International Journal of Food Science and Technology 2019, 54, 149–160 149

Original article
Fatty acids, volatile compounds and microbial quality
preservation with an oregano nanoemulsion to extend the shelf
life of hake (Merluccius hubbsi) burgers

Claudia M. Asensio,1 Patricia R. Quiroga,1 Qingrong Huang,2 Valeria Nepote3 & Nelson R. Grosso1*
1 Qu
ımica Biol
ogica, Facultad de Ciencias Agropecuarias, Instituto Multidisciplinario de Biolog
ıa Vegetal (IMBIV), Consejo Nacional de
Investigaciones Cient
ıficas y T
ecnicas (CONICET), Universidad Nacional de C
ordoba (UNC), Av. Valpara
ıso s/n, CC 509, X5016GCA,
C
ordoba, Argentina
2 Department of Food Science, School of Environmental and Biological Sciences, Rutgers, State University of New Jersey, 50 Dudley Rd,
New Brunswick 08901, NJ,
3 Facultad de Ciencias Exactas, F
ısicas y Naturales, UNC, IMBIV, CONICET, Av. Velez Sarsfield, 1611, C
ordoba, Argentina

(Received 9 May 2018; Accepted in revised form 23 July 2018)

Summary Hake (Merluccius hubbsi) is the most important fish of Argentinean industry. The preservative effects of
an oregano essential oil (EO) nanoemulsion (NE) on hake burgers (FB) during 15 days at 4 °C were stud-
ied. Fatty acids determined by gas chromatography coupled with mass spectrometry (CG-MS), volatile
compounds determined by solid phase microextraction (SPME) coupled with CG-MS, total volatile basic
nitrogen (TVBN), and microbial count was measured. ANOVA and DCG tests were performed. After
14 days, significant differences were detected among samples (P ≤ 0.05). FB with EO (EO-FB) and FB
with NE containing EO (NEEO-FB) had the highest content of EPA (eicosapentaenoic acid) (191.82 and
198.42 g kg
1) and DHA (docosahexaenoic acid) (107.97 and 110.87 g kg
1, respectively). Also, NEEO-
FB had the lowest microbial count (7 log CFU g
1). The incorporation of the oregano essential oil into a
nanoemulsion improves the preserving activity in hake fish burgers.
Keywords Hake, nanoemulsion, oregano essential oil, quality.

bonds. Consequently, these positions are the thermo-

Introduction
dynamically favoured sites for the attack by lipid per-
The Argentine hake (Merluccius hubbsi) is crucial to oxyl radicals (Erickson, 2008). Moreover, it is
the Argentinean fishing industry, representing more established that during cold storage, lipid hydrolysis
than 50% of the total fish catch in the region (Marrari also occurs and PUFA contents substantially decrease
et al., 2016). Hake is mostly commercialised as fillets (Saldanha & Bragagnolo, 2008).
and exported frozen. From January to September Once caught, fish deteriorate rapidly and undesirable
2016, Argentina exported 67 887 tons of M. hubbsi compounds are produced. Autolytic reactions con-
(representing US$ 158 397 000) and its processing trolled by endogenous enzymes are established first,
industry had grown 30.1% (INDEC 2016). followed by enzymatic reactions of bacterial origin
Fish are a rich source of polyunsaturated fatty acids (Garc
ıa-Soto et al., 2013). The chemical measurement
(PUFAs), particularly, eicosapentaenoic acid (EPA, of total volatile basic nitrogen (TVB-N) in fish has
C20:5n-3) and docosahexaenoic acid (DHA, C22:6n- long been used as an indicator of fish spoilage, either
3). Terrestrial animals have a limited ability to synthe- by bacterial or enzymic degradation. Moreover, solid
sise these FAs. The consumption of PUFAs has health phase microextraction coupled with gas chromatogra-
benefits. (Uauy & Valenzuela, 2000). However, PUFAs phy/mass spectrometry (SPME–GC/MS) can be used
are highly susceptible to oxidation (Saldanha & Bra- to study volatile organic compounds (VOCs) in sea-
gagnolo, 2008). Carbon-hydrogen bond dissociation food, providing valuable information about the extent
energies of a FA are lowest between adjacent double of spoilage (Parlapani et al., 2015).
Essential oils (EOs) are natural antioxidant and
*Correspondent: Fax: 0054-0351-4334103; antimicrobial preservatives, used as flavouring and
e-mail: [email protected] functional ingredients in food (Asensio et al., 2014,

doi:10.1111/ijfs.13919
© 2018 Institute of Food Science and Technology
150 Oregano nanoemulsion: hake burger quality preserver C. M. Asensio et al.

2015a,b). The quality preservation capacity (antioxi- Emir C ß oban, 2017). However, few studies included the
dant, antimicrobial and sensory) of different Argen- use of a generally recognised as safe (GRAS) product
tinean oregano EO in various food matrix has been as oregano essential oil, many less include the formula-
deeply studied. Oregano EOs were found to have pre- tion of them in a nanoemulsion to prolong the shelf
serving effects on the chemical, sensory and microbio- life of sea products. The objective of this study is to
logical quality of: ricotta cheese, cottage cheese, cream evaluate preserving effects of an oregano NE in the
cheese, olive oil, roasted peanuts, fried peanuts, sun- quality of hake burgers during 14 days of refrigerated
flower kernels among others (Asensio et al., 2013; Clau- storage.
dia M Asensio et al., 2014, 2015b; Olmedo et al., 2013,
2008; Quiroga et al., 2013). However, due to their vola-
Materials and methods
tile nature, EOs are susceptible to degradation by heat,
pressure, light and oxygen (Mohammadi et al., 2015).
Essential oil extraction and analysis
What is more, their direct incorporation into food is
limited not only because of their hydrophobicity that Leaves and flowers of Origanum vulgare ssp hirtum
limits their adequate dispersion but also because of high (oregano clone Criollo) (crop 2015) were purchased in
concentrations cause undesirable flavour changes, and Villa Dolores (C
ordoba, Argentina) and hydro-dis-
these may be toxic (Bhargava et al., 2015; Topuz et al., tilled. The EO was extracted and analysed according
2016a; Artiga-Artigas et al., 2017). to Asensio et al. (2015a), (Asensio et al., 2015a).
Nanoemulsions (NEs) are used to encapsulate, pro-
tect and deliver lipophilic ingredients to liquid or mini-
Nanoemulsion preparation, characterisation and physical
mally processed foods (Acevedo-fani et al., 2015;
stability
Almadiy et al., 2016). They offer high thermodynamic
stability against aggregation, flocculation, coalescence An aqueous solution of soy lecithin (Alcolec PC75,
and Ostwald ripening. Furthermore, aside from pro- American Lecithin Co., Oxford, MS) was prepared
tecting the lipophilic active from deleterious interac- and stirred for 30 min. The oil phase consisted of
tions with food components and the environment, the medium chain triglycerides (MCT) (Neobee 1053, Ste-
NE allows increasing the distribution of the active in pan Co., Northfield, MN) and oregano EO (1:1 w/w).
food matrices into which the NE is added (Herculano The final NE composition was 81% water, 3% soy
et al., 2015). The incorporation of a nanoemulsion lecithin and 16% oil phase. The final concentration of
containing essential oil was studied to preserve the EO in the NE was 160 mg mL
1. Both phases were
quality of chicken pate (Moraes-Lovison et al., 2017), high-speed homogenised with an Ultra-Turrax T25
fresh lettuce (Bhargava et al., 2015), rainbow trout fil- (IKA Works Inc., Wilmington, NC) at 14 489 g for
lets (Shadman et al., 2016), low fat cut cheese (Artiga- 3 min. These were homogenised with a high-pressure
Artigas et al., 2017) and green beans (Dons
ı et al., homogeniser (EmulsiFlex-C3, Avestin Inc., Ottawa,
2015)”. Canada) for six cycles at pressure of 150 MPa. A
The quality preservation of fish fillets and burgers, blank nanoemulsion (B-NE) was prepared with the
including hake, has been studied. Most researches same concentration of water (81%), soy lecithin (3%)
included the use of modified atmosphere packaging and MCT (16%) as the oil phase (without EO). Sam-
(MAP), different freezing conditions, the incorporation ples (NE-EO and B-NE) were stored in 20 mL glass
of an antimicrobial products (polymers and/or natural vials (Supelco Analytical) in two different storage con-
products) or the binary and tertiary combination of ditions: room temperature (23 °C) and fridge (4 °C)
them (Hassoun & Emir C ß oban, 2017; Bainy et al., for 12 days.
2015; Otero et al., 2017; Fern
andez-Saiz et al., 2013; The average particle size (mean diameters, nm) and
Rodr
ıguez et al., 2004; Schelegueda et al., 2016; Del polydispersity index (PDI) were monitored using a
Nobile et al., 2009). The combination of chitosan, dynamic light scattering (DLS) instrument (Brookha-
nisin and sodium lactated combined with MAP ven BIC 90 plus) equipped with a Brookhaven BI-
extended the shelf life of Argentinean hake burgers up 9000AT digital correlator (Brookhaven Instrument
to 30 days (Schelegueda et al., 2016). In a different Corp, New York, NY). The mean diameters of emul-
study, the combined effect of thymol, grapefruit seed sions were determined by cumulant analysis of the
and lemon extracts with MAP could extend the shelf intensity-intensity autocorrelation function, G (q, t).
life of blue fish burgers up to 22 days at 4 °C (Del
Nobile et al., 2009). In a recently published review
Storage study and chemical analysis of stored hake
concerning fish and other sea products preservation,
burgers
EOs are considered a promising option due to their
antioxidant and antimicrobial effects and to the con- Hakes (total 9 kgs) were purchased fresh from a local
sumer0 s demand for clean label products (Hassoun & market. Pure EO, nanoemulsion containing EO (NE-EO)

International Journal of Food Science and Technology 2019 © 2018 Institute of Food Science and Technology
 Oregano nanoemulsion: hake burger quality preserver C. M. Asensio et al. 151

and B-NE, were added at 0.05 (g/100 g) to filets and divinylbenzene/carboxen/polydimethylsiloxane (DVB/
minced in a grinder (AD5663C9, Moulinex, Colombia) CAR/PDMS) (Supelco, SIGMA, St. Louis, MO) fibre
for 5 min. A control sample (C) without any addition was used. Vials were heated at 70 °C for 20 min with-
was treated identically. A total of four fish burgers out stirring. The fibre was exposed to the vial head-
treatments were prepared: control fish burgers without space for 10 min and then injected into the CG-MS.
any addition (C-FB), fish burgers with EO (EO-FB), an Elite-5MS column (Perkin Elmer) was used. Identi-
fish burgers with NE-EO (NEEO-FB) and a fish fication and quantification were performed according
burger with B-NE (BNE-FB). Three replicates of each to Olmedo et al. (2014) and Asensio et al. (2017).
treatment were set. A silicone mould for hamburger
preparation was used. Burgers were put in foam trays
Total volatile basic nitrogen (TVBN)
covered with food wrap film and stored at 4 °C for
14 days. Each foam tray was considered a replicate. A The procedure was followed from the official method
total of 60 foam trays were stored (4 treatments, 3 of the Commission of the European Communities
replicates, 5 periods of time). The experimental design (European Commission, 1995). TVBN was titrated
consisted of 4 treatments (C-FB, EO-FB, NEEO-FB, with hydrochloric acid in a boric acid solution and
and BNE-FB) 9 3 replicates (burgers) of each treat- expressed as mg N/100 g sample.
ment 9 4 chemical/microbiological variables 9 5 periods
of times.
Microbiological count
Changes in the FA, headspace volatile compounds,
TVBN and total mesophilic bacteria count were mea- Samples of 10 g of fish burgers were analysed. Total
sured every 3 days. aerobes were investigated by deep inoculation on plate
count agar (PCA). Plates were incubated for 4 days at
25 °C (FAO 2016).
Lipid extraction
Briefly, homogenised samples (10.0  0.5 g) were
Statistical analysis
extracted with 300 mL chloroform: methanol (2:1, v/v),
mixed in an orbital shaker for 20 min and filtered Experiments were carried out three times and results
using Whatman No. 1 filter paper. The chloroform were expressed as mean  standard deviations. Analy-
lower layer was collected, and the lipid fraction was sis of variance (ANOVA, a = 0.05) and DCG multiple
recovered after evaporating the solvent under vacuum range tests were performed to determine significant dif-
using a rotary evaporator. ferences between means. Linear regression equations
from changes of FA during the storage study were
obtained. Principal component analysis (PCA) was
Fatty acid methyl esters (FAME)
performed on the correlation matrix of the standard-
Fatty acid methyl esters were prepared from extracted ised (normalised) data from storage study (Asensio
lipids by transmethylation (Asensio et al., 2015b). The et al., 2015a). Associations between samples chemical
FAME were analysed in a Perkin Elmer Clarus 600 and microbiological variables were assessed by PCA.
GC (Shelton, CT) equipped with a flame ionisation A biplot from PCA was obtained. Vectors and points
detector. An Elite-wax (60 m, 0.25 mm internal diame- represented dependent variables and treatments,
ter, 0.25 lm phase thickness) capillary column was respectively. The angle formed between vectors indi-
used. The column temperature increased from 100 to cates correlation between variables. Data were anal-
200 °C(10 °C min
1) and then, from 200 to 250 °C ysed using the InfoStat software, version 2016 (Di
(5 °C min
1). The carrier gas was helium (1 mL min
1 Rienzo et al., 2015).
flow rate). The separated fatty acid methyl esters were
identified by comparing their retention times with
Results and discussion
those of an ampoule C4-C24 FAME mix (SUPELCO,
Sigma, St Louis, MO). Quantitative fatty acid analysis
NE characterisation and physical stability
was performed using heptadecanoic acid methyl ester
(Sigma Chemical Co.) as internal standard (Asensio In the current study, B-NE kept larger mean droplet
et al., 2015b). diameters than EO-NE throughout the storage dura-
tion (146.9 nm at day 14) (Table 1). EO-NE showed a
significant increase in the mean diameter through the
Volatile compounds analysis
storage (from 42.8 to 62.9 nm). Surprisingly, no effect
Volatile compounds were studied to analyse the release of the temperature (RT vs F) was registered for both
of the EO and the quality changes. Briefly, 2 g of sam- samples after 14 days of storage. The EO contains sur-
ple were weighted into 20 mL capacity vials. A SPME face-active substances, endearing the EO-NE prone to

© 2018 Institute of Food Science and Technology International Journal of Food Science and Technology 2019
152 Oregano nanoemulsion: hake burger quality preserver C. M. Asensio et al.

Ostwald ripening (Acevedo-fani et al., 2015). Previous

Table 1 Means and standard deviations (n = 3) of effective diameter and polydispersity index of blank (B-NE) and essential oil containing (EO-NE) nanoemulsions stored at

1
1
1
1

1
1
1
1
‡
studies demonstrated that a ripening retarder could

a
a
a
b
b
c

b
†
contravene the Ostwald ripening effect (Liang et al.,

4.93
2.66
2.18
2.35

0.01
0.01
0.01
0.01
SD 2012; Moghimi et al., 2016). The retardant may be of
relatively high molecular weight, as MCT (Topuz

0.29
0.28
0.31
0.32
Mean

146.9

62.9
62.8
et al., 2016a). A NE containing oregano EOs increase
148
14

its droplets size from less than 50 nm (at day zero), up

to 169.30, after 7 days of storage at room temperature
1
1
1
1

1
2
1
1
‡

(Guerra-Rosas et al., 2017).

a

a
a
a
a
b
b
c
†

Regarding the PDI, no significant changes were

2.78
2.17
2.18
1.92

0.01
0.01
0.01
0.01
observed for this parameter between days 0 and 14 of
SD

storage for both the formulas (B-NE and EO-NE) and

63.47

0.25

0.31

Different numbers in each column indicate significant differences between storage temperatures for each sample (ANOVA, DCG test, a = 0.05).
storage conditions (RT and F) (Table 1). A low PDI
Mean

141.9
144.2

62.6

0.3

0.3 value, indicated a narrow particle size distribution

12

(Liang et al., 2012). NEs with a PDI < 0.30 were

2
1
1
1

1
2
1
1
‡

reported to possess a relatively narrow range of size

a
a
a

a
a
a
a
c
†

distribution (Topuz et al., 2016a). In the current study,

2.78
2.66
2.18
1.92

0.01
0.01
0.01
0.01

PDI values were under 0.30, mostly.

SD

53.23

0.25
0.28
0.29
0.32
Mean

149.7
131.5

59.6

EO chemical composition and compounds captured by

the SPME fibre
9

1
1
1
1

1
1
1
1
‡

A total of 30 compounds were identified. Seven major

Different letters in each raw indicate significant differences between samples (ANOVA, DCG test, alpha 0.05).
c
d

b
a

a
a
a
a

compounds were identified (Table 2). They were

†

terpinolene (186.2 g kg
1), thymol (168.7 g kg
1), c-

2.27
2.17
2.67
1.92

0.01
0.01
0.01
0.01
SD

terpinen (150.8 g kg
1), o-cymene (110.8 g kg
1), 4-

terpineol (92.9 g kg
1), a-terpinene (40.3 g kg
1) and
52.97

0.27
0.28
0.28
0.28
Mean

177.6
175.9
42.9

b-pinene (38.1 g kg
1). Previously, 33 components

7

were found in Criollo EO (Quiroga et al., 2011), and

c-terpinene (18.2%), thymol (14.9%) and trans-sabi-
1
1
1
1

1
1
1
1
‡

nene hydrate (6.3%), were reported as major com-

b
b

b
a

a
a
a
†

pounds.
2.78
2.17
2.67
1.92

0.01
0.01
0.01
0.01
SD

The release of EO volatile compounds in FB was

evaluated (Table 2). At day 0, 20 compounds were
49.35

0.31

0.29
Mean

162.3
152.5

48.9

0.3
0.3

captured and identified from EO-FB, whereas only 14

were detected from NEEO-FB. The major compounds
5

and the major/minor volatiles ratio of EO-FB were

1
2
1
1

1
1
1
1
‡

similar to the pure EO sample. It is generally

b
c

b
a
a

a
a
a
†

accepted that the smaller the molecular size, the faster

2.27
2.17
2.18
1.92

0.01
0.01
0.01
0.01

the molecule diffuses through a polymeric medium

SD

(Muriel-Galet et al., 2015). In EONE-FB, the major/

0.31
0.29

0.29

minor volatiles ratio was significantly higher

Mean

0.3
160.9
166.5
40.7
40.7
23 °C a (RT) and at 4 °C (F) during 14 days

(P ≤ 0.001) (6.23).Terpinolene (242.9 g kg
1), 4-teri-

2

neol (201.7 g kg
1) and thymol (176.1 g kg
1) were

1
1
1
1

1
1
1
1
‡

the main compounds released from EO-FB, whereas

4-Terpineol (594.1 g kg
1) in EONE- FB. Carvacrol,
b

b
a

a
a

a
a
a
†

2.27
2.17
2.67
2.35

0.01
0.01
0.01
0.01

thymol, and c-terpinene were the major released com-

SD

pounds in an oregano NE (Moraes-Lovison et al.,

2017).
0.31
0.31
0.28
0.28
Mean

150.8
150.8
42.8
42.8

At 14 days of storage, the number of released com-

Polydispersity index
0

pounds increased significantly (28 and 24 compounds

Effective diameter
Con.

detected in EO-FB and NEEO-FB, respectively). The

RT

EO-NE RT

RT

EO-NE RT
F

concentration of the main volatile components of an

EO-pectin NE gradually decreases during storage time
Sample

B-NE

B-NE

(Guerra-Rosas et al., 2017), as found in the current

study. Several factors have been documented to
†

International Journal of Food Science and Technology 2019 © 2018 Institute of Food Science and Technology
 Oregano nanoemulsion: hake burger quality preserver C. M. Asensio et al. 153

Table 2 Oregano essential oil chemical composition and oregano volatiles compounds captured by SPME from hake burgers prepared with
oregano essential oil and with nanoemulsion containing oregano essential oil

EOa EO-FBa NEEO-FBa

Day 0 Day 0 Day 14 Day 0 Day 14

Compound (g kg
1)   SD
X b   SD
X b   SD
X c   SD
X b   SD
X c

1 a-Phellandrene 10.2  0.4 b 0  0 a 4.4  0.1 1 0  0 a 3.5  0.7 1

2 a-Pinene 7.6  0.1 b 0  0 a 3.3  0.6 1 0  0 a 1.5  0.7 1
3 Camphene 3.4  0.1 b 0  0 a 1.3  0.4 1 0  0 a 9  0 2
4 b-Pinene 38.1  0.2 c 15.58  0.29 b 15.3  0.5 1 0  0 a 34.2  1.13 2
5 3-Octanone 16.4  0.2 c 6.9  0.8 b 5.1  0.4 2 0  0 a 0  0 1
6 b-Myrcene 2.5  0.1 a 3.27  4.3 a 7.3  0.8 2 0  0 a 0.5  0.7 1
7 a-Terpinene 40.3  0.5 b 15.6  3.37 a 17.9  0.7 1 0  0 a 24.5  0.49 1
8 0rto-cymene 110.8  1.6 c 65.2  2.3 b 42.9  0.6 1 10  1.4 a 41.5  0.49 1
9 b-trans-Ocimene 10.2  0.4 c 7.4  0.5 b 16.5  1.3 1 0.5  0.1 a 13.8  4.2 1
10 c-Terpinen 150.8  0.3 c 68  7.1 b 67.7  0.4 1 33.5  4.9 a 79.3  17 1
11 cis Sabinene hidrate 25.2  1.6 a 37.8  16.3 a 23.7  0.1 1 15.5  1.9 a 27.2  2.1 1
12 b-cis-Ocimene 8.5  0.3 c 5.23  0.5 a 7.1  0.2 1 3  0.1 a 6.5  0.7 1
13 Terpinolene 186.2  5.3 b 242.9  3.22 c 127.2  3 2 95  7.1 a 57  4.2 1
14 b-Phellandrene 8.1  0.4 a 13.8  1.7 a 6.9  0.1 1 10.5  0.6 a 7  0 1
15 Camphor 0.2  0.1 a 8.6  1.4 b 1  0 1 0  0 a 1  0 1
16 Borneol 5.1  0.4 a 14.4  5.2 a 3.7  0.1 2 6.5  0.6 a 2  0 1
17 4-Terpineol 92.9  2.2 a 201.7  3.7 b 96.9  2.6 1 594.1  9.05 c 163.5  1.2 2
18 a-Terpineol 22.8  1 a 42.8  3.7 b 21.4  0.9 1 64.5  7.8 c 21.5  2.1 1
19 Thymol methyl ether 10.2  0.3 a 17.8  9 b 9.2  0.1 1 16.9  1.4 b 8  1.4 1
20 Carvacrol methyl ether 3.1  0.5 a 10.9  1.2 b 3.7  0.1 1 13  2.8 b 6  1.4 1
21 Thymol 168.7  0.78 a 176.1  3.86 a 95.6  0.83 1 115.3  1.56 b 90.2  1.4 1
22 Carvacrol 8.3  0.9 a 6.8  0.3 a 6.9  2.9 1 5.2  2.1 a 7.8  1.4 1
23 Tetradecane 0.1  0 a 1.4  0.8 a 0.6  0 1 0  0 a 0  0 1
24 b-Bourbonene 1.3  0.1 b 0.4  0.1 b 0.3  0 1 0  0 a 0  0 1
25 Caryophyllene 17.7  0.3 c 3.9  0.2 b 4.8  0.7 1 0  0 a 4  1.4 1
26 Germacrene D 8.5  0.1 c 0.5  0 b 1.6  0.1 2 0  0 a 1  0 1
27 c-Gurjunene 11.4  0.3 b 3.7  03 a 0  0 1 0  0 a 1.5  0.7 1
28 d-Cadinene, (+)
1  0.1 b 0.7  0.01 b 0  0 1 0  0 a 0  0 1
29 Aromadendrene, dehydro- 2.3  0.2 b 1.17  0 a 0  0 1 0  0 a 0  0 1
30 Lanceol, cis 1.1  0.2 b 1  0 a 0  0 1 0  0 a 0  0 1
31 9-Eicosyne 0  0 a 0  0 a 1.8  1.1 1 0  0 a 2.5  0.35 1
32 Hexadecane 0  0 a 0  0 a 0.6  0.1 2 0  0 a 0  0 1
EO id. comp. (g kg
1)d 973.3  4.2 a 973.6  5.13 a 594.7  3.4 1 983.5  11.8 a 614.5  16.4 1
Major compounds (g kg1)e 787.4  1.9 a 785.1  16.6 a 463.5  4.2 1 847.5  25.6 b 489.5  10.4 2
Minor compounds (g kg
1)f 185.9  2.4 a 188.5  20.7 a 131.2  0.8 2 136  2.87 b 125  5.5 1
Major/Minor ratiog 4.23  0.6 a 4.16  0.55 a 3.53  0.05 1 6.232  1.56 b 3.916  0.09 2
Tot. id. comp. (g kg
1)h 979.9  4.2 a 974  4.2 a 943.3  2.9 1 983.5  1.88 a 914.6  2.06 1

a
Samples: EO, oregano essential oil; EO-FB, fish burger prepared with EO; and NEEO-FB, fish burger prepared with nanoemulsion containing EO.
b
Different letters in each raw indicate significant differences between samples at day 0 (ANOVA, DCG test, a = 0.05).
c
Different numbers in each row indicate significant differences between samples at day 14 of storage (ANOVA, DCG test, a = 0.05).
d
EO id. Comp.: Chemical compounds of oregano EO detected by CG/MS with direct injection or SPME fibre.
e
Major compounds: sum of compounds with concentrations higher than 30 g kg
1 in the EO (bold letters).
f
Minor compounds: sum of compound with concentrations lower than 30 g kg
1 in the EO.
g
Major/Minor ratio: ratio between major and minor compounds.
h
Tot. id. Comp.: Total identified compounds of each sample by CGMS (including EO and volatile compounds detected during the storage stability
study).

decrease the concentration of the main volatiles: they major and minor components are different (Guerra-
might be able to migrate from the oil droplets into the Rosas et al., 2017). In EO-FB, thymol decreased
water phase and be volatilised, or they might be oxi- from 176.1 to 95.6 g kg
1 and in NEEO-FB, from
dised to unstable forms, or the boiling points of the 115.3 to 90.2 g kg
1. This oxygenated monoterpene is

© 2018 Institute of Food Science and Technology International Journal of Food Science and Technology 2019
154 Oregano nanoemulsion: hake burger quality preserver C. M. Asensio et al.

(a) (b)
Eq: β1 : –0.05 R20.44 b; β1 : –0.09 R20.69 b; β1 : –0.15 R20.74 a; β1 : –0.18 R20.91 a Eq: β1 : 0.05 R20.4 c; β 1 : –0.08 R20.44 c; β1 : –0.22 R20.75 b; β1 : –0.6
123.41 217.46

115.21 19.405
194.05 C
20:5 w3 (g kg–1 oil)

22:6 w3 (g kg–1 oil)

C
B
C

107.01 170.63

98.82 147.22
A
A

90.62 123.81
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Refrigerated time (days) Refrigerated time (days)

(c) (d)
Eq: β 1 : 0.17 R20.43 c; β 1 : –0.38 R20.95 c; β1 : –0.70 R 20.95 b; β1 : –1.29 R20.96 a
Eq: β1 : 1.09 R20.94 c; β1 : 0.56 R20.88 b; β1 : 0.36 R20.66 ab; β 1 : 0.15 R20.4 a
693.33 496.03 D
Total unsaturated (g kg–1 oil)

Total saturated (g kg–1 oil)

639.03 C
449.01
C
C

584.72 401.99 B
B

530.42 354.97

476.12 307.95
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Refrigerated time (days) Refrigerated time (days)
(e) (f)
Eq: β1 : –0.25 R20.69 c; 2
β1 : –0.29 R 0.81 c; 2
β1 : –0.53 R 0.89 b; 2
β1 : –0.96 R 0.97 a Eq: β1 : 0.03 R2 0.92 b; β1 : 0.16 R20.92 b; β1 : 0.009 R20.68 a; β1 : 0.004 R20.58
43.472
434.72 1.00
C
Saturated/Unsaturated ratio

39.436
394.46 0.86
PUFAs (g kg–1 oil)

C
C
B

35.400
354.00 0.72
B
A

A
31.364
313.64 0.58

273.28
27.328 0.44
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Refigerated time (days) Refrigerated time (days)

Figure 1 Changes in fatty acid composition and regression equations (Eq): (a) 20:5 x3 (EPA), (b) 22:6 x3 (DHA), (c) saturated, (d) unsatu-
rated, (e) PUFAs content and in (f) saturated/unsaturated ratio of hake fish burgers stored during 14 days.

characteristic of oregano species and its antioxidant were released and captured, from NEEO-FB than
activity (Dambolena et al., 2010; Quiroga et al., 2011; from EO-FB at the end of the storage. It was pre-
Asensio et al., 2015a). Consequently, the antioxidant sumed that these compounds were retained for more
activity of the NE was expected to decrease. It was time in the emulsion in contact with the burger matrix.
reported in oregano microcapsules that the volatile
compound release affected the antioxidant capacity of
Storage study of fish burgers
all formulas (Asensio et al., 2017). The ratio of the
major/minor volatile compounds also decreased in Changes in the FA profile
both treatments. EO-FB had the lowest ratio (3.53). A The effects of refrigerated storage on the FA composi-
lower amount and number of EO volatile compounds tion of hake burgers are presented in Fig. 1. At day 0

International Journal of Food Science and Technology 2019 © 2018 Institute of Food Science and Technology
 Oregano nanoemulsion: hake burger quality preserver C. M. Asensio et al. 155

(a) (b)
Dimethyl amine (g kg–1) 105.0 855.75

Trimethyl amine (g kg–1)

78.75
7.875 64.181
641.81

5.250
52.50 42.788
427.88

26.25
2.625 21.394
213.94

0.000
0.00 0.00
0.000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days Days
(c) (d)

2.52
25.20 12.285
122.85

Dimethyl-disulphide (g kg –1)
18.90
1.89 9.214
92.14
Benzene (g kg–1)

12.60
1.26 6.143
61.43

0.63
6.30 30.71
3.071

0.00
0.00 0.00
0.000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days Days
(e)
3.465
34.65
3-methyl butanol (g kg–1)

2.599
25.99

1.732
17.32

0.866
8.66

0.00
0.000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days

Figure 2 (a) dimethylamine, (b) trimethylamine, (c) benzene, (d) dimethyl-disulfide and (e) 3-methyl butanol (g kg
1)(captured by SPME) of
hake fish burgers stored during 14 days.

of storage, 19 FAs were identified. Palmitic acid (16:0) acid (Saldanha & Bragagnolo, 2008). In a different
(221.3 g kg
1) was followed by 22:6 x3 (DHA; with study, hake was reported to have higher quantities of
211.5 g kg
1) and oleic acid (18:1; with 160.1 g kg
1). palmitic (21.7 g/100 g), oleic (9.65 g/100 g), 20:5 x3
c-Linolenic acid (18:3x6) was also present at a high (12.91 g/100 g), 22:6 x3 (22.08 g/100 g) (Huynh and
amount (57.1 g kg
1). In frozen Atlantic hake fillets, Kitts 2009). The contents and composition of fish
the day 0 of storage, the main FAs were oleic (28.9 g/ lipids varied according to the species, age, location,
100 g), 22:6 x3 (13.0 g/100 g), 20:5 x3 (6.79 g/100 g), species origin and environmental conditions (Huss
palmitic (12.2 g/100 g)and palmitoleic (8.70 g/100 g) et al., 2004). The sum of the total saturated FAs was

© 2018 Institute of Food Science and Technology International Journal of Food Science and Technology 2019
156 Oregano nanoemulsion: hake burger quality preserver C. M. Asensio et al.

328.5 g kg
1 (the lowest percentage of the storage sugars, with the production of ethyl acetate, and
study) and the PUFAs totalled 422.9 g kg
1 (the high- anaerobic respiration by certain bacteria, can also
est value for this parameter in the storage study). induce TMAO reduction to TMA (Ashie et al., 1996;
After 14 days of refrigerated storage, significant dif- Jaffres et al., 2011). In the current study, TMA levels
ferences were detected (P ≤ 0.01). Total unsaturated of C-FB and BNE-FB increased from day 0 to 10, fol-
FAs and PUFAs decreased, whereas the total saturated lowed by a decrease. In comparison, the TMA levels
and the saturated/unsaturated FA ratio (S/U) increased of NEEO-FB and EO-FB increased from day 0 to 14
in all samples (Fig. 1). The control sample presented the but at a considerably lower rate (Fig. 2b). TMA is
lowest amounts of 18:3x3 (7.41 g kg
1), 18:3x6 considered a good indicator of hake quality, there is a
(32.96 g kg
1), 20:4 (5.42 g kg
1) and 22:6 high correlation with sensory changes and microbial

1
(136.02 g kg ) at the end of storage. The PUFAs con- loads during iced storage (Orban et al., 2011). In
tent in C-FB, decreased 69.29%, whereas the total satu- Argentine hake burgers MAP packed, TMA levels and
rated FAs increased 45.30%. The extreme sensitivity of the odour score remained lower than air packed sam-
n-3 PUFAs to lipid oxidation is well-recognised. Gener- ples after 15 days of storage (Schelegueda et al., 2016).
ally, the greater the number of double bonds in the FA, In another study, Serratia liquefaciens and P. phospho-
the greater the ease of its oxidation. For instance, DHA reum were found to produce TMA in cold-smoked
is estimated to be 50-fold more sensitive to oxidation and raw salmon (Mac
e et al., 2013).
than oleic acid (Polavarapu et al., 2011). TMAO may decompose to dimethylamine (DMA)
The preservation of 22:6 x3 and 20:5 x3 is particu- and formaldehyde (Ashie et al., 1996). In C-FB, the
larly important. Moreover, as these compounds are
typical of seafood, and hakes contain high amounts, (a)
their losses need to be studied (Saldanha & Bragag- 23.73 b
C-FB b
nolo, 2008). The samples NEEO-FB and EO-FB had EO-FB
a
TVB-N (mg N/100 g sample)

the highest content of 20:5 x3 (107.97 and BNE-FB a

110.87 g kg
1, respectively) and 22:6 x3 (191.82 and 17.84 NEEO-FB

198.42 g kg
1, respectively) at 14 days of storage c

(Fig. 1a,b). This behaviour is consistent with the total bc
PUFAs content in both treatments (375.04 and 11.95 ab

373.48 g kg
1, respectively), which decreased an

b
ab
a
ab a
11.68% and 11.33% in EO-FB and in NEEO-FB
throughout storage, respectively. The S/U ratio 6.05 b ab ab
a
remained lower in EO-FB (0.58) and NEEO-FB (0.64)
than in the rest of the treatments. Furthermore, no sig- a a a a

nificant differences were found between these two 0.16

0.00 3.00 7.00 10.00 14.00
treatments for most of the FA slopes. Therefore, EO- Days
FB and NEEO-FB had the lowest slopes for S/U ratio (b)
(0.009 and 0.004, respectively). Interestingly, the con- 10 c
c b b b
tent of EO in EONE-FB was 5.65 times lower than in d
b b
b
EO-FB. Consequently, the incorporation of the EO 9 C-FB
a
into a NE seemed to improve its lipid oxidation pre- EO-FB d a
a
BNE-FB
serving activity in hake fish burgers. 8 c
log UFC g–1

NEEO-FB b
a
Volatile analysis 7
Fresh fish aroma compounds are typically depleted
throughout storage (Parlapani et al., 2017). In the cur- 6 a aa a

rent study, only volatile compounds that exhibited a

consistent trend and were associated with bacteria 5
spoilage were reported (Fig. 2).
Trimethylamine is already known to be responsible 4
0.00 3.00 7.00 10.00 14.00
for strong fishy and ammonia-like off-odours. TMA is
Days
produced by either endogenous enzymes or spoilage
bacterial enzymes, from trimethylamine oxide Figure 3 (a) Total volatile basic nitrogen (TVBN) and (b) total aer-
(TMAO). Mostly, Shewanella putrefaciens and Pseu- obic count (log10 UFC/g) of hake burgers during 14 days of storage.
domonas phosphoreum use TMAO, instead of oxygen, Means (n = 3) and standard deviations are indicated by bars. For
as the final electron acceptor in their respiratory meta- each time of storage, different letters indicate significant differences
bolism (Parlapani et al., 2015). The fermentation of (P < 0.05) between treatments.

International Journal of Food Science and Technology 2019 © 2018 Institute of Food Science and Technology
 Oregano nanoemulsion: hake burger quality preserver C. M. Asensio et al. 157

7.00
20:5 EPA 20:0
Dimethyl trisulfide
24:0
22:0 UCF/g
thymol
3.50 cis sabinene hidrate
Terpinolene 3-Octanone 18:1
α-Terpineol γ-Terpinen Monounsaturated C-FB
β-trans-Ocimene α-Pinene Benzene
α-Terpinene tetradecane 16:1 Saturated/Unsaturated
Saturated/Unsaturated Ratio
Ratio

CP 2 (13.6%)
0rto-cymene 4-terpineol 3-methyl butanol
carvacrol Saturated
0.00 NE EO TVBN Dimethyl-disulfide
EO
TrimethylamineNitrous
Nitrous Oxide
Oxide
Unsaturated 18:0

Figure 4 Biplot from the principal compo- Poliunsaturated 18:3 w6

16:0
nent analysis. Vectors: dependent variables NE Blank
analysed during storage study of hake fish –3.50 22:6 DHA

burgers. Points: treatments: C-F (Control 20:4

fish burger), EO-F (Fish burger with essen- 18:3 w3

22:1
tial oil), BNE-F (fish burger with blank Dimethyl amine
nanoemulsion), NEEO-FB (fish burger with –7.00
the addition of nanoemulsion with essential –7.00 –3.50 0.00 3.50 7.00
oil). CP 1 (77.8% )

DMA levels increased from day 7 compared to day 10 this compound was registered in NEEO-FB
onwards, in the remaining samples (Fig. 2a). At the (1.55 g kg
1) at the end of storage. 3-Methyl butanol
end of storage, significant differences were observed has been considered a potential chemical spoilage indi-
for this compound, such that NEEO-FB showed the cator and has been detected in raw salmon fillets, gut-
lowest amount of DMA (115.0 g kg
1), followed by ted sea bass, ice-stored sea bream, Nordic peeled
EO-FB (193.5 g kg
1). shrimps and yellowfin tuna (Jaffres et al., 2011; Parla-
Benzene levels increased constantly from day 0 to 14 pani et al., 2015).
in C-FB. At the end of storage, all samples contained
benzene (Fig. 2c). However, the samples with the low- TVBN and bacteria count
est amount of this compound were NEEO-FB Total volatile basic nitrogen was demonstrated to be
(3.45 g kg
1) and EO-FB (3.33 g kg
1). Benzene levels more representative of fish spoilage rather than fish
were previously reported in raw salmon fillets spoiled freshness (Orban et al., 2011). Increases in this indicator
with P. phosphoreum (Mac
e et al., 2013). are caused by bacterial degradation of fish muscular tis-
The transformation of sulphur-containing amino sue and non-protein nitrogenous compounds producing
acids by microbial activity produces dimethyl-disulfide ammonia, DMA, TMA and other volatile, nitrogen-
(DMDS). The levels of DMDS increased in C-FB containing compounds (Parlapani et al., 2015). In
from storage day 3. The rest of the samples underwent general, TVB-N increased in all samples (Fig. 3a),
a rapid increase in DMDS, from day 10 (Fig. 2d). significant differences (P < 0.001) were found. At day 0,
Once more, the highest values were observed in C-FB the TVB-N values range from 1.35  0.44 (EO-FB) to
(111.0 g kg
1) and BNE-FB (113.48 g kg
1). DMDS 1.48  0.35 mg N/100 g sample (C-FB) between treat-
is an important contributor to the aroma of numerous ments. C-FB presented the highest concentrations of
food products, due to its low sensory threshold value. TVB-N (days 3, 7, 10 and 14). At day 14, EO-FB
This compound has been found in various seafood showed the lowest TVB-N content (17.2 mg N/100 g
products (Giri et al., 2010)and, furthermore, has been sample), followed by NEEO-FB (19.23 mg N/100 g
associated with Pseudomonas and Shewanella activity sample). In tuna fish preserved with liquid smoke nano-
(Mac
e et al., 2013; Parlapani et al., 2017). capsules, when prolonging the storage time, the TVB-N
Three-methyl butanol can be attributed to the values increased (Saloko et al., 2014), as observed in the
oxidative deamination of the free amino acids (Giri current study. The European Commission (EC) Direc-
et al., 2010; Parlapani et al., 2017). In general, 3- tive 95/149 limits the TVB-N to 35 mg/100 g fish. This
methyl butanol levels increased from day 3, in all sam- limit would be reached in 23 days by the control treat-
ples (Fig. 2e). C-FB and BNE-FB experienced a high ment, and in 29 and 33 days, in treatments with EO and
increment in 3-methyl butanol between days 3 and 7, NEEO, respectively.
and kept high levels of this compound throughout the Figure 3b illustrates the growth of aerobic meso-
storage. Samples with EO/NEEO had lower levels of philic bacteria in all treatments, during storage. At
this compound throughout storage. The lowest level of day 3 of storage, significant differences (P < 0.001)

© 2018 Institute of Food Science and Technology International Journal of Food Science and Technology 2019
158 Oregano nanoemulsion: hake burger quality preserver C. M. Asensio et al.

were observed between the control and the rest of microbial spoilage exhibited a better performance.
the treatments. NEEO-FB did not exhibit counts Regarding lipid oxidation, the behaviour was similar
above 7 log CFU g
1, even at 14 days of storage pre- in both treatments (EO and NEEO). However, the
senting the best fish microbial quality. The antimicro- concentration of EO per unit of mass (kg) was 5.65
bial activity of oregano clone Criollo EO, was times lower in the NEEO samples.
previously demonstrated. This EO presented the low- In this research, a single mechanism of preservation
est MIC values against L. helveticus, S. termophyllus, was tested in a complex food matrix. The addition of
B. cereus, S. aureus, Salmonella sp. and E. coli, being a nanoemulsion with oregano essential oil protects
statistically different to other oregano EOs. More- hake burgers from deterioration extending their shelf
over, after 30 days of storage, ricotta flavoured with life. The implementation of this natural preserving
Criollo EO had the lowest total bacteria count. mechanism should be considered by the food industry
(Asensio et al., 2014). In a different study, the same as a new alternative to preserve fresh fish products.
oil showed good bioactivity against yeast and nema-
todes (Asensio et al., 2015a). However, in compar- Acknowledgment
ison, the current results, showed a significant
inhibitory effect on the growth of aerobic mesophilic We thank Consejo Nacional de Investigaciones Cient
ıfi-
bacteria, by the NE containing this EO. cas y Tecnicas (CONICET, PIP#11220120100371CO)
who supported this study.

Principal component analysis (PCA)

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