1600035 (1 of 9) Eur. J. Lipid Sci. Technol.

2017, 119, 1600035

Short Communication
Inuence of freezing and oxygen-free packaging methods
on lipid oxidation and other esh quality parameters of
Norway lobster (Nephrops norvegicus)

Gioacchino Bono, Charles Odilichukwu R. Okpala, Cinzia V. Badalucco, Giacomo Milisenda

and Sergio Vitale

Istituto per l Ambiente Marino Costiero, Consiglio Nazionale delle Ricerche (IAMC-CNR), Mazara del Vallo, Italy

By adapting oxygen-free systems, the use of modied atmosphere packaging (MAP) appears increasingly
attractive and of commercial global interest for (sea)foods products. Moreover, when combined with
other preservation methods, MAP continues to show high promise in tackling a wide range of
deterioration aspects affecting shery products. This study was therefore purposed to apply freezing and
oxygen-free (vacuum as well as modied atmospheres of 50% N2-50%CO2- and 100% N2) packaging
methods onto Norway lobster (NL) (Nephrops norvegicus) and resultant data compared with
conventional preservative treatment of sulphite together with control (untreated). The specic objective
was to determine the inuence of the (above-mentioned) treatments on lipid oxidation (thiobarbituric
acid {TBA}) and other esh quality parameters (total volatile base (TVB) nitrogen, pH, free amino acids
(FAAs), proximal composition, as well as melanosis scores) during the inspected storage period of up to
12 months. The result showed that across treatments, whilst the TBA values were little coincidental with
signicantly changing pH plus supercially uctuating proximate composition(s), the TVB-N values
seemed unaffected except some increases in 100% N2-treated NL samples at the second half of frozen
storage. Whilst individual FAAs per treatment considerably varied, the trends of total FAAs between
treatments seemed relatively unvaried. Additionally, the treatments of 50% N2-50%CO2- and 100% N2-
to apparently delay the melanosis formation in NL samples indicated oxygen-free treatment methods
herein with high promise to replace the sulphite ones. To best of our knowledge, this is the rst study
about freezing combining oxygen-free packaging treatment methods specically applied to NL samples.

Practical applications: Norway lobster (NL) is among crustacea species receiving increasing global
interest given its economic value together with the promising market status. But lipid damage together
with other freshness restraints continually affects its post-harvest shelf. Modied atmosphere packaging
(MAP) adapts oxygen-free system which when combined with other preservation methods offers real-
time and tested food storage success. Amid the advances in MAP to-date, relevant information about
freezing combined with oxygen-free packaging atmospheres applied to Norway lobster species has not
been found. Occupying a space within the state-of-the-art of extant knowledge, this current study has
showed that freezing and oxygen-free packaging atmospheres can capably inuence the lipid oxidation
and other esh quality attributes of a crustacean product such as NL samples.
Keywords: Crustacea / Lipid autoxidation / Modified atmosphere packaging / Quality attributes / Sub-zero
temperature storage
Received: January 22, 2016 / Revised: May 17, 2016 / Accepted: May 31, 2016
DOI: 10.1002/ejlt.201600035

Correspondence: C. O. R. Okpala, Istituto per l Ambiente Marino Costiero, 1 Introduction

Consiglio Nazionale delle Ricerche (IAMC-CNR), 91026 Mazara del Vallo,
Italy
E-mail: charlesokpala@gmail.com
Abbreviations: FAA, free amino acid; MAP, modied atmosphere
packaging; MDA, malonaldehyde; NL, Norway lobster; PPO, polyphenol
oxidase; PUFA, polyunsaturated fatty acids; TBA, thiobarbituric acid;
TVB-N, total volatile basic-nitrogen
Recent times, there has been increasing emphasis on
freshness and sensorial attributes of shery products in
many parts of the globe. Not only as useful source of
protein, crustaceans products particularly are of economic
importance given by the never-ending consumer demand
coupled with its constituent non-protein nitrogenous (NPN)

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Eur. J. Lipid Sci. Technol. 2017, 119, 1600035 Freezing and oxygen-free packaging of Norway lobster
compounds as well as polyunsaturated fatty acids (PUFAs),
both latter in considerable quantities. Compared to other
species included in the Decapoda order, Norway lobster
(NL) (Nephrops norvegicus) is highly priced specically in
Europe and occupies promising market status within the ice
chilled/frozen supply chain. On the other hand, it faces post-
harvest setbacks such as rapid lipid breakdown/oxidation as
well as quick black spot/melanosis formation, which result in
decreased quality and market acceptability. The signicant
presence of PUFA in crustacea esh/muscle makes the
latter additionally vulnerable to autoxidation. Actually, it is
the reaction between oxygen and free radicals within this
biochemical milieu gives rise to hydroperoxides that further
releases off-avour volatiles such as aldehydes and ketones.
In addition, the increases in concentration of hydroperoxide
obtainable at crustacean postharvest have always been
indicative of advances of oxidation stages [112]. Moreover,
melanosis in NL samples has been of concern although it
fronts no danger to human health. Specically, oxidative
reactions involving enzyme polyphenol oxidase (PPO) can
further progress even under ice/refrigerated storage. Whilst
pH range 78 is believed to facilitate formation of black
spotting/melanosis in crustacea during cold storage, quanti-
fying quality parameters such as pH, total volatile base
(TVB-N) nitrogen, as well as thiobarbituric acid (TBA) also
helps in the monitoring of post-harvest degradation. Besides,
some free amino acids (FAAs) have been considered capable
to oppose the quality of shery products, e.g., production of
biogenic amines [24, 1119].
The drive towards improved food preservation makes
non-thermal technologies such as high hydrostatic pressure,
ozone treatment, modied atmosphere packaging (MAP)
and so on to continually evolve. Specically, MAP appears
increasingly attractive and of commercial global interest
for (sea)foods products given its capacity to offer real-time
and tested storage success. Moreover, it adapts oxygen-
free systems that when combined with other preservation
methods show high promise to robustly tackle the overall
deterioration of shery products [29, 1122]. But even with
the advances of MAP to-date, relevant information specic
to freezing combined with oxygen-free packaging atmos-
pheres applied to Norway lobster species has not been
found. In view of the changing market geography in which
seafood products are consumed distance(s) away from the
harvest point as well as increase in consumer demand for
improved shery product quality, the protective atmospheres
packaging co-adjuvant with refrigeration has become in-
creasingly convenient especially for retail packaging and
bulk storage. In fact, regulating gas mixtures (such as
nitrogen, oxygen and CO2) of modied atmospheres also
delivers effective preservative control prospect to shery
products [2, 4, 11, 1721]. In addition to lipid oxidation
and on other hand, some concepts such as enzymatic
reactions (lipolysis, proteolysis and browning), dehydration
and protein aggregation are among important quality shelf
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1600035 (2 of 9)
aspects to consider during frozen storage of sh/shellsh.
Furthermore, the preservative success of freezing, commonly
demonstrated by colder temperatures of approximately

18C and beyond that is widely applied to shery products,
is believed to depend on age, muscle structure as well as
size of shery product [2, 17, 22]. In that, previous studies
have provided evidences of MAPs promising product
appearance, consumer acceptability as well as its drive
towards sustained product development, the oxygen-free
gas mixtures (N2 and N2/CO2) combined with frozen
temperatures can strongly improve the overall shery product
quality [2, 4, 12, 1619, 21].
This study was therefore purposed to apply freezing and
oxygen-free (vacuum as well as modied atmospheres of
50% N2-50%CO2- and 100% N2) packaging methods to NL
(N. norvegicus) and resultant data compared with conventional
preservative treatment of sulphite together with control
(untreated). Thus, the specic objective was to determine
the inuence of the (above-mentioned) treatments on lipid
oxidation and other esh quality parameters during storage
period of up to 12 months. To best of our knowledge, this is
the rst study about freezing combining oxygen-free packaging
treatment methods specically applied to NL samples.
2 Materials and methods
2.1 Sample collection, treatment protocol and
packaging procedures
Directly on board and offshore of shing trawler, NL samples
(carapace length 34.6  3.5 mm) were harvested during a
spring season in the Strait of Sicily (Central Mediterranean)
at 480  40 mL of depth. These samples were washed in
owing seawater and rapidly pre-chilled (1  0.5C) by
dipping using 1:1 mixture of seawater and ice. After about
15 min, when the core temperature of NL had been deemed
equal with that of ice-seawater mixture, the NL were allowed
to drip and randomly divided into ve lots of approximately
10 kg for each lot, to accord with the following treatment
protocols: (i) the rst (of the ve lots) has been divided into
13 sub-lots and each sub-lot constituting about 700 g
(similarly applied to the other treatments subsequently
detailed below), placed in high oxygen barrier trays and
frozen in a blast freezer room at
35C. After 8 h, when the
water at the thermal centre of sample had been converted into
ice, each bag was packed in N2 (100%) using the semi-
automatic-modied atmosphere packaging (MAP) system
(Mondini, Brescia, Italy); (ii) another 13 sub-lots of NL were
frozen in a blast freezer room at
35C for 8 h and thereafter
packaged under vacuum using the semi-automatic MAP
system (Mondini, Brescia, Italy); (iii) a third 13 sub-lots of
NL were packed in an N2 CO2 gas mixture (5050%) using
the semi-automatic MAP system (Mondini, Brescia, Italy)
and subsequently frozen in a blast freezer room at
35C for
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8 h. This was deemed necessary as it was expected to allow for
the full dissolution of CO2 into all NL sample tissues [20];
(iv) a fourth 13 sub-lots of NL were dipped in a seawater
solution (4% w/v) of commercial anhydrous sodium sulphite
(the sample-to-dipping solution ratio was 1:4) according to
the preservation techniques and materials usually employed
by Mediterranean eet crews. The dipping time was no more
than 20 min. The samples after dripping were packaged with
the help of semi-automatic MAP system then subject to
blast freezer room of
35C for 8 hours; (e) Finally, a fth 13
sub-lots of NL were frozen in a blast freezer room at
35C
without any treatment and these served the purpose of
control. Notably, while the N2 and vacuum-packed samples
were frozen prior to packaging under MAP, the sample under
N2-CO2 was packaged under MAP before frozen storage.
As the MAP operations completed, all NL samples were
subject to frozen storage (
18C) for 12-month period
consistent with the widely accepted European Union (EU)
regulation prescribed for frozen foods.
All NL samples were packed using transparent A.PET/
EVOH/PE carrier trays (tray size: 290  200 mm; bag
volume: 1800 cc; laminate density: 1.39 g/cm3; thickness:
500 mm) with manufacturer-labelled oxygen (O2) and water
vapour permeability of 1.8 cm3 m
2 day
1 atm
1 and 4 g
m
2 day
1, respectively (Arcoplastica srl, Andenzeno, Italy).
Heat-sealed trays employed Multiex OPP/EVOH/PE lm
(weight: 72 g/m2; thickness: 75 mm) of maximum O2, CO2
and water vapour permeabilities of 3 cm3 m
2 day
1 atm
1,
10 cm3 m
2 day
1 atm
1 and 3 g m
2 day
1, respectively
(Cryovac, Sealed Air Corp., Italy). Gas-to-product ratio
(v/w) was 2.25:1 for modied atmosphere packaging of
NL samples. To minimize samples horn damaging the
multiex lm, an additional anti-pinhole headspace lamina
(1.35 g/cm3) has been inserted.
2.2 Study design
This study has been designed to evaluate differences in lipid
oxidation and other esh quality parameters of NL samples
as affected by oxygen-free atmospheres in comparison to
conventional preservative treatment of sulphite together
with control (untreated) during investigated frozen storage
of up to 12 months. Furthermore, the lipid oxidation was
determined in terms of thiobarbituric acid (TBA) whereas
the other esh quality parameters were determined in
terms of total volatile base (TVB) nitrogen, pH, free amino
acids (FAAs), proximal composition, as well as melanosis
scores. All experimental analyses commenced at the third day
postharvest because it was the best time that allowed for the
arrival of samples to the laboratory. Thereafter, these
analyses continued at the scheduled times that lasted up to
the end of the inspected storage period. Prior to the
laboratory analyses, the NL samples were thawed, then
beheaded, peeled and deveined. Unless otherwise indicated,
laboratory/experimental analyses were conducted in triplicates
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Eur. J. Lipid Sci. Technol. 2017, 119, 1600035
and independently on different NL samples already allocated
as per treatment unit/sub-lot. All chemicals and reagents
employed were of analytical grade.
2.3 Experimental methods
2.3.1 Determination of lipid damage, pH and total
volatile basic nitrogen
Capably detecting malonaldehyde (MDA), thiobarbituric
acid (TBA) usefully quanties oxidative rancidity of shery
products [6]. A TBA method previously described [23] was
used to evaluate (secondary) lipid breakdown in NL esh and
presented in milligram malonaldehyde equivalents per one
kilogram (mg MDA/kg). While pH measurement employed
digital pH meter (WTW GmbH & Co. KG, Weilheim,
Germany), the total volatile basic nitrogen (TVB-N) was
determined by distillation method as previously de-
scribed [24] and presented in mg TVB-N/100 g.
2.3.2 Determination of free amino acid (FAA)
From extraction up to chromatography as previously
described [17, 25] with some modications, FAA (expressed
in mg/100 g) of NL samples were determined. Aliquots
(10 g) of homogenised NL esh mixed with 40 mL of
extracting solvent (75% methanol in distilled deionised
water) were transferred to 100 mL volumetric ask, thereaf-
ter stored at 4C (60 min to 2 h), after which contents were
transferred into a 50 mL tube for centrifuging (15 000 rpm/
40 min/4C). Prior to derivatisation, supernatant has been
ltered on PTFE 0.2 mm lter membrane (Gelman Scien-
ces). By way of o-phthaldialdehyde (OPA) pre-column
derivatisation, the OPA Thiol Reagent (OPT) was prepared
24 h prior to use by dissolving 27 mg of o-phthaldialdehyde
in 500 mL of absolute alcohol. Then, 5 mL of 0.1 mL
sodium tetraborate (Na2B4O7, 10H2O) (pH 9.5) was added,
followed by 50 mL of mercaptoethanol and then mixed and
kept in the dark. Amino acid standards stock solution was
prepared by dissolving the equivalent of 2500 nmol of each
amino acid in 0.05 mL NaH2PO4 buffer (pH 5.5) and then
diluted for calibration. Further, to 100 mL of amino acid
standard or 189 diluted sample supernatant at high pressure
liquid chromatography (HPLC), 400 mL of OPT was added,
followed by manual injection of sample into the HPLC
column/system. Mobile phase A constituted 0.05 mL sodium
phosphate buffer (pH 5.5), methanol and THF (80:19:1).
Mobile phase B constituted 80% methanol and 20% of the
0.05 mL NaH2PO4 buffer. The pH of phosphate buffer was
adjusted to 5.5. The mobile phases were ltered under
0.2 mm lter membranes (Gelman Sciences, Ann Arbor,
MI, USA) and degassed by vacuum for 5 min. The HPLC
column was an Ultrasphere ODS 5 mm particle size,
4.6 mm  25 cm (Beckman Instruments, Inc., Fullerton,
CA). The gradient elution was generated using a Elite
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Eur. J. Lipid Sci. Technol. 2017, 119, 1600035
LaChrom equipped with a L-7100 pump (LaChrom,
Hitachi), oven L-7350 (LaChrom, Merck), programmable
uorescence detector with excitation monochromator setting
of 330 nm as well as an emission cutoff lter of 418 nm.
Chromatographic data were processed by software
EZChrom Elite (Agilent Tech., Santa Clara, CA, USA).
2.3.3 Determination of proximate composition
The proximate composition (g/100 g) represented by mois-
ture, protein, total lipids, ash and carbohydrate contents
as per AOAC method [26] of NL samples has been performed
at the start and end of frozen storage.
2.3.4 Assessment of melanosis development
To assess the melanosis development of both treated and
control NL samples, trained panel of four experienced judges
visually inspected different sample units per treatment/sub-
lots and independently. This was performed using a ve-
point descriptive scoring scale as previously described [2].
Melanosis scoring employed numerical values, namely: 0
indicated either natural or absence of discolouration,
1 indicated yellowing, 2 indicated slight black spotting,
3 indicated moderate black spotting, and nally, 4
indicated intense black spotting.
2.4 Statistical analysis
Analysis of co-variance (ANCOVA) was used to determine
signicant differences between treatment groups relative
to control concurrent with storage time. The level of
signicance has been dened at P < 0.05. In most cases,
results have been expressed as mean of already replicated
measurements  standard deviation (SD), the latter highlight-
ing minima, maxima and pooled values. To determine
signicant differences in means, Fishers post-hoc least
signicant difference (LSD) was applied.
3 Results and discussion
3.1 Effects on lipid autoxidation
Figure 1 shows that during the rst 6 months, the TBA
of vacuum-packaged, sulphited, 50% N2-50%CO2 and
100% N2-treated NL samples compared to control some-
what uctuated within the range of between 0.10 and
0.25 MDA/kg. Apparently, the result was in contrast to the
remaining months. Whilst the progress of lipid oxidation is
among the major quality concerns affecting postharvest
crustacea, it is still the less of limiting factors of product shelf
given its slow incidence/rate [6, 8, 9]. PUFA, largely present
in crustacean products, is understood to account for the
increases in secondary lipid oxidation products, which is
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Freezing and oxygen-free packaging of Norway lobster 1600035 (4 of 9)
Figure 1. Changes in lipid oxidation (in terms of thiobarbituric acid
{TBA}) of vacuum-packaged, sulphited, 50%N2-50%CO2 and
100% N2-treated Norway lobster samples relative to control during
frozen storage. Error bars represent the standard deviation (n 3).
Information of standard deviation (SD) data of TBA of all samples
with storage, include: minima SD 0.06; maxima SD 1.61;
pooled SD 0.59. Legends represented as follows: (&) vacuum;
(~) N2-CO2; (D) N2; (*) sulphited; (&) control.
generated in such a manner that it obtain a steep rise under
ice/cold storage situations well before the primary lipid
oxidation products would do so [8, 9]. The detectability of
TBA should be reiterated here also because this very lipid
oxidation parameter is considered less sensitive compared
to para-anisidine (p-AnV) [8, 22]. Yet, both parameters
authoritatively help in quantifying (different) secondary lipid
oxidation products in seafood [59, 17, 22]. It is important to
highlight the baseline report of (another) untreated crusta-
cean product stored on ice. Therein, quantities of (second-
ary) lipid oxidation products were increasingly generated
during storage period (of 12 days) [1]. However, the context
of above-cited baseline (secondary) lipid oxidation data is
unalike compared with those of frozen-stored NL samples
already herein submitted to preservative treatments. Besides,
different crustacean products would show different fatty acid
proles. The latter, in addition, would account for differ-
ences in rates of formation of oxidation products [8].
Quantifying such evidences as that of TBA quality limit
of 58 mg MDA/kg as well as objectionable odor emanating
beyond 2 mg MDA/kg provided for cold/frozen shery
products [6, 15], not only would the NL samples herein be
considered acceptable throughout the inspected frozen
storage period of up to 12 months, but also, that both
freezing and oxygen-free packaging methods would be
deemed useful candidates to delay the progress in secondary
lipid oxidation. Besides, our TBA data compares favorably
only with those untreated NL samples of range between
0.12 and 0.21 mg MDA/100 g previously reported by
Martinez-Alvarez and colleagues during their 13-day chilled-
storage study [27]. Contextualizing and quantifying the lipid
oxidation versus NL samples herein, the thick exoskeleton
of NL species possibly plays a protective function to control
the degree of penetration of oxygen into its esh, which might
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 1600035 (5 of 9) G. Bono et al.
limit the progress of oxidative degradation even under frozen
storage.
3.2 Effects on total volatile basic nitrogen (TVB-N)
Whilst perceived to be rather unspecic due to evolution of
NH3, dimethylamine (DMA) and trimethylamine (TMA)
and yet affected by various factors such as microbial growth/
autolysis, the use of TVB-N determination still helps in the
estimation of freshness and quality status of shery
products [15, 7, 8]. Figure 2 shows that TVB-N of vacuum
packaged, sulphited and, 50% N2-50%CO2-treated NL
samples exhibited minor differences during frozen storage,
except some increases at 100% N2-treated samples com-
pared to the others. And even with this, the TVB-N values
ranged between 25 and 38 mg/100 g. In this context
therefore, it appears that excluding oxygen totally from the
headspace might alone itself not be sufcient enough to
control the TVB-N production in crustacean samples such as
NL even after the second half of frozen storage [2, 17] of
this study. Furthermore, although our TVB-N values would
seem rather high, it still agrees well with the high quality
status of other previously reported NL samples [2729].


Notably, Martinez-Alvarez et al. [27] reported TVB-N
values of 25 mg/100 g for high quality freshly harvested NL
samples. Further, G okalp et al. [30] dened a quality control
scheme of between 35 and 100 TVBN mg/100 g for spoiled
frozen meat and meat-related products whereby approaching
its lower limit of 35 TVB-N mg/100 g would not necessarily
indicate spoilage. And contextualizing the NL versus
TVB-N values provided by Boziaris et al. [31], the further
attening with storage time of already increasing pattern of
Figure 2. Changes in total volatile base (TVB) nitrogen of vacuum-
packaged, sulphited, 50%N2-50%CO2 and 100% N2-treated
Norway lobster samples relative to control during frozen storage.
Error bars represent the standard deviation (n 3). Information
of standard deviation (SD) data of TVB of all samples with
storage, include: minima SD 0.001; maxima SD 0.047; pooled
SD 0.003. Legends represented as follows: (&) vacuum; (~)
N2-CO2; (D) N2; (*) sulphited; (&) control.
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Eur. J. Lipid Sci. Technol. 2017, 119, 1600035
TVB-N can be deduced as the storage-temperatures was
decreasing in the orders of 20, 5 and 0C. These evidences


of Martinez-Alvarez et al. [27], Gokalp et al. [30] and
Boziaris et al. [31] seem to support the temperature-TVB-N
relationship of NL samples kept under chilled/cold storage
particularly during the rst week, as reported elsewhere [13,
28, 29]. Consequently, placing Figs. 2 and 6 of this current
study side-by-side, the 100% N2-treated NL samples having
TVB-N value of 35 mg/100 g at the end of frozen storage is
showed with signicantly lower melanosis scores compared
to vacuum packaged and control samples. So what can be
deduced of the context(s) of the signicantly lower melanosis
scores of for example the 100% N2-treated NL samples?
Firstly, the TVB-N with peak of 38 TVB-N mg/100 g to
approach end of frozen storage can be argued to be within
the acceptable quality limits. Secondly, the TVB-N values of
our NL samples remained considerably lower compared with
those other previously reported similar species that had been
kept under chilled/cold conditions especially from day 7 and
onwards. Therefore, all these above-highlighted contexts of
TVB-N versus NL sample clearly demonstrate that the
widely used acceptable limit of 30 mg/100 g should not apply
to the NL samples together with other related crustacea
species that would exhibit similar TVB-N outcomes. Thus, a
re-evaluation of TVB-N acceptability limits of such crustacea
species under frozen storage is warranted. Further, the use of
TVB-N as quality parameter to assess biochemical spoilage
in NL species particularly during frozen storage should be
applied/handled with some caution.
3.3 Effects on pH
Figure 3 shows that the pH changed noticeably (P < 0.05)
comparing all treated NL samples with control during frozen
storage. In addition, the pH trends seemingly followed a
Figure 3. Changes in pH of vacuum-packaged, sulphited, 50%N2-
50%CO2 and 100% N2-treated Norway lobster samples relative to
control during frozen storage. Error bars (n 3) are smaller than
symbols. Information of standard deviation (SD) data of pH of
all samples with storage, include: minima SD 0.00; maxima
SD 0.04; pooled SD 0.00. Legends represented as follows: (&)
vacuum; (~) N2-CO2; (D) N2; (*) sulphited; (&) control.
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similar pattern. In view of higher alkalinity of Na2SO3 (pH

of 8 and higher) associated with sulphite treatment of
crustacea [17] and contrary to our expectations, the changes
in pH of sulphite-treated NL samples resembled the
chemical-free ones. The hard exoskeleton characterising this
crustacea species might probably be preventing the penetra-
tion of sulphite contents into the muscle/tissue. Regardless
of treatments, even though pH of treated NL samples
somewhat uctuated during the rst 6 months, some
increases would likely occur thereafter to depict the
accumulation of alkaline compounds such as NH3. Subse-
quent increases in pH during cold storage may also be
facilitating the formation of (other) bio-compounds that are
probably activated by endogenous enzymes. Besides, if pH of
8 were to be exceeded, crustacean species would have to be
rejected [10, 14]. Further, the slight alkalinity characterising
such shery products might also be accounting for its high
non-protein/nitrogen contents [2]. In the light of this
scenario and placing Figs. 2 and 3 side-by-side, the trends
of pH of 100% N2-treated NL samples seemingly resembled
its TVB-N. Moreover, the pH of treated NL samples ranging
between 6.7 and 7.6 ostensibly performs more stable
compared to respective peak pH of 7.9 of NL during 10 days
of (only) chilled storage as reported by Boziaris et al. [31] as
well as 8.3 of NL during 12 days of chilled storage as reported Figure 4. Different free amino acids (FAAs) of vacuum-packaged,
sulphited, 50%N2-50%CO2 as well as 100% N2-treated Norway
by Lopez-Caballero et al. [28], which was already treated
lobster samples with control. Details of FAA acronyms include:
with sulphite and 4-hexylresorcinol-based formulations.
ASP aspartate; GLU glutamic acid; SER serine; ASN
asparagine; GLY glycine; GLN glutamine; HIS histidine;
3.4 Effects on FAAs THR threonine; ARG arginine; ALA alanine; PRO proline;
TYR tyrosine; VAL valine; MET methionine; ILE isoleucine;
Figure 4 shows that the individual FAAs of treated NL LEU leucine; LYS lysine; PHE phenylalanine; error bars
samples with frozen storage ranged between <50 (ASP) and represent the standard deviation (n 5, representing ve different
950 (ARG) mg/100 g. The result appears not comparable storage time measurements). Information of standard deviation
with the FFA data reported by Ruiz-Capellas and Moral [11] (SD) data of FAAs with storage, include: minima SD 3.70;
for NL samples, which was probably caught outside the maxima SD 263.44; pooled SD 64.33. Legends represented as
Mediterranean Sea (at a different shing ground and depth follows: ( ) vacuum; (&) N2-CO2; ( ) N2; ( ) sulphited; (&) control.
range). Such detectable FFA differences might be accounted
for, not only by specic habitat factor(s) associated with NL
species, but also by the wide geographical distribution amply
stretching from Iceland up to Egyptian waters [32]. Across
treated-NL groups herein, there seemed no distinct differ-
ence between individual FAAs. Yet, some (individual) FAAs
seem equalled, such as ASN  TYR  MET  PHE  ILE;
GLY  PRO; GLU  THR as well as SER  VAL. In
particular, the ARG obtained highest whereas the HIS
obtained least mean values. Fairly, the FFA values of
GLY seemed higher over either GLN or ALA. Besides,
crustaceans are reported to possess relatively high alanine
(ALA), arginine (ARG), proline (PRO), as well as glycine
(GLY) values [11]. In the situation where per treatment and Figure 5. Changes in total free amino acids (FAAs) obtained of
storage time the individual FFAs of (processed) crustacean vacuum-packaged, sulphited, 50%N2-50%CO2 as well as 100%
samples would resemble, to sum-up such values would offer a N2-treated Norway lobster samples relative to control during frozen
better holistic perspective by way of total FFAs [17]. Herein, storage. Note, the total FAAs comprise of sums of individual FAAs
Fig. 5 shows that the pattern/trend of total FAAs of different per treatment. Legends represented as follows: (&) vacuum; (~)
treated-NL groups appear relatively unvaried with control N2-CO2; (D) N2; (*) sulphited; (&) control.

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during frozen storage. In fact, between months 0 and 2, total

FAA of all NL samples had some increases except the
sulphite-treated ones. Between months 2 and 8, total FAA
had different rates of decreases. Between months 8 and 12, as
total FFAs of vacuum packaged and 50%N2-50%CO2
treatments seemingly decreased, those of 100% N2-,
sulphite-treated and control NL samples seemingly in-
creased. Generally, increases and decreases in FAAs seem
not new in Lobster species, such as in Spiny lobster
(Palinuridae) during chilled storage [33]. Besides, decreases
and increases in FAA might respectively be owed to either
enzymatic degradation or hydrolysis [11, 17].

3.5 Effects on proximate composition

Table 1 shows that differences in proximal composition of

vacuum-packaged, sulphited, 50% N2-50%CO2 and 100%
N2-treated NL samples seemed trivial during frozen storage.
The mean values of moisture, protein, total lipids, ash and
carbohydrate contents ranged between 77.2 and 79.1 g/100 g,
17.8 and 19.8 g/100 g, 0.4 and 0.8 g/100 g, 2.1 and 2.6 g/100 g
and 0.1 and 0.2 g/100 g, respectively. Notably, the NL samples
with above-mentioned proximate composition data were
collected/harvested within spring season. It is interesting
that our proximate composition values are comparable with
another NL samples collected/harvested within winter sea- Figure 6. (a) Changes in melanosis scores of vacuum-packaged,
son [29]. Besides, lipid contents of NL samples (and other sulphited, 50%N2-50%CO2 and 100% N2-treated Norway lobster
related crustaceans) may vary by age/sexual maturity, harvest specimens relative to control during frozen storage. Error bars
season and diet/feed composition [34, 35]. represent the standard deviation (n 4). Information of standard
deviation (SD) data of melanosis scores of all samples with storage,
include: minima SD 0.000; maxima SD 0.957; pooled SD 0.346.
3.6 Effects on melanosis development
Legends are represented as follows: (&) vacuum; (~) N2-CO2; (D)
N2; (*) sulphited; (&) control; (b) Pictorial examples of different
Figure 6(a) shows that during frozen storage, mean melanosis advances in black spotting/melanosis of inspected NL samples
scores of NL samples began from absence of discolouration specic to the end of frozen storage, that is, month 12.
(score 0) without clearly surpassing the intense black
spotting (score 4). Melanosis status of sulphite NL

Table 1. Changes in proximal composition (moisture, ash, protein, total lipids and carbohydrates contents) of vacuum-packaged, sulphited,
50%N2-50%CO2 as well as 100% N2-treated Norway lobster (NL) samples relative to control (untreated) as determined only at the
beginning (month 0) and end (month 12) of frozen storage

Treatments Control Vacuum Sulphited 50%N2 50%CO2 N2 (100%)

Storage time
(months) 0 12 0 12 0 12 0 12 0 12

Moisture (g/100 g) 78.6 (0.6) 77.3 (0.5) 77.5 (0.5) 77.3 (0.6) 78.0 (0.6) 77.0 (0.5) 77.2 (0.5) 77.5 (0.5) 79.1 (0.7) 78.8 (0.7)
Ash (g/100 g) 2.1 (0.2) 2.5 (0.2) 2.1 (0.1) 2.5 (0.2) 2.2 (0.1) 2.5 (0.2) 2.2 (0.2) 2.6 (0.3) 2.3 (0.1) 2.6 (0.2)
Protein (g/100 g) 18.9 (0.9) 19.4 (1.0) 19.8 (0.9) 19.5 (0.9) 19.4 (0.9) 19.6 (1.0) 19.7 (1.1) 19.2 (0.9) 17.8 (0.8) 17.9 (0.8)
Total lipids (g/ 0.4 (0.1) 0.8 (0.2) 0.5 (0.1) 0.7 (0.1) 0.4 (0.0) 0.7 (0.1) 0.8 (0.2) 0.7 (0.2) 0.8 (0.2) 0.7 (0.1)
100 g)
Carbohydrate (g/ N.R N.R 0.1 (0.0) N.R. 0.0 (0.0) 0.2 (0.0) 0.1 (0.0) N.R. N.R. N.R.
100 g)

Standard deviation (SD) values inserted in brackets. N.R., not recorded.

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Eur. J. Lipid Sci. Technol. 2017, 119, 1600035 Freezing and oxygen-free packaging of Norway lobster
samples, apparently absent at earlier stages, exhibited some
yellowing more evidently at later stages. Apparently, the
melanosis scores of oxygen-free (50% N2-50%CO2- and
100% N2-) packaged samples would closely follow those of
chemical-treated (i.e., sulphited). Heretofore, the oxygen-
free packaged treatment delaying post-mortem black spot
development of NL samples is highly promising to replace
the sulphite ones. Further, comparing the control and
vacuum-packaged NL samples, the slight melanosis that
initially (corresponding to the third day after catch) tended
to intensify, remained unchanged from month 4 onwards.
Figure 6(b) shows black spotting/melanosis of different
inspected NL samples visualized at the 12th month. The
incomplete inhibition of black spot is clearly seen more at
vacuum-packaged and control NL samples compared to the
others. Specic to vacuum packaged ones, it is highly
probable that some traces of atmospheric air (i.e., oxygen)
still remained within the package headspace during frozen
storage period [17] for these inspected NL samples. Hence,
this could be a discernible shortcoming specic to this
packaging method [17] and as such, even when it is combined
with freezing storage and yet appears otherwise physically
complete, it would likely still produce incomplete delay/
inhibition of black spotting/melanosis formation in these
crustacea samples. To include an oxygen-absorber in (similar)
vacuum packaging system(s) may well improve and make
more robust the oxygen-free headspace performance [2, 17].
4 Conclusions
In this communication, the inuence of freezing and oxygen-
free (vacuum- as well as modied atmospheres of 50% N2-
50%CO2- and 100% N2) packaging methods on lipid
oxidation and other esh quality parameters of NL samples
was investigated. The resultant data has been compared with
those of conventional preservative treatment of sulphite
and control samples. Evidently, the detected low/trivial
MDA amounts suggest freezing combined with oxygen-free
packaging methods as highly promising to delay the progress
in (secondary) lipid oxidation. Some other esh quality
parameters would also be preserved, given by the respective
lowering and controlling of evolving melanosis and (some)
biochemical aspects such as proximate components, TVB-
N, pH as well as FAAs. Based on these observations, the
oxygen-free packaging system can serve as a good replace-
ment for the sulphite method. To best of our knowledge, this
is the rst study about differences in (secondary) lipid
oxidation and other esh quality parameters of NL samples
as specically affected by freezing combining oxygen-free
packaging treatment methods. Importantly, the widely used
TVB-N limit of acceptability of 30 mg/100 g seems not
applicable to evaluate the freshness of NL samples during
frozen storage. Thus, future studies should consider both
microbial and sensorial assessment of this crustacean species
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1600035 (8 of 9)
under similar treatment conditions to help justify this
proposal. Further, future studies should apply freezing and
oxygen-free treatment conditions used herein to other
economically important related/relevant species in the view
to supplement existent information.
Financial support from the Italian Ministero delle Politiche
Agricole Alimentari e Forestali, Italian Ministero dellIstruzione,
dellUniversit
a e della Ricerca, PON R&C 20072013 (project
PESCATEC) and PNR 20012013 (project RITMARE) are
gratefully acknowledged.
The authors have declared no conicts of interest.
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