Accepted Manuscript

Characterization of virgin walnut oils and their residual cakes

produced from different varieties

Rosa M. Ojeda-Amador, María Desamparados Salvador, Sergio

Gómez-Alonso, Giuseppe Fregapane

PII: S0963-9969(18)30256-4
DOI: doi:10.1016/j.foodres.2018.03.066
Reference: FRIN 7503
To appear in: Food Research International
Received date: 22 December 2017
Revised date: 22 March 2018
Accepted date: 25 March 2018

Please cite this article as: Rosa M. Ojeda-Amador, María Desamparados Salvador, Sergio
Gómez-Alonso, Giuseppe Fregapane , Characterization of virgin walnut oils and their
residual cakes produced from different varieties. The address for the corresponding author
was captured as affiliation for all authors. Please check if appropriate. Frin(2018),
doi:10.1016/j.foodres.2018.03.066

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 ACCEPTED MANUSCRIPT

Characterization of virgin walnut oils and their residual cakes produced

from different varieties

Rosa M. Ojeda-Amadora, María Desamparados Salvadora, Sergio Gómez-

Alonsob, Giuseppe Fregapanea*
a
Departamento de Tecnología de Alimentos, Facultad de Ciencias Químicas,
Universidad de Castilla-La Mancha, Ciudad Real, Spain

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b
Instituto Regional de Investigación Científica Aplicada, Universidad de Castilla-
La Mancha, Ciudad Real, Spain

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*Corresponding author: Giuseppe Fregapane, giuseppe.fregapane@uclm.es
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Other authors e-mails:
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Rosa M. Ojeda-Amador: rosamaria.ojeda@alu.uclm.es

Sergio Gómez Alonso: sergio.gomez@uclm.es

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María Desamparados Salvador, amparo.salvador@uclm.es

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Abstract

This study addresses the composition and properties of different walnut

varieties (Chandler, Hartley and Lara), in particular their virgin oils and residual

cakes obtained by screw pressing employing different cultivars. Among nuts,

walnut (Juglans regia L.) exhibits interesting nutritional value, mainly due to

their high content in linoleic acid, phenolic and tocopherol compounds, which

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show antioxidant and other healthy properties. Valuable results related to fatty

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acid profile and minor components were observed. Virgin walnut oil is a rich

source in linoleic acid (60 – 62 %) and γ-tocopherol (517 – 554 mg/kg).

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Moreover, walnuts show a very high content in total phenolic compounds
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(10,045 – 12,474 mg/kg; as gallic acid), which contribute to a great antioxidant

activity (105 – 170 mmol/kg for DPPH, and 260 – 393 mmol/kg for ORAC),
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being the hydrolysable tannins (2132 - 4204 mg/kg) and flavanols (796 - 2433

mg/kg) their main phenolic groups. Aldehydes account for the highest
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contribution to aromatic volatiles in virgin walnut oil (about 35% of total). As

expected, polar phenolic compounds concentrate in the residual cake, after the
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separation of the oily phase, reaching a content of up to 19,869 mg/kg, leading

to potential added value and applications as source of bioactive compounds to

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this by-product.
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Keywords: walnut; virgin oil; residual cake; composition; phenolics; antioxidant

capacity.
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1. INTRODUCTION

A great interest in acquiring more knowledge regarding the health promoting

effects ascribed to nuts exists among the scientific community, since there are

increasing evidences from prospective observational studies that nut intake

lowers the risk of cardiovascular disease (Kris-Etherton, Zhao, Binkoski, Coval

& Etherton, 2001) and produce a beneficial impact on other conditions such as

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hypertension, diabetes, inflammation and cancer (Salas-Salvadó et al., 2008;

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Grosso, Yang, Marventano, Micek, Galvano & Kales, 2015), as observed in the

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PREDIMED study (Prevention with Mediterranean Diet) and other

epidemiological or clinical trials. These described benefits are probably mainly

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due to the content in unsaturated fatty acids and phytochemicals like phenolics,

tocopherols, sterols and other biological active compounds (Kamal-Eldin &

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Moreau, 2010). Among nuts, walnuts are known to be a good source of

essential linoleic acid, and therefore of PUFA, which decreases LDL-cholesterol

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and increases HDL-cholesterol (Davis et al., 2007); moreover, they also

possess phytosterols, dietary fiber, phenolics and tocopherols that may be

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beneficial for health (Anderson, Teuber, Gobeille, Cremin, Waterhouse &

Steinberg, 2001; Amaral, Alves, Seabra & Oliveira, 2005).

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Walnut (Juglans regia L.) is a plant of the Juglandaceae family native to Central

Asia, the western Himalayan chain and Kyrgyzstant (Fernández-López, Aleta &

Alías, 2000) and was introduced in Europe before Roman times (1000 B.C.)

and spreads later to many other regions with Mediterranean type ecosystems

throughout the world such as USA and North of Africa. China is the major crop

producer followed by the USA, Iran, Turkey and France (Martínez, Labuckas,
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Lamarque & Maestri, 2010). In Spain, 7811 hectares are registered with a

production of 14,230 tons/year, mainly in the regions of Galicia, Extremadura

and Andalucía with over 15% of the national production each one of them;

Castilla-La Mancha contributes with 1,159 tons/year (about 8% of the total

amount). Hartley and Chandler are the main cultivars in Spain (MAGRAMA

2013, Spanish Ministry of Agriculture), both varieties are characterized in this

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study.

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Apart from nuts themselves, a growing interest towards virgin vegetable oils has

also appeared in recent years, which goes beyond the virgin oil for excellence,
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that produced from olive. The main reasons for this trend are marked by

consumers that increasingly appreciate the taste and smell of oils related to the
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raw materials from which they come from as well as by their potential nutritional

properties, resulting in novel oils with added value for the consumers as
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compared to the most common industrial refined vegetable oils (Kamal-Eldin et

al., 2010), being the denomination “virgin” or “cold pressed” oils generally
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recognized as a synonym for quality and enjoyment by consumers (Matthäus,

2008). According to FAO-WHO Codex Stan 210, the difference between these
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two categories is that “cold pressed” oils are obtained without the application of
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heat, whereas the “virgin” oils can be produced by application of heat. A recent

project for amending this standard for named vegetable oils (CX/FO 15/24/11,

2015) is under study and consideration with the purpose of incorporate

composition standards for cold pressed oils including virgin nut oils (e.g. walnut,

pistachio, hazelnut and avocado). Walnut oil presents a 4:1 ratio between n-6

and n-3 polyunsaturated fatty acids that helps to prevent diseases, although
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makes the oil more susceptible to oxidation; being the oxidative deterioration

reduced by the high content in natural antioxidants that also contributes to

health (Gharibzahedi, Mousavi, Hamedi & Khodaiyan, 2014).

These virgin oils obtained from seeds and nuts are considered as specialty oils

due to their flavour and potential nutritional properties and are generally

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produced in small size mills with a low production and mainly employed in

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gastronomy (Kamal-Eldin et al., 2010), as well as in cosmetic formulations

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(Hannon, 1997). As well known, industrial edible vegetable oils are mainly

manufactured by solvent extraction, which require a refining process that

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remove a great amount of minor components like antioxidants and aromatics.

On the contrary, the employment of mechanical processes, such as expelling or

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pressing – allow the maintenance of high levels of bioactive compounds with

health-promoting and functional properties (Ramadan & Elbanna, 2017).

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Mechanical process generates a partially defatted by-product (denominated

residual cake) which retains nutrients and bioactive compounds present in their
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kernels, being of great interest their valorisation as potential functional

ingredients (Santos, Corrêa, Carvalho, Costa, França & Lannes, 2013).

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The purpose of the present work is the evaluation of the fatty acid profile and

minor components of walnuts and in particular of their virgin oils and

corresponding residual cakes produced by screw pressing, employing three

different nut varieties in order to advance in the characterization of these

products and show possible cultivars driven differences, mainly regarding their

minor components profiles – phenolics, tocopherols and volatiles - and their

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antioxidant properties. A comparison with several commercial walnut virgin oils

has also been performed.

2. MATERIALS AND METHODS

2.1. Walnut samples.

Three walnut varieties (Chandler, Hartley and Lara; 3 kg each) were supplied by

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the company “Nueces de Navarra” (Navarra, Spain).

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2.2. Virgin walnut oils

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The oil was extracted using a screw press (Komet Screw Oild, Expeller CA59G-
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CA563, IBG Monforts Oekotec GmbH & Co. KG, Germany) employing a nozzle

of 6 mm of diameter and a screw speed of 30 rpm. The screw press was first
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run empty for 10-15 min to raise the screw-press barrel temperature to the

minimum required for extracting the oil (about 50ºC) using the electrical
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resistance ring attached around the press barrel. The resulting crude oil

(approximately 1.5 L of each variety) was centrifuged at 5,000 rpm in order to

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remove the residual plant material. Oil samples were stored in amber bottles

without headspace to protect them from light and the residual cakes were place
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in labelled pouches and vacuum packed to prevent any oxidative degradation.

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All samples were stored at 4°C in darkness until analysed.

Furthermore, five commercial virgin walnut oils have also been studied and

acquired from “Oleonucis” (Cuenca, Spain), “Azada” (Tarragona, Spain), “La

Tourangelle” (Loire Valley, France), “Cauvin” (Saint-Gille, France) and “Clovis”

(Reims, France).
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2.3. Moisture and fat content

The water content of the walnut and residual cake was determined by

desiccation in a vacuum oven at 100°C to a constant weight (ISO 662: 2016).

The fat content was determined by Soxhlet extraction, according to the AOAC

method 920.39.

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2.4. Fatty acid composition

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The methyl esters were prepared by vigorous shaking of a solution of oil in

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heptane (0.5 g in 3 ml) with 0.5 ml of 2 N methanolic potassium hydroxide and

then analysed by GC using an Agilent Technologies (HP 6890; Santa Clara,

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CA) chromatograph equipped with a FID detector. A fused silica capillary

column (60 m length x 0.25 mm i.d.) coated with SP TM-2380 phase (0.2 μm
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thickness; Supelco, Madrid, Spain) was used. Helium was used as the carrier

gas at a flow rate of 1 ml/min. The injector and detector temperature were set at
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220 and 250 °C, respectively. The oven temperature was maintained at 185°C

and the injection volume was 1 μl. Individual fatty acid were expressed as a
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percentage of the total, according to the European Regulation (EC) 2568/91

and subsequent amendments, corresponding to the American Oil Chemists’

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Society (AOCS) method Ch 6-91.

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2.5. Tocopherols were determined after double extraction, according to the

AOCS Method Ce 8-89. Briefly, 0.4 g of ground nut kernel or residual cake in n-

hexane (6 + 4 mL) was vortexed for 2 min, followed by 5 min of ultrasound and

then centrifugation at 2000 g for 10 min. Then, the combined extracts were

filtered prior to analysis. For nut oils, a solution of 0.1 g in 10 mL n-hexane was
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prepared and analysed on an Agilent Technologies HPLC (1100 series) using a

silica gel LiChrosorb Si-60 column (particle size 5 μm, 250 mm x 4.6 mm i.d.;

Sugerlabor, Madrid, Spain), with n-hexane/2-propanol (98.5:1.5) at a flow rate

of 1 ml/min, as the eluent. A fluorescence detector (Waters 470, Milford, MA)

was used with excitation and emission wavelengths set at 290 and 330 nm,

respectively.

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2.6. Total phenolic compounds

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The total polar phenolic (TPP) content was analysed following the method

described by Vázquez, Janer & Janer (1973) and Gutfinger (1981). Briefly, a
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double extraction 0.4 g of the solid residue obtained from the n-hexane

extraction described above was performed in 20 mL MeOH:H2O:HCOOH

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(80:20:0.1; 10 + 10 mL), with 2 min vortex, followed by 5 min ultrasound and

centrifugation at 2000 g for 10 min. The combined extracts were filtered prior to
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analysis.

Virgin oil (5 g) was dissolved in n-hexane (10 mL) and 10 ml of

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MeOH:H2O:HCCOH (80:20:0.1). The mixture was vortexed for 2 min, followed

by 5 min of ultrasound and then centrifuged at 2000 g for 10 min. Finally, the
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polar fraction was isolated and filtered. Suitable aliquots (100500 µl) of the
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polar extracts were transferred into a 10-ml volumetric flask. Water (up to 8 ml)

and the Folin–Ciocalteu reagent (0.5 ml) were added. After 3 min, 1.5 ml of

saturated (20%, w/v) sodium carbonate solution was added to the reaction

mixture. After 30 min, the absorbance of the solution was measured at 725 nm

against a blank solution with a UV–visible spectrophotometer (Agilent

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Technologies 8453; Santa Clara, CA). A calibration curve was established using

gallic acid as the external standard.

2.7. Individual phenolic compounds by HPLC-DAD-MSn

Individual phenolic compounds were determined by an HPLC-DAD-ESI-MS/MS

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method adapted from conditions previously described (Castillo-Muñoz et al.,

2009). The analysis was performed on an Agilent 1100 series system (Agilent,

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Waldbronn, Germany) equipped with a photodiode array detector (DAD) and an

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LC/MSD Trap VL electrospray ionization mass spectrometry (ESI-MS/MS), both

coupled with an Agilent ChemStation for data processing.

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Aliquots (1.75 mL) of phenolic extracts were evaporated in a rotary evaporator
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at 35 °C under vacuum, and then were dissolved in 200 µL of methanol/water

(20:80, v/v) by sonicating (5min.) and vortexed (2 min) before injecting 20 µL in

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the HPLC system. Separation was achieved on a narrow-bore column Zorbax

Eclipse XDB-C18 (2.1 x 150 mm; 3.5 µm particle; Agilent), with pre-column
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Zorbax Eclipse XDB-C8 (2.1 x 12.5 mm; 5 µm particle; Agilent), both

thermostated at 40 ⁰ C by using a ternary gradient and a 0.19 mL/min flow

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rate. Eluents were (A) acetonitrile/water/formic acid (3:88.5:8.5 v/v/v), (B)

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acetonitrile/water/formic acid (50:41.5:8.5 v/v/v), and (C) methanol/water/formic

acid (90:1.5:8.5 v/v/v). The linear solvent´s gradient was as follows: zero min,

98 % A 2 % B and 0% C; 8 min, 98 % A 2% B and 0% C; 40 min, 70 % A 17 %

B and 13 % C; 54 min, 50 % A 30% B and 20 % C; 54.5 min, 30 % A 40 % B

and 30 % C; 59 min, 0 % A 50 % B and 50 % C; 60 min, 0 % A 50 % B and 50

% C; 67 min, 98 % A 2 % B and 0 % C. For identification, an Ion Trap ESI-

MS/MS detector was used in negative ion mode, setting the following
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parameters: dry gas N2, 8 L/min; drying temperature, 325 °C; nebulizer, N2, 50

psi; scan range, 50-1,200 m/z. Identification was based on spectroscopic data

(UV-Vis and MS/MS) obtained from authentic standard or data previously

reported in literature. Quantification was made using the DAD chromatograms

recorded at 280 nm and calibration curves for the analysed compounds were

prepared from pure standards (Sigma-Aldrich, Steinheim, Germany). Gallic acid

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derivates were quantified as gallic acid, glansreginin, vescalagin and ellagic

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acid derivates were quantified as ellagic acid, catechin was expressed as

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catechin, procyanidin was quantified as procyanidin B2. Unknown compounds

were quantified as gallic acid and named by their Retention Time (RT).
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2.8. Antioxidant activity was evaluated by measuring the radical scavenging
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effect of the methanolic extract toward the synthetic radical 2,2-diphenyl-1-

(2,4,6-trinitrophenyl)hydrazyl (DPPH), as reported previously by Brand-Williams,

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Cuvelier & Berset (1995) and Sánchez-Moreno, Larrauri & Saura-Calixto

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(1998). Briefly, 100 µl of the polar extract, also used for TPP determination, was
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added to a methanolic DPPH solution (2.9 ml, 6 × 10-5 M) and stored in the dark

for 30 min. The decrease in absorbance of the resulting solution was then
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measured at 515 nm using an Agilent 8453 spectrophotometer. A calibration

curve was constructed using Trolox as the external standard.

Furthermore, the oxygen radical absorbance capacity (ORAC) was assessed

(Dávalos, Gómez-Cordovés & Bartolomé, 2004) by preincubation of 20 µl of

polar extract and 120 µl of fluorescein (70 nM) at 37°C for 15 min. Next, 60 µl of

24 mM 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) was added and

the mixture incubated at 37°C, with the kinetics measured every minute for 80
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min, with a pre-measured agitation at maximum intensity for 10 s. The

experiment was carried out in Nunclon black 96-well flat-bottom plates (Sigma-

Aldrich, Madrid, Spain) and measurements acquired using a plate reader with

emission and excitation filters set at a wavelength of 528 and 485 nm,

respectively. The fluorescence curves were normalized with respect to the blank

curve (without antioxidant). The calibration curve was prepared using Trolox as

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the external standard.

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2.9. Volatile Compounds

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A solid-phase microextraction (SPME) technique, followed by GC, was used to
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analyse the volatile compounds in the nut virgin oil samples, using a method

adapted from Vichi, Castellote, Pizzale, Conte, Buxaderas & López-Tamames

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(2003). Virgin oil (1.5 g) was spiked with 4-methyl-2-pentanol (as the internal

standard) to 1.5 mg/kg and placed in a 10-mL vial fitted with a silicone septum.
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The SPME sampling was performed by exposing the DVB/Carboxen/PDMS

fiber (50/30 µm, 2 cm long; Supelco) in the headspace of the sample

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maintained at 40 °C, for 30 min. It was then retracted into the needle and

immediately transferred and desorbed for 5 min in the injection port of an

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Agilent 6850 gas chromatograph equipped with an Agilent 5975C mass detector
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(Agilent Technologies). Compounds were separated on a DB-Wax column (30

m x 0.25 mm x 0.25 μm; Agilent Technologies) under the following conditions:

injection port temperature, 260 °C; helium flow, 0.8 mL/min; oven temperature

ramp, 35 °C for 10 min, 3 °C/min to 160 °C and then 15 °C/min to 200 °C

(maintained for 5 min). Volatile compounds were identified using the NIST MS
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library and by comparing Kovats retention index and mass spectra of standard

substances (Sigma-Aldrich) added to the refined olive oil.

2.10. Pigments

Chlorophyll and carotenoid (expressed as -carotene) compounds were

determined at 670 and 457 nm, respectively, in cyclohexane using specific

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extinction values, following the method described by Minguez-Mosquera, M.I.,

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Rejano-Navarro, L., Gandul-Rojas, B., Higinio Sanchez-Gomez, A.H., Garrido-

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Fernandez, J. (1991).
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2.11. Color

The color of ground walnut, the residual cakes and virgin nut oils was measured
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using a Konica Minolta CR-400 colorimeter (Tokio, Japan) to obtain the CIELAB

chromatic coordinates L* (lightness), a* (redness) and b* (yellowness). The C

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illuminant and a standard tile for calibration was used.

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All solvents used were of analytical, HPLC or spectroscopic grade, and were

supplied by Merck (Darmstadt, Germany). FAME mixture (C14 - C22, certified

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reference material), (+)-α-tocopherol (from vegetable oil), (+)-γ-tocopherol

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(≥96%, HPLC), tocopherols mixed (mixture of D-α, D-β, D-Δ, and D-γ-

tocopherols), Gallic acid, Procianidin B2, Catechin, Ellagic acid, Quercetin-3-

glucoside, Folin-Ciocalteu reagent, Trolox, DPPH, and 4-methyl-2-pentanol

were purchased from Sigma-Aldrich (Steinheim, Germany).

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All experiments and analytical determinations were carried out at least in

duplicate.

2.12. Statistical analysis

Anova and PCA were performed using XLSTAT 19.5 statistical software

(Addinsoft, Paris, France). One-way ANOVA was carried out using the Duncan

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test. Means were considered statistically different at p < 0.05.

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3. RESULTS AND DISCUSSION

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3.1. Fat composition of walnut, virgin walnut oil and its by-product
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Walnut is one of the highest oil content nut (Venkatachalam & Sathe, 2006);

indeed, varieties studied in this work possessed an average oil content of 63%,
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with no statistically significant difference observed between them (Table 1) and

in agreement with literature (Ozkan & Koyuncu, 2005; Gharibzahedi et al.,

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2014). As concerned the partially defatted cake obtained after screw pressing

oil extraction, its residual oil content was 6-10%. Whilst, moisture of the by-
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product (5.4-6.3%) was always higher than the corresponding kernel (1.7-

3.2%), since the separation of the oil concentrate the other components in the
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residual cake.
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The fatty acids profile of the obtained Virgin Walnut Oils (VWO) is shown in

Table 1, being remarkable the high amount of healthy essential linoleic acid

(59.6-62.4%), higher than many other seed and nut oils (e.g. 9-12% palm oil;

48-59% soy oil; 16-37% pistachio oil; 15-34%, almond oil; Codex Stan 210,

1999, FAO-WHO and its project for amending CX/FO 15/24/11, 2015). Lara

VWO cultivar had the highest (62.4%) and Chandler the lowest content (59.6%),
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showing statistic differences among the three cultivars studied. Oleic and

linolenic acids were the second and third in abundance with small but

statistically differences among cultivars (15-16% and 13-15% respectively; Tab.

1), showing Lara a higher proportion in linoleic acid (62.4%) whilst Chandler

contributing with a major proportion of linolenic acid (14.9%), as has been also

previously reported in the literature (Martínez et al.; 2010). Five commercial

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walnut virgin oils have also been analysed (Tab. 1), without information on

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cultivars used reported in their labels; from their fatty acids profile it is not

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possible to establish which variety cultivar was used for their manufacturing,

due to the similar amounts of fatty acids observed in the studied cultivars.
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3.2. Minor components and antioxidant properties

Virgin walnut oils (VWO) exhibit a high tocopherols content, mainly in the form
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of its γ-tocopherol isomer; being Hartely the richest (554 mg/kg; quantified as

α-tocopherol as established by AOCS Ce8-89), followed by Chandler and Lara

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(527 and 517 mg/kg; Table 2). This form represents about 85% of the total

tocopherols in the VWO, and the other main isomers, δ and α-tocopherols,
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account for 56-67 mg/kg and 24-26 mg/kg respectively (tab. 2), similarly to what
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reported in literature (Martínez et al. 2010; Górnas et al., 2014). Other virgin nut

oils such as those obtained from pistachio, almond and hazelnut exhibit a

similar tocopherols concentration: 500-750 mg/kg in pistachio oils, 320-550

mg/kg in almond oils and 200-600 mg/kg in hazelnut oils (Robbins, Shin,

Shewfelt, Eitenmiller & Pegg, 2011; Ojeda-Amador, Fregapane & Salvador,

2018), although almond and hazelnut oils show a different isomers distribution,

being α-tocopherol the most abundant.

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Partially defatted residual cakes contain a much lower amount in tocopherols

due to the apolar characteristics of these compounds, which remain in the oily

phase during the extraction, ranging from 43 to 88 mg/kg (tab. 2) for Hartley

and Lara respectively, depending on the residual oil in the cake, as compared to

their kernels content (414-480 mg/kg). VWO is therefore a good source of these

nutrients (vitamin E) with recognized antioxidant activity – both in vivo and in

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food stuff (Burton, 1994); with this respect, it is worth to remark that γ-

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tocopherol is recognized as a more efficient food lipids antioxidant than α-

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tocopherol (Wagner, Kamal-Eldin & Elmadfa, 2004).

Walnut is the nut with the highest content in phenolic compounds (Kornsteiner
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et al., 2006; Yang, Liu & Halim, 2009). Indeed, as shown in Table 2, the total

concentration of these compounds measured by Folin-Ciocalteu, ranged from

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10045 to 12474 mg/kg for kernels (showing Hartley cultivar a higher content,

and Chandler and Lara a lower, without statistical difference between the last
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two), in agreement with other researches (Kornsteiner et al., 2006; Figueroa,

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Marhuenda, Zafrilla, Martínez-Cachá, Mulero & Cerdá, 2016). Contrary to

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tocopherols, phenolics are more concentrated in the residual cake, due to their

polarity properties, leading to added value and potential applications of this by-
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product as functional ingredient for the food and nutrition industry. Statistically

significant differences have been observed between the three cultivars studied,

showing a content range from 16557 mg/kg (Chandler) to 19869 mg/kg

(Hartley; tab. 2).

As a consequence of the phenolics polarity, VWO exhibit a low content in these

compounds, 14 – 26 mg/kg (by Folin as gallic acid), in agreement with previous

studies (Górnas et al., 2014), but similar to other virgin seed oils (e.g. 1030
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mg/kg as caffeic acid in soybean, sunflower, rapeseed, corn and pumpkin;

Siger, Nogala-Kalucka & Lampart-Szczapa, 2008).

To measure the antioxidant activity of the polar phenolic extracts two different

methods have been used, DPPH and ORAC, as reported in Table 2. The

Hartley walnut cultivar showed the highest antioxidant activity (170 mmol/kg for

DPPH and 393 mmol/kg for ORAC assay), as expected due to its highest

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content in phenolics as compare to the others two cultivars studied, close to

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results reported by Figueroa et al., 2016 (about 180 mmol/kg for DPPH and 250

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mmol/kg for ORAC). Besides, residual cakes show the highest antioxidant

capacity, making this by-product as an excellent source of bioactive

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compounds, giving values of 143-185 mmol/kg for DPPH and 455-520 mmol/kg

for ORAC (tab. 2). The correlation coefficient between DPPH or ORAC assay in
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methanolic extracts with polar phenolic compounds was measured, showing a

reasonable correlation (0.72 and 0.92 respectively).

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Antioxidant activity of the virgin walnut oil was very low (tab. 2), as compared to
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kernel and cake, but similar to other virgin nut oils (Miraliakbari & Shahidi,
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2008); indeed, it is generally expressed as µmol/kg and not as mmol/kg, in

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agreement with the reduced content of phenolics as discussed above.

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Commercial virgin walnut oils show a wider composition range for minor

components as compared to the studied cultivars. For example, a lower

minimum value of 390 mg/kg of γ-tocopherol was observed, as compared to

527 mg/kg for Chandler; as well as a higher maximum of 52 mg/kg of total polar

phenolics, as compared to 26 mg/kg for Chandler cultivar – although, except

one the other commercial oils showed similar phenolic range (8-28 mg/kg) -,

that as a consequence increased the maximum values of antioxidant activity

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measured (i.e. 5.6 mmol/kg for ORAC). These differences may be explained by

the varieties employed but in particular to different and more drastic extraction

conditions like higher process temperature and/or pressure with the purpose of

increasing oil yields.

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3.3. Individual phenolic compounds

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The main phenolic families found in walnuts kernel are hydrolysable tannins

(Table 3), constituted by nine identified compounds and accounting from 60%

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(Hartley) to 80% (Chandler) of the total phenolics. Its most abundant compound
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is Di-HHDP glucose (Di-Hexahydroxydiphenoyl-glucose) with a concentration

range from 918 mg/kg (Lara) and up to 2226 mg/kg (Hartley). Glansreginin A - a
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dicarboxylic acid derivate – follows with highest content in Lara cultivar (391

mg/kg). Slatnar, Mikulic-Petkovsek, Stampar, Veberic & Solar (2015) reported a

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similar contribution for this family (about 70% of total). Flavanols is the second

family in importance, ranging from 26% (796 mg/kg Chandler) to 35% of the
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total content (2433 mg/kg Hartley; tab. 3), being procyanidin dimer its most

relevant compound reaching values from 753 mg/kg to 2366 mg/kg in Chandler
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and Hartley cultivars respectively, in agreement to Slatnar et al. (2015) who

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found up to 996 mg/kg of flavonols in the Franquette walnut cultivar. Regarding

ellagic acid derivates (137-365 mg/kg) they account less than 5% of the total

amount of phenolic compounds determined in walnut kernels, with ellagic acid

pentoside as the mayor components (69 mg/kg for Lara and 166 mg/kg for

Hartley cultivars). Hartley cultivar showed statistically significant difference with

regards to the other two walnut cultivars studied, containing a much higher
 ACCEPTED MANUSCRIPT

concentration for all the phenolic families identified and consequently for the

total amount (more than double than Chandler and Lara; tab. 3).

All the phenolic compounds identified in the kernel also appear in the

corresponding residual cake and as expected concentrated due to the

separation of the oily phase (about 60% of total weight) during the extraction

process. Indeed, flavonols and ellagic acid derivates almost duplicate their

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concentration in the corresponding by-products, with contents from 1253 mg/kg

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(Lara) to 4877 (Hartley) and 247 mg/kg (Lara) to 533 mg/kg (Hartley)

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respectively, as reported in Table 3; hydrolysable tannins also increased their

concentration. As a result, a total concentration of phenolics up to 5245 mg/kg

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for Lara and 10075 mg/kg for Hartley walnut cultivar. Due to these measured

very high contents in phenolic compounds it is worth to stress again the great
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interest in employing these by-products as potential functional food ingredients

with interesting biological and health promoting effects, which require more
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advanced and deep pre- and clinical research.

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Four different phenolic compounds have been identified in virgin walnut oils (di-
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HHDP glucose, glansreginin A, glansreginin B and HHDP galloyl glucose 1), all
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belonging to the hydrolysable tannins´s family (ranging between 0.91 and 2.91
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mg/kg, accounting Hartley and Chandler with the lowest and highest

concentration; tab. 3). Glansreginin A apparently is the main compound with a

concentration from 0.37 to 0.85 mg/kg. Several peaks could not be positively

identified in VWO, which generally accounted for more than 50% of total content

(unknowns were quantified as gallic acid and named by their Retention Time -

RT; tab.3). The highest total content was observed in Chandler VWO cultivar

(5.50 mg/kg) resulting statistically different than Lara (3.11 mg/kg) and Hartley
 ACCEPTED MANUSCRIPT

(2.34 mg/kg) cultivars. In general terms commercials VWO show higher

maximum phenolic contents for each of them (e.g. 10.6 mg/kg of hydrolysable

tannins in commercial oils as compared to 2.9 mg/kg for Chandler; tab. 3),

especially in the case of one specific sample. This observation reinforces the

assumption than more drastic extraction conditions are used in the

manufacturing of some of these industrial oils.

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3.4. Virgin walnut oil colour and aromatic compounds

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A similar concentration of chlorophylls and carotenoids is found in the studied
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virgin walnut oils, as depicted in Table 4, being generally slightly higher the

amount of chlorophylls than of carotenoids (i.e. 9.9 vs. 7.3 mg/kg in Chandler
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respectively; or 8.5 mg/kg vs. 6.9 in Lara), in agreement with Górnas et al.

(2014).
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Regarding the CIE L*a*b* colour space, in virgin walnut oils, the ‘b*’ parameter

(yellow-blue component) ranged from 25.5–30.9 and from -2.9 to -3.8 for ‘a*’
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parameter (red-green component), resulting in a clear yellow tonality common

for the cultivars studied, in agreement with other authors (Górnas et al., 2014;
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Ling et al., 2016). However, commercial oils showed quite different colour
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parameters for a* and b* components (-0.5 - -2.1 and 10.5-13.9 respectively;

tab. 4), which could be due to the use of different extraction process conditions,

which also results in a lower amount of pigments’ content (1.2-12.9 mg/kg

chlorophylls and 1.6-5.1 mg/kg carotenoids; tab. 4).

As known volatile compounds in vegetable oils are mainly generated by the

oxidative pathways of linoleic acid, due to the activity of the lipoxygenase (LOX)
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which catalyses the oxidation of polyunsaturated fatty acids (Robinson, Zecai,

Claire & Rod, 1995; Martínez & Maestri, 2008). Due to the known limitations of

the SPME analytical method used to measure the aroma components of edible

oils, the data reported must be taken into consideration as semi-quantitative or

qualitative data and no firm conclusions on basis these data can be obtained.

The profile of the studied virgin walnut oils is characterised by aldehydes (31-

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37%), acids (20-38%) and alcohols (16-36%) with different proportions

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depending on the cultivar, as reported in Table 5.

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The major aldehyde component present in the head-space of virgin walnut oils

are theoretically related with hydroperoxide precursors, which ones produces

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hexanal, 2-heptenal, octanal, nonanal, 2-decenal and 2-undecenal (Torres,

Martínez & Maestri, 2005; Choe & Min, 2006). Chandler VWO cultivar showed
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the highest aldehydes concentration (3.16 mg/kg) followed by Lara (2.19 mg/kg)

and Hartely (1.37 mg/kg), showing significant statistically differences between

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them (tab. 5). Hexanal, related to green descriptor (Morales et al., 2005), is the

major contributor within the aldehyde´s family: 1.79 mg/kg in Chandler VWO
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and 0.60 mg/kg in Lara cultivar (56% and 27% of total respectively); being on

the contrary nonanal - associated with citrus odour (Kochhar, 1993) - the main
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compound of this family in Hartley cultivar (0.38 mg/kg, 28%), but also high in
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Chandler (17%) and Lara VWOs (27%); similarly to what reported by Martínez

et al. (2008).

Volatiles’ acids and alcohols showed closed percentages in the headspace

composition of the studied VWOs, with a concentration range of 0.87-3.19

mg/kg for the acids (observing three different statistical groups; Hartley and

Chandler showing the lowest and highest amount; tab. 5) and 1.25-1.55 mg/kg
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(no statistic differences observed) for alcohols. Within the acids´ family, acetic

acid accounts from 5% to 26% of the total (0.25-2.53 mg/kg, depending on the

cultivar), which agrees with studies reported by Bail et al., 2009 and Uriarte,

Goicoechea & Guillen, 2010, who found acetic acid and hexanal as the main

headspace components of the VWO. Although the concentration in acetic acid

(expressed as the internal standard 4-methyl-2-pentanol) is apparently high, it is

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actually closed to its odour detection threshold and therefore, it could be not

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perceived. Regarding alcohols, 1-dodecanol and 1-nonen-3-ol are the most

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important compounds found at levels of 0.56-0.88 mg/kg and 0.15-0.29 mg/kg

respectively (tab. 5). Moreover, 1-pentanol (about 5% of this family and 1% of

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the total volatiles, in agreement to Martínez et al., 2008) - related to fruity notes

(Gandul-Rojas, Gallardo-Guerrero, Roca & Aparicio-Ruiz, 2013) - is also

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present in this kind of oils, due to the degradation of linoleic acid (Choe et al.,

2006).
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Other families of volatiles, such as terpenes and ketones are present in much

less concentration, only accounting for 7-13% and 2-3% each one respectively
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of the total volatile compounds in virgin walnut oils studied.

Regarding the five commercial VWO oils analysed, some of them showed a
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closed volatile profile to the virgin walnut oils prepared by screw pressing in this
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experimental work, but on the contrary others showed very different

concentrations. Some of these oils contained a much higher concentration in

aldehydes (13 mg/kg as maximum; tab. 5), mainly due to hexanal (7.36 mg/kg)

and 2-heptenal (3.62 mg/kg). Moreover, other volatile compounds such as

pyrazines (up to 13% of totals) and furans (up to 10%) have been also identified

in these samples, which are formed when a high temperature is used during the
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extraction process or when roasting nuts are used, in agreement with the

observed behaviour of other constituents like tocopherols and phenolics as

commented above.

3.5 Principal Component Analysis of VWO chemical characteristics.

Several chemical variables were selected on the basis of their ANOVA p-values

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and relative concentration within the 8 VWO samples studied (3 experimental

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and 5 commercial oils) to perform a Principal Component Analysis (PCA),

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depicted in Figure 1.

Principal component 1 (F1 in Fig. 1) is mostly related to some volatiles - acetic

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acid (factor loading: 0,982), hexanal (0,921) and 2-methyl-2-butenal (0,902) -,

phenolics - glansreginin A (0,974) and di-HHDP glucose (0,866) -, saturated

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fatty acids - palmitic acid (C16:0; -0,826) and stearic acid (C18:0, -0,965) and α-

tocopherol (0,952). Whereas, in the second component (F2), unsaturated fatty

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acids – linoleic (C18:2, 0,986), linolenic (C18:3, -0,879) and oleic (C18:1, -

0,845) -, phenolics – RT 44,7 (0,836) and RT 4,5 (-0,807) – and δ-tocopherol (-

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0.924), mainly explained the variance.

As reported in figure 1, the three experimental VWO cultivars studied are well
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separated in the PCA axes: Chandler oil is mostly related to the positive zone of
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the F1-axis, whereas on the contrary Hartley with the negative zone of the same

component; finally, Lara cultivar is associated with the F2-axis. Three

commercial oils resulted located close to Hartley and a forth one to Chandler.

Commercial oils data were treated as supplementary observations in the PCA

procedure.
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Conclusions

The chemical characterization carried out highlights that virgin walnut oil (VWO)

is a rich source of essential n-6 linoleic fatty acid, being Lara the richest (62.4%)

and Chandler the poorest (59.6%) showing statistic differences among the

cultivars studied, and of n-3 linolenic acid (12-15%), contributing Chandler with

a major proportion (14.9%). VWO exhibit a high γ-tocopherol content, being

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Hartely the richest (554 mg/kg), followed by Chandler and Lara (527 and 517

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mg/kg).

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The profile of the studied VWO is characterised by aldehydes (31-37%), acids

(20-38%) and alcohols (16-36%) with different proportions depending on the

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cultivar. Hexanal - related to green descriptor - is the major contributor within

the aldehyde´s family: 1.79 mg/kg in Chandler VWO and 0.60 mg/kg in Lara
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(56% and 27% of total respectively); being on the contrary nonanal - associated

with citrus odour - the main compound of this family in Hartley (0.38 mg/kg,
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28%).

Nuts and especially their residual cakes possess a very high content in phenolic
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EP

components (e.g. 5245 mg/kg for Lara and 10075 mg/kg for Hartley), which

taking into account the corresponding high antioxidant activity (460 and 520
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mmol/kg ORAC, respectively) result of a great interest for employing these by-
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products as potential functional food ingredients. The main phenolic families

found are hydrolysable tannins, constituted by nine identified compounds and

accounting from 60% (Hartley) to 80% (Chandler) of the total phenolics. Its most

abundant compound is Di-HHDP glucose (Di-Hexahydroxydiphenoyl-glucose)

with a concentration range from 918 mg/kg (Lara) and up to 2226 mg/kg
 ACCEPTED MANUSCRIPT

(Hartley). Glansreginin A - a dicarboxylic acid derivate – follows with highest

content in Lara cultivar (391 mg/kg).

Acknowledgement

This research project was supported by the Junta de Comunidades de Castilla-

La Mancha and the European Regional Development Fund (FEDER; ref. POII-

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2014-003-P).

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T ED
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TABLE 1. Walnuts’ oil content (% ) and fatty acids profile (% ) in virgin

walnut oils from different cultivars and commercial samples (n=5).

TABLE 2. Tocopherols (mg/kg*), polar phenolic compounds (mg/kg**),

antioxidant activity (mmol/kg***) and oxygen radical absorbance capacity

(mmol/kg****) in walnuts, their virgin oils and corresponding residual

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cakes from different cultivars and commercial cold-press samples (n=5).

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TABLE 3. Mean content of individual phenolic compounds (mg/kg) in

walnut kernels, their virgin oils and by-products from different cultivars.
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TABLE 4. Pigments (mg/kg; chlorophylls and carotenoids) and

colorimetric parameters (L, a*, b*) in virgin walnut oils from different
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cultivars and commercial samples (n=5).

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TABLE 5. Volatile compounds (mg compound/kg oil*) in walnut oils from

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different cultivars and commercial samples (n=5).

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FIGURE 1. PCA biplot of virgin walnut oil from different cultivars.

Commercial oils data were treated as supplementary observations in the PCA

procedure.
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TABLE 1. Walnuts’ oil content (% ) and fatty acids profile (% ) in virgin

walnut oils from different cultivars and commercial samples (n=5).

Chandler Hartley Lara Com (range)

Oil content 63.1 ± 1.5 64.2 ± 2.0 62.2 ± 0.9 –
C16:0 6.7 ± 0.1a 7.3 ± 0.1b 7.3 ± 0.1b 6.7 – 7.3
C16:1 0.1 ± 0.0 a
0.1 ± 0.0a
0.1 ± 0.0 a
0.1 – 0.1
C18:0 2.4 ± 0.1a 2.7 ± 0.2a 2.6 ± 0.1a 2.1 – 2.9
C18:1 16.1 ± 0.2 b
15.5 ± 0.3 ab
15.1 ± 0.1 a
16.0 – 23.8

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C18:2 59.6 ± 0.1a 60.3 ± 0.1b 62.4 ± 0.2c 56.2 – 61.5
C18:3 14.9 ± 0.2 c
13.6 ± 0.1 b
12.5 ± 0.1 a
10.5 – 12.7

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MUFA 16.2 ± 0.2 b
15.6 ± 0.3 ab
15.2 ± 0.1 a
15.2 – 23.9
PUFA 74.5 ± 0.1 b
73.8 ± 0.1 a
74.8 ± 0.3 b
66.8 – 73.6

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SFA 9.2 ± 0.1 a
10.2 ± 0.1 b
10.1 ± 0.2 b
9.1 – 10.1

Values in the same row with different lower-case letters (a – c) are significantly different
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at p<0.05 by Duncan test. Com, commercial oils.
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TABLE 2. Tocopherols (mg/kg*), polar phenolic compounds (mg/kg**),

antioxidant activity (mmol/kg***) and oxygen radical absorbance capacity
(mmol/kg****) in walnuts, their virgin oils and corresponding residual
cakes from different cultivars and commercial cold-press samples (n=5).

Chandler Hartley Lara Com (range)

a b a
α-T 19.2 ± 0.1 22.3 ± 5.4 17.0 ± 3.0 –
-T Nut 414.2 ± 2.2 442.3 ± 57.9 414.7 ± 56.3 –
a b a
δ-T 50.0 ± 3.3 57.9 ± 17.4 49.8 ± 6.6 –
α-T 23.6 ± 0.1 26.3 ± 1.4 24.0 ± 1.5 21.2 – 69.0
a b a
-T 390 – 607

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Oil 527.4 ± 0.0 553.6 ± 1.9 516.6 ± 10.0
b b a
δ-T 63.1 ± 0.9 66.6 ± 1.1 55.6 ± 1.0 16.1 – 78.7
a a b
α-T 3.4 ± 0.2 3.6 ± 0.2 9.8 ± 0.7 –

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a a b
-T Cake 53.3 ± 2.0 42.5 ± 2.0 88.2 ± 5.7 –
a a b
δ-T 6.3 ± 0.6 5.0 ± 0.8 10.4 ± 1.0 –

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a b a
Nut 10 045 ± 524 12 474 ± 391 10 862 ± 689 –
Total polar c b a
Oil 26.0 ± 0.6 17.5 ± 1.9 13.8 ± 0.3 4.1 – 51.8
phenols a c b
Cake 16 557 ± 277 19 869 ± 325 18 978 ± 262 –
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a b a
Nut 104.7 ± 2.5 170.2 ± 37.0 106.0 ± 17.6 –
b a a
DPPH Oil 0.10 ± 0.01 0.08 ± 0.01 0.07 ± 0.00 0.03 – 0.43
a b a
Cake 142.9 ± 13.9 184.5 ± 2.5 149.0 ± 2.5 –
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ab b a
Nut 319.5 ± 25.2 392.7 ± 124.0 259.7 ± 21.3 –
c b a
ORAC Oil 1.00 ± 0.08 0.72 ± 0.08 0.50 ± 0.07 0.92 – 5.57
a b a
Cake 454.9 ± 36.9 519.6 ± 11.8 457.7 ± 34.8 –
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Values in the same row with different lower-case letters (a – c) are significantly different
at p<0.05 by Duncan test. *Quantified using α-tocopherol as standard. **Quantified by
Folin using gallic acid as standard. ***Quantified by DPPH using Trolox as standard.
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****Quantified by ORAC using Trolox as standard. Com, commercial oils.

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TABLE 3. Mean content of individual phenolic compounds (mg/kg) in

walnut kernels, their virgin oils and by-products from different cultivars.
-
[M-H] ;
Chandler Hartley Lara 2
MS
Compound Kernel Cake Kernel Cake Kernel Cake (m/z)
Catechin 43 ± 20ab 80 ± 34αβ 67 ± 3b 126 ± 1β 22 ± 1a 38 ± 3α 289; 245
Procyanidin dimer 1750 ± 16α 2366 ± 4750 ± 1216 ± 577; 425,
753 ± 84a 825 ± 54a
143 316β 48α 407
Σ Flavanols 796 ± 1830 ± 51α 2433 ± 4877± 1253 ±
847 ± 56a
104a 140b 314β 52α
Ellagic acid 79 ± 5α 287 ± 12β 86 ± 1α 301; 257,
35 ± 1a 150 ± 24b 39 ± 14a
229

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Ellagic acid derivate 23 ± 3 10 ± 1 22 ± 7 595; 433,
10 ± 1ab 8 ± 1a 12 ± 1b
301
Ellagic acid hexoside 21 ± 1a 52 ± 7β 39 ± 2b 52 ± 1β 19 ± 4a 31 ± 4α 463; 301

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β α
Ellagic acid hexosyl 18 ± 4 9±0 13 ± 0αβ 585;
3±1 3±0 6±2
pentoside
Ellagic acid pentoside 123 ± 14αβ 184 ± 31β 108 ± 4α 433; 301,
70 ± 0a 166 ± 5b 69 ± 16a

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257
Σ Ellagic acid 277 ± 1α 533 ± 43β 247 ±
137 ± 2a 365 ± 26b 144 ± 37a 15α
derivates
Di-HHDP glucose 1094 ± 1446 ± 4α 2226 ± 2456 ± 1636 ± 783; 481,
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918 ± 87a
82a 393b 257β 56α 130
Galloyl bis HHDP 372 ± 36 461 ± 15 392 ± 26 935; 633,
230 ± 10a 359 ± 23b 245 ± 65a
glucose 1 301
Galloyls bis HHDP 30 ± 3α 39 ± 3α 51 ± 2β 935; 633,
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21 ± 8 32 ± 3 39 ± 19
glucose 2 301
Glansreginin A 875 ± 94β 358 ± 14β 784 ± 592; 403,
265 ± 26 265 ± 107 391 ± 52
18α 343
Glansreginin B 153 ± 12β 171 ± 12α 98 ± 1β 565; 403,
59 ± 6a 163 ± 39b 43 ± 10a
343
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HHDP digalloyl glucose 286 ± 19α 517 ± 43β 302 ± 785; 301,
171 ± 12a 462 ± 15b 156 ± 19a
1 23α 419
HHDP digalloyl glucose 156 ± 18α 308 ± 13β 165 ± 3α 785; 301,
92 ± 17 232 ± 84 89 ± 17
2 419
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Vescalagin isomer 1 234 ± 17 369 ± 15 247 ± 9 933; 451,

181 ± 22 240 ± 155 190 ± 54
631
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Vescalagin isomer 2 51 ± 6 79 ± 4 55 ± 4 933; 451,

62 ± 6 175 ± 57 64 ± 28
631
Σ Hydrolysable 2475 ± 3584 ± 4204 ± 4656 ± 2132 ± 3731 ±
tannins 189a 172α 508b 292β 351a 26α
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Total 3108 ± 5708 ± 7001 ± 10075 ± 3123 ± 5245 ±

86a 127α 674b 21β 444a 92α
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Oil Com
Chandler Hartley Lara
(range)
Di-HHDP glucose 0.84 ± 0.03b 0.11 ± 0.02a 0.17 ± 0.02a 0.13 – 3.18
Glansreginin A 0.85 ± 0.10b 0.37 ± 0.01a 0.74 ± 0.00b 0.06 – 2.13
b a a
Glansreginin B 0.17 ± 0.02 0.03 ± 0.00 0.01 ± 0.00 0.00 – 3.18
HHDP digalloyl glucose c b a 0.02 – 2.13
1.05 ± 0.01 0.39 ± 0.00 0.21 ± 0.02
1
Σ Hydrolysable 0.21 – 10.6
2.91 ± 0.07c 0.91 ± 0.00b 1.14 ± 0.04a
tannins
RT 4.5 0.57 ± 0.11b 0.26 ± 0.06a 0.10 ± 0.01a 0.02 – 1.34
RT 16.3 0.22 ± 0.02 0.19 ± 0.01 0.20 ± 0.01 0.03 – 1.78
RT 29.0 0.20 ± 0.03b 0.05 ± 0.00a 0.06 ± 0.00a 0.02 – 1.35
b a ab
RT 29.8 0.25 ± 0.01 0.20 ± 0.02 0.23 ± 0.02 0.05 – 0.25
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RT 30.8 0.26 ± 0.01b 0.23 ± 0.00a 0.22 ± 0.01a 0.00 – 2.50

b a
RT 44.7 0.35 ± 0.01 0.18 ± 0.00 0.53 ± 0.03c 0.04 – 1.03
RT 45.9 0.17 ± 0.01b 0.01 ± 0.00a 0.13 ± 0.01b 0.00 – 0.24
Σ Unknow ns 2.59 ± 0.17b
1.43 ± 0.09a ab
1.98 ± 0.30 0.29 – 7.31
Total 5.50 ± 0.23b 2.34 ± 0.09a 3.11 ± 0.34a 0.51 – 17.9

Values in the same row with different lower-case letters (a – c, kernel and oil; α-γ,
cake) are significantly different at p<0.05 by Duncan test. Com, commercial oils.
Unknown compounds are quantified as gallic acid and named by their Retention Time
(RT).

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TABLE 4. Pigments (mg/kg; chlorophylls and carotenoids) and

colorimetric parameters (L, a*, b*) in virgin walnut oils from different
cultivars and commercial samples (n=5).

Chandler Hartley Lara Com (range)

Pigments
Chlorophylls 9.9 ± 0.7b 3.3 ± 0.1a 8.5 ± 1.2b 1.2 – 12.9
Carotenoids 7.3 ± 0.1b 4.2 ± 0.0a 6.9 ± 0.8b 1.6 – 5.1
Color
L* 46.8 ± 0.0b 45.5 ± 0.0a 47.5 ± 0.0c 42.8 – 47.0
a* -3.9 ± 0.0a -3.8 ± 0.0b -2.9 ± 0.0c -0.5 – -2.1

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b* 30.9 ± 0.0c 29.7 ± 0.0b 25.5 ± 0.2a 10.5 – 13.9

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TABLE 5. Volatile compounds (mg compound/kg oil*) in walnut oils from

different cultivars and commercial samples (n=5).

Compound Com
Chandler Hartley Lara
(range)
c a b
Acetic acid 2.53 ± 0.17 0.25 ± 0.02 1.16 ± 0.01 0.29 – 11.93
Hexanoic acid 0.59 ± 0.07 0.55 ± 0.04 0.68 ± 0.06 0.00 – 2.00
Pentanoic acid 0.06 ± 0.00 0.06 ± 0.01 0.07 ± 0.00 0.02 – 0.96
c a b
Σ Acids 3.19 ± 0.23 0.87 ± 0.06 1.92 ± 0.05 0.61 – 14.88
b c a
1,3-Butadienol 0.03 ± 0.00 0.06 ± 0.00 0.01 ± 0.00 0.00 – 0.04
a b a
2,3-Butadienol 0.02 ± 0.00 0.08 ± 0.00 0.03 ± 0.01 0.00 – 0.12
a b a
1-Dodecanol 0.67 ± 0.07 0.88 ± 0.07 0.56 ± 0.04 0.05 – 0.91

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b a b
2-Ethyl-1-hexanol 0.06 ± 0.00 0.04 ± 0.00 0.06 ± 0.00 0.04 – 0.35
b a a
2-Methyl-1-butanol 0.17 ± 0.00 0.14 ± 0.01 0.13 ± 0.00 0.00 – 0.20
b a c
2-Methyl-1-undecanol 0.04 ± 0.00 0.03 ± 0.00 0.09 ± 0.00 0.00 – 0.06
b a b
1-Nonen-3-ol 0.24 ± 0.01 0.15 ± 0.01 0.29 ± 0.03 0.00 – 0.66

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b b a
1-Pentanol 0.12 ± 0.00 0.11 ± 0.01 0.04 ± 0.00 0.00 – 1.98
a c b
2-Propil-1-heptanol 0.01 ± 0.00 0.08 ± 0.01 0.03 ± 0.01 0.00 – 0.33

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Σ Alcohols 1.37 ± 0.10 1.55 ± 0.12 1.25 ± 0.07 0.53 – 2.94
a b b
Benzaldehyde 0.18 ± 0.00 0.24 ± 0.01 0.25 ± 0.01 0.00 – 0.79
a ab b
2-Decenal 0.13 ± 0.00 0.15 ± 0.01 0.16 ± 0.04 0.00 – 0.26
b a b
2-Heptenal 0.09 ± 0.00 0.03 ± 0.00 0.08 ± 0.01 0.00 – 3.62
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c a b
Hexanal 1.79 ± 0.04 0.29 ± 0.02 0.60 ± 0.01 1.92 – 7.36
b a b
2-Methyl-2-butenal 0.26 ± 0.01 0.08 ± 0.00 0.26 ± 0.01 0.00 – 0.54
b a b
Nonanal 0.52 ± 0.01 0.38 ± 0.03 0.59 ± 0.04 0.00 – 0.54
a b a
Octanal 0.08 ± 0.00 0.09 ± 0.00 0.08 ± 0.00 0.00 – 0.08
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a a b
2-Undecenal 0.12 ± 0.00 0.12 ± 0.01 0.15 ± 0.01 0.01 – 0.14
c a b
Σ Aldehydes 3.16 ± 0.07 1.37 ± 0.10 2.19 ± 0.02 2.93 – 13.01
a a b
6-Methyl-5-hepten-2-one 0.13 ± 0.01 0.11 ± 0.01 0.21 ± 0.01 ND
a a b
Σ Ketones 0.13 ± 0.01 0.11 ± 0.01 0.21 ± 0.01 ND
b a b
Longifolene 0.20 ± 0.00 0.14 ± 0.01 0.23 ± 0.02 0.03 – 0.16
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b a b
α-Terpineol 0.43 ± 0.00 0.34 ± 0.02 0.52 ± 0.05 0.32 – 0.79
b a b
Σ Terpenes 0.63 ± 0.01 0.58 ± 0.03 0.75 ± 0.07 0.36 – 0.95
c a b
Total 8.47 ± 0.40 4.38 ± 0.32 6.32 ± 0.17 7.61 – 31.74
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Values in the same row with different lower-case letters (a – c) are significantly different
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at p<0.05 by Duncan test. *Quantified using 4-methyl-2-pentanol as internal standard.

Com, commercial oils.
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FIGURE 1. PCA biplot of virgin walnut oil from different cultivars.

Biplot (axes F1 and F2: 100,00 %)

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10 C18:2
rt 44,7
nonanal

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benzaldehyde longifolene
1-nonen-3-ol
C16:0
5 LARA
2-methyl-2-butenal

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C18:0
glansreginin A
F2 (44,73 %)

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acetic acid
α-tocopherol HARTLEY
CHANDLER hexanal
COM 2 COM 3
-5 COM 4 di-HHDP glucose
COM 5
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γ-tocopherol HHDP digalloyl glucose 1
1-dodecanol rt 4,5
C18:3 C18:1
δ-tocopherol
-10 COM 1
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-15
-15 -10 -5 0 5 10 15
F1 (55,27 %)
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Active variables active observations Supplementary observations

Commercial oils data were treated as supplementary observations in the PCA

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Graphical abstract
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Highlights

 Three walnut cultivars, their virgin oils and cakes were fully characterized

 Walnuts/cakes: high phenolics content (hydrolysable tannins as main family)

 Residual cakes: potential application as functional ingredient due to high

phenolics

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 Virgin walnut oils: rich in essential linoleic acid and γ-tocopherol

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 Aldehydes, main family of virgin walnut oil aroma components

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