Microsoft Word - 274-2068-2-LE-rev ACTA IMEKO  ISSN: 2221‐870X  April 2016, Volume 5, Number 1, 22‐31    ACTA IMEKO | www.imeko.org  April 2016 | Volume 5 | Number 1 | 22  Variations of phenolic compounds and sensory properties of  virgin olive oils from the variety “Istrska belica”  Milena Bučar‐Miklavčič 1 , Terezija Golob 2 , Vasilij Valenčič 1 , Erika Bešter 1 , Bojan Butinar 1 , Ana   Miklavčič Višnjevec 1   1  UP ZRS IZO; LABS d.o.o.,  Zelena ulica 8 c, Izola, Slovenia  2  Univerza v Ljubljani,  Biotehniška fakulteta, Jamnikarjeva 101, Slovenija      Section: RESEARCH PAPER   Keywords: olive oil; phenolic compounds; sensory properties; “Istrska belica”  Citation: Milena Bučar‐Miklavčič, Terezija Golob, Vasilij Valenčič, Erika Bešter, Bojan Butinar, Ana  Miklavčič Višnjevec, Variations of phenolic compounds and  sensory properties of virgin olive oils from the variety “Istrska belica”, Acta IMEKO, vol. 5, no. 1, article 6, April 2016, identifier: IMEKO‐ACTA‐05 (2016)‐01‐06  Section Editor: Claudia Zoani, Italian National Agency for New Technologies, Energy and Sustainable Economic Development affiliation, Rome, Italy  Received May 29, 2015; In final form November 10, 2015; Published April 2016  Copyright: © 2016 IMEKO. This is an open‐access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits  unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited  Funding: This work was supported by EU founded project Uelije  Corresponding author: Ana M. Višnjevec, e‐mail: ana.visnjevecmiklavcic@zrs.upr.si    1. INTRODUCTION  The olive (Olea europaea L.) variety “Istrska belica” is the most widely spread olive variety in Slovenian olive groves. The quality of the olive oil from “Istrska belica” is distinguished by its rich aroma, which reminds the user of the fresh, optimal ripe olive fruit, combined with freshly mown grass. The high content of phenolic compounds gives the “Istrska belica” oil its characteristic bitter taste and pungent tactile sensation. Diets containing the phenolic compounds found in olive oil can have health benefits, which include the reduction of risk factors for coronary heart disease, protection against several types of cancers, and modification of immune and inflammatory responses [1]-[19]. According to European Union Regulation (UE) 432/2012, phenolic compounds can be cited according to the indication: “Olive oil phenolic compounds contribute to the protection of blood lipids from oxidative stress”. This claim demonstrates the importance of these phenolic compounds. The phenolic compounds in olive oil are secondary metabolites that arise through the conversion of complex substances produced by olive trees, and they can be classified as lignans, flavonoids and secoiridoids. Virgin olive oil contains at least 30 different phenolic compounds [1]. The most common lignans in olive oil are pinoresinol, acetoxypinoresinol and hydroxypinoresinol [11], and the most common flavonoids are luteolin and apigenin [20]. While lignans and flavonoids are also in other foods, such as wine, secoiridoids are specific for olive ABSTRACT  The olive variety “Istrska belica” is well known for its numerous positive properties, such as resistance to low temperature and high oil  content.  The  aim  was  to  determine  the  variations  in  the  levels  of  phenolic  compounds  and  sensory  properties  during  storage  of  “Istrska belica” virgin olive oil. The profile of the phenolic compounds and sensory properties of “Istrska belica” olive oil were further  compared with those for other varieties,  including “Leccino” and “Maurino”. The content of phenolic compounds of the olive oils  decreased  after  1  year  and  2  years  of  storage.  After  2  years  of  storage,  the  levels  of  oleuropein  and  the  ligstroside  derivatives  significantly decreased, while the end‐stage compounds tyrosol and hydroxytyrosol increased. These data show that after 1 year of  storage, the “Istrska belica” olive oil preserves similar intensities for bitterness and pungency, and similar oleuropein and ligstroside  derivatives levels. In contrast to the other oils, the intensities of bitterness and pungency of “Istrska belica” olive oil decreased greatly  only after 2 years of storage. Moreover, the phenolic compounds content, and oleuropein and ligstroside derivatives levels, and the  intensities of bitterness and pungency were highest in fresh “Istrska belica” olive oil, compared to other olive oils analysed. Overall,  “Istrska belica” olive oil has important advantages over olive oil from other varieties that are grown in the Istria region.  ACTA IMEKO | www.imeko.org  April 2016 | Volume 5 | Number 1 | 23  oil [21], [22]. The two main secoiridoids in olive oil are ligstroside and oleuropein, and their conversion products give olive oil its unique aroma and taste. During the olive-pressing process or if the drupes are injured, ligstroside and oleuropein in the fresh drupes can enter different transformation-reaction pathways, such as their enzymatic and chemical transformation to aldehyde or hydroxy forms [23]. One possible transformation pathway is autooxidative decay as a consequence of the protection of phenolic compounds against harmful oxidative changes [24]. This conversion is gradual and continues in the olive oil throughout its use. During olive oil storage, hydrolytic mechanisms that lead to the release of simple phenols, such as hydroxytyrosol and tyrosol, from the complex phenols, such as the secoiridoids, can occur [25]-[27]. The newly formed substances from these secoiridoids have amphiphilic characteristics, and are thus partitioned between the oily layer and the vegetation water, and are concentrated in the water fraction through their polar functional group [1]. As long as these secoiridoids do not get transformed into their final forms (i.e., the aromatic alcohols tyrosol and hydroxytyrosol), the olive oil preserves its freshness, fruitiness and harmony. It should be noted that when the transformation pathway is reaching its end and the olive oil has already lost its freshness and antioxidative properties, the content of total phenolic compounds can be relatively high. Therefore, it is crucial to determine the relative amounts of each of these compounds, and not just the total sum of all of the phenolic compounds. Oxidation of olive oil begins as soon as it has been extracted. Endogenous enzymes in the olive fruit are involved in the phenolic profile and in its qualitative and quantitative modification during the processing of virgin olive oils [28]. However, olive oil is more severely affected during its storage. The decomposition of the phenolic compounds depends on oxygen, light, temperature, metals, pigments, unsaturated fatty acid content and composition, and quality and kind of natural antioxidants present [29], [30]. Part of the low quantity of water in virgin olive oil is free and available for chemical and enzymatic reactions. This low quantity of water keeps hydrophilic phenols in solution, which is where the decomposition process can occur for the phenols and triacylglycerols during olive oil storage [31], [1]. Furthermore, according to Frankel [30], the hydrophilic antioxidants, such as the polar phenols that are oriented at the air-oil interface, can better protect against oxidation compared to the lipophilic antioxidants, like the tocopherols, which remain in solution in the olive oil. Moreover, Aparicio et al. [32] measured the correlation between the oxidative stability of virgin olive oil and several compositional variables. They showed that phenols, o- diphenols, and the oleic/ linoleic ratio have the highest stability values, followed by chlorophylls, total tocopherols and caratenoids. According to Tsimidou et al. [33], hydroxytyrosol is the most active antioxidant compound in virgin olive oil. Carrasco-Pancorbo et al. [34] showed that the hydroxytyrosol oleuropein-aglycone di-aldehyde (3,4-DHPEA-EDA; i.e., elenoic acid linked to hydroxytyrosol) and oleuropein aglycon have the strongest antioxidant power. Elenoic acid, which does not have a phenolic ring, was one of the compounds investigated in virgin olive oil that has the weakest antioxidant activity. Extra virgin olive oil can be characterized by a unique combination of aroma and taste that is highly appreciated [35], [36]. The method of sensory evaluation of virgin olive oils, introduced in 1991 by Commission Regulation (EEC) No. 1348/2013 annex XII (revision of Reg. EEC 2568/91), lays down the procedure for evaluating the sensory attributes of virgin olive oils and quality classification (categorization). It specifies the criteria for sensory evaluation of virgin olive oil, as well as providing a special vocabulary and standardized conditions for evaluation. However, the panel test, performed according to EU procedures, it is an expensive process that requires accredited assayers and is not easily accessible to the companies that have limited production, and therefore alternative tools have been developed [37]. Many studies have tried to clarify the relationships between the sensory attributes in virgin olive oil and the phenolic compounds that are responsible for its aroma and taste [1], [36], [38]-[41]. Some studies have suggested that secoiridoid derivatives of hydroxytyrosol are the main contributors to olive oil bitterness [1]. Caponio et al. [38] showed that the bitter to pungent taste can be ascribable to oleuropein aglycon. Furthermore, oleuropein and its aglycon decrease as the ripening of olives progresses [38]. Rotondi et al. [39] confirmed the relationship between the decrease in bitterness and pungency and the reduction in total phenols and diphenol levels. In particular a positive correlation between the content of oleuropein and ligstroside derivatives and the bitterness and pungency was shown. Frank and co-authors [40] reported that when an isomer (or isomers) of oleuropein aglycon was prepared by β- glucosidase hydrolysis of oleuropein isolated from olives and evaluated by assessors, it was defined as bitter. Using the same evaluation technique, no bitterness was observed for hydroxytyrosol or elenolic acid. According to Andrewes et al. [41], the dialdehyde form of decarboxymethyl ligstroside aglycone (p-HPEA-EDA) is the key source of the pungent sensation found in olive oil, while 3,4-DHPEA-EDA produces very little burning sensation. Moreover, Beauchamp et al. [5] assessed the pungent intensity of p-HPEA-EDA isolated from different virgin olive oils, and confirmed that p-HPEA-EDA is the principal agent responsible for throat irritation. Gutierrez- Rosales et al. [42] concluded that the chromatographic peaks corresponding to 3,4-DHPEA-EDA, oleuropein-aglycone mono-aldehyde (3,4-DHPEA-EA) and p-HPEA-EDA are mainly responsible for the bitter taste of virgin olive oil. Overall, some phenols mainly define the bitterness of olive oil, while others define the perception of pungency, and these might be related to the olive variety. In the present study, the levels of 14 phenolic compounds and the total sum of all of the phenolic compounds included were determined in 167 samples. In all of the samples, an International Olive Council (IOC) recognized panel tested the sensory properties, as the bitterness, pungency, olive fruity, defects and sensations that resemble olive fruit, green-leaf, tomato, almond, artichoke and vanilla. The aim of the present study was to determine the changes in the phenolic compounds and sensory properties during the storage of the oil of the “Istrska belica” variety, which is specific and very important for the region of Istria. The phenolic profile and sensory properties of “Istrska belica” oil were further compared to the phenolic compounds and sensory properties determined for the oil of other varieties grown in the study area. In addition, the aim was to define the correlations between the content of phenolic compounds and the sensory properties of the fresh oil, and after 1 year and 2 years of storage. To the best of our knowledge, this is the first investigation into the effects of storage on the content of the phenolic compounds and the ACTA IMEKO | www.imeko.org  April 2016 | Volume 5 | Number 1 | 24  sensory properties as tested for the “Istrska belica” variety, the oil of which has a characteristic high content of phenolic compounds and a bitter taste. 2. METHODS  2.1. Collection and storage of samples  More than 80 samples of fresh olive oil were randomly collected from Slovenian producers. During this 3-year project, the determination of the phenolic compounds and the sensory analyses were carried out on both fresh oil and stored oil produced in Slovenia. Therefore, the first year samples (2011) were analysed after 1 year and 2 years of storage, and the second year samples (2012) were analysed after 1 year of storage. Olive fruit samples were hand-picked from olive trees at the same optimal maturity level for each cultivar taking into consideration the index of maturity and the hardness of the fruit [43]. Healthy fruits, without any kind of infection or physical damage, were used for the oil production. The collected samples of extra virgin olive oils were produced using modern production technology with a two-phase decanter. During the production process the temperature was monitored and maintained at 27 °C. The oils were produced according to good manufacturing practice guide [44]. The chemical analyses of fresh olive oil samples were performed after the extraction process in each particular year. All the olive oil samples were stored at 20°C in the closed dark bottles in the same place. 2.2. Sensory analysis  The sensory characteristics were determined by a panel composed of eight trained assessors. The sensory properties of fruitiness, bitterness, pungency and other specific characteristics of selected oils were assessed based on the method defined in Annex XII of EEC Regulation No 2568/91, 640/2008, which includes the use of a 10-cm linear scale for intensity determination. 2.3. Determination of phenolic compounds  The phenolic compounds in the olive oil, such as the natural and oxidised derivatives of oleuropein and ligstroside, lignans, flavonoids and phenolic acids, were extracted using 60 % (w/w) aqueous methanol solution, and analysed by reverse phase high- performance liquid chromatography (HPLC), according to COI/T.20/Doc No 29 [42]. The HPLC system (Agilent 1100) was equipped with a thermostated autosampler, a binary pump system (BinPump G1312A), and a diode array detector (G1315B). A Phenomenex Synergi 4 µm Hydro-RP 80 Å column (250 × 4.6 mm i.d.; Torrance, CA, USA) was used. The analyses were performed according to a modified method published by the IOC [42]. Detection was at 280 nm, with the exception of the flavonoids luteolin and apigenin, which were detected at 340 nm. Calibration curves for tyrosol (mass fraction from 30 to 800 mg/kg; y = 0.0811a) were constructed using standard compounds. All of the phenolic compounds were determined according to the IOC publication and quantified using the response factor for tyrosol [42]. In this study, the following groups of phenolic compounds were determined: 1) The sum of oleuropein and the ligstroside derivatives; 2) the sum of tyrosol and hydroxytyrosol; 3) the sum of oleuropein, the ligstroside derivatives, tyrosol, hydroxytyrosol, lignans and phenolic acids; and 4) the total phenolic compounds (mg/kg). The term “total phenolic compounds” refers to the “biophenolic minor polar compounds” determined according to the IOC method [45]. 2.4. Statistical analysis  All of the data were analysed using the STATA13/SE software. The distribution of the total phenolic compounds, oleuropein, the ligstroside derivatives, tyrosol and hydroxytyrosol determined in the fresh olive oils and after 1 year and 2 years of storage are given as box plots. The normality of the variable distributions was determined using Shapiro–Wilk tests. The correlations between the levels of the determined phenolic compounds and the sensory parameters were evaluated. Spearman rank correlations were used for bivariate comparisons. Due to the significant correlations between the variables of the different phenolic compounds, factor analysis was applied. After running the factor analysis, rotation of the factor loads was performed to provide a clearer pattern. From the different concentraions of the specific phenolic compounds, new variables were created (n=5) for “eigenvalues” >1. Wilcoxon–Mann–Whitney tests were applied for comparisons of two different groups, and Kruskal Wallis tests were applied for comparisons of three different groups. The level of statistical significance was set to p <0.05. 3. RESULTS AND DISCUSSION   3.1. Phenolic compounds  The phenolic compounds were quantified using the response factor for tyrosol [41]. The sum of oleuropein, the ligstroside derivatives, tyrosol, hydroxytyrosol, lignans and phenolic acids (1), the sum of oleuropein and the ligstroside derivatives (2), and the sum of tyrosol and hydroxytyrosol (3) determined in all the fresh olive oils from three years of sampling varied from 145 mg/kg to 966 mg/kg (median, 417 mg/kg), 83 mg/kg to 584 mg/kg (median, 251 mg/kg), and 2 mg/kg to 97 mg/kg (median, 9 mg/kg), respectively. It is important to note that the composition of the phenolic compounds can vary widely according to each olive variety. The contents of the flavonoids luteolin and apigenin in the fresh olive oil from “Istrska belica” were in the ranges of 2.6 mg/kg to 5.8 mg/kg (n = 20) and 0.9 mg/kg to 1.9 mg/kg (n = 20), respectively. The same flavonoids contents for “Leccino” oil ranged from 1.5 mg/kg to 4.3 mg/kg (n = 11) and 0.3 mg/kg to 0.8 mg/kg (n = 11), and for “Maurino” oil they ranged from 0.8 mg/kg to 2.0 mg/kg (n = 4) and 0.2 mg/kg to 0.6 mg/kg (n = 4). The “Istrska belica” oil lignans ranged from 22 mg/kg to 70 mg/kg (n = 20), with “Leccino” oil as 11 mg/kg to 33 mg/kg (n = 15), and “Maurino” oil as 46 mg/kg to 51 mg/kg (n = 6). The dialdehyde forms of both decarboxymethyl oleuropein aglycone (DMO-Agl-dA) and decarboxymethyl ligstroside aglycone (DML-Agl-dA) for “Istrska belica” oil varied from 23 mg/kg to 124 mg/kg (n = 20), with “Leccino” from 17 mg/kg to 274 mg/kg (n = 15), and “Maurino” from limit of detection (LOD) to 119 mg/kg (n = 6). The oxidised aldehyde and hydroxyl forms of oleuropein aglycone (O-Agl- dA) and ligstroside aglycone (L-Agl-dA) were lower compared to DMO-Agl-dA and DML-Agl-dA, and for “Istrska belica” oil they varied from 5.6 mg/kg to 87 mg/kg (n = 20), with “Leccino” from 1.5 mg/kg to 21 mg/kg (n = 15), and “Maurino” from