatives, was recently approved by EC Regulation 432/2012. As no official method exists so far for the complete separation of all forms of hydroxytyrosol and tyrosol present in the oil, it is of utmost importance to find a simple, reproducible, and undisputable protocol to protect consumers and avoid unfair competition. In this study two hydrolysis protocols for the complex forms either in the polar fraction of the oil (protocol 1) or directly in the oil (protocol 2) were comparatively applied to a series of extra virgin olive oils. Protocol 1 gave at least 50% higher levels of total hydroxytyrosol and tyrosol. Nevertheless, the minimum amount of 5 mg/20 g oil of phenols was fulfilled only when suitable correction factors were introduced in the calculations to account for the differences in the mass between simple and complex phenols. Changes in the terminology used in the health claim are also proposed.