Accepted Manuscript Olive oil phenolic compounds affect the release of aroma compounds Alessandro Genovese, Nicola Caporaso, Veronica Villani, Antonello Paduano, Raffaele Sacchi PII: DOI: Reference:

S0308-8146(15)00288-5 http://dx.doi.org/10.1016/j.foodchem.2015.02.097 FOCH 17196

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Food Chemistry

Received Date: Revised Date: Accepted Date:

3 November 2014 9 February 2015 19 February 2015

Please cite this article as: Genovese, A., Caporaso, N., Villani, V., Paduano, A., Sacchi, R., Olive oil phenolic compounds affect the release of aroma compounds, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/ j.foodchem.2015.02.097

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OLIVE OIL PHENOLIC COMPOUNDS AFFECT THE RELEASE OF AROMA COMPOUNDS

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Alessandro Genovese *, Nicola Caporaso, Veronica Villani, Antonello Paduano, Raffaele Sacchi

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Department of Agriculture, University of Naples Federico II, Via Università 100, 80055 Portici (NA), Italy

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*Corresponding author. Tel.: +39 081 2539352.

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E-mail address: [email protected] (A. Genovese)

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ABSTRACT

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Twelve aroma compounds were monitored and quantified by dynamic headspace analysis after their

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addition in refined olive oil model systems and extra virgin olive oil (EVOO) biophenols to

31

simulate EVOO aroma. The influence of polyphenols on aroma release was studied under simulated

32

mouth conditions by using human saliva, and SPME-GC/MS analysis. While few differences were

33

observed in orthonasal assay (without saliva), interesting results were obtained for retronasal aroma.

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Biophenols caused generally the lowest headspace release of almost all volatile compounds.

35

However, only ethyl esters and linalool concentrations were lower in retronasal than orthonasal

36

assay. Saliva also caused higher concentration of hexanal, probably due to hydrodroperoxide lyase

37

(HPL) action on linoleyl hydroperoxide. Epicatechin was compared to EVOO phenolics and the

38

behaviour was dramatically different, likely to be due to salivary protein‒tannin binding

39

interactions, which influenced aroma headspace release. These results were also confirmed using

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two extra virgin olive oils.

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Keywords: Virgin olive oil; SPME-GC/MS; Phenolic compounds; Human saliva; RAS; Volatile compounds.

46 47

Abbreviations used:

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GC: gas-chromatography; SPME: solid-phase microextraction technique; VOO: virgin olive oil;

49

RAS: retronasal aroma simulator; ROO: refined olive oil; ROOP: refined olive oil with added olive

50

oil polyphenols; ROOC: refined olive oil with added catechins (epicatechin); EVOO: extra virgin

51

olive oil.

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1. Introduction

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Virgin olive oil (VOO) is the oil obtained from the fruit of the olive tree solely by mechanical or

56

other physical means under conditions, particularly thermal conditions, that do not lead to

57

alterations in the oil, and which has not undergone any treatment other than washing, decantation,

58

centrifugation and filtration (EC 2568/91). It is a complex food, from a sensory and a chemical

59

point of view. During oral processing, olive oil odorants can interact with odour receptors by

60

moving from the mouth to the nasal cavity via the nasopharynx (retronasal route). The sensations of

61

orthonasal and retronasal odours differ in the level of perception, even though they involve the same

62

mechanisms (Bojanowski & Hummel, 2012). Such differences are due to the fact that salivation,

63

mouth size, breathing, and temperature are factors able to change the volatility of olive oil odorants

64

and consequently VOO odour when it enters the mouth (Van Ruth & Roozen 2000; Van Ruth,

65

Grossmann, Geary & Delahunty, 2001). In fact, when olive oil is put in the mouth, the odorants are

66

affected by different factors. Initially, saliva has a hydration effect and can produce a water-in-oil

67

emulsion in which aroma compounds are partitioned between the water and oil phase.

68

Subsequently, they are transported from the two liquid phases to the air phase in the mouth and then

69

they reach the olfactory receptors located in the nose. Primarily, a molecular property such as

70

hydrophobicity (polarity), expressed as log Po/w (octanol–water partition coefficient), could be

71

responsible for the mass transfer from two different liquid phases such as the organic phase of olive

72

oil and water phase of saliva.

73

The activity of salivary proteins (mucins, albumin and proteins rich in proline) and enzymes

74

(amylase, lipase, and lysozymes) are also responsible for emulsion destabilisation (Vingerhoeds,

75

Blijdenstein, Zoet & Van Aken, 2005). These changes in the structure of the emulsion have also an

76

impact on the final perception (Arancibia, Jublot, Costell & Bayarri, 2011). In addition, respiration

77

and the dilution effect of saliva make oral processing a "dynamic process" which cause a

78

continuous change in terms of volume, composition, and viscosity of the foods. This is the reason 3

79

why VOO tasters frequently aspirate the olive oil in the mouth. In fact, this procedure favours

80

volatilization by increasing the surface area contact and enhances retronasal detection. Therefore,

81

there are two major factors that control the release of volatile flavor compounds from food

82

products: the volatility of the compounds in the product base (thermodynamic factor) and the

83

resistance to mass transfer from product to air (kinetic factor). Only the kinetic factor is affected by

84

the texture and this becomes apparent only under dynamic (non-equilibrium) conditions (de Roos,

85

2003).

86

Van Ruth and co-workers (2001) studied a model system of sunflower oil and reported a salting

87

out effect of some volatile compounds after the addition of artificial saliva (salts, mucin, and α-

88

amylase). The authors stated that this effect was due to the hydrophilicity of the molecules. It is also

89

known that mucins have binding sites available to trap and decrease volatiles (Friel & Taylor,

90

2001). In fact, mucin can bind specific aroma compounds, principally aldehydes (Friel & Taylor,

91

2001; Van Ruth & Roozen, 2000), probably to form Schiff bases. Other evidence has been found

92

for the hydrolysis of ethyl esters, according to their chemical structures, and for the oxidation of

93

some thiols due to the enzymes in human saliva (Buettner, 2002a, 2002b; Genovese, Piombino,

94

Gambuti & Moio, 2009).

95

Another important factor is the non-volatile matrix of a food, which could determine chemical

96

and/or physic interactions with aroma compounds. These interactions may alter the food‒air

97

partitioning (volatility) of the aroma compounds. Therefore, they could also affect the aroma release

98

(Van Ruth & Roozen, 2010; Van Ruth, King & Giannouli, 2002). For other food or beverages, like

99

red wine, a possible interaction between some phenolic and volatile compounds was reported and

100

these interactions influenced the aroma release of wine (Pozo-Bayon & Reineccius, 2009). In

101

addition, when saliva enters in contact with polyphenols (De Freitas & Mateus, 2001; Bennick,

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2002), their interaction could affect the extent of this effect in red wine (Genovese et al., 2009;

103

Lorrain, Tempere, Iturmendi, Moine, de Revel & Teissedre, 2013; Munoz-Gonzalez et al., 2014a).

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Some correlations between the level of phenolic compounds and aroma compounds with sensory

105

descriptors were also found in VOO. In particular, higher concentrations of 1-penten-3-one and

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phenolic compounds causes the increase of leaf odour, and an increase in the phenolics

107

concentration causes the increase of walnut husk odour (Angerosa, Mostallino, Basti &Vito, 2000).

108

Although the volatile and phenolic compounds in VOO have been widely studied (for reviews see

109

Angerosa et al., 2004; Kalua, Allen, Bedgood, Bishop, Prenzler & Robards, 2007; Carrasco-

110

Pancorbo, Cerretani, Bendini, Segura-Carretero, Gallina-Toschi & Fernandez-Gutierez, 2005;

111

Bendini et al., 2007), and some studies have aimed to investigate the aroma release of volatile

112

compounds in vegetable oil model solutions or model emulsions (Van Ruth & Roozen, 2000; Van

113

Ruth et al., 2001; Van Ruth et al., 2002; Arancibia et al., 2011), so far, no study has aimed to verify

114

the retronasal perception of VOO volatiles in the presence of phenolic compounds. Moreover, the

115

majority of these in vitro studies employed artificial saliva. This could be a limit because the effect

116

of numerous enzymes and proteins present in human saliva are not considered (Salles et al., 2011).

117

Since VOO odour plays an important role in the quality, as well as in the classification of the

118

virgin olive oil commercial categories, it could be very important to understand the retention and/or

119

the release of volatile compounds from olive oil-in-water emulsion produced in mouth with human

120

saliva in relation to VOO phenolic compounds, which could affect the aroma and the sensory

121

perception.

122

Therefore, the aim of the present study was to investigate the effect of phenolic compounds on

123

aroma release of VOO under simulated mouth conditions by using a RAS (retronasal aroma

124

simulator) device. For this purpose, we set up a "two-phase" model system without saliva,

125

composed of a liquid phase (olive oil) and air phase (headspace), with the aim of simulating the

126

orthonasal conditions, i.e. olive oil odour. The second system simulating retronasal condition, i.e.

127

olive oil aroma, consisted of a "three-phase" system after saliva addition: the lipid phase (olive oil),

128

water phase (human saliva), and air phase (headspace).

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2. Material and Methods

131 132

2.1 Samples, standards and reagents

133 134

The refined olive oil (ROO) and extra virgin olive oil (EVOO) from Coratina cultivar were

135

supplied by IOBM (Industria Olearia Biagio Mataluni, Montesarchio, Benevento). Two EVOO

136

from Ravece cultivar were provided by APOOAT Soc. Coop arl (Avellino, Italy) in 250 mL green

137

glass bottles. The olive oil samples were stored under suitable conditions avoiding light exposure

138

and high temperatures in order to prevent oxidation and were used within eight months from their

139

production (November 2013).

140

Ethyl isobutyrate (99%), ethyl butyrate (99%), ethyl 2-methylbutyrate (99%), hexyl acetate

141

(99%), cis-3-hexenyl acetate (98%), hexanal (98%), trans-2-pentenal (95%), trans-2-hexenal

142

(98%), 1-hexanol (99%), cis-3-hexen-1-ol (99%), linalool (97%), and 1-penten-3-one (97%) were

143

supplied by Sigma–Aldrich (St. Louis, MO). The following reagents were used for the analysis:

144

hexane (95%), methanol (99.9%), glacial acetic acid, trifluoroacetic acid, acetonitrile, diethyl ether,

145

distilled water, supplied by Romil (Cambridge, England). Potassium iodide and sodium carbonate

146

were provided by AppliChem (Darmstadt, Germany). Ammonium acetate, Folin-Ciocalteu solution

147

and sodium hydroxide were purchased from Sigma-Aldrich. Sodium hydroxide, phenolphthalein

148

and starch were provided by Titolchimica S.p.A. (Rovigo, Italy). Sodium thiosulfate was supplied

149

by Fluka (Buchs, Switzerland), and chloroform was supplied by LabScan (Dublin, Ireland). The (-)-

150

epicatechin (90%) was supplied by Sigma-Aldrich.

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2.2 Sample preparation

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To study the effect of phenolic compounds on the release of olive oil aroma, the experimental

155

plan reported in Figure 1 was applied. Six model systems were set up in order to use known 6

156

amounts of aroma compounds in refined olive oil with added phenolic extract (Figure 1A). In order

157

to verify our results, obtained by using model systems, two EVOO were also analysed (Figure 1B).

158

Human saliva was added to the model systems and the samples were subsequently analysed by

159

using a retronasal aroma simulator, a dynamic headspace device simulating mouth conditions

160

(RAS). In the systems without saliva, 20 glass balls were added to obtain the same headspace

161

volume in all the samples.

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2.2.1 Preparation of the refined olive oil sample with added virgin olive oil phenolic compounds (ROOP)

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The phenolic extract was obtained from extra virgin olive cultivar Coratina, typically known for

167

its high content of phenolic compounds. An aliquot of the oil sample (200 g) was dissolved in

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hexane (200 mL) and vigorously shaken for 10 s. A subsequent extraction was carried out by using

169

a water/methanol mixture (40/60 v/v) in a separating funnel. This step was repeated three times by

170

using a total of 420 mL solvent. Subsequently, the obtained hydro-alcoholic extract was washed

171

with hexane to remove any oil contamination and was centrifuged for 10 min at 3500 rpm (PK-120;

172

ALC International s.r.l., Milan, Italy). The organic phase was removed from the sample, and the

173

hydro-alcoholic phase was collected in the flask and evaporated under vacuum in a rotary

174

evaporator at 40 °C (VV 2000; Heidolph, Schwabach, Germany). The phenolic compounds were

175

suspended using 40 mL methanol. An aliquot of the extract was used for the HPLC and Folin-

176

Ciocalteau analyses. An amount of 2 mL phenolic extract was adjusted to volume using refined

177

olive oil in a 100-mL volumetric flask and the oil mixture was treated in an ultrasonic bath for 15

178

min. Then, methanol was evaporated using a vacuum evaporator (Heidolph VV 2000) at 38 °C for

179

15 min (Garcia-Mesa, Pereira-Caro, Fernandez-Hernandez, Garcia-Ortiz Civantos, & Mateos,

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2008). The amount of phenolic compounds added to refined olive oil (334 mg kg‒1) was chosen on

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the basis of the average levels reported in the literature for extra virgin olive oil, equivalent to about 7

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260 mg kg‒1 total phenolic compounds according to Bayram, Esatbeyoglu, Schulze, Ozcelik, Frank

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and Rimbach (2012), and 220‒340 mg kg‒1 (slight bitterness taste of VOO) as indicated by Beltrán,

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Ruano, Jiménez, Uceda, and Aguilera (2007). The composition of the biophenols added to our

185

model system was as follows: hydroxytyrosol 1.0 ± 0.1 mg kg‒1, tyrosol 6.8 ± 0.1, dialdehydic form

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of elenoic acid linked to hydroxytyrosol 60.7 ± 1.3, dialdehydic form of elenoic acid linked to

187

tyrosol 138.0 ± 0.1, pinoresinol/acetoxypinoresol 49.0 ± 0.8, aldehydic form of elenoic acid linked

188

to hydroxytyrosol 56.7 ± 1.7, aldehydic form of elenoic acid linked to tyrosol 32.8 ± 0.3.

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2.2.2 Preparation of the refined oil sample with added (‒)-epicatechin (ROOC)

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Thirty-four mg of (-)-epicatechin were added to refined olive oil to reach a final volume of 100

193

mL to obtain a final concentration of 329.1 mg kg-1. The procedure applied was the same as

194

previously described for olive oil phenolic compounds.

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2.2.3 Preparation of the refined olive oil sample (ROO)

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In the control sample (ROO) phenolic extract was not added; 2 mL methanol were adjusted to

199

volume using refined olive oil in a 100-mL volumetric flask. Then, the oil mixture was subjected to

200

the same protocol, previously described, for the phenolic compounds addition.

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2.2.4 Preparation of aroma solutions

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The most abundant and significant volatile compounds of virgin olive oils were considered in

205

our study for preparing the solutions of aroma compounds, according to the literature (Kalua et al.,

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2007); they included 5 esters, 3 aldehydes, 2 alcohols, 1 ketone and 1 terpene (Table 1).

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Volatile compounds were dissolved in 10 mL refined olive oil and homogeneously mixed. The

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aroma solution was obtained by diluting 4 mL of each volatile compound in oil to 100 mL refined

209

oil. The aroma solution was added to oil sample 1 h before the analysis, in order to allow its

210

stabilisation. The final concentration for each volatile compound in oil sample was reported in

211

Table 1. The concentrations were chosen to stay within the range typically found in extra virgin

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olive oil (Angerosa et al., 2004; Kalua, et al., 2007).

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2.3 Human saliva sampling

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Mixed whole unstimulated saliva (about 150 mL) was separately collected from fourteen

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panellists 2 h after breakfast and thorough cleaning of teeth. Panellists (eight males and six females)

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were non-smoking volunteers (23‒48 years of age) from the Department of Agriculture, University

219

of Naples Federico II, with no known illnesses at the time of saliva donation and with normal

220

olfactory and gustatory functions. Before sampling, each panellist rinsed his/her mouth several

221

times with tap water to avoid any contamination from traces of toothpaste and/or microbial growth,

222

which might have caused the formation of extraneous volatile compounds (Buettner, 2002a; 2002b).

223

Each donor provided approximately 10‒12 mL saliva, which was combined and homogenised. It

224

was then separated into aliquots of 2 mL and finally frozen at ‒20 ˚C. Before being used, saliva

225

samples were put in a thermal bath at 37 ˚C and shaken in order to dissolve any suspension.

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2.4 Free acidity, peroxide value, and specific ultraviolet absorbance K232, K270 analyses

228 229

Refined olive oil and EVOOs (from Ravece cultivar) were analysed to determine their acidity

230

levels, peroxide value (PV), K232, K270 and ∆K according to UE Reg. 2568/91. Acidity was

231

expressed as oleic acid percentage (%), while PV was expressed as meq O2 per kg‒1 oil. For the

9

232

analysis of spectrophotometric indices a UV-visible Shimadzu UV-1601 spectrophotometer

233

(Shimadzu, Kyoto, Japan) was used. All analyses were performed in triplicate.

234 235

2.5 Extraction and analysis of phenolic compounds

236 237

The phenolic compounds were analysed both in ROO and EVOO Ravece samples. The

238

extraction and analysis of phenolic compounds were carried out according to Sacchi, Caporaso,

239

Paduano, and Genovese (2014). The quantification of phenolic compounds was carried out by using

240

the Folin-Ciocalteau and HPLC methods. The analyses were performed in triplicate for each

241

extraction.

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2.6 Release of aroma compounds in model mouth system

244 245

The measurement of aroma compound headspace release was performed under dynamic

246

conditions using an experimental RAS, as previously reported (Roberts & Acree, 1995; van Ruth &

247

Roozen 2000; Van Ruth, O’Connor & Delahunty, 2000; Genovese, Piombino, Gambuti & Moio,

248

2009; Genovese, Caporaso, Civitella & Sacchi, 2014). Aroma solutions (10 µL) were added to 10

249

mL of oil sample and the solution was left to react for 1 h. In the case of the system with extra-

250

virgin olive oils (Ravece), this addition was not performed. Subsequently, the solution was

251

transferred to the RAS device (100 mL), and maintained in a bath (Analytical Control De Mori,

252

Milan, Italy) at 37 °C. Ten microlitres internal standard (500 mg L‒1 isobutyl acetate 99.8% purity;

253

Fluka, Buchs, Switzerland), and 2 mL human saliva, previously kept at 37 ˚C were added to the

254

system. In the systems without saliva, 20 glass balls were added to obtain the same headspace

255

volume in all the samples.

256

The SPME fibre was inserted through the septum in the RAS Teflon and exposed to sample

257

headspace. Nitrogen flow (20 mL s-1), passed through the sample for 4 min. During that time, the 10

258

volatile compounds were trapped on the fibre. The exposure time was chosen according to data

259

previously published by other authors (Van Ruth et al., 2000).

260 261 262

2.7

Dynamic

headspace-solid phase microextraction

and

gas chromatography/mass

spectrometry analysis (SPME-GC/MS)

263 264

The SPME device (Supelco, Bellefonte, PA) was equipped with a 50/30 µm thickness divinyl-

265

benzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fibre coated with 2-cm length

266

stationary phase. Volatile compounds were analysed by GC-MS using a QP5050A (Shimadzu,

267

Kyoto, Japan) equipped with a Supelcowax-10 capillary column (60 m × 0.32 mm i.d., 0.5 µm

268

thickness; Supelco). Temperature was set at 40 °C for 4 min, followed by an increase of 3.5 °C

269

min‒1 up to 240 °C, and held for 3 min at maximum temperature. The injector was kept at 250 °C.

270

Helium was used as a carrier gas (1.4 mL min‒1). Volatile compounds thermal desorption was

271

carried out by exposing the SPME fibre in the injector for 10 min. The compounds identification

272

was performed by comparing retention times and mass spectra obtained by analysing pure reference

273

compounds in the same conditions. Moreover, the identification was confirmed by comparing mass

274

spectra with those of the NIST database. Mass spectra were recorded at 70 eV. Source temperature

275

was 200 °C and the interface temperature was 250 °C. Before using it, the fibre was conditioned at

276

270 °C for 1.5 h. A blank test was performed before each analysis to prevent the release of

277

undesirable compounds. All the analyses were performed in triplicate.

278 279

2.8 Quantitative analysis

280 281

The quantitative analysis of olive oil volatile compounds was carried out using selected ion

282

monitoring (SIM). The selected ions for each volatile compound are listed in Table 1. Peak areas of

283

each compound were normalised with respect to the area of the internal standard peak and 11

284

interpolated on the calibration curve. A calibration curve for each molecule was constructed by

285

preparing a solution containing known amounts of analyte in the oil and the internal standard. The

286

oil was diluted in order to obtain 1‒7 solutions with decreasing values of the concentration of

287

analytes and each of these solutions was analysed by SPME/GC-MS in selected ion monitoring, by

288

applying the same conditions, previously reported (see sections 2.6 and 2.7). The concentration

289

range considered for the calibration curve of each molecule (Table 1) was within the values

290

typically found in virgin olive oil (Angerosa et al., 2004; Kalua et al., 2007). Peak areas were

291

calculated by using the Lab Solutions acquisition system (GCMS Solutions version 1.20; Shimadzu,

292

Kyoto, Japan).

293 294

The linear regression coefficient (r2) for the studied volatile compounds was satisfactory, higher than 0.9975 (Table 1).

295 296

2.9 Estimation of physicochemical properties of the volatile compounds

297 298

The logarithm of octanol/water, octanol/air and air/water partition coefficients (log P), and

299

vapour pressure of the volatile compounds were calculated by using EPI Suite v.4.1 software, U.S.

300

Environmental Protection Agency and Syracuse Research Corp.

301 302

2.10 Statistical analysis of data

303 304

Significant differences among the different model systems were determined for each compound

305

by one-way ANOVA statistical analysis. Tukey’s test was used to discriminate among the means of

306

the variables. Differences with p < 0.05 were considered significant. Data elaboration was carried

307

out using XLStat (version 2009.3.02), an add-in software package for Microsoft Excel (Addinsoft

308

Corp., Paris, France).

309 12

310

3. Results and discussion

311 312

Table 2 reports the free acidity, peroxide value, ultraviolet indices (K232, K270, ∆K), and total

313

phenolic compounds in refined and EVOOs from Ravece cultivar. Free acidity, peroxide value,

314

K232, K270, and ∆K of all olive oil samples were within the legal limits of the category they belong

315

to, i.e. extra virgin olive oil (EC 2568/91). Total phenolic compounds of EVOOs, determined by

316

colorimetric measurement using Folin-Ciocalteau reagent, were 431 and 410 mg kg‒1, respectively

317

in EVOO 1 and EVOO 2. In the refined olive oil, phenolic compounds were about 44.0 mg kg‒1,

318

while in the refined olive oil with added phenolic extract of VOO, their level was 328 mg kg‒1.

319 320

3.1 Effect of phenolic compounds addition on aroma release in oil model systems

321

In order to verify the effect of phenolic compounds on VOO aroma release, a model system

322

(ROOP) was produced by adding VOO phenolic extract to ROO, to obtain a final concentration of

323

328 mg kg‒1. Aroma compounds were then added both to ROO and ROOP, and allowed to interact

324

with human saliva (Figure 1A). A third model system with added catechins (ROOC) was also

325

produced as a reference.

326

Figure 2 shows the headspace concentration of ethyl esters, acetates, and linalool in model

327

systems with and without saliva. Ethyl esters and linalool showed lower concentration in their

328

headspace in the three-phase system (retronasal simulation essay), obtained when saliva was added

329

to olive oil. This decrease of esters and linalool was statistically significant only when olive oil

330

phenolic compounds were added to such a system, while this difference was not observed for

331

acetates, when comparing retronasal and orthonasal essays.

332

In the retronasal essay a series of interrelated partition effects determine the distribution of

333

aroma compounds among the different phases in the RAS device (Van Ruth et al., 2002). Aroma

334

compounds distribute between olive oil and saliva, as well as between the liquid phases and air

335

phase. Generally, molecular weight and hydrophobicity are important factors influencing aroma 13

336

release in model mouth conditions and volatile compounds with similar structure are expected to

337

partition similarly. The hydrophobicity of a molecule, expressed as log Po/w (octanol‒water partition

338

coefficient), could be a useful means to understand the retention and/or the release of volatile

339

compounds from the system in the sample headspace. A high octanol‒water partition coefficient

340

indicates that a particular compound preferentially partitions into organic phase rather than water;

341

thus, it is inversely related to the solubility of a compound in water. On the contrary, log Po/a and

342

log Pa/w are useful to understand the liquid‒air partitioning (Reineccius, 2006). Therefore, our

343

results are in accordance with literature data, with the sole exception for acetates, which were also

344

those having the highest molecular weight as well as the highest hydrophobicity.

345

Figure 3 reports the headspace concentration of aldehydes, alcohols, and 1-penten-3-one in

346

model systems with and without saliva. In this case, the most hydrophilic compounds and those

347

with the lowest molecular weights were trans-2-pentenal and 1-penten-3-one. Both compounds

348

showed higher headspace release when saliva was added to olive oil in the absence of phenolic

349

compounds, with respect to orthonasal essay. However, only for trans-2-pentenal was the higher

350

release statistically significant. This result is in accordance with van Ruth and co-workers (2001),

351

where a salting out effect was reported for hydrophilic compounds, due to the binding interaction of

352

hydrophobic compounds with salivary proteins, in a similar system set up using sunflower oil.

353

However, there was not always a similar behaviour in the release or retention of volatile compounds

354

based exclusively on their hydrophobicity. Probably, this effect could be caused by other factors

355

such as non-volatile food matrix components, which could affect the release of volatiles, since they

356

are responsible for direct interactions with aroma compounds or with salivary constituents.

357

While the addition of VOO phenolic compounds and catechins caused little or few modifications

358

in the levels of headspace volatile compounds, the addition of human saliva in the systems caused

359

stronger effects (Figures 2 & 3). Catechin reactivity is due its numerous hydroxyl functional groups

360

and its aromatic ring, and it is characterised by a molecular weight of about 290 Da. It has been

361

reported that phenolic compounds, including catechin, could exhibit weak interactions with some 14

362

aroma compounds (e.g., hydrophobic interaction, π-π), with a consequent important role in flavour

363

release (Pozo-Bayón & Reineccius, 2009). Olive oil phenolic compounds are oleuropein and

364

ligstroside derivatives (Carrasco-Pancorbo et al., 2005). The simplest molecular forms of a mixture

365

of biphenyl alcohols are tyrosol and hydroxytyrosol (Bendini et al., 2007). Weak interactions

366

between olive oil phenolic compounds and proteins have been reported, these interactions being

367

weaker than tannic acid in their binding action toward food proteins (Pripp, Vreeker & van

368

Duynhoven, 2005). This information could allow us to hypothesise that olive oil phenolic

369

compounds are likely to interact also with salivary proteins. However, no data have been published

370

so far on the interaction between olive oil phenolics and volatile compounds.

371

No significant differences were found in the orthonasal system on the headspace release of volatile

372

compounds with the addition of olive oil phenolic compounds or catechins. The sole exception was

373

represented by trans-2-pentanal that showed higher release in the presence of catechin. Lorrain and

374

co-workers (2013) recently reported a study investigating the effect of catechin on odour release of

375

some esters in wine. The authors reported that the most apolar esters were the most affected by

376

catechin addition, and suggested that steric hindrance would also reduce the magnitude of

377

interaction. In our study catechins led to a general increase of the headspace release for ethyl esters

378

and acetates, even if this increase was not statistically significant. Moreover, other literature data

379

based on in vivo study of wine retronasal aroma, reported an enhancing effect of wine polyphenols

380

on the release of some volatile compounds (Munoz-Gonzalez, Martin-Alvarez, Moreno-Arribas &

381

Pozo-Bayón, 2014b). In our experiment the addition of saliva to the system with catechins, caused a

382

significant increase of cis-3-hexen-1-ol, trans-2-pentenal and trans-2-hexenal. On the contrary, the

383

presence of olive oil phenolic compounds always caused significant lower headspace concentrations

384

of all volatiles, with the exception of hexanal and trans-2-pentenal. These results suggest different

385

interactions among volatile compounds, saliva, and catechins, or VOO phenolic compounds.

386

Evidence for a weak binding effect of catechins and salivary constituents directly on volatile

387

compounds, which would result in a strong action of lowering the headspace release of volatile 15

388

compounds, has already been reported (Pozo-Bayón & Reineccius, 2009; Buettner, 2002a;

389

Genovese et al., 2009; Van Ruth & Roozen, 2000; Friel & Taylor, 2001; Munoz-Gonzalez et al.,

390

2014b). A strong interaction between catechins and salivary constituents is also expected (De

391

Freitas & Mateus, 2001; Bennick, 2002), with an effect that counterbalances the other phenomena

392

previously described.

393

The final effect was a slightly higher release of volatile compounds in the presence of catechins. In

394

the case of olive oil phenolic compounds, a different interaction could occur. In fact, the

395

saliva‒volatiles interaction is expected to be similar, and probably of the same magnitude, as

396

hypothesised for the system with catechin. On the contrary, phenolics‒saliva interaction is supposed

397

to be lower than catechin‒saliva binding, as previously reported by Pripp, Vreeker and Van

398

Duynhoven (2005) for food proteins, where a weak binding interaction was found. It would result in

399

a higher saliva effect and a consequent lower release of volatile compounds from olive oil.

400

However, these interactions do not fully explain the results found in our model systems, because if a

401

higher effect of saliva had occurred, we would have obtained a headspace release similar to ROO.

402

In fact, in ROO sample phenolic compounds were not added and, therefore, the differences should

403

be only due to saliva. Therefore, it is possible to hypothesise a direct interaction also of VOO

404

phenolics with aroma compounds. The magnitude of this binding depends upon the chemical

405

characteristics of each molecule, e.g., functional group, molecular weight, hydrophobicity, vapour

406

pressure, etc.

407

Retronasal release of hexanal increased about 3.2 times when saliva was added to olive oil

408

(Figure 3). This increase did not depend upon the presence of phenolic compounds, as no statically

409

significant difference was observed between ROO, ROOP, and ROOC models. This result was

410

unexpected, since it is known that mucin can bind aldehydes to form Schiff bases and their level

411

may consequently decrease (Friel & Taylor, 2001). The increase in hexanal concentration could be

412

explained by different phenomena. The first possible explanation of this effect is that the available

413

time for interaction between mucin and saliva (4 minutes, as reported by literature data) is too short, 16

414

as it occurs for the esterase and carboxylase enzymes, which interact with ester and acetate

415

compounds (Seuvre, Philippe, Rochard & Voilley, 2007). Therefore, hexanal reduction could not

416

have occurred in this case. The second explanation of this effect is that the results could be due to

417

the salting out effect as described by Van Ruth and co-workers (2001) in a model system of

418

sunflower oil after addition of artificial saliva (containing salts, mucin, and α-amylase) or by simply

419

adding water (Van Ruth & Roozen, 2010). The authors explained this phenomenon as due to the

420

hydrophilicity of the molecules. However, it does not explain why the concentration of other

421

hydrophobic molecules did not increase in our systems. Another possible reason could be the action

422

of other salivary enzymes, different from those ones previously considered. These enzymes, such as

423

acyl hydrolases (AH), lipoxygenase (LOX), or hydroperoxide lyases (HPL) are found in the mouth

424

and could interact with lipids to form hexanal (Salles et al., 2011). It is also assumed that they may

425

play an important role in fat perception (Neyraud, Palicki, Schwartz, Nicklaus & Feron, 2012).

426

Hexanal is the main end-product of lipid oxidation in vegetable oils, in which linoleic acid is the

427

most abundant oxidative substrate. Linoleic fatty acid is oxidised by LOX to form hydroperoxide,

428

which is subsequently degraded by HPL to form hexanal (Kalua et al., 2007). The lingual lipases

429

are released continuously and ranged from 0 to 12 mmol fatty acids min per litre, with a median

430

activity of 2 mmol (Stewart, Feinle-Bisset, Golding, Delahunty, Clifton & Keast, 2010), which

431

occurs within 1–5 s. Generally, this short time is enough to perceive fat (Kawai & Fushiki, 2003).

432

trans-2-Hexenal could also derive biosynthetically from lipase action, by the isomerisation of cis-3-

433

hexenal. Its level did not increase significantly in our sample, probably because it requires longer

434

interaction times or because of the absence of the necessary isomerase in human saliva.

435 436

3.2 Retronasal aroma release in EVOO systems

437 438

In order to compare our model systems with a real food system, we used two commercial

439

EVOOs from Ravece cultivar, by analysing headspace concentration of the volatile compounds 17

440

considered in our study. The approach was similar to that described for model systems, with the

441

exception that volatile and phenolic compounds were not added but were constitutively present in

442

the matrix. Similarly to model systems, saliva addition did not cause significant changes in the

443

release of volatile compounds, with the exception of hexanal, cis-3-hexen-1-ol and trans-2-

444

pentenal, the latter two compounds being different only in the case of sample 2 (Figure 4).

445

The effect of phenolic compounds on headspace release of ethyl esters and linalool was not

446

significant, being mainly present at trace level in EVOO. While the presence of linalool, as well as

447

terpenic compounds, could depend on the olive variety (Vichi, Guadayol, Caixach, Lopez-Tamames

448

& Buxaderas, 2006), the level of ethyl esters could be related to VOO defects. In fact, at low levels,

449

ethyl esters contribute to olive oil flavour (Kalua et al., 2007), while at higher levels they are

450

associated with fusty and musty defect of virgin olive oil (Morales, Luna & Aparicio, 2005).

451

Hexanal has been similarly related to rancid defect in virgin olive oils at high concentrations

452

(Morales et al., 2005). In our samples, retronasal release was lower than those obtained for rancid

453

VOOs. However, a higher increase was observed in sample 1 (about five times) than sample 2

454

(about two times). Probably, this difference could be due to a higher oxidation of sample 1. In fact,

455

it showed a higher level of hydroperoxides, 11.8 meq of O2 kg‒1, compared to 7.8 meq of O2 kg‒1 in

456

sample 2 (Table 2). These findings suggest that the direct action of hydroperoxide lyases (HPL) on

457

hydroperoxides, obtained from linoleic acid oxidative degradation, should be the main cause of

458

hexanal release from VOO in mouth conditions.

459

The concentration of trans-2-pentenal was significantly higher in sample 2 in retronasal

460

simulation essay, while oil 1 showed no statistical difference. This result was in accordance with

461

our model system for EVOO 1, while in the case of EVOO 2, no difference was observed.

462

Therefore, considering both experimental systems, it is possible to state that VOOs with the

463

same concentration of "defective" volatile compounds (associated with fusty and musty defects)

464

could be partially masked by a high concentration of natural phenolic compounds. Our results also

465

suggest that higher levels of rancidity would probably be better perceived by tasters via retronasal 18

466

rather than orthonasal in vivo analysis (sensory assessment). Further studies could apply a

467

promising technique called BOSS (buccal odour screening system), which includes the intra-oral

468

extraction of odour compounds after food consumption under optimised in vivo sampling

469

conditions, together with analysis via high resolution gas chromatography-olfactometry (Buettner et

470

al., 2004).

471 472

4. Conclusion

473 474

The presence of VOO phenolic compounds resulted in a lower headspace concentration of

475

almost all the volatile compounds studied, in simulated retronasal conditions (with saliva addition),

476

while this difference was not observed in the case of catechins. Very little differences were

477

observed in orthonasal simulation essay on the volatile compounds, also by considering the

478

presence of phenolic compounds and catechins. The main reason for these results is the dynamic

479

equilibrium and the factors influencing it, particularly the two-phase orthonasal system and three-

480

phase system in the simulated retronasal condition. A great influence is attributed to the

481

physicochemical interactions due to salivary components, but also to the food matrix and

482

specifically to the presence of phenolic compounds. Our results suggest that the low reactivity of

483

olive oil phenolic compounds toward mucin causes a higher binding activity of this latter compound

484

with volatile compounds, with respect to catechins. In addition, it is possible to hypothesise a direct

485

interaction of phenolic compounds with aroma compounds. Independently from the presence of

486

phenolic compounds, hexanal concentration clearly was higher in retronasal assay than orthonasal,

487

indicating lipid oxidation by salivary enzymes.

488

The consequent practical application of our work could be interesting, both from a sensory point

489

of view and at industrial level. Firstly, because of the possible interacting effect of phenolic

490

compounds on the aroma release and perception, which could influence the score given by

491

panellists for EVOOs with slight defective notes, which would not be perceived in very bitter19

492

pungent samples because of a "masking effect". In such a case, it is not the "distracting effect" of

493

kinesthetic sensation that would result in a possible lower score for these aromatic attributes, but

494

our data suggest that there is a physicochemical trapping of some volatile compounds in the matrix.

495

This is particularly true for some typical EVOOs, where peculiar sensory notes are perceived and

496

are required by law, in order to label them as Protected Designation of Origin (PDO). As a

497

consequence, industry would also benefit from our research when designing a food product with

498

high phenolics content because of the possible implications, positive or negative, on the level of

499

some aroma compounds. While the presence of catechin in a food preparation could imply no

500

change or a slightly higher headspace release retronasally, with probably a consequent higher aroma

501

perception, in the case of VOO phenolic compounds a lower release is expected.

502

However, the extent of these effects could be higher during consumption than those found in the

503

present experiment, because of the in vitro conditions we used. This could occur because in vivo

504

consumption represents a more dynamic process in which saliva is continuously produced and

505

replenished. Further sensorial studies are needed to confirm our findings and better understand

506

whether and to what extent the matrix composition and the dispersed water affect volatile release

507

during VOO tasting.

508 509

Acknowledgements

510

Prof. Marco Genovese is acknowledged for proofreading the English manuscript. This research

511

was not supported by any specific grant or funding from public institutions or private companies.

512

All authors declare no competing financial interests.

513 514

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516

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632 633 634

Figure captions:

635 636

Figure 1. Experimental plan applied for the Retronasal Aroma Simulator (RAS) analysis of model

637

olive oil (A) and extra virgin olive oil (B) systems.

638

25

639

Figure 2. Headspace concentration of ethyl esters, acetates, and linalool in model systems of

640

Refined Olive Oil (ROO), ROO added with VOO phenolic compounds (ROOP), and added with

641

(‒)-epicatechin (ROOC).

642

Different letters indicate statistically significant differences (p < 0.05) between phenolic

643

compounds, catechin, and blank samples. Asterisks indicate significant differences between

644

saliva/no saliva runs. Error bars indicate standard deviation (n = 3).

645 646

Figure 3. Headspace concentration of aldehydes, alcohols, and 1-penten-3-one in model systems of

647

refined olive oil (ROO), ROO added with VOO phenolic compounds (ROOP), and with added (‒)-

648

epicatechin (ROOC).

649

Different letters indicate statistically significant differences (p < 0.05) between phenolic

650

compounds, catechin, and blank samples. Asterisks indicate significant differences between

651

saliva/no saliva runs. Error bars indicate standard deviation (n = 3).

652 653

Figure 4. Headspace concentration of volatile compounds in two EVOO samples.

654

Different letters indicate statistically significant differences (p < 0.05) between samples. Asterisks

655

indicate significant differences between saliva/no saliva runs. Error bars indicate standard deviation

656

(n = 3).

657 658 659

26

Figure 1

Figure 2

Figure 3

Figure 4

Table 1. Chemical standards, concentration added, odour descriptor, physicochemical properties, and MS fragments used for quantitative analysis. Compound

Concentration added (mg kg-1)

Odour descriptora

Molecular weight

Carbon atoms

log Pb o/w

o/a

a/w

Vapour pressure (Pa at 25 °C)b

Ion fragmentsc (m/z)

r²

Range of calibration curve (mg kg‒1)

n

Ethyl esters Ethyl isobutyrate Ethyl butyrate Ethyl-2-methylbutyrate Hexyl acetate cis-3-Hexenyl acetate

0.1172 0.1070 0.0998 0.1817 1.2747

FruityI CheesyI, fruityII FruityI I, II Fruity , sweetI, floralI Banana-likeI, greenI, II, fruityI, II , floralI, esterI

116 116 130 144 142

6 6 7 8 8

1.77 1.85 2.26 2.83 2.61

3.546 3.637 3.912 4.494 4.195

‒1.78 ‒1.79 ‒1.65 ‒1.66 ‒1.59

3226 1946 1071 193 152

71-116-88 71-88 57-102 43-56-84 43-67-82

0.9990 0.9992 0.9999 0.9991 0.9992

0.0058‒1.4060 0.0053‒1.2841 0.0049‒1.1970 0.0089‒2.1808 0.0627‒15.2964

6 6 5 6 6

Aldehydes Hexanal trans-2-Pentenal trans-2-Hexenal

1.3322 0.1013 5.0726

GreenI, green appleI, cut grassI, II GreenI, II, appleI, grassyII, pleasantI, floralI GreenI, II, apple-likeI, bitter almondI, cut grassI, II

100 84 98

6 5 6

1.8 1.09 1.58

3.84 3.607 4.279

‒2.06 ‒2.52 ‒2.70

1276 2466 629

56-57-72 55-84-83 55-69-83

0.9990 0.9996 0.9995

0.0085‒15.9859 0.0050‒1.2154 0.2493‒60.8713

5 6 7

C6-Alcohols 1-Hexanol cis-3-Hexen-1-ol

0.1716 0.1734

FruitI, grassI, II, floralII, aromaticI Leaf -likeI, greenI, II, pungentI, bananaI, herbalII

102 100

6 6

1.82 1.61

5.185 4.808

‒3.16 ‒3.20

117 125

56-55-69 67-82-55

0.9994 0.9996

0.0084‒2.0589 0.0085‒2.0812

7 6

Others Linalool 1-Penten-3-one

0.0718 0.0945

LilacII, lavenderII Green , pungent , sweetI, strawberryI, sharpI, metallicI

154 84

10 5

3.38 0.9

6.026 3.748

‒3.06 ‒2.85

11 5092

71-93-121 55-84

0.9975 0.9997

0.0088‒0.8615 0.0046‒1.1345

4 7

I

I

n = Numbers of calibration points. r² = linear regression coefficient. a The odour descriptors were indicated as reported in the literature: IAngerosa, Servili, Selvaggini, Taticchi, Esposto, & Montedoro, 2004; II Kesen, Kelebek & Selli, 2014. b The logarithm of octanol/water, octanol/air and air/water partition coefficients (log P) and vapour pressure of the volatile compounds were calculated using EPI Suite v.4.1 software, U.S. Environmental Protection Agency and Syracuse Research Corp. c Bold numbers indicate quantifier ions.

Table 2. Legal quality indices for olive oil and total phenolic compounds for refined olive oil (ROO) and two extra virgin olive oils (EVOO) from Ravece cultivar. ROO

EVOO 1

EVOO 2

ROO with added phenolic extract

EVOO accepted values

ROO accepted values

Acidity 0.09 ± 0.01 0.21 ± 0.03 0.21 ± 0.02 ≤ 0.80 ≤ 0.30 Peroxide value 0.80 ± 0.04 11.79 ± 0.37 7.81 ± 0.86 20.0 ≤ 5.0 K232 1.820 ± 0.050 2.177 ± 0.007 1.984 ± 0.024 ≤ 2.50 K270 0.694 ± 0.020 0.160 ± 0.003 0.159 ± 0.005 ≤ 0.22 ≤ 1.10 ∆K 0.078 ± 0.004 ‒0.065 ± 0.007 ‒0.050 ± 0.000 ≤ 0.01 ≤ 0.16 Total phenolic compounds 44.0 ± 7.1 431.1 ± 93.2 410.3 ± 34.1 328.0 ± 59.1 Acidity was expressed as oleic acid equivalent. Peroxide value was expressed as meq O2 kg‒1 oil. Total phenolic compounds were expressed according to the Folin-Ciocalteu method and expressed as mg kg‒1. Values are the average of three replicate analyses (n = 3).

660 661


Volatiles release in model systems simulating virgin olive oil (VOO) was studied

662


Mouth conditions were simulated by addition of human saliva and SPME-GC/MS analysis

663


The presence of VOO phenolics caused lower headspace release of many volatiles

664


Hexanal headspace concentration was always increased in retronasal compared to orthonasal

665

assay

666 667 668 669 670 671

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