LWT - Food Science and Technology 146 (2021) 111345

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LWT
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Shelf life of extra virgin olive oil manufactured with combined microwaves
and megasonic waves at industrial scale
Miguel Amarillo a, *, Adriana Gámbaro a, Ana Claudia Ellis a, Bruno Irigaray a, Jimena Lázaro a,
Antonia Tamborrino b, Roberto Romaniello c, Alessandro Leone c, Pablo Juliano d
a
Department of Food Technology, University of Uruguay, Uruguay
b
Department of Agricultural and Environmental Science, University of Bari Aldo Moro, Bari, Italy
c
Department of the Science of Agriculture, Food and Environment, University of Foggia, Foggia, Italy
d
CSIRO Agriculture and Food, Werribee, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Previous research has demonstrated that the innovative application of combined microwaves and megasonic
Olive oil waves for preconditioning olive paste enabled olive oil production in continuous mode, while delivering higher
Extra virgin olive oil yields. The present work further examines the impact of these technologies on the chemical and sensory
Megasonic waves
stability of the oils obtained in previous studies, with reference to international extra virgin olive oil standards.
Microwaves
Shelf life
Olive oil samples obtained after traditional malaxation (Control), microwave conditioning (MW), traditional
malaxation assisted with megasonics (MS), and the combination of both microwave conditioning followed by
megasonics (MW + MS) in a commercial olive oil plant at 350 kg/h, were characterised and evaluated across 1
year at 3-month intervals. All oil samples evaluated met the international extra virgin olive oil standard pa­
rameters across the shelf life study. Ethyl esters and waxes increased uniformly across storage indicating no effect
of MW or MS on fermentation processes during conditioning. The application of these technologies enhanced the
phenolic content in the oils across one-year, with fluctuations among technologies, in comparison to the tradi­
tional control process (Control < MW ≈ MS ≈ MW + MS). Overall, this study demonstrates that both MW and MS
conditioning technologies and their combination, applied industrially, can create higher quality extra virgin olive
oils across storage.

1. Introduction recovery. Leone, Tamborrino, Romaniello, Zagaria, and Sabella (2014),

The olive oil extraction process requires physical methods for an oil
to be commercially classified as “virgin”, without chemical in­
terventions during extraction, according to the International Olive
Council (IOC, 2019b). The process consists of the crushing of olives,
malaxation of the paste and centrifugation. Malaxation is considered the
most important step, for extraction yield and olive oil quality (Difonzo
et al., 2021). However, the olive paste malaxation itself is a batch pro­
cess where heating is not uniform across the vessel, resulting in over­
heated paste, which often affects olive oil quality and stability after the
first extraction step.
Therefore, in recent years novel technologies applying low frequency
ultrasound (Clodoveo & Hachicha, 2013), microwaves (Jiao et al., 2013)
and pulse electric fields (Puértolas & Martínez, 2015) have been intro­
duced to improve olive oil malaxation and extra virgin olive oil
developed a 4-magnetron (2.45 GHz, 24 kW, 28.8 kJ/kg) microwave
(MW) tube prototype that allows continuous and rapid heat condition­
ing of 3000 kg/h paste without the need for olive paste malaxation. The
prototype provided uninterrupted flow and better heat uniformity than
traditional malaxation, while avoiding overheating of residual products.
The system was shown to reduce thermal conditioning from 40 min
during conventional malaxation to a few seconds without compromising
oil extractability and providing similar oil extractabilities to the tradi­
tional malaxation process.
Microwave localised heating causes the rupture of cell wall materials
through the formation of internal vapour pressure flow, facilitating the
release of cell wall components such as tissue disrupting enzymes, while
enabling the paste conditioning (Aguilera & Stanley, 1999; Jiao et al.,
2013). Leone, Romaniello, Tamborrino, Xu, and Juliano (2017) have
demonstrated that microwave heating reduces paste viscosity, which

* Corresponding author.
E-mail address: amarillom@fq.edu.uy (M. Amarillo).

https://doi.org/10.1016/j.lwt.2021.111345
Received 17 December 2020; Received in revised form 18 March 2021; Accepted 19 March 2021
Available online 25 March 2021
0023-6438/Crown Copyright © 2021 Published by Elsevier Ltd. All rights reserved.
M. Amarillo et al. LWT 146 (2021) 111345

prepares the paste for adequate oil separation during centrifugation.

Application of high frequency ultrasound standing waves, mega­
sonics (MS), to the olive paste post-malaxation has also been tested
industrially as a process intervention to enhance olive oil recovery
(Juliano, Agustin, Xu, Mawson, & Knoerzer, 2016; Juliano et al., 2017).
Extractability improvements of up to 2.5% olive oil (expressed on an oil
in fruit basis) were seen after testing the technology at 350 kg/h. Unlike
microwaves or low frequency ultrasound (Tamborrino, Romaniello,
Caponio, Squeo, & Leone, 2019), megasonics promotes oil release
through the small formation of expansion and compression bubbles
across the vegetable structure (Amarillo et al., 2019). This megasonic
phenomenon was also shown to reduce paste viscosity, enabling higher
yields (Leone et al., 2017).
Given the separate process benefits of microwaves and megasonics,
their combined application (MW + MS) towards the development of a
novel continuous olive oil process that provides higher yield and does
not require malaxation, has been evaluated (Leone et al., 2017).
Previous work establishes that the pre-conditioning microwave
equipment tested by Leone et al. (2018), in various settings did not
impart negative sensory effects on the virgin olive oil quality. Moreover,
MW-conditioned oils obtained did not change the headspace profile but
provided a higher content of volatile compounds as C6 aldehydes
responsible for the “green” sensory notes (Leone et al., 2018; Tambor­ Fig. 1. Industrial processing plant set up encompassing (1) malaxer, (2) MW
rino, Romaniello, Zagaria, & Leone, 2014). generator and tube, (3) megasonic equipment including transducers. (4)
decanter centrifuge, (5) balance tank, as described in detail be Leone et al.
The application of megasonics on the malaxed paste has also shown
to yield an oil of acceptable sensory and chemical virgin olive oil quality
characteristics (Leone et al., 2018). Furthermore, this study reported power of 6.0 kW.
increased concentration of phenolic compounds after malaxation + MS The megasonic reactor developed by Juliano et al. (2017), was the
or MW + MS application, whereas no significant increase in oil pheno­ first prototype for large scale trials and is composed by a stainless-steel
lics was found with MW-only preconditioning. However, little is known flow through reactor (200 L). Four transducers operating at 400 and 600
on the effects of MW-only, malaxation + MS, or MW + MS conditioning kHz (Sonosys Ultraschallsysteme GmbH, Germany) were attached to the
of olive paste on the sensory and chemical quality stability of the ob­ reactor walls across the length of the vessel consuming 2 kW (20 kJ/kg).
tained olive oil during storage. A set of generators transformed electrical energy into high frequency
It is also unknown whether or not MW-only, malaxation + MS or sound pressure waves for each frequency required.
MW + MS pre-conditioning can influence fermentative processes of Olive oil extraction trials were carried out using the following pro­
bacteria naturally present in the paste, which may enhance the forma­ cess configurations for the following samples:
tion of ethanol and generate undesirable ethyl esters, respectively
(Bendini, Cerretani, Valli, Lercker, & Mazzini, 2009; Pérez-Camino, (a) oil obtained by the traditional malaxation process (Control, 30
Moreda, Mateos, & Cert, 2002). No study follows the impact of ultra­ min malaxation + 30 min residence time in the megasonic
sound on fermentation of olives. Furthermore, previous studies do not reactor) following the sequence 1-3-4-5 in Fig. 1 with ultrasound
track formation of esters or waxes following neither a megasonic con­ in the reactor OFF.
ditioning of the malaxed olive paste nor a MW + MS conditioning. (b) oil obtained with the traditional malaxation process followed by
This work therefore aims to assess the sensory-chemical profiles of a megasonic treatment (malaxation + MS, 30 min malaxation +
Coratina olive oils, extracted using different paste conditioning pro­ 30 min residence time in the megasonic reactor, 2 kW, 20.5 kJ/kg
cedures during a 12-month storage period. Of particular interest is the – 400 and 600 kHz), following the sequence 1-3-4-5 in Fig. 1 with
evaluation of phenolic and tocopherol compounds in the oil and the ultrasound in the reactor ON.
ability of the novel preconditioning methods to maintain higher poly­ (c) oil obtained from extraction using microwaves for conditioning
phenol values than the traditional malaxation process during storage. of the paste without malaxation (MW-only, 63 s residence time, 6
Furthermore, this study follows the formation of ethyl esters during kW, 61.7 kJ/kg, 2.45 GHz) by following the sequence 1-2-4-5 in
storage to establish the influence of these preconditioning technologies Fig. 1.
on fermentation processes. This is the first study that considers sensory (d) oil obtained from extraction using MW prototype for condition­
and chemical stability of olive oil as a result of implementing combined ing of the paste, without malaxation, followed by a megasonic
novel processing interventions in the pre-conditioning stage at industrial intervention (MW + MS, conditions as per (b) and (c)), following
level. the sequence 2-3-4-5 in Fig. 1.

2. Materials and methods
2.1. Olive oil conditioning methods
Oil samples from Coratina variety (Olea europaea L.) were obtained
for each treatment in the plant described in Fig. 1, which shows the
specific process equipment. The microwave equipment (EMITECH s.r.l
(Corato, Italy)) has been designed for continuous processing and in­
cludes a reverberant chamber with a polypropylene tube inside, a
generator head connected to a power supply and coupled to a set of
magnetrons. The system operates at 2.45 GHz consuming a maximum
Each extraction configuration was run in triplicate but in random­
ized order. Extractability data is discussed above in the Introduction.
2.2. Olive oil sampling for stability study
Oil samples were taken directly from the decanter after each trial.
Each triplicate oil obtained for each preconditioning method was pooled
and fractionated into five 250 mL amber glass bottles ensuring protec­
tion during storage as depicted in Fig. 2, to be evaluated at different time
points. Bottles were totally filled, leaving no headspace and remained
stored at room temperature in the absence of light.

2
M. Amarillo et al.
Fig. 2. Oil sampling methodology carried for the storage study.
2.3. Olive oil stability study
Commercial olive oil manufacturers target at least a one-year shelf
life (Irigaray, Martinez, Feller, Amarillo, & Grompone, 2016). Samples
were taken every three months for up to 12 months to evaluate chemical
parameters and the sensory profile by following the IOC standard (IOC,
2019b) as described below.
2.4. Sensory evaluation
As explained in Leone et al. (2018); sensory profile of the oils was
obtained through the evaluation of the samples by a panel of judges
accredited for this purpose, certified by the IOC. The sensory panel was
composed of 9 olive oil panellists. All attributes (positive or defective)
were rated in a 10 cm unstructured scale (IOC, 2018). Table 1 provides
information about each sensory attribute and their respective de­
scriptors as reported by Fernandez, Ellis, Gámbaro, and
Barrera-Arellano (2018). Each sample was presented in monadic form in
blue tasting cups (15 mL), standardized, and coded with one letter and
two random numbers. During trials, the four treatment samples were
presented randomly to each panellist together with a form that includes
the defects (rated or qualitative), attributes and positive descriptors
listed in Table 2. The sensory laboratory was equipped with five indi­
vidual cabins at controlled temperature (22 ◦ C–24 ◦ C) and ventilation
(IOC, 2007).
2.5. Chemical quality determination
The chemical quality of the obtained oils included analyses listed and
required by the IOC standard (IOC, 2019b). The parameters established
under the IOC norm studied are legally and scientifically validated as a
requirement for EVOO quality assessment to determine whether or not
the olive oil is extra virgin. All the chemical analyses were carried out by
triplicate. The current literature on olive oil quality and shelf life focuses
on flavour and aroma aspects without considering olive oil appearance
and colour (Andreou et al., 2016; Di Serio, Giansante, Di Loreto, & Di
Giacinto, 2018; Irigaray et al., 2016) and the IOC norm establishes that
flavour and aroma descriptors are the guiding parameters for shelf life
quality.
2.5.1. Acidity
Acidity determination was performed according to IOC (2017b).
Samples were weighed and dissolved in ethylic alcohol-diethyl ether
3
LWT 146 (2021) 111345
(1:1). Phenolphthalein was added as indicator and titrated with potas­
sium hydroxide 0.1 N.
2.5.2. Peroxide value
It was determined according to IOC (2017c). Weighed samples were
dissolved in acetic acid-isooctane (3:2). Saturated potassium iodide so­
lution is added and left in the dark for 1 min. Then, after adding 30 mL of
water, the solution is titrated with 0.1 N sodium thiosulphate using
starch as indicator.
2.5.3. K232 and K270 coefficient
In accordance with IOC (2019a), samples were weighed and dis­
solved in isooctane. Then, absorbance was determined at wavelengths
232 nm and 270 nm in a Shimadzu, Mini-UV 1240 model.
2.5.4. Ethyl esters and waxes content
This analysis was performed according with IOC (2017d), “Deter­
mination of the content of waxes, fatty acid methyl esters and fatty acid
ethyl esters by capillary gas chromatography”. Samples were dissolved
in hexane, a solution of methyl ester heptadecanoic and a solution pal­
mityl palmitate was added as internal standards for quantification of
ethyl esters and waxes respectively. Ethyl esters were separated together
with waxes by solid phase chromatography (SPE) using hexane-diethyl
ether (99:1) as elution solvent. The extracts were analyzed in a gas
chromatograph (Model 2010; Shimadzu, Japan) using a GC column
(RXI-5MS, Restek, US).
2.5.5. Polyphenols content
The extraction was performed in accordance with IOC (2017a),
“Determining olive oil biophenols through HPLC”. Samples were dis­
solved in a mixture of MeOH-water (80:20) with addition of syringic
acid as internal standard, sonicated in an ultrasound bath for 10 min and
centrifuged for 25 min at 5000 rpm. Then, an aliquot of the supernatant
was taken with glass syringe, filtered with a 0.45 μm PVDF syringe filter
and 20 μL were injected in the HPLC (Model 20A, Shimadzu, Japan)
using a reverse phase column (Phenomenex C18, USA) with a particle
size of 5 μm, 250 mm long and 4.6 mm diameter. The mobile phase was
composed of phosphoric acid 0.2 %-water (Solvent A), methanol (Sol­
vent B) and acetonitrile (Solvent C). The gradient was modified as fol­
lows: initial mobile phase 96% A/2% B/2% C, 50% A/25% B/25% C at
40 min, 40% A/30% B/30% C at 45 min, 0% A/50% B/50% C at 50 min
remaining at this concentration for 10 min. Then returned to the initial
mobile phase and allowed to elute for 10 min. The total running time
M. Amarillo et al. LWT 146 (2021) 111345

Table 2

Tomato. Fruity can be described by green or ripe fruitiness

Tactile sensation referred to a spiciness sensation that can

be perceived in the whole mouth cavity, especially in the
List of defects, positive attributes and positive descriptors (rated or qualita­

Quince jam, metallic in mouth, pencil graphite, pencil

tive) employed to describe the oil samples according to IOC presented in the

Astringent, Banana, Fig tree, Green, Nuts, sweet and

form.

Fermented olive, pizza olives, cheese crust, rotten

In most cases linked to sensory descriptors Apple,

Vinegar, ethanol, nail polish remover, acetic acid

Rated Defects Fusty/Muddy Sediment

Basic taste compared to caffeine or quinine

Musty/Humid/Earthy
Winey/Vinegary and Acid/Sour
Frostbitten Olives (Wet Wood)
Main positive and negative sensory attributes of virgin olive oil according to the IOC standard including the causes of these attributes, chemical compounds and the related sensory descriptors.

Mold, moisture, earth, green nuts

Rancid
Examples of Sensory Descriptors

Qualitative Defects Metallic

Dry Hay
Grubby
Brine

Rancid oil or butter

Heated of Burned
Vegetable Water
Esparto
Cucumber
throat

Greasy

wood
Rough
Positive Attributes Fruity
Bitter
Pungency
Combination of tyrosol (p-HPEA) and hydroxytyrosol (3,4-
DHPEA) with the dialdehydic form of elenolic acid (EDA),

Ethyl 2-methyl butanoate, ethyl propanoate, pentanal and

Positive Descriptors Green
Alcohols, esters, ketones, aldehydes and hydrocarbons.

Fig Tree
C8 primary and secondary alcohols and C8 ketones

Apple
Five-carbon branched alcohols, 2-ethyl-butyrates

Banana
resulting in p-HPEA-EDA and 3,4-DHPEA-EDA
Mainly C5–C6 compounds from LOX pathway

Nuts
Others
Sweet
Ethanol, ethyl acetate and acetic acid

Astringency
p-HPEA-EDA and 3,4-DHPEA-EDA

was 82 min. The flow rate was 1 mL/min throughout the analysis.
Hydroperoxide fragments

Detection of polyphenols was performed using the diode array detector

Chemical Compounds

(DAD) set at 280 nm. The test determines presence and concentration of
20 phenolic compounds. The total polyphenol value includes the sum of
all detected polyphenolic compounds. Individual and total phenolic
octanal

compounds are expressed as ppm in tyrosol. For this, an external cali­

bration solution of tyrosol and syringic acid was used.

2.5.6. Tocopherol content

Volatile compounds formed during the extraction, crushing and malaxation

Anaerobic fermentation during olive storage (before extraction), when pile

of badly stored, or during oil storage if the organic matter (small fragments

Characteristic flavor of oils extracted from olives which have been injured
Fungi and yeast development when olives are stored in humid conditions
steps. Action of lipoxygenase (LOX) pathway over C18 unsaturated fatty

Aerobic fermentation occurred on the top of pilled olives or even in the

Tocopherols content was determined according to Andrikopoulos,

Brueschweiler, Felber, and Taeschler (1991) HPLC analysis of phenolic
paste residue left when the production line is not properly cleaned

antioxidants, tocopherols and triglycerides. Oils were weighed and

dissolved in isopropanol and tocopherols were determined by HPLC
of fruits and water) is not completely removed from the oil

(Model 20A, Shimadzu, Japan) equipped with column (Phenomenex

C18, USA) with a particle size of 5 μm, 250 mm long and 4.6 mm
Phenolic compounds naturally present in the olives

Phenolic compounds naturally present in the olives

Oxidation process mainly during the storage of oils

diameter.
over a long period. Olives harvested from soil

The mobile phase was composed of 5% acetic acid in water (Solvent

A), acetonitrile (Solvent B), methanol (Solvent C), and isopropanol
(solvent D).
The gradient was modified as follows: initial mobile phase 69% A/
18% B/13% C/0% D, at 25 min 0% A/58% B/42% C/0% D and was
maintained to 30 min, at 45 min 0% A/23% B, 17% C, 60% D, at minute
by frost while on the tree

46 0% A/0% B/100% D and was maintained to 48 min, at minute 48,5

69% A/18% B/13% A, total running time was 57 min. The solvent flow
was 1 mL/min. Detection of tocopherols was performed by fluorescence
detector (Shimadzu RF-20A XS) at the following wavelengths: 290 nm
excitation and 330 nm emission. For tocopherols quantification, a cali­
Causes

acids

bration curve was used with alpha tocopherol as external standard

supplied by Sigma-Aldrich (USA). The method provided individual to­
copherols and only total tocopherols are reported for simplicity.
Frostbitten Olives
Winey/Vinegary
Musty/Humid/

and Acid/Sour
Fusty/Muddy

(Wet Wood)
Pungency

Sediment

2.6. Data analysis

Rancid
Earthy
Fruity

Bitter
Sensory Attribute

MATLAB statistics toolbox (The MathWorks, Inc., Natick, MA, USA)

was the software employed for calculation of averages, to carry out
attributes

Analysis of Variance (ANOVA) using Tukey’s HSD, calculated with a

Positive

Defects
Table 1

95% confidence interval. Given that the oil samples were pooled for
each conditioning treatment after completing triplicate trials, significant

4
M. Amarillo et al.
differences were assessed in terms of testing method variability. For the
sensory attributes, an ANOVA test was carried out using data of each
attribute intensity from each judge to compare the mean values of the
four samples at time 0 as well as individual time points during storage. A
two-way ANOVA was also carried out to determine effects on poly­
phenol changes.
3. Results and discussion
3.1. Paste conditioning effects on olive oil extractability
Table 3 summarises that extractabilities obtained after four paste
conditioning configurations at a scale of 350 kg/h, previously published
by Leone et al. (2018): (a) traditional malaxation (Control), (b) tradi­
tional malaxation followed by a megasonic treatment (malaxation +
MS), (c) microwave preconditioning without malaxation (MW-only),
and (d) microwave preconditioning followed by a megasonic treatment
(MW + MS). Results show that the application of MW + MS provided the
highest extractability among the four paste preconditioning methods,
significantly (p < 0.05) recovering 2.4% extra virgin olive oil compared
to malaxation alone. The application of MW to the non-malaxed paste
provided similar extractability to the traditional batch malaxation pro­
cess, indicating the potential for the application of MW to develop a
continuous process (Leone et al., 2014). The application of MS to the
traditionally malaxed paste also provided an enhanced recovery of 2.2%
oil, compared to the traditional malaxation process. Therefore, results in
the MW + MS configuration prove the concept for a potential high yield
continuous malaxation process.
As reported in Leone et al. (2014), the introduction of new machines
in a production plant involves new economic investments and new
management costs, but in the case of the industrial use of the two
combined technologies studied in this scientific paper this leads to an
increase in the extractability of the oil (Leone et al., 2017). Further
validation of the economic feasibility is required with new prototype
designs that operate at larger scales for a medium sized olive oil plant
beyond 1 tonne per hour in future studies.
3.2. Virgin olive oil characterisation of untreated and treated samples at
time zero
Leone et al. (2018), reported the sensory characteristics of four olive
oils obtained after applying the four paste conditioning methods, i.e.,
Control, malaxation + MS, no malaxation + MW, and MW + MS at the
beginning of the stability study. The sections below present a more
detailed characterisation of the oil samples from both sensory and
chemical perspective.
3.2.1. Sensory profile
As observed in Table 4, at the beginning of the study, none of the oils
presented sensory defects and, therefore, all four conditioning methods
produced oils that belonged to the extra virgin category (IOC, 2019b).
Table 3
Extractability of olive oils for each paste conditioning treatment. Different let­
ters for each treatment indicate significant differences according to Tukey’s Test
(p < 0.05).
Treatment Extractability (g oil/100 g olive paste)
Control 84.7 c
Malaxation + MS 86.7 a,b
MW-only 85.2 b,c
MW + MS 86.9 a
p-value 0.0348
*Control – traditional malaxation; Malaxation + MS – traditional malaxation
followed by a megasonic treatment; MW-only – microwave preconditioning
without malaxation; MW + MS – microwave preconditioning followed by
megasonic treatment.
5
LWT 146 (2021) 111345
Our previous work reported the volatile compounds as measured by the
GC/MS, among which no volatile compounds associated with defects
were reported (Leone et al., 2018).
The oils evaluated were characterized as balanced because the dif­
ference between the evaluated sensory attributes fruity, bitter, and
pungency, was below 2 points in the sensory scale, as suggested by the
standard (IOC, 2018). All oils presented a green fruity flavour with ripe
notes of medium to high intensity, while consistently showing bitter and
pungent flavours of medium to high intensity. Our previous work indi­
cated that C5 and C6 alcohols and aldehydes, linked to the LOX pathway
developed in the paste that provides green fruity flavours in the oil, did
not significantly vary due to the tested conditioning technologies (Leone
et al., 2018). Furthermore, all oils showed high complexity, and there­
fore required several positive descriptors to characterise it (Table 2). No
significant difference (p > 0.05) was found in any sensory descriptor in
this particular point in time, indicating that the studied technologies did
not affect the sensory profile of the obtained oils.
3.2.2. Chemical quality
Analytical results for peroxide value (Fig. 3a), K232 and K270
(Fig. 3b and c), ethyl esters (Fig. 4a), waxes (Fig. 4b), and acidity
(Fig. 4c) at time zero show that the four oils obtained by the tested
conditioning methods belong to the extra virgin category. All mentioned
figures show that their respective time zero values are below the limit
established by the IOC standard (IOC, 2019b).
Even though the panel rated a zero rancid defect on all oils (Table 4),
it is worth noting that the Control (malaxation only) oil was significantly
higher in peroxide value (Fig. 3a), compared to the oils obtained after
MW and MS treatments, although it is not reflected by the UV extinction
coefficients K232 and K270 (Fig. 3b and c). This might be linked with the
increase in total polyphenols detected in all oils obtained with malax­
ation + MS, MW-only, and MW + MS, which have antioxidant proper­
ties. However, the tested paste conditioning MW and MS treatments did
not alter the tocopherol values for any oil.
This work reports the individual polyphenols, tyrosol, hydroxytyr­
osol, vanillic acid, oleuropein, and lutein measured with the methods
suggested by the IOC, which differ to the methods utilised by Leone et al.
(2018). Both methods indicate that all individual polyphenols also dis­
played higher content in the oils obtained after MW and MS condi­
tioning, with respect to the Control. Rupturing effects of MW and
localised bubbles created at tissue level by MS, enhanced the release of
oleuropein that is precursor compound of the above-polyphenolics
tyrosol and hydroxytyrosol. Even though these compounds are
enhanced, the sensory panel did not pick-up variations in bitter and
pungency notes in oils obtained from MW and MS conditioning.
Ethyl esters in oils obtained by the said technologies are for the first
time reported in this work, and were found below the standard limits
(IOC, 2019b). This shows, that no fermentative processes have occurred
during the paste conditioning with MW or assisted by MS. The formation
of ethyl esters is related to the esterification of free ethanol (possibly
formed by a fermentation in olives prior or during oil extraction)
resulting in acidity products (free fatty acids). Absence of fermentative
processes is supported by the zero wax content values for all oils, which
is also indicative of the absence of reactions between alcohols derived
from fermentative processes with esters (Pérez-Camino et al., 2002).
Waxes are formed by high molecular mass alcohols esterification with
fatty acids (Irigaray et al., 2016). According toSamaniego-Sanchez,
Quesada-Granados, Lopez-Garcia de la Serrana, and Lopez-Martinez
(2010), C40, C42, C44 and C46 are the most frequent waxes found in
olive oils.
Therefore, aerobic fermentative processes occurring during paste
conditioning, leading to winey, vinegary and acid sour tones did not
occur (Table 4) (Gómez-Coca, Moreda, & Pérez-Camino, 2012;
Pérez-Camino et al., 2002; Samaniego-Sanchez et al., 2010). Other
anaerobic fermentation processes that may start in the olives, did not
manifest into fusty notes (Cayuela, Gomez-Coca, Moreda, &
 M. Amarillo et al.
Table 4
Evolution of sensory profiles of oil samples derived from the tested preconditioning technologies during the 12-month study. Different letters and bold values for each row indicate significant differences according to
Tukey’s Test (p < 0.05) between oil samples at a given month.
Month Technologies Defects Positive Attributes Positive Descriptors

Fusty/Muddy Musty/ Winey/Vinegary Frostbitten Olives Rancid Fruity Bitter Pungency Apple Astringent Banana Fig Green Nuts Sweet Tomato
Sediment Humid/ and Acid/Sour (Wet Wood) Tree
Earthy

0 Control 0.0 0.0 0.0 0.0 0.0 5.8 a 5.2 a 5.7 a 0.8 a 2.6 a 4.0 a 0.6 a 5.3 a 3.4 1.3 a 3.0 a
a
Malaxation + 0.0 0.0 0.0 0.0 0.0 5.7 a 5.6 a 5.7 a 0.6 a 1.9 a 4.1 a 0.4 a 5.1 a 3.0 1.6 a 3.7 a
MS a
MW-only 0.0 0.0 0.0 0.0 0.0 5.4 a 5.2 a 5.2 a 1.2 a 2.6 a 3.8 a 0.1 a 5.2 a 2.7 0.9 a 3.3 a
a
MW + MS 0.0 0.0 0.0 0.0 0.0 5.8 a 5.3 a 5.7 a 0.0 a 2.4 a 4.0 a 0.4 a 5.3 a 3.1 1.6 a 3.1 a
a
3 Control 0.0 0.0 0.0 0.0 0.0 5.2 a 4.7 a 5.1 a 0.0 a 1.8 a 3.8 a 0.3 a 4.5 a 3.2 1.4 a 2.8 a
a
Malaxation + 0.0 0.0 0.0 0.0 0.0 5.1 a 5.2 a 4.9 a 0.3 a 1.8 a 3.0 a 0.6 a 4.5 a 1.6 1.2 a 2.6 a
MS a
MW-only 0.0 0.0 0.0 0.0 0.0 5.0 a 4.8 a 4.3 a 0.3 a 2.8 a 3.1 a 0.4 a 4.7 a 2.8 1.4 a 3.1 a
a
MW + MS 0.0 0.0 0.0 0.0 0.0 5.3 a 5.4 a 5.1 a 0.3 a 2.7 a 2.9 a 0.0 a 4.9 a 2.4 0.9 a 2.7 a
a
6 Control 0.0 0.0 0.0 0.0 0.0 4.8 a 4.5 a 5.5 a 0.0 a 1.7 a 2.9 a 1.0 a 4.1 a 3.1 0.8 a 2.6 a
6

a
Malaxation + 0.0 0.0 0.0 0.0 0.0 4.8 a 4.5 a 5.5 a 0.0 a 1.7 a 2.9 a 1.0 a 4.1 a 3.1 0.8 a 2.6 a
MS a
MW-only 0.0 0.0 0.0 0.0 0.0 4.9 a 4.5 a 4.5 a 0.0 a 2.0 a 3.0 a 0.6 a 4.4 a 3.2 0.6 a 2.7 a
a
MW + MS 0.0 0.0 0.0 0.0 0.0 5.2 a 5.0 a 5.1 a 0.3 a 2.1 a 3.3 a 0.0 a 4.6 a 3.1 0.6 a 3.6 a
a
9 Control 0.0 0.0 0.0 0.0 0.0 4.8 a 4.8 a 5.4 a 0.0 a 1.7 a 2.9 a 1.0 a 4.1 a 3.0 0.8 a 2.5 a
a
Malaxation + 0.0 0.0 0.0 0.0 0.0 4.9a 4.7 a 5.5 a 0.0 a 1.7 a 3.1 a 1.0 a 4.3 a 2.8 0.6 a 3.1 a
MS a
MW-only 0.0 0.0 0.0 0.0 0.0 4.8 a 4.7 a 4.7 a 0.0 a 2.0 a 3.1 a 0.6 a 4.5 a 3.1 0.6 a 2.6 a
a
MW + MS 0.0 0.0 0.0 0.0 0.0 5.5 a 5.1 a 5.1 a 0.3 a 2.1 a 3.3 a 0.0 a 4.6 a 3.1 0.6 a 3.6 a
a
12 Control 0.0 0.0 0.0 0.0 0.0 3.8 a 4.8 a 4.4 a 0.0 a 1.2 a 2.1 a 0.8 a 3.2 a 2.3 0.8 a 2.1 a
a
Malaxation + 0.0 0.0 0.0 0.0 0.0 3.9 a 3.7 a 4.2 a 0.0 a 1.7 a 2.8 a 1.0 a 3.4 a 2.3 0.6 a 2.7 a
MS a
MW-only 0.0 0.0 0.0 0.0 0.0 4.3 a, 4.4 a 4.4 a 0.0 a 1.8 a 2.8 a 0.6 a 4.1 a 2.7 0.6 a 2.3 a
b a
MW + MS 0.0 0.0 0.0 0.0 0.0 4.7 b 4.4 a 4.3 a 0.3 a 1.8 a 2.9 a 0.0 a 3.8 a 2.9 1.3 a 3.0 a
a

LWT 146 (2021) 111345

*Control – traditional malaxation; Malaxation + MS – traditional malaxation followed by a megasonic treatment; MW-only – microwave preconditioning without malaxation; MW + MS – microwave preconditioning
followed by megasonic treatment.
M. Amarillo et al. LWT 146 (2021) 111345

Fig. 3. Evolution of peroxide value, K232 and K270 in tested oils during the 12-month storage study. Error bars represent the respective standard deviation for
triplicate measurements at each time point. Labels indicate, Control – traditional malaxation; Malaxation + MS – traditional malaxation followed by a megasonic
treatment; MW-only – microwave preconditioning without malaxation; MW + MS – microwave preconditioning followed by megasonic treatment. Different letters
reflect significant differences (p < 0.05) between technologies at specific points in time where such differences were detected.

Fig. 4. Evolution of ethyl esters, waxes and acidity in tested oils during the 12-month storage study. Error bars represent the respective standard deviation for
triplicate measurements at each time point. Labels indicate, Control – traditional malaxation; Malaxation + MS – traditional malaxation followed by a megasonic
treatment; MW-only – microwave preconditioning without malaxation; MW + MS – microwave preconditioning followed by megasonic treatment. Different letters
reflect significant differences (p < 0.05) between technologies at specific points in time where such differences were detected.

Perez-Camino, 2015).
The present study has evaluated chemical compounds that validate
sensory parameters studied as per the IOC standard (IOC, 2019b) which
do not include the evaluation of colour compounds such as carotenoids
or chlorophyll. As mentioned earlier, sensory descriptors do not include
appearance and colour as the norm requires trained panels to evaluate
the oils in a coloured tasting cup to assess aroma and flavour only.
Furthermore, little or none has been reported in the literature on the
correlation between colour compounds and sensory descriptors. Even
though the current literature describing the use of microwave processing
of olive paste as a malaxation aid did not study the residual concen­
tration of chlorophyl and carotenoids in olive oil, it is an area worth

7
M. Amarillo et al. LWT 146 (2021) 111345

considering for future studies. There is also evidence that the nature of Table 5
the mild treatment given by megasonic waves at high frequency and Individual polyphenol evolution in oil samples derived from the tested pre­
high specific energy does not impart an effect of additional leaching of conditioning technologies during the 12-month study. Different letters for each
chlorophyl to the olive oil (Juliano et al., 2017) or carotenoid com­ row indicate significant differences according to Tukey’s Test (p < 0.05) be­
pounds to the palm oil (Juliano et al., 2013). tween oil samples at a given month.
Tyrosol (ppm) Control Malaxation MW- MW + p
3.3. Stability study of untreated and treated samples along the 12-month + MS only MS

storage T0 29 a A 43 b A 51 c A 54 c A <0.0001
T3 26 a A 41 b A 42 b A,B 47 b A,B 0.0003
T6 26 a A 48 b A 41 b A,B 44 b B,C 0.001
Both sensory and chemical stability parameters for the olive oils
T9 22 a A 46 b A 39 b A,B 40 b B,C 0.0056
obtained after applying the four paste conditioning methods, will be T12 19 a A 38 b A 33 a,b B 36 a,b A 0.0407
described in the sections below on oil samples measured every three p 0.1414 0.271 0.0415 0.0007
months up to 12 months.
Hydroxytyrosol Control Malaxation MW- MW + p
(ppm) + MS only MS
3.3.1. Sensory stability
T0 42 a A 59 b A 61 b A 63 b A 0.0005
Table 4 shows that zero defects were found in any of the oils along T3 30 a A 63 b A 47 a,b C 53 a,b A, 0.0035
the 12-month study. Therefore, all oil samples evaluated by the sensory B
panel belonged to the extra virgin category (IOC, 2019b). T6 43 a A 54 b A 59 b B 52 b B,C 0.0017
In this case, no defect related to fermentative processes or oxidative T9 48 b A 53 b A 56 b B 57 b B 0.0203
T12 32 a A 49 b A 47 a,b C 51 b C 0.03026
rancidity effects are attributed by the panel to the tested oils across the p 0.496 0.1544 0.0001 0.0002
12-month storage period with descriptor values remaining at zero.
The evolution of the positive attributes fruity, bitter and pungent of Vanillic acid Control Malaxation MW- MW + p
the 4 samples is shown in Table 4. Even though there is a significant (ppm) + MS only MS
decrease of the intensity of these attributes from zero to 12 months, T0 1.9 a A 2.2 a A 2.9 b A 3.5 b A 0.0002
T3 1.7 a A 2.0 a A 1.8 a B 3.1 b A, 0.0025
significant difference (p < 0.05) was found at 12 month having MW +
B
MS the higher fruity intensity comparing to Malaxation oil and Malax­ T6 1.4 a A 2.3 b A 2.7 b A 2.5 b B,C 0.0014
ation + MS. Previous studies demonstrate the decrease of these attri­ T9 41 b A 41 b A 2.3 b A, 2.4 b B,C 0.0219
butes with time (Di Serio et al., 2018). B
Likewise, some positive descriptors including apple, fig tree and nuts T12 1.5 a A 1.6 a A 1.7 a B 1.8 a C 0.8917
p 0.3028 0.1528 0.0009 0.0013
did not significantly change across all tested oils during the storage
trials, mainly due to the panel rating these notes at the lower end of Oleuropein Control Malaxation MW- MW + p
score therefore making it difficult to pick up the changes. The decrease (ppm) + MS only MS
in banana and green notes further supports to overall decrease in T0 180 a 201 a,b A 213 b A 222 b A 0.0065
fruitiness. T3 152 a A, 187 b A,B 171 a,b 192 b B 0.0231
B B
T6 137 a A, 162 a,b B,C 182 b B 203 b A, 0.0051
3.3.2. Chemical stability results B B
Analytical results for peroxide value (Fig. 3a), K232 and K270 T9 123 aB 155 a,b C 170 b,c 192 c B 0.0009
(Fig. 3b and c), ethyl esters (Fig. 4a), waxes (Fig. 4b) and acidity (Fig. 4c) B
T12 117 a,b 135 b C 109 a C 122 a,b 0.0476
at months 3, 6, 9, and 12, also support that the four oils obtained by the
B C
tested conditioning methods belonged to the extra virgin category. All p 0.0064 0.0003 <0.0001 <0.0001
mentioned figures show that their respective values are below the limit
established by the IOC standard across the one-year study (Di Serio Lutein (ppm) Control Malaxation MW- MW + p
et al., 2018). + MS only MS
The oxidative behaviour of all oils during storage is shown in Fig. 3 T0 5.1 a A 8.4 b A 9.8 c A 8.9 b,c A <0.0001
T3 4.7 a A 6.7 a,b B 8.3 b B 8.0 b A
through the peroxide value, and UV coefficients K232 and K270, which
<0.0001
T6 4.5 a A 7.2 b B 7.6 b B,C 8.1 b A <0.0001
significantly increased during storage for all samples (p < 0.05). In T9 4.0 a A 6.9 a,b B 7.0 b C 7.3 b A 0.0188
parallel, both natural antioxidants polyphenols and tocopherols, T12 3.7 a A 5.3 b C 4.8 a,b D 5.0 a,b B 0.0239
significantly decreased during storage (Table 5 and Fig. 5). The total p 0.7070 <0.0001 <0.0001 0.0002
polyphenol content in the Control remained consistently below the other
Total Control Malaxation MW- MW + p
oils obtained with the MW and MS conditioning methods, while the
polyphenols + MS only MS
differences between malaxation + MS, MW-only and MW + MS fluctu­ (ppm)
ated across storage. The total tocopherols (Fig. 5) maintained similar T0 539 a A 613 bA 619 b A 658 c A <0.0001
values among all treated oils during storage until month 6. Similar T3 502 a B 601 b A,B 609 b A 644 c A 0.0001
T6 507 a B 579 b CD 606 c A 615 c B 0.0001
trends to total tocopherols (Fig. 5) were observed for individual to­
T9 495 a B 575 bC 603 c A 598 b,c <0.0001
copherols (alpha, beta, gamma) and these are therefore not shown. Iri­ B
garay et al. (2016) reported similar values of total tocopherols T12 449 a C 434 b D 505 b B 509 c C <0.0001
throughout the storage in comparison to those obtained here. p <0.0001 <0.0001 0.0001 <0.0001
During the first 6 months, the peroxide values of the Control samples *Control – traditional malaxation; Malaxation + MS – traditional malaxation
were significantly (p < 0.05) higher than the oils obtained from MW and followed by a megasonic treatment; MW-only – microwave preconditioning
MS conditioning (Fig. 3a). However, peroxide values of all oils nearly without malaxation; MW + MS – microwave preconditioning followed by
coincide at months 9 and 12. This is supported by the uniform decrease megasonic treatment.
in both polyphenols and tocopherols in all oils (Table 5 and Fig. 5),
through reactions with free radicals created during storage as reported
in olive oil storage studies elsewhere (Irigaray et al., 2016). The diverse
behaviour in oxidation of the oils is also supported by the differentiated
increase in the K232 (Fig. 3b), which shows a more pronounced increase

8
M. Amarillo et al.
Fig. 5. Evolution of total tocopherols in tested oils during the 12-month storage
study. Error bars represent the respective standard deviation for triplicate
measurements at each time point. Labels indicate, Control – traditional
malaxation; Malaxation + MS – traditional malaxation followed by a megasonic
treatment; MW-only – microwave preconditioning without malaxation; MW +
MS – microwave preconditioning followed by megasonic treatment. Different
letters reflect significant differences (p < 0.05) between technologies at specific
points in time where such differences were detected.
for the oils obtained from MW-only and MW + MS conditioning. How­
ever, the changes in K232 did not reflect on the rancidity values
described by the sensory panel at month 12. Di Serio et al. (2018) re­
ported higher K232 values than the IOC threshold for Coratina olive oil
extracted by traditional malaxation at month 12 of storage without
identifying sensory defects on rancidity.
Baccouri et al. (2008), found a good correlation between the
oxidative stability (measured in Rancimat) of various olive oils and the
concentration of total polyphenols and tocopherols. The consistent
higher content of phenolic compounds in oils produced through MW and
MS conditioning, is reflected in the oil concentration of the individual
main phenolic compounds found, tyrosol, hydroxytyrosol, vanillic acid,
oleuropein, lutein until month 6 (Table 5). Tyrosol, hydroxytyrosol, and
lutein content were superior in all MW and MS conditioned samples
until month 12, while oleuropein was only superior for the malaxation
+ MS and MW + MS samples. Therefore, MW affected the degradation of
specific and total polyphenols over storage time (Table 5), also being
reflected in the K232 values. The megasonic treatment, however, may
have protected the breakdown of complex secoiridoid compounds such
as oleuropein, which have been reported to be unstable during storage
(Lozano-Sanchez et al., 2013). The content of individual polyphenols
was similar to those reported elsewhere and their degradation during
storage followed a similar behaviour to those evaluated by Di Serio et al.
(2018) and Irigaray et al. (2016).
Several authors have stated that polyphenols are the most important
antioxidant in olive oil being the main contributors to the protection of
olive oil from oxidation (Carrasco-Pancorbo et al., 2005; Franco,
Galeano-Díaz, Sanchez, De Miguel, & Martin-Vertedor, 2014; Loz­
ano-Sanchez et al., 2013). Increased phenolics in the MW derived oil
samples (MW-only and MW + MS) is also reflected in consistently higher
(although not significant) astringency from month 3 until the end of the
12-month study. The low values rated on the sweet descriptor, trans­
lated into very short times to picking up pungency and bitter flavours by
the panel, which decrease uniformly for all oil samples, as previously
noted for both polyphenols and tocopherols and interplay of the various
reactions controlling oil oxidation.
9
LWT 146 (2021) 111345
Ethyl esters content in all oils did not differ between technologies
until reaching month 12, while fluctuating through time across tech­
nologies (Fig. 4a). There is, however, ethyl ester values that are
consistently lower for MW derived oils (MW-only and MW + MS),
indicating a potential impact of the MW heating mechanism on
fermentative enzymes.
In addition, the wax formation processes have been also altered by
the preconditioning technologies with MW and/or MS from month 6
onwards (Fig. 4b). It could be possible that the formation of base alco­
hols via enzymatic processes that contribute to the esterification reac­
tion with fatty acids to form large molecular weight waxes could be
diminished. This is an area that requires further research, although it is
well known that microwaves and ultrasound can modulate the enzy­
matic activity. For example, microwaves can affect the enzymatic
pathway of the lipoxygenase enzyme, which mainly generates volatile
compounds responsible for fruity sensory tones in olive oil (Caponio,
Leone, Squeo, Tamborrino, & Summo, 2019). Moreover, low frequency
ultrasound in general has shown to promote fermentation processes e.g.;
Chang and Chen (2002) applied 20 kHz in wine rice ageing for one week
and found that it was similar to one year with no sonication. Earlier
studies have shown the impact of high frequency ultrasound on enzymes
and fermentation of fruits and vegetables (Novoa-Díaz et al., 2014; Ojha,
Mason, O’Donell, Kerry, & Tiwari, 2017; Terefe, Sikes, & Juliano, 2016).
As it can be observed in Fig. 4c, acidity remained practically constant
until month 9, when it significantly increased (p < 0.05). However, all
the values are much lower than the standard limit of 0.8% (ref) even at
month 12, and do not reflect on sensory defects including fusty (Table 4)
(IOC, 2017b). Acidity increases are mainly due to lipase activity, which
may have increased during storage (Cayuela et al., 2015). Lower values
on the oil obtained after MW-only and MW + MS may be related to
enzyme inactivation due to rapid heating of MW (Baştürk, 2019).
While the IOC standard, and olive oil shelf life studies in the litera­
ture (Di Serio et al., 2018), focus on flavour and aroma aspects, they do
not consider olive oil appearance and colour or specific colour com­
pounds, which are worthy of future research.
In summary, all MW and MS derived oils had a positive impact on the
quality of the extra virgin oils during the 12 months of the study. At
present the authors are not aware of shelf life studies on olive oils ob­
tained through novel technologies combinations, other than the work of
Andreou et al. (2016); who pretreated olives with high pressure pro­
cessing and pulsed electric fields before olive oil extraction. Further
work must evaluate the MW and MS technologies and their combination
for conditioning pastes produced with other olive varieties and with
other ripeness indices. This work supports the further development of
the combined MW and MS technologies to develop a continuous
extraction process, while avoiding the 60–90 min batch malaxation step.
4. Conclusions
The present work demonstrated that conditioning olive paste from
Coratina olives with microwaves and megasonics to improve olive oil
extractability, does not negatively impact on the sensory quality nor the
chemical quality (fermentative and oxidative) parameters of the ob­
tained oils for at least 12 months. Furthermore, the oils displayed higher
phenolic compounds for all pastes with both single and combined mi­
crowaves and megasonic conditioning without significantly impacting
on the oil’s sensory quality. These results open the door to the possible
development of new devices that allow both technologies to be com­
bined and in turn, promotes the continuous operation of the olive oil
extraction process.
CRediT authorship contribution statement
Miguel Amarillo: Investigation, Data curation, Writing – original
draft, preparation, Writing – review & editing. Adriana Gámbaro: Su­
pervision. Ana Claudia Ellis: Investigation. Bruno Irigaray:
M. Amarillo et al.
Investigation. Jimena Lázaro: Investigation, Writing – review & edit­
ing. Antonia Tamborrino: Investigation, Methodology. Roberto
Romaniello: Investigation, Methodology. Alessandro Leone: Investi­
gation, Methodology. Pablo Juliano: Conceptualization, Investigation,
Methodology, Supervision, Writing – review & editing.
Acknowledgements
This study was funded by Puglia Region “Guidelines for Research and
Experimentation in Agricultura 2012–2014”, MICROLIO research proj­
ect (Grant number: PRS_011), “Determinazione del dirigente servizio
agricoltura” 31 March 2014, n.100, by CSIRO Julius Career Award grant
(Grant number: WBS R90070) and Agencia Nacional de Investigación e
Innovación (National Agency for Research and Innovation, ANII). Also,
we would like to mention Pedeciba Química for its contribution.
We would especially like to remember Dra. María Antonia Grompone
for her invaluable contributions, advice and motivation. RIP.
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