The results of quality parameters and composition of the VOOs after clarification by VCS and CBST at 24 and 48 h are shown in Table 2. For the first and third crop years the HSSB oils showed lower moisture content, and then, the oil clarified by CVS and CBST, showed similar residual moisture. However, when the HSSB oil showed higher moisture (as for the second crop year), the clarification achieved by each system was different. VCS was more efficient since the oil moisture was reduced until 0.51%. CBST at 24 h produced oils with higher moisture (1.03%) although after settling 48 h moisture values were close to those obtained by VCS. Solid organic impurities were removed by both clarification systems until similar values for the three crop years.
For clarification efficiency calculation has been used the sum of moisture and organic solid impurities (MSOI). VCS was more efficient as HSSB oil had higher MSOI content (second year), obtaining a clarification efficiency of 96.6%. Those HSSB oils with lower MSOI content achieved clarification efficiencies comprised between 70.63 and 56.48% for the first and third crop year, respectively. Clarification by CBST at 24 h showed similar efficiency than VCS for those HSSB oils with lower MSOI CBST after 48 h of settling showed a slight increase of clarification efficiency regarding to clarification by CBST at 24 h. In general, for both systems the oil clarification degree was conditioned
by MSOI content of the initial HSSB oil, reaching higher clarification efficiency valúes for those oils with higher MSOI content.
Regarding to the quality parameters (FA, PV, K232, and K270), all the clarified oils, were classified into the category «extra-virgin» as established by the EU regulation (EUC, 2013). K232 and K270 indices were not affected by the clarification systems. These results did not agree with the marked increase of the oxidative parameters reported when the oil was clarified by vertical centrifugation [6]. The clarification using VSC, with minimal water addition, did not affect significantly the oxidative state of the VOO comparing with the CBST clarification systems applied in this work.
FAEEs content have been adopted recently as quality parameter to distinguish EVOOs from those VOOs of lower quality. According to the limits established in the last modification of the Regulation no 2568/91/EEC [19] HSSB oil as much as the clarified oils were classified into the category extra-virgin (<3 mg/kg) for this parameter.
In general, oil tocopherol content did not show significant differences between the clarification systems assayed. The a-tocopherol was the major tocopherol detected in «Picual» VOO according to Beltrán et al. [20]. These compounds are natural antioxidants present in the VOO and are included in the health claims by European Union 1924/2006 [21].
The pigments (carotenoids and chlorophylls) are mainly responsible for the color of virgin olive oil and play an important role in the VOO oxidative stability [22]. The carotenoids are effective inhibitors of photo-oxidation by quenching singlet oxygen and chlorophyll can transfer energy from light to triplet oxygen, producing thus singlet oxygen, which then reacts with the unsaturated fatty acids [23-24]. These compounds were determined in the oils from the two last crop years. The carotenoid content was not affected by the clarification systems studied. However, the chlorophylls showed different behavior for each year. Whereas, for the second crop year differences between clarification systems were not observed, for the last year oil clarified by CBST showed a reduction of the chlorophylls content at 24 and 48 h. Nevertheless, for this last year, the chlorophylls were not affected after clarification by VCS as described by Masella et al. [6].
In general, VOO clarification produced a phenol content reduction. VCS gave oils with higher phenol content achieving significant differences respect to the CBST oils, differences between settling time were not observed. Oils clarified by VCS showed slight phenol losses (<15%) whereas, for those settled for 24 h the losses varied between 42 and 13%for the first and third crop years. Oil settling for 48 h did not produce additional phenol losses. Jiménez et al. [5] described a decrease of the phenol content as water addition to vertical centrifugation as water addition was higher (1:1- oil:water). This reduction can be explained by the partition coefficients of phenols between oil and water [25]. Because in this work the water amount added to VCS was reduced until the minimum for processing, it may explain the slight reduction of oil phenolics observed. These results agreed with Masella et al. [6] that reported a similar behavior, when water addition was reduced until 0.25Lwaterper liter of oily must. For natural decantation previous works were not published for our best of knowledge. However, settling may sweep along VOO phenolics explaining their reduction for longer settling time.
The bitterness was evaluated by absorbance at 225nm, VCS oil showed higher bitterness than CBST oils at 24 h. Longer settling time did not affect oil bitterness. The reduction of bitterness of the clarified oils can be explained by the reduction of phenols since they are related [26].
Individual phenols and secoiridoids content of both HSSB and clarified oils for the third crop year are shown in Fig. 2. In general, for simple phenols (Fig. 2A), oil clarification by VCS generated a slight increase respect to HSSB oils. CBST clarification reduced simple phenols, showing higher reductions for hydroxytyrosol and tyrosol. As settling time was longer their reduction was more important.
For secoiridoids (Fig. 2B), clarification by VCS did not affect their concentration. However CBST produced a reduction for the compounds identified. Only for 3,4- DHPEA-EDA was observed significant differences between clarification systems, achieving losses up to 66 and 67% after 24 and 48 h of settling, respectively. Longer settling time did not affect to oil secoiridoids concentration. The reduction of secoiridoids, during the natural settling, may be explained due to hydrophilic character of the phenols, which were washed by the water during the settling process by a drag effect. However, the phenols content was not affected when the oil was clarified by VCS, since the water amount was reduced until the minimum for processing, and then, the washing effect could be avoided. By contrast, Masella et al. [6] reported a decrease in the simple phenols (Hydroxytyrosol and Tyrosol) content in oils clarified by vertical centrifugation, although higher water amounts were used in their experiments.
Figure 2. Effect of clarification systems (VCS, CBST at 24 h, and CBST at 48 h) on VOOs (A) simple phenols and (B) secoiridois content.
The phenolic compounds are strongly related to the VOO shelf life because of their antioxidant ability and bioactive activities. They are responsible of the inclusion of the VOO on the nutrition and health claims made on foods by European Union [21]. The VCS showed a better phenolic preservation than CBST, obtaining oils with higher quality.
The sensory characterization and classification of HSSB and clarified oils according to EU regulation and clarified oils are shown for the second and third crop year (Table 3). The oils taken from HSSB were classified as «extra virgin» category for both years. For the second crop year the clarified oils by VCS were classified as «extra virgin», whereas those clarified by CBST at 24 and48 h were classified as «virgin,» since «winey- vinegary» defect was detected for an intensity of 2 and 2.5, respectively. For the last crop year, clarified oils by VCS, CBST at 24 h, and CBST 48 h were classified as «extra virgin» category. In general, the effect of clarification on sensory characteristics was different depending of the system used. For VCS the fruity intensity did not change whereas, bitterness and pungency showed a slight increase. Vertical centrifugation gave higher intensities of fruity, bitterness and pungent than CBST oils at 24 h, especially for the 2011/2012 crop year. Longer settling time (48 h) gave oils with positive attributes at intensities similar to those clarified by VCS.
Table 3. Effect of clarification systems (VCS, CBST at 24h and CBST at 48h) on the main sensory attributes of the HSSB oils and category according to UE regulation for the three crop years.
Attention should be paid in the detection of the winey/vinegary sensory defect in the oils clarified by CBST for the second crop year. Its intensity increased for longer settling time. The appearance of this defect may be explained by the high MSOI content still remained that could not be removed by the purges. The MSOI may help to fermentations and therefore to the appearance of this sensory defect.
4. Conclusions
The VCS with a minimal water addition can be considered a better option to produce VOO of improved quality, since carried out a quick and efficient separation of the MSOI, reducing the contact between oil and the remanent water and impurities during the storage stage. Concerning the VOO quality parameters and sensorial characteristics no significant difference were observed between both clarification systems and no alteration was noted. The oil composition (tocopherols and pigments) was not affected by the VCS and CBST, however the phenolic compounds were affected by the clarification systems, and more precisely by CBST were those compounds decreased between 13 and 42% especially for the hydroxytyrosol, tyrosol and secoiridoids. While, the VCS allowed the conservation of these compounds, which could lead to prolong its shelf-life during the storage.
Acknowledgements
This work was supported by, a fellowship from Ministry of Science and Innovation (Spain) associated to the project FPI-INIA RTA2009-00002-00-0, the grant CAICEM11- 67 with the company Pieralisi España SL and the project ‘PI 26323’ from ‘Consejería de Innovación, Ciencia y Empresa’ of the ‘Junta de Andalucía’ (Spain). The authors gratefully acknowledge their financial support.