Research Article. Improvement of sensorial and volatile profiles of olive oil by addition of olive leaves. 1 Introduction

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1 Eur. J. Lipid Sci. Technol. 2017, 119, (1 of 11) Reserch Article Improvement of sensoril nd voltile profiles of olive oil y ddition of olive leves Ricrdo Mlheiro 1,2, Nuno Rodrigues 1, Cmil Bissro 3, Fernnd Leimnn 3, Susn Csl 2, Els Rmlhos 1 nd Jose Alerto Pereir 1 1 Centro de InvestigSc~o de Montnh (CIMO), ESA, Instituto Politecnico de BrgnSc, Cmpus de Snt Apoloni, BrgnSc, Portugl 2 REQUIMTE/LAQV/Lortorio de Bromtologi e Hidrologi, Fculdde de Frmci, Universidde do Porto, Porto, Portugl 3 Technologicl Federl University of Prn, Cmpus Cmpo Mour~o, Cmpo Mour~o, Prn, Brzil The chnges cused y the ddition of olive leves (0, 5, nd ) during the extrction of olive oil nd mlxtion time (20, 30, nd ) in the voltile profile nd sensory ttriutes of olive oil from cv. CornScos were studied. To investigte such trnsformtions, centrl composite designs from the Response Surfce Methodology (RSM) ws used, retrieving 13 runs comining lef percentges nd mlxtion times. Ech run ws extrcted in triplicte (39 olive oils overll). Sensory ttriutes were improved to leves ddition, minly green nd fruitiness ttriutes in olfctory nd gusttory-olfctory senstions, ut high mlxtion times (>) reduced pungent nd itter notes. Leves ddition incresed the mounts of totl voltiles, prticulrly the GLV s (green leves voltiles) (E)-2-hexenl, (Z)-3-hexenl, nd (Z)-3-hexenyl cette, directly correlted with the improved sensory ttriutes. RSM models showed positive liner effect with leves ddition, ut negtive effect with mlxtion time. These results suggest the use of olive leves s effective odornts for the olive mill industry, while enling the reduction of mlxtion times nd y-product mounts. Prcticl pplictions: The results otined clerly open new lines of reserch to use olive leves, su-product of olive oil extrction, in vlule wy. Olive leves cn e used s nturl sources of odornts for olive oils. Furthermore, their use during the extrction of olive oils from overmture olives my lso led to n improvement of the voltile frction nd provide enhnced sensory properties to the consumers, thus conferring n dded vlue to these oils. Another importnt prcticl ppliction is the extrction process. In our work, we dvise to optimize oth the percentge of leves nd the mlxtion time s much s possile, s they fcilitte oth sensory nd voltile frctions of the extrcted olive oils. Keywords: olive leves / mlxtion / olive oil / sensoril / voltiles / Response Surfce Methodology Received: April 12, 2017 / Revised: June 25, 2017 / Accepted: June 27, 2017 DOI: /ejlt Introduction Olive leves represent n importnt y-product of the olive oil extrction industry, immeditely fter olive mill wstewters. With stedy increse in production worldwide, Correspondence: J.A. Pereir, Centro de InvestigSc~o de Montnh (CIMO), ESA, Instituto Politecnico de BrgnSc, Cmpus de Snt Apoloni, BrgnSc, Portugl E-mil: jpereir@ip.pt Fx: þ prticulrly in the lst decde [1], the olive oil industry is seeking for effective lterntives to these industril residues. According to Bouziz et l. [2], out 3 to of the totl mss tht enters the olive mills corresponds to olive leves tht re collected together with olives. In generl, these leves re discrded, or prtilly used for composting nd niml feed. While no effective vloriztion is given to this suproduct, their primry fte is destruction, representing n extr finncil effort for this industry. However, severl strtegies re eing ttempted to vlorize olive leves within the food sector. One of those strtegies includes their use (s

2 (2 of 11) R. Mlheiro et l. Eur. J. Lipid Sci. Technol. 2017, 119, fresh leves or s n extrct) to improve the qulity, composition, stility, nd properties of olive oil nd other vegetle oils [3, 4]. Indeed, the use of fresh olive leves during extrction hs improved the chemicl composition of olive oils extrcted from overmture olives [5] y incresing pigments (lutein, -crotene, chlorophyll, nd pheophytin ) nd vitmin E contents. This leds to n increse in their oxidtive resistnce nd, consequently, shelf life. Other uthors hve lso reported n increse in the numer of ntioxidnts nd ntioxidnt properties [6], s this effect is minly ttriuted to the extrction of phenolic compounds from the olive leves, minly oleuropein [7, 8] nd flvonoids [9]. Nevertheless, other chemicl clsses lso contriute to the overll qulity of olive oils nd need to e studied under the ddition of olive leves. The sensory properties nd voltile frction re two spects tht need to e tken into ccount, s they ct directly nd indirectly, respectively, in the commercil clssifiction of olive oils. In prticulr, the use of olive leves to improve olive oil chrcteristics cn cuse sensory defects tht re not permitted in the extrvirgin olive oil ctegory, in ccordnce with Europen regultions [10]. Furthermore, nother importnt spect tht influences the sensory nd voltile profiles of olive oils is mlxtion, minly the dulistic feture time-temperture used during this extrction step [11, 12]. Excessive mlxtion times nd tempertures led to the reduction of the formtion of voltile compounds connoted with plesnt ttriutes, like green, fresh-cut grss formed during the lipoxygense pthwy, while rising the potentil formtion of off-flvors nd the development nd promotion of moleculr mechnisms, nmely the conversion of mino cids [13]. Therefore, in the present work, we intend to investigte if the ddition of fresh olive leves, comined with different mlxtion times, could improve the sensory properties nd voltile composition of olive oils from cv. CornScos, widespred olive cultivr in Portugl. To chieve such gol, three mss percentges of olive leves (0, 5, nd of fresh weight) nd three mlxtion times (20, 30, nd t 258C) were studied using centrl composite design to retrieve the numer of runs nd fctors comintions. A trined pnel lso ssessed the sensory profile, nd the voltile composition ws determined y hedspce solidphse microextrction (HS-SPME) gs-chromtogrphy with mss spectrometry (GC/MS). mturtion index 3 4 (ccording to Hermoso et l. [14]) were collected, together with pproximtely 3 kg of leves. The olives were immeditely trnsported to the lortory nd visully inspected to discrd ll olives nd leves with symptoms of ttcks of pests nd incidence of diseses. Once inspected, helthy olives were seprted in 39 su-smples of t lest 1 kg for extrction. 2.2 Experimentl design In order to study the impct of olive leves ddition nd mlxtion time on the sensory nd voltile profiles of olive oils from cv. CornScos, one of the most representtive of the Portuguese olive cultivrs, Response Surfce Methodology (RSM) y Minit 1 softwre ws used to retrieve the numer of events nd comintions to e studied. A one lock fce-centered ( ¼ 1) centrl composite design (CCD) ws constructed. The two independent fctors studied were the quntity of olive leves dded (X 1 :0 w/w) nd the mlxtion time (X 2 : 20 ). The mounts of olive leves were selected sed on previous study crried out in the sme cultivr [5]. The mlxtion times were within those commonly used, ut selected in order to void prolonged exposure of olive pste to ir, reducing the oxidtion of olive oil. The response vriles were the min voltile components identified nd quntified, s detiled elow, nmely, (E)-2-hexenl, (Z)-3-hexenl, nd totl voltiles, in the olive oils otined. Ech one of the vriles to e optimized ws coded t three levels: 1, 0, þ1, s presented in Tle 1. In totl, 13 runs were crried out with five replictions in the centrl point (. Runs 1, 5, 6, 12, nd 13). In ech run, three independent olive oils were extrcted, with totl of 39 independent olive oil extrctions. To reduce systemtic errors, the order of the olive oil extrctions ws rndomly estlished. The experiments performed in the centrl point llowed the estimtion of the influence of the experimentl error, wheres the other experiments llowed the clcultion of the regression coefficients of the model. The experimentl dt from the CCD ws fitted to secondorder polynomil model, presented in Eq. (1): Y ¼ 0 þ X2 i¼1 i X i þ X2 i¼1 ii X 2 i þ 12 X 1 X 2 ð1þ 2 Mteril nd methods 2.1 Smples collection Olives nd olive leves from cv. CornScos were collected in trditionl olive grove locted in SuSc~es (Mirndel N W), Trs-os-Montes (Northest Portugl), in Novemer Aout 50 kg of olives t Tle 1. Independent vriles nd their coded nd uncoded vlues Coded vlue Olive lef (%, w/w) Mlxtion time (min)

3 Eur. J. Lipid Sci. Technol. 2017, 119, Olive leves improve olive oil voltiles nd sensory profiles (3 of 11) where Y is the predicted response; 0 is the constnt (intercept); i is the liner coefficient, ii is the qudrtic coefficient nd 12 is the interction coefficient. X 1 nd X 2 re coded independent vriles (lef percentge nd mlxtion time, respectively). The dequcy of the models ws determined y evluting the lck of fit, the coefficient of determintion (R 2 ), nd the djusted-r 2 otined from the nlysis of vrince (ANOVA) generted y the softwre. The sttisticl significnce of the model nd model vriles ws determined t the proility level (¼0.05). 2.3 Olive oils extrction The olive oils were extrcted within 24 h fter olives nd leves were hrvested, in pilot extrction plnt using n Aencor nlyzer (Comercil Aengo S.A., Seville, Spin) with three min units: A mill, thermoeter where mlxtion tkes plce t controlled temperture, nd centrifuge. Olive smples were enriched with freshly chopped leves t the defined mounts (,, nd w/w) nd then milled together. The olive pste otined ws homogenized, nd out 700 g were trnsferred to the thermoeter unit for mlxtion t pre-determined times (20, 30, nd ) in thermosttic wter th t 258C. In the finl 5 min of ech mlxtion, 100 ml of wter t 358C ws dded to id in the olive oil seprtion. The mixture ws centrifuged nd decnted, nd the olive oil ws collected nd stored in 100 ml drk ottles. 2.4 Sensory nlysis The sensory nlysis ws performed ccording to the methodology descried in the stndrds of Interntionl Olive Council (IOC), nmely COI/T.20/Doc. No. 15/Rev. 8 [15], nd y using the profile sheet provided in the COI/ T.30/Doc. No. 17 [16]. A tem of four trined pnel memers ssessed the 39 olive oil smples. The numer of trined pnelists ws decided ccording to the mount of olive oil ville, conjugting lso with the chemicl prmeters smple needs. Ech trined pnelist evluted olfctory, gusttory-retronsl, nd olfctory-gusttory senstions, in tht order, ll ccounting for totl of 100 points. The following ttriutes were evluted in the olfctory senstions (mximum of 35 points): olive fruitiness (0 7); other fruits (0 3); green (grss/leves) (0 2); other positive senstions (0 3); nd hrmony (0 20). For the gusttoryretronsl senstions, the following prmeters were ssessed (mximum of 45 points): olive fruitiness (0 10); sweet (0 4); itter (0 3); pungent (0 3); green (grss/leves) (0 2); other positive senstions (0 3); nd hrmony (0 20). For the olfctory-gusttory senstions (mximum of 20 points), two prmeters were ssessed: Complexity (0 10) nd persistence (0 10). 2.5 Voltile chrcteriztion y HS-SPME-GC-MS The chrcteriztion of the voltile frction of the olive oils ws performed y hedspce solid-phse microextrction (HS-SPME) gs-chromtogrphy-mss spectrometry (GC/ MS). In 50 ml vils, 3 g of olive oil ws spiked with n ccurte mount of internl stndrd (2-methyl-4-pentnol) nd voltiles dsored to SPME fier coted with divinylenzene/cronex/polydimethylsiloxne (DVB/CAR/PDMS 50/ 30 mm) (Supelco, Bellefonte, USA). The vils were conditioned for 5 min t 508C for n incisive relese of the voltile compounds. After this period, the SPME fier ws exposed for, t the sme temperture, for the compound dsorption from the hedspce. The conditions used for the voltile extrction were those optimized y Peres et l. [17]. The totl procedure ws mde in duplicte per olive oil smple, with control smples (empty vils regulrly with internl stndrd). The retined compounds were eluted from the fier y therml dsorption for 1 min in the injection port (2208C). The fier ws mintined for further 10 min in the injector port of the chromtogrphy system for clening nd conditioning for further nlyzes. The gs chromtogrpher used ws Shimdzu GC-2010 Plus equipped with mss spectrometer Shimdzu GC/MS-QP2010 SE detector. A TRB-5MS (30 m 0.25 mm 0.25 mm) column (Teknokrom, Spin) ws used. The injector ws set t 2208C nd the mnul injections were mde in splitless mode. The moile phse consisted of helium (Prxir, Portugl) t liner velocity of 30 cm/s nd totl flow of 24.4 ml/min. The oven tempertures were the following: 408C/1 min; 28C/min until 2208C (). The ioniztion source ws mintined t 2508C with n ioniztion energy of 70 ev nd n ioniztion current of 0.1 kv. All mss spectr were cquired y electron ioniztion. The MS spectr frgments were compred with those otined from dtse (NIST 11), nd with those of commercil stndrds cquired from diverse producers. For qulittive purposes, ech smple ws injected in duplicte. The res of the chromtogrphic peks were determined y integrting the re-constructed chromtogrm from the full scn chromtogrm using the ion se (m/z intensity 10) for ech compound. For semi-quntifiction purposes, voltile mounts were clculted y the rtio of ech individul se ion pek re to the re of the internl stndrd se ion pek re nd converted to mss equivlents sed on the internl stndrd mss dded. 2.6 Sttisticl nlysis Anlysis of vrince An nlysis of vrince (ANOVA) with Type III sums of squres ws performed using the GLM (Generl Liner Model procedure) of the SPSS softwre, version 22.0 (IBM

4 (4 of 11) R. Mlheiro et l. Eur. J. Lipid Sci. Technol. 2017, 119, Corportion, New York, U.S.A.). The fulfilment of the ANOVA requirements, nmely the norml distriution of the residuls nd the homogeneity of vrince, were evluted y mens of the Kolmogorov-Smirnov with Lilliefors correction (if n > 50) or the Shpiro-Wilk s test (if n < 50), nd the Leveneś tests, respectively. All dependent vriles were nlysed using one-wy ANOVA with or without Welch correction, depending on whether the requirement of the homogeneity of vrinces ws fulfilled. The min fctors studied were the voltile composition nd the ttriutes ssessed in the sensory nlysis of the olive oils extrcted with olive leves nd different mlxtion times. If sttisticl significnt effect ws found, mens were compred using Tukeyś honestly significnt difference multiple comprison test or Dunnett T3 test, lso depending on whether equl vrinces could e ssumed. All sttisticl tests were performed t significnce level. 3 Results nd discussion 3.1 Sensory nlysis Sensory nlysis ws performed t three levels: Olfctory, gusttory-olfctory, nd olfctory-gusttory senstions. An importnt result ws the totl sence of negtive ttriutes (defects) in the 39 smples, independent of the tested mlxtion times. The results otined in the olive oils for ech senstion re detiled in Tle 2. Strting with the olfctory senstions, nd considering the men vlues of the centrl point events ( ), the vlues of fruitiness vried etween 5.5 ( nd ) nd 6.0 ( ). According to Europen legisltion, these olive oils could e considered extr-virgin olive oils (EVOO) sed only on sensory nlysis, due to their zero defects nd medin vlue of fruitiness higher thn zero [10]. The prmeter nmed other fruits mesured the presence of fruit senstion in the olive oil sensory profile. This prmeter vried etween 1.2 ( ) nd 1.8 (-20 nd ), suggesting tht n elevted quntity of olive leves nd higher mlxtion times reduce the perception of other fruits in the olive oils. The prmeter green received lower scores from the pnelists in the oils extrcted without leves for ( ) (Tle 2). In contrst, oils with leves (20 nd ) were mong the smples with higher scores. Generlly, the ddition of olive leves improved the green senstions, very importnt ttriute for consumers. Concerning other senstions smples with nd those with reported lower mount (men of 1.2), compred to smples with (men of 1.8). Evidently, the ddition of olive leves with incresed mlxtion times could e responsile for reduction of other senstions perceived. Hrmony, mesuring the equilirium etween ll the perceived prmeters, vried etween 16.1 nd 16.8 points. Oils with were more hrmonious thn those extrcted without leves ( ) or with higher mlxtion times in the presence of leves ( ) (Tle 2). This mens tht the ddition of olive leves with low mlxtion time improves the equilirium nd hrmony of the olive oils t olfctory senstions. However, s oserved in Tle 2, if the mlxtion time is incresed from 20 to ( leves), the hrmony immeditely reduces from 16.8 to 16.1 points. After olfctory senstions, the gusttory-olfctory senstions were ssessed (Tle 2; Fig. 1). Regrding fruitiness, vlues vried etween 6.4 ( ) nd 7.0 (, nd ). To otin higher fruitiness, mlxtion time should e incresed, since higher scores were otined t. However, no significnt differences were verified with the percentge of olive leves nd mlxtion time (P ¼ 0.306; Fig. 1). The sweet senstion ws highly scored in the oils with nd with score of 2.5, while smples extrcted with nd received score of 3.1. Evidently, cler pttern ws not noticed for sweetness regrding the ddition of leves nd the increse of the mlxtion time, since no significnt differences were verified (P ¼ 0.360). Bitter senstion ws highly scored in the oils with, while the smples,, nd retrieved lower scores, with 1.1 points. Regrding pungent senstion, it ppered tht the comintion of mlxtion time nd ddition of olive leves reduced this ttriute, since oils with presented the lowest score of 1.5, while nd reported the highest pungent senstion with 2.3. Bitter nd pungent senstions re minly relted to the extrction of phenolic compounds, minly secoiridoids [8, 18]. Therefore, incresed extrction times re essentil to improve these positive ttriutes, s they were oserved minly in the smples with. However, the ddition of leves my not increse considerly these two ttriutes, since different types of phenolic compounds re extrcted from the olive leves nd olive pste, minly secoiridoids in the former, s oleuropein nd oleuropein derivtives, like oleuropein glycon, demethyloleuropein, nd the dildehydic form of decroxymethyl elenolic cid linked to hydroxytyrosol nd tyrosol from the olive pste [19, 20]. Therefore, mlxtion time is more importnt for these two ttriutes rther thn the quntity of leves dded, proly due to the different phenolic components extrcted from leves nd from olive pste. Green gusttive senstions were higher in olive oils with nd with score of 1.5 (Tle 2; Fig. 1). This mens tht y dding olive leves mlxtion timecnereducey20min,otiningthesmegreen senstion in the finl olive oil. The lowest scores were reported for oils extrcted with nd, with 1.0 (Tle 2; Fig. 1). With respect to other gusttive senstions, higher scores were otined in the oils with nd. Evidently,

5 Eur. J. Lipid Sci. Technol. 2017, 119, Olive leves improve olive oil voltiles nd sensory profiles (5 of 11) Tle 2. Sensory profile of olive oils from cv. CornScos extrcted with different mounts of olive leves (0, 5, nd ) nd mlxtion times (20, 30, nd ) (n ¼ 3; men with the vlue of stndrd devition represented in rckets) Run Leves (%) Extrction time (min) P-vlue Olfctory senstions Fruitiness 5.6, 5.8, 5.5, 5.9, 5.4, , 6.0, , 5.5, 6.0, 5.8, (0.66) (0.41) (0.55) (0.20) (0.66) (0.42) (0.66) (0.00) (0.42) (0.20) (0.55) (0.00) (0.41) Other fruits , 1.8, 1.7, 1.8, 1.4, 1.7, 1.3, 1.3, 1.3, 1.5, 1.6, (0.26) (0.41) (0.27) (0.41) (0.27) (0.20) (0.26) (0.26) (0.27) (0.26) (0.45) (0.00) (0.26) Green 1.3 -c ,c 1.4,c 1.4,c 1.2 -c 1.4,c 1.5 c 1.5 c 1.4,c 1.4,c 1.1, 1.4,c <0.001 (0.26) (0.00) (0.20) (0.20) (0.20) (0.26) (0.20) (0.00) (0.00) (0.20) (0.20) (0.00) (0.20) Other senstions , , , 1.5, 1.4, , 1.2 <0.001 (0.27) (0.26) (0.26) (0.00) (0.27) (0.20) (0.26) (0.26) (0.00) (0.20) (0.26) (0.35) (0.26) Hrmony 16.6, , , , 16.5, , 16.1 <0.001 (0.49) (0.20) (0.49) (0.26) (0.00) (0.49) (0.20) (0.41) (0.32) (0.26) (0.26) (0.35) (0.20) S of olfctory 27.1 c , senstions,c -c -c -c -c -c,c -c,c -c (1.07) (0.80) (0.49) (0.52) (0.68) (0.68) (0.80) (0.45) (0.55) (0.42) (1.02) (0.71) (0.68) Gusttory-Olfctory senstions Fruitiness (0.52) (0.49) (0.00) (0.49) (0.00) (0.55) (0.63) (0.41) (0.42) (0.42) (0.41) (0.71) (0.00) Sweet (0.75) (0.20) (0.55) (0.00) (0.45) (0.61) (0.66) (0.26) (0.27) (0.38) (0.55) (0.71) (0.38) Bitter 1.8 c,d d ,c 1.6 -c 1.8 c,d 1.8 c,d 2.0 c,d 1.3, < d -d (0.26) (0.20) (0.52) (0.20) (0.26) (0.20) (0.41) (0.26) (0.00) (0.42) (0.26) (0.00) (0.20) Pungent 2.2 -c 1.6, 2.3 c 1.8 -c 2.0 -c 1.9 -c 2.0 -c 1.7 -c 1.9 -c 1.6, 2.3,c 1.8 -c (0.26) (0.58) (0.26) (0.42) (0.45) (0.38) (0.45) (0.26) (0.38) (0.38) (0.27) (0.35) (0.32) Green , 1.3, , 1.3, , <0.001 (0.38) (0.00) (0.00) (0.00) (0.00) (0.27) (0.26) (0.00) (0.00) (0.26) (0.26) (0.35) (0.26) Other senstions 1.7 c c ,c 1.6,c 1.6,c 1.2, 1.5 -c c 1.5 -c 1.1 <0.001 (0.26) (0.20) (0.27) (0.20) (0.20) (0.20) (0.20) (0.26) (0.00) (0.20) (0.27) (0.35) (0.20) Hrmony (0.55) (0.00) (0.49) (0.20) (0.41) (0.41) (0.41) (0.00) (0.20) (0.20) (0.55) (0.71) (0.00) S of gusttoryolfctory e 30.9, , <0.001 senstions d,e (0.80) (0.86) (1.13) (0.49) -e (0.92) -d (1.29) -e (1.10) -d (0.52) -e (0.32) (1.48) c-e (0.93) -e (1.77) -c (0.38) Olfctory-gusttory senstions Complexity (0.52) (0.41) (0.41) (0.32) (0.41) (0.20) (0.41) (0.00) (0.00) (0.00) (0.41) (0.71) (0.00) Persistence (0.52) (0.41) (0.52) (0.41) (0.49) (0.63) (0.89) (0.45) (0.20) (0.20) (0.66) (0.00) (0.20) S of olfctorygusttory senstions (0.82) (0.63) (0.75) (0.41) (0.49) (0.66) (1.17) (0.45) (0.20) (0.20) (0.61) (0.71) (0.20) Finl score e , d,e -d,e -d -e -e -e -c c-e -e (2.27) (1.75) (1.72) (0.66) (1.68) (2.07) (2.56) (0.75) (0.58) (1.57) (1.58) (3.18) (1.08) <0.001 For ech sensory ttriute ssessed, within the sme line, men vlues with different letters differ significntly (P < 0.05); P < 0.05, y mens of Levene s test. P vlues re those from one-wy Welch ANOVA nlysis. Mens were compred y Dunnett T3 s test, since equl vrinces could not e ssumed; P > 0.05, y mens of Levene s test. P vlues re those from one-wy ANOVA nlysis. Mens were compred y Tukey s test, since equl vrinces could e ssumed.

6 (6 of 11) R. Mlheiro et l. Eur. J. Lipid Sci. Technol. 2017, 119, A P = D c -c 0.0 B ,c 1.5, 0.8, -c P < c c P = E, C 1.5,,c 2.3 c c,c, c P < 0.001, P < Figure 1. Chromtogrphic profiles of olive oils from cv. CornScos extrcted with different percentges of olive leves t of mlxtion time (I.S. internl stndrd). Voltile compounds: 1 (Z)-3-hexenl; 2 (E)-2-hexenl; 3 (Z)-3-hexen-1-ol; 4 hexnol; 5 2-heptnone; 6 (E,E)-2,4-hexdienl; 7 3-ethyl-1,5-octdiene (I); 8 3-ethyl-1,5-octdiene (II); 9 enzldehyde; 10 (Z)-3-hexenyl cette; 11 hexyl cette; 12 limonene; 13 enzyl lcohol; 14 -ocimene; 15 octnl; 16 nonnl; 17 phenylethyl lcohol; 18 dodecne; 19 decnl; 20 -copene; 21 cryophyllene; 22 -frnesene. with the ddition of of leves, we cn lso reduce the mlxtion time in to chieve the sme score for other senstions. However, regrding gusttive hrmony, higher scores were otined with lower mlxtion times,, with nd without leves (0 nd, respectively) (Tle 2). Incresing mlxtion time generlly reduces hrmony, since some other ttriutes my e extrcted in higher mounts, like green senstion (Tle 2). Reltive to finl senstions, only two prmeters were evluted: Complexity nd persistence, s shown in Tle 2. Complexity presented the highest nd lowest scores in the oils without leves ddition, depending on the mlxtion time, with higher complexity t nd score of 7.3, which ws reduced to 6.8 for of mlxtion. Smples with leves ddition ll hd higher scores thn the control ones, vrying etween 7.0 nd 7.2 points, independent of the mlxtion times. Regrding persistence, higher scores were reported in oils without leves nd higher mlxtion times ( ), with 7.7, while the oils with 5 nd leves, oth with, reported lower persistence (7.1). Knowing tht persistence is mostly ssocited with itter nd pungent senstions, the higher scores t were expected. This oservtion, s lredy explined, is relted to the different types of phenolic compounds extrcted from the olive leves nd olive pste. Overll, nd pertining to the totl finl scores of the pnelists, vlues rnged from 69.8 ( ) nd 75.4 ( nd ). Within the oils with leves ddition, those extrcted with of leves during presented the highest score of 74.5 (Tle 2). This result suggests tht the ddition of olive leves my llow reduction on the mlxtion time without loss of sensory ttriutes, ut tht higher mlxtion times might compromise the chieved ttriutes. The ddition of olive leves to Itlin olives lso improved the sensory chrcteristics of olive oils [21]. The min improvements were verified in the green, fruity, nd itter tste, in ccordnce with our work. Furthermore, these

7 Eur. J. Lipid Sci. Technol. 2017, 119, Olive leves improve olive oil voltiles nd sensory profiles (7 of 11) Tle 3. Voltile profile (mg/kg of oil; vlues in old re expressed s mg/kg of oil) of olive oils from cv. CornScos extrcted with different mounts of olive leves nd mlxtion times (n ¼ 3; men with the vlue of stndrd devition represented in rckets) Run Leves (%) Extrction time (min) P-vlue (Z)-3-hexenl c 2.45, c d d 4.66 c,d d 4.55 c,d c < d -d -d (0.28) (0.12) (0.79) (1.56) (0.35) (1.74) (0.74) (0.30) (0.18) (0.90) (2.15) (1.18) (0.50) (E)-2-hexenl 8.83, 8.67, , 9.37, 8.78, 8.91, 11.7, 11.1, 12.5, 10.9, 11.9, (1.56) (2.36) (1.04) (3.00) (1.95) (1.20) (0.41) (3.03) (3.51) (3.15) (4.12) (4.31) (3.74) (Z)-3-hexen-1-ol 0.09, 0.09, , , <0.001 (0.02) (0.02) (0.01) (0.02) (0.03) (0.01) (0.02) (0.02) (0.03) (0.04) (0.03) (0.04) (0.04) Hexnol (0.03) (0.10) (0.07) (0.15) (0.06) (0.05) (0.01) (0.20) (0.15) (0.11) (0.10) (0.15) (0.11) 2-Heptnone 0.31, 0.32, 0.35, 0.30, 0.32, 0.32, , 0.28, 0.29, 0.33, 0.28, 0.27 <0.001 (0.02) (0.03) (0.04) (0.05) (0.01) (0.04) (0.16) (0.04) (0.01) (0.03) (0.04) (0.03) (0.03) (E.E)-2,4-hexdienl 1.21 d , 0.26, 0.30,c 0.25, 0.20, 0.26, 0.26, 0.34,c 0.35,c 0.44 c <0.001 (0.05) (0.01) (0.03) (0.10) (0.03) (0.06) (0.02) (0.08) (0.09) (0.13) (0.08) (0.13) (0.06) 3-Ethyl-1,5-octdiene (I) 1.07, c c-e 1.39 d,e 1.21 c c c c c 1.41 e 1.24 c,d 1.18,c <0.001 (0.05) (0.10) (0.08) (0.14) (0.08) (0.03) (0.04) (0.05) (0.04) (0.09) (0.07) (0.05) (0.04) 3-Ethyl-1,5-octdiene 1.16, , c 1.23, 1.20, 1.16, 1.15, c 1.26, 1.21, <0.001 (II) (0.04) (0.07) (0.08) (0.14) (0.19) (0.04) (0.06) (0.09) (0.06) (0.10) (0.08) (0.04) (0.03) Benzldehyde c c d d c 1.90 c,d 2.02 d 2.07 d 1.44, c 1.86 < d (0.08) (0.10) (0.37) (0.32) (0.12) (0.29) (0.18) (0.20) (0.12) (0.05) (0.34) (0.17) (0.31) (Z)-3-hexenyl cette c 1.79, c 3.29,c c 3.19,c 3.73 c c c 1.82, 3.48,c 3.73,c 1.23 <0.001 (0.71) (0.58) (0.49) (1.22) (0.70) (1.52) (1.54) (0.99) (0.49) (0.89) (0.42) (1.26) (0.23) Hexyl cette c 0.13, d 0.12, d c c,d 0.09, < d -d c-d -d (0.05) (0.03) (0.04) (0.08) (0.06) (0.10) (0.11) (0.00) (0.03) (0.07) (0.08) (0.06) (0.01) Limonene , 0.06, 0.07, 0.06, , 0.07, 0.07, 0.07, , 0.08 <0.001 (0.03) (0.02) (0.02) (0.03) (0.03) (0.02) (0.02) (0.02) (0.02) (0.03) (0.00) (0.00) (0.02) Benzyl lcohol c c d d c c.d 0.55 c. 0.33, d 0.60 d < d d (0.03) (0.12) (0.10) (0.06) (0.06) (0.10) (0.06) (0.12) (0.08) (0.07) (0.12) (0.09) (0.19) -ocimene , d d 0.35 d 0.34 d c c,d < d -d -d -d -d (0.03) (0.07) (0.06) (0.10) (0.08) (0.06) (0.04) (0.07) (0.04) (0.03) (0.02) (0.08) (0.06) Octnol c d 0.02, e d c-e 0.03 d, e 0.03 e < d -d e -d (0.00) (0.00) (0.00) (0.00) (0.00) (0.01) (0.00) (0.00) (0.00) (0.00) (0.01) (0.00) (0.01) Nonnl c , 0.08, d c c 0.16 d, c 0.14 c,d 0.22 e < d e (0.01) (0.01) (0.02) (0.02) (0.00) (0.04) (0.01) (0.03) (0.01) (0.02) (0.03) (0.02) (0.07) Phenylethyl lcohol d d c-e 0.20, 0.35 d,e 0.43 e < d (0.02) (0.03) (0.04) (0.06) (0.03) (0.08) (0.02) (0.05) (0.07) (0.06) (0.02) (0.07) (0.09) Dodecne 0.05, 0.05, , 0.04, 0.05, 0.04, , 0.04, 0.19 c <0.001 (0.01) (0.02) (0.01) (0.02) (0.00) (0.02) (0.02) (0.00) (0.02) (0.01) (0.01) (0.01) (0.06) Decnl n.d c n.d 2.42, c c c c 2.61, 4.25 c c 3.94,c c <0.001 (1.06) (0.75) (0.47) (1.35) (0.41) (0.88) (0.71) (1.06) (0.50) (1.20) (0.88) -Copene c 0.02 c 0.01 c 0.02 d 0.02 d c 3.92 <0.001 (0.00) (0.21) (1.90) (0.63) (2.81) (0.00) (0.00) (0.00) (0.00) (0.00) (1.06) (0.00) (1.37) Cryophyllene 5.50, 5.86, 5.25, c 0.01 d,e 0.01 e,f 6.44, 0.01 c-e 0.02 f < d -d -d (0.42) (0.99) (1.66) (1.91) (0.81) (1.85) (0.68) (1.20) (0.00) (0.00) (1.46) (0.00) (0.00) -Frnesene 0.14, 0.15, , 0.16, 0.15, 0.33 c 0.44 c,d 0.48 d 0.10, c <0.001 (0.02) (0.04) (0.00) (0.04) (0.01) (0.03) (0.02) (0.12) (0.09) (0.08) (0.04) (0.01) (0.13) For ech voltile compound, within the sme line, men vlues with different letters differ significntly (P < 0.05); P < 0.05, y mens of Levene s test. P vlues re those from one-wy Welch ANOVA nlysis. Mens were compred y Dunnett T3 s test, since equl vrinces could not e ssumed; P > 0.05, e mens of Levene s test. P vlues re those from one-wy ANOVA nlysis. Mens were compred y Tukey s test, since equl vrinces could e ssumed.

8 (8 of 11) R. Mlheiro et l. Eur. J. Lipid Sci. Technol. 2017, 119, uthors found tht 3% of olive leves ddition retrieved higher orgnoleptic evlution, compred to 1, 2, nd. In the cse of Tunisin olive cultivrs, the ddition of 3% olive leves to olives lso improved the fruity, itter, nd green senstions [3]. In our study, the most influenced prmeters tht were improved y the ddition of olive leves were those from olfctory nd gusttory-olfctory senstions, showing tht the voltile compounds hve key role on the cceptility nd clssifiction of the olive oils. Therefore, the voltile frction of cv. CornScos olive oils extrcted with different percentges of olive leves nd with different mlxtion times ws lso evluted, s detiled in the next section. 3.2 Voltile composition The voltile composition of cv. CornScos olive oils extrcted with olive leves nd different mlxtion times ws chrcterized, nd the profile otined is presented in Tle 3. Overll, 22 voltiles (Fig. 2; Tle 3) were found in the olive oils, distriuted y seven chemicl clsses: Aldehydes, lcohols, esters, ketones, terpenes (monoterpenes nd sesquiterpenes hydrocrons), lknes, nd lkenes. The ddition of olive leves nd the ppliction of different mlxtion times influenced quntittively, ut not qulittively, the voltile compounds present in the olive oils (Fig. 2; Tle 3). Totl voltiles rnged from 18.5 mg/kg ( ) nd 26.3 mg/kg ( ). Evidently, for totl voltiles, the min fctor ws the percentge of leves dded; for ll the mlxtion times tested, the ddition of leves positively nd significntly influenced the totl voltiles (P ¼ for ; P < for ; nd P ¼ for of mlxtion). Among the 22 voltiles, only the content of hexnol ws not ffected y the tested conditions (P ¼ 0.461), s this lcohol is minor component of the voltile profile of the olive oils otined, rnging from 0.59 nd 0.73 mg/kg. The voltile composition of the otined olive oils ws minly composed y GLV s (green lef voltiles), minly (E)- 2-hexenl. This compound content vried etween 7.7 nd 14.1 mg/kg, respectively, in oils extrcted with nd (Tle 3), s the increse ssocited with the presence of leves ws significnt for oth nd mounts (P ¼ 0.004) (Fig. 3A). This ldehyde comprised etween 40 nd 53% of the totl voltiles mss quntified in the 39 olive oils nlyzed. At of mlxtion, oils with of leves reported nerly doule the mount of (E)- 2-hexenl (14.1 mg/kg) compred to oils (7.68 mg/kg) (x10,000,000) TIC I.S. 2 leves (x10,000,000) TIC 0.75 I.S. 2 leves (x10,000,000) TIC 1.00 I.S. 2 leves Figure 2. Gusttory-olfctory senstions of cv. CornScos olive oils extrcted with different percentges of olive leves nd mlxtion times. A Fruitiness; B Sweet; C Bitter; D Pungent; E Green. In the sme sensory ttriute, different letters differ significntly (P < 0.05). 22

9 Eur. J. Lipid Sci. Technol. 2017, 119, Olive leves improve olive oil voltiles nd sensory profiles (9 of 11) A C (E)-2-hexenl (mg kg -1 ) (Z)-3-hexenyl cette (mg kg -1 ) A 10.9 A 11.9 A 12.5 A 10.4 A 10.0 A 8.8 A 8.7 A 7.7 A 3.7 B 3.3,A 3.5 B 2.8,A 2.1 A 2.4 A 1.8,A 1,8 A 1.2 A B (Z)-3-hexenl (mg kg -1 ) D Totl voltiles (mg kg -1 ) B 8 4.2,B 4.6 A,B B A 2.5 A 2.8 A 2.9 A 1.9 A A 27.2 A 26.3 A A 24.0 A 24.0 A 20.9 A 19.1 A 18.5 A Figure 3. Voltile chnges in (E)-2-hexenl (A), (Z)-3-hexenl (B), (Z)-3-hexenyl cette (C), nd totl voltiles (mg/kg) (D) in olive oils from cv. CornScos extrcted with different mounts of olive leves nd mlxtion times. Men vlues of different percentges of olive leves with specific mlxtion time with different cpitl letters differ significntly (P < 0.05). Men vlues of specific percentge of leves t different mlxtion times with different minor letters differ significntly (P < 0.05). Similr oservtions were verified y Di Giovcchino et l. [21] with of leves nd 60 min of mlxtion. Ammr et l. [3] reported n increse of out 3 in (E)-2-hexenl y dding 3% of leves to Chemlli olives. The increse of(e)-2-hexenl is relted to the LOX pthwy (lipoxygense), y which polyunsturted ftty cids (PUFA) re oxidized nd ltter cleved y the hydroperoxide lyse, leding to the formtion of ldehydes [22]. The min ldehydes formed during LOX re hexnl nd (Z)-3-hexenl, ut through the ction of isomerse (Z)-3-hexenl yields (E)-2-hexenl. Therefore, through the ddition of olive leves, the enzymtic production of (E)-2-hexenl is enhnced. The inclusion of olive leves during olive oil extrction my increse the levels of PUFA in the olive pste, since olive leves re rich in PUFA [23], thus enhncing the enzymtic ction nd LOX pthwy. Another ldehyde tht ws influenced y the ddition of olive leves nd the mlxtion time ws (Z)-3-hexenl (Tle 3), lso formed under the LOX pthwy. The cv. CornScos olive oils (Z)-3-hexenl content ws significntly influenced y the ddition of leves with 20 nd of mlxtion time (P ¼ nd P < 0.001, respectively), rnging from 1.9 mg/kg ( ) to 5.6 mg/kg ( ) (Tle 3 nd Fig. 3B). Mlxtion time did not ffect the (Z)-3-hexenl content in the control olive oils (P ¼ 0.565), ut significnt reduction (P < nd P ¼ 0.019, respectively) ws oserved with nd of leves. In the oils with of leves, losses of 1 mg/kgwere oserved from 20 to of mlxtion, nd of 1.7 mg/kg from 30 to (Fig. 3B). (E)-2-hexenl nd (Z)-3-hexenl re connoted with green leves, grssy, green, pple-like, lef-like, nd fresh cut grss senstions [24]. Therefore, their presence in olive oil is importnt for the overll sensory chrcteristics of the oil. Such hypothesis ws lso verified y Di Giovcchino et l. [21], reporting tht leves ddition improved the fresh-cut grss senstion in the finl olive oil. Additionlly, these compounds cn e used s sustrte for the formtion of other voltiles [25 27]. When (Z)-3-hexenl is reduced y lcohol dehydrogense, it forms (Z)-3-hexen-1-ol. This lcohol, lso GLV, ws present in low mounts in our olive oils, rnging from 50 nd 140 mg/kg (Tle 3). However, it is importnt ecuse, in the LOX pthwy, this lcohol leds to the formtion of (Z)-3-hexenyl cette y esterifiction, due to the ction of lcohol cetyltrnsferse. (Z)-3-hexenyl cette is one of the voltiles responsile for the chrcteristic odor of green nn in some olive oils, s well s fruity nd green leves [24]. This ester content vried etween 1.2 mg/kg ( ) nd 3.7 mg/kg ( ) (Fig. 3C). Depending on the mlxtion times pplied, the ddition of olive leves cused vrile results: (Z)-3-hexenyl cette content incresed significntly with the ddition of leves t 20 nd (P ¼ nd P ¼ respectively), while for of mlxtion, the ddition of leves significntly reduced this ester content (P ¼ 0.011). Regrding the mlxtion times, only in the oils with of leves ws significnt chnge in ccordnce with the mlxtion time verified (P < 0.001), while in the control oils nd leves oils, no significnt differences were detected

10 (10 of 11) R. Mlheiro et l. Eur. J. Lipid Sci. Technol. 2017, 119, (P ¼ nd P ¼ respectively). From the results otined, it cn e retined tht high mlxtion times reduced the presence of (Z)-3-hexenyl cette in the finl olive oil when leves were dded. Y ðeþ-2-hexenl ¼ 10:152 þ 1:951X 1 ð2þ 3.3 Response surfce models Figure 4A C present the response surfce plots, showing the effect of mlxtion time nd percentge of leves mss on the mjor voltiles of olive oils, prticulrly (E)-2-hexenl nd (Z)-3-hexenl, s well s on totl voltiles. The fitted second-order polynomil equtions for (E)-2-hexenl, (Z)-3- hexenl, nd totl voltiles re presented in Eqs. (2), (3), nd (4), respectively, fter neglecting the non-significnt terms t of significnce, with coefficients of determintion (R 2 )of 0.781, 0.846, nd 0.885, respectively, nd dj-r 2 of 0.624, 0.737, nd 0.802, respectively. Y ðzþ-3-hexenl ¼ 3:990 þ 0:874X 1 0:811X 2 0:699X 1 X 2 Y Totl Voltiles ¼ 24:055 þ 3:680X 1 with X 1 ¼ percentge of leves nd X 2 ¼ mlxtion time. Considering the R 2 vlues, our results indicte tht more thn 78% of vrition in the contents of these compounds cn e explined y the independent vriles studied, nmely, percentge of leves nd mlxtion time. Furthermore, the lcks-of-fit were not significnt, with P-vlues of 0.773, 0.281, nd 0.937, respectively, indicting the suitility of the models to ccurtely predict the vrition. The regression nlysis of the experimentl dt showed tht the percentge of leves lwys hd significnt positive liner effect on the contents of (E)-2-hexenl, (Z)-3-hexenl, nd totl voltiles, in line with the previously oserved dt (Fig. 3A, B nd D). On the contrry, mlxtion time hd significnt negtive liner effect on (Z)-3-hexenl, s significnt negtive interction ws lso found etween oth independent vriles. Moreover, for this compound, the effects of mlxtion time nd percentge of leves were similr due to the similrity of the solute vlues of oth fctors coefficients (0.874 vs ). These results showed tht the contents of (E)-2-hexenl, (Z)-3-hexenl, nd totl voltiles increse s lef percentge increses from 5 to, while mlxtion time did not hve pronounced effect, with the exception of (Z)-3-hexenl. ð3þ ð4þ 4 Conclusions Figure 4. Response surfce plots of (E)-2-hexenl (A), (Z)-3- hexenl (B), nd totl voltiles (C), in function of percentge of olive leves dded during the extrction process nd mlxtion time. With the present work, it ws concluded tht the ddition of olive leves to olives during the extrction process of olive oil ws not responsile for the ppernce of sensory defects, nd improved oth olfctory nd gusttive-olfctory senstions, minly fruity nd green ttriutes. It ws concluded tht this oservtion ws relted to the improvement of the voltile frction of olive oils, minly (E)-2-hexenl, (Z)-3-hexenl, nd (Z)-3-hexenyl cette. Mlxtion time hs determinnt prt in the finl voltile content nd sensory ttriutes, prticulrly in the presence of leves, with incresed contct times inducing potentil voltiles loss, for instnce, of (Z)-3-hexenyl cette. The ddition of olive leves to olives for the extrction of differentited nd sensoril improved olive oils could e considered t percentges etween 5 nd. However, mlxtion time should e kept for up to, since cuses the loss of importnt sensory ttriutes in the finl olive oils. Olive leves, su-product of olive mill industry, could e used s powerful odornts for olive sector,

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