International Journal of Food Microbiology 135 (2009) 281–287

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Combined effect of MAP and active compounds on fresh blue fish burger
M.A. Del Nobile a,b,⁎, M.R. Corbo a,b, B. Speranza a, M. Sinigaglia a,b, A. Conte a,b, M. Caroprese b,c
a
Department of Food Science, University of Foggia, Via Napoli, 25, 71100 Foggia, Italy
b
Istituto per la Ricerca e le Applicazioni Biotecnologiche per la Sicurezza e la Valorizzazione dei Prodotti Tipici e di Qualità, University of Foggia, Via Napoli, 25, 71100 Foggia, Italy
c
Department of Production Sciences and Innovation of Applied Agricultural Mediterranean Systems, University of Foggia, Via Napoli, 25, 71100 Foggia, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The combined effects of three essential oils [thymol, lemon extract and grapefruit seed extract (GFSE)] and
Received 4 May 2009 modified atmosphere packaging conditions (MAP) on quality retention of blue fish burgers was studied and
Received in revised form 15 July 2009 discussed. In particular, samples were packaged in air and in three different gas mix compositions: 30:40:30
Accepted 20 July 2009
O2:CO2:N2, 50:50 O2:CO2 and 5:95 O2:CO2. During a 28-day storage period at 4 °C, the nutritional,
microbiological and sensorial quality of the packed products was assessed. The potential development of
Keywords:
biogenic amines was also evaluated.
Blue fish burger
Essential oil The obtained results highlight the possibility to improve the microbial quality of blue fish burgers by using
MAP very small amount of thymol (110 ppm), GFSE (100 ppm) and lemon extract (120 ppm) in combination with
Shelf life MAP. Based primarily on microbiological results, the combined use of the tested natural preservatives and a
packaging system characterized by a high CO2-concentration, was able to guarantee the microbial
acceptability of fish burgers until the 28th day of storage at 4 °C. On the other hand, results from sensory
analyses showed that sensorial quality was the sub-index that limited the burgers shelf life (to about 22–
23 days), even if the proposed strategy was also effective in minimizing the sensory quality loss of the
product having no effect on its nutritional quality.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction
Seafood is an important part of healthy diet (Altekruse et al., 1995;
Trondsen et al., 2003). Among the numerous species available,
nutritionists often highlight the importance of blue fish for human
nutrition. Blue fish is characterized by protein composition of high
biological value and fatty acid composition especially rich in
polyunsaturated fatty acids, in particular omega-3. Although blue
fish has a high nutritional value, the consumer choice shows a
preference for medium-fine species that are scarce in the Mediterra-
nean Sea and, therefore, mostly imported. As a consequence, blue fish
are actually considered as fishing waste. The lack of consumption of
particular fish species depends on the poor knowledge of their
nutritional characteristics, their optimal conditions to clean and cook
and, above all, on the useful storage conditions to prolong the shelf life
of these products. Fresh fish is, in fact, a highly perishable product due
to its biological composition (Haard, 1992; Gram and Dalgaard, 2002):
during storage, the quality of fish quickly degrades as a result of
complex processes in which several forms of deterioration are
implicated (Amanatidou et al., 2000; González-Fandos et al., 2005).
The main cause of deterioration is the microbial activity of typical
⁎ Corresponding author. Department of Food Science, University of Foggia, Via
Napoli, 25, 71100 Foggia, Italy. Tel./fax: +39 881 589 242.
E-mail address: ma.delnobile@unifg.it (M.A. Del Nobile).
spoilage seafood microorganisms that limits the shelf life of packed as
well as unpacked fresh fish (Dalgaard, 1995; Fraser and Sumar, 1998;
Gram and Dalgaard, 2002; Gram and Huss, 1996). Considerable
research has been directed toward using various preservation
strategies to preserve or prolong the shelf life of fresh fish and the
most suitable technology appears to be the modified atmosphere
packaging (MAP) (Debevere and Boskou, 1996; Boskou and Debevere,
2000; Boknaes et al., 2002; Masniyom et al., 2002; Sivertsvik et al.,
2002; Arvanitoyannis et al., 2005; Corbo et al., 2005; Poli et al., 2006;
Torrieri et al., 2006; Sivertsvik, 2007). These reports clearly suggest
that MAP in combination with refrigeration could provide a
substantial shelf life extension of fresh fish. Nevertheless, it is worth
noting that the specific shelf life extension depends on raw material
(species, fat content, initial microbiological populations, etc.), tem-
perature, gas mixture and packaging systems used (Davies, 1997;
Sivertsvik et al., 2002).
In order to increase shelf life of fresh fish, low levels of salt and/or
natural preservatives (antimicrobials and antioxidants) have been
also used. Thus, to this aim, oregano, thyme, garlic, bay leaf, rosemary,
marjoram, clove, etc., or their extracts, known as essential oils (EOs),
have been used alone or in combination with other preservation
methods such as MAP, salting, irradiation etc. (Lòpez-Malo et al.,
2000; Nychas and Tassou, 2000; Scandamis and Nychas, 2001; Burt,
2004; Devlieghere et al., 2004; Gimenez et al., 2004; Chouliara et al.,
2005; Corbo et al., 2009b). Despite the numerous studies in the

0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2009.07.024
282 M.A. Del Nobile et al. / International Journal of Food Microbiology 135 (2009) 281–287
literature on the antibacterial activity of EOs and their subsequent
effect on minimally processed fish-based products shelf life (Amana-
tidou et al., 2000; Mejlholm and Dalgaard, 2002; Altieri et al., 2005;
Mahmoud et al., 2004, 2006, 2007; Goulas and Kontominas, 2007;
Corbo et al., 2008, 2009a), no data are available, to the best of our
knowledge, on blue fish-based burgers.
In order to valorise fishing waste and move towards blue fish
consumption, in this study a new seafood product, represented by a
blue fish-based burger, was developed and studied. In previous
related works (Corbo et al., 2008, 2009a), among different EOs tested,
thymol, lemon extract and grapefruit seed extract (GFSE) were
identified as successful active natural preservatives to improve the
microbial stability of minimally processed fresh fish burgers. In
particular, an optimal composition of these three antimicrobial
compounds (110 ppm of thymol, 100 ppm of GFSE and 120 ppm of
lemon extract), able to increase (by about 40%) the microbiological
acceptability limit of fish burgers stored under refrigeration and
packaged in air, was determined (Corbo et al., 2008).
This study was mainly initiated to evaluate the combined effect of
the MAP and the above-mentioned EOs (at their optimal composition)
on the quality retention of blue fish burgers stored at 4 °C, in order to
identify a suitable preservation strategy. In particular, quality decay of
the investigated products was assessed by monitoring three quality
sub-indices: nutritional, microbiological and sensorial.
2. Materials and methods
2.1. Raw material
Specimens of mackerel (Scomber japonicus) and hake (Merluccius
merluccius), caught in the Gulf of Manfredonia in the Adriatic Sea,
were obtained from a local farm (Cooperativa Santa Lucia, Manfre-
donia, Foggia, Italy). Fish were slaughtered by immersion in ice-cold
water (hypothermia) and packed in insulated polystyrene boxes with
ice. Then they were delivered to the laboratory within 2 h from the
moment of harvest. Once at the laboratory, fish were decapitated,
cleaned, filleted and skinned.
2.2. Antimicrobial compounds
The three natural antimicrobial compounds used were thymol
(Sigma, Poole, UK), grapefruit seed extract (GFSE, Biocitro, Probena s.l,
Zaragoza, Spain) and lemon extract (Spencer Food Industrial,
Amsterdam, The Netherlands). A working active solution containing
the three tested compounds was prepared with higher compound
concentrations (2750 ppm of thymol, 2500 ppm of GFSE and
3000 ppm of lemon extract), to enable greater subsequent dilution
of the samples. In order to enhance their water solubility, the
compounds were dissolved in ethyl alcohol (95%) and then diluted
with distilled water (50%, v/v). The active solution was freshly
prepared before use and sterilized by filtering through membranes
(0.20 µm pore size; Minisart, Sartorius, Goettingen, Germany).
2.3. Mini fish burgers preparation
Skin-off fillets of both species were weighed and mixed in order to
obtain a mackerel-hake ratio of 70:30 (w/w). Mincing was performed
by a domestic food processor (Multichef, Ariete, Firenze, Italy). The
obtained fish patty was homogenized in a bowl mixer with a spiral
dough hook for 5 min and separated into two batches. An aliquot of
the antimicrobial compounds solution was added to one batch in
order to obtain a final concentration of 110 ppm of thymol, 100 ppm
of GFSE and 120 ppm of lemon extract (ACT samples), and then mixed
again for 5 min, according to our previous work (Corbo et al., 2008a).
As control samples, the same amount of 50% hydro-alcoholic solution
was added to the second batch (CNTR samples). Mini fish burgers
were prepared by hand (25 g, 50–60 mm diameter) and packed in
Nylon/Polyethylene bags (95 µm, Tecnovac, San Paolo D'Argon,
Bergamo, Italy) by means of S100-Tecnovac equipment. The bags
were 170 mm × 250 mm long, with O2 permeability of 50.65 cm3/m2
day atm and water vapor transmission rate of 1.64 g/m2 day, as
specified by the manufacturer. The samples were packaged in air and
in three different gas mix compositions: 30:40:30 O2:CO2:N2, 50:50
O2:CO2 and 5:95 O2:CO2. During storage at 4 °C, nutritional analyses
and determination of biogenic amines were performed after 0, 14 and
28 days. Microbiological, sensory analyses and determination of pH
were made after 0, 1, 2, 5, 8, 12, 15, 21 and 28 days.
2.4. Nutritional analysis
A minimum of 6 samples from each fresh fish burger were
analyzed for nutritional analysis. The water content of burgers was
determined using a 5 g sample by heating at 105 °C to constant weight
(AOAC, 2000). Fat was determined by the Soxhlet method, using
Soxtec 2055 (Foss, Denmark) according to AOAC procedures (AOAC,
2000). Total nitrogen was determined by the Kjeldahl procedure in
Kjeltec (Foss, Denmark) and converted to crude protein by multi-
plying by 6.25 (AOAC, 2000). Ash was determined as the remnant
weight after calcination of an 8 g sample at 550 °C during 3 h (AOAC,
2000). The fat and protein contents were expressed as percentage on
dry matter (%). Lipid extraction followed the Bligh and Dyer method
(Bligh and Dyer, 1959). Methyl esters were prepared by transmethy-
lation using 2 M KOH in methanol according to IUPAC method (IUPAC,
1987). Fatty acid methyl esters (FAME) were quantified by gas-
chromatography. The fatty acids composition was analyzed by GC
6890N (AGILENT, USA), equipped with a flame ionization detector,
automatic injector and a fused silica capillary AGILENT HP88 column
(100 m × 0.25 mm, 0.2 μm). The carrier gas was helium at constant
flow of 0.8 ml/min; the injector and the detector temperature were
set at 240 °C and 260 °C, respectively. The column temperature was
60 °C, held for 5 min, raised to 180 °C at a rate of 25 °C/min and
further up to 230 °C at a rate of 6 °C/min. The split used was 1:50. Fatty
acids were identified by comparing the retention times of FAME with
a standard 37 component FAME mixture (Supelco, Milan, Italy). The
results were expressed in g/100 g of total fatty acids.
2.5. Microbiological analyses and determination of pH
For microbiological analyses, mini fish burgers (25 g) were diluted
with 225 ml of 0.1% peptone water with salt (0.85% NaCl) in a
Stomacher bag (Seward, London, England) and homogenized for
1 min in a Stomacher Lab Blender 400 (Seward). Serial dilutions of fish
homogenates were plated on the surface of the appropriate media in
Petri dishes. The media and the conditions used were: Plate Count
Agar (PCA) incubated at 30 °C for 48 h for aerobic plate count (APC)
(International Commission on Microbiological Specifications for
Foods, ICMSF, 1986); Pseudomonas Agar Base (PAB), with added
Cephaloridine Fusidin Cetrimide (CFC) supplement, incubated at
25 °C for 48 h for Pseudomonas spp.; pour plated Iron Agar (IA),
incubated at 25 °C for three days, for hydrogen sulphide-producing
bacteria (HSPB); spread plated chilled IA, supplemented with 5 g/l
NaCl and incubated at 15 °C for 7 days, for psychrotolerant and heat
labile aerobic bacteria (PHAB). The conditions used during the counts
of HSPB and PHAB were those suggested by the Nordic Committee on
Food Analyses, with regard to fish and fishery products (NCFA, 2006).
All media were supplied from Oxoid (Milan, Italy). Microbiological
data were log-transformed and expressed as the average of two
replicates. The variability coefficient, expressed as a percentage ratio
between the standard deviation and the mean value was less than 7%.
The measurement of pH, conducted in duplicate, was performed
on the first homogenized dilution of the fish samples with a pH meter
(Crison, Barcelona, Spain).
 M.A. Del Nobile et al. / International Journal of Food Microbiology 135 (2009) 281–287
2.6. Biogenic amines analysis
The biogenic amines were separated and quantified by High
Performance Ion Chromatography system (HPIC), as described by
Cinquina et al. (2004). Two grams of homogenized fish were extracted
with 5 ml of 20 mM methanesulfonic acid (MSA) for 10 min and
centrifuged at 1300 ×g for 20 min at 4 °C removing the supernatant.
This procedure was repeated three times and the combined super-
natant was made up to 20 ml with 20 mM MSA. The obtained extracts
were first filtered through Whatman filters N.1 (Maidstone, England)
and then filtered again through a 0.45 µm PTFE filter (Teknokroma,
Dublin, CA, USA), before injecting into HPIC.
All biogenic amines standards (putrescine, cadaverine, spermidine,
spermine, agmatine) and methanesulfonic acid were from Sigma-
Aldrich (Milan, Italy).
HPIC analyses were performed by Dionex Model DX-500 (Dionex,
Sunny Ale, CA) apparatus equipped with a GP50 gradient pump
(Dionex, Sunny Ale, CA) and an electrochemical detector ED50
(Dionex, Sunny Ale, CA) in the conductivity mode. The suppressor
current was set at 74 mA. The eluent was MSA and the gradient
conditions consisted of: 3 to 18 mmol/l from 0 to 10 min, 18 mmol/l
for 4 min, 18 to 25 mmol/l from 14 to 19.5 min, 3 mmol/l for 5 min.
The column was a weak ion-exchange IonPac CS17 (250 mm × 4 mm,
particle size 7 μm). Each sampling was replicated three times.
2.7. Sensory analysis
The sensory evaluation panel consisted of 10 panelists aging
between 22 and 38 years (students and researchers of the Department
of Food Science, Faculty of Agricultural Science, University of Foggia).
Using a scale ranging from 0 to 5 (where 0 = very poor and 5 =
excellent), the sensorial overall quality of the burgers was determined.
Panelists were asked to base their decision on the sample overall quality
only taking into account its odor, texture and drip loss. A score of 2 was
used as the threshold for product acceptability. During the evaluation
sessions, the samples were coded by a letter and presented in random
order.
2.8. Modeling and statistical analysis
The experiments were performed on two independent batches.
Nutritional data were tested for normality using Shapiro–Wilk test
(Shapiro and Wilk, 1965). Then, data were processed by ANOVA, using
the GLM procedure of Statistical Analysis System Version 8.1 (SAS
Inst., Cary, USA) for repeated measures, using the treatment as
repeated factor. When significant effects were found (at P b 0.05), the
Student t-test was used to locate significant differences between
means.
To assess the effectiveness of investigated packaging strategies on
the microbial stability of fish burgers, the viable cell concentration at
28 days of storage (i.e., the extent of the period of observation) was
calculated for each microbial group, according to the Gompertz
equation as re-parameterized by Corbo et al. (2006):
 
 
λ − 28
logðNðtÞÞ = logðN28 Þ− A⋅exp −exp ðμmax ⋅2:71Þ⋅ + 1
A
 
 
λ−t
+ A⋅exp −exp ðμmax ⋅2:71Þ⋅ + 1 ð1Þ
A
where: N(t) is the viable cell concentration (CFU/g) at time t; N28 is
the viable cell concentration at 28 days storage (CFU/g); A is related to
the difference between the decimal logarithm of bacterial load
attained at the stationary phase and the decimal logarithm of the
initial value of cell concentration; μmax is the maximal specific growth
rate (Δlog CFU/g/day); λ is the lag time (day); t is the storage time
283
(day). The log N28 value was determined by fitting Eq. (1) to the
experimental data.
Regarding sensory analysis, the Sensory Acceptability Limit (SAL),
defined as the time at which the fresh fish burger overall sensorial
quality reaches its threshold value, was also calculated by using the
re-parameterized Gompertz equation (Corbo et al., 2006):
( (" # ))
Q λQ − SAL
Q
SAðtÞ = SAmin − A ⋅exp −exp ðμ max ⋅2:71Þ⋅
+ 1
AQ
( (" # ))
Q Q λQ − t ð2Þ
+ A ⋅exp −exp ðμ max ⋅2:71Þ⋅ + 1
AQ
where: SA(t) is the fresh fish burgers sensory attribute at time t; AQ is
related to the difference between the value of the sensory attribute
attained at the stationary phase and the initial value of the sensory
attribute; µQ Q
max is the maximal rate at which SA(t) changes; λ is the
lag time; SAmin is the threshold value of the sensory attribute; SAL is
the time at which SA(t) is equal to SAmin; and t is the storage time. The
value of SAmin is set up to 2.
3. Results and discussion
In this study the nutritional, microbiological and sensorial quality
sub-indices were monitored to assess the quality loss of blue fish-
based burgers. In the following, the above-mentioned quality sub-
indices are presented and discussed separately.
3.1. Nutritional quality
Blue fish demonstrates an exceptional nutritional value in the
human diet being rich in minerals, vitamins and polyunsaturated fatty
acids (PUFA) (Karakoltsidis et al., 1995). Nutritional quality of fish
burgers was unaffected by MAP and active compounds. As shown in
Table 1, no differences emerged from analyses of moisture, fat, protein
content and fatty acid composition among samples.
Marine lipids contain high level of PUFA, especially eicosapentae-
noic acid (EPA, C20:5n3) and docosahexaenoic acid (DHA, C22:6n3)
(Ackman, 1989). The fatty acid profile generally exhibits a dominance
of two classes: saturated fatty acids (SFA) and PUFA. The last-
mentioned are reported to improve the nutritional value and protect
against diseases (Moreira et al., 2001). PUFA/SFA ratio found in the
analyzed fish burgers ranged from 1.01 to 1.34; the minimum value of
PUFA/SFA ratio recommended is 0.45 (Department of Health, 1994),
which is lower than those obtained from all fish burgers species
studied in this work. The n6/n3 ratio was affected by treatments, even
if such difference may be ascribed to the variability among the
samples because neither packaging treatment nor time of treatment
modified fatty acid composition of fish burgers. The UK Department of
Health recommends an ideal ratio of n6/n3 of 4.0 at maximum
(Department of Health, 1994). Values higher than the maximum value
are harmful to health and may promote cardiovascular diseases
(Moreira et al., 2001). In the current study, the ratio of n6/n3 was
found to range from 0.35 and 0.20 (Table 1), lower than the maximum
threshold (Department of Health, 1994).
3.2. Microbial quality
Fig. 1 shows the APC viable cell concentration plotted as a function
of storage time for all investigated samples. As it can be inferred, the
highest APC viable cell concentration was observed for the sample
without antimicrobial compounds (control, CNTR), suggesting that
the proposed packaging strategies were all effective in inhibiting the
APC growth. To quantitatively determine the efficiency of the
proposed packaging strategies, Eq. (1) was fitted to the experimental
284 M.A. Del Nobile et al. / International Journal of Food Microbiology 135 (2009) 281–287

Table 1
Values (Means ± Standard Error) of chemical composition of CNTR samples (fish burgers without antimicrobial compounds) and ACT samples (fish burgers with antimicrobial
compounds), packed in AIR and in three different MAP (30:40:30 O2:CO2:N2; 50:50 O2:CO2; 5:95 O2:CO2).

Parameter Day CNTR ACT- CNTR- ACT- CNTR- ACT- CNTR- ACT- SE Effects, P
(control) AIR 30:40:30 30:40:30 50:50 50:50 5:95 5:95 Treatment
Moisture, % 0 74.97 73.60 74.97 73.60 74.97 73.60 74.97 73.60 2.30 NS
28 72.10 74.96 74.49 77.20 74.41 75.11 71.03 75.00
Fat/DM, % 0 0.12 0.12 0.11 0.12 0.11 0.12 0.11 0.12 0.04 NS
28 0.10 0.09 0.09 0.10 0.08 0.10 0.07 0.10
Protein/DM, % 0 0.97 0.94 0.97 0.94 0.97 0.94 0.97 0.94 0.03 NS
28 0.90 0.90 0.92 0.93 0.91 0.85 0.84 0.87
Ash, % 0 1.26 1.22 1.26 1.22 1.26 1.22 1.26 1.22 0.06 NS
28 1.43 1.26 1.37 1.28 1.27 1.45 1.40 1.26
C20:5n3 (EPA), g/100 g 0 10.82 10.86 10.82 10.86 10.82 10.86 10.82 10.86 0.92 NS
28 11.15 11.59 10.21 11.57 11.6 11.43 11.31 12.62
C22:6n3 (DHA), g/100 g 0 23.4 22.49 23.4 22.49 23.4 22.49 23.4 22.49 1.92 NS
28 20.59 19.8 18.65 19.74 20.81 20.8 20.48 23.51
P/S 0 1.03 1.05 1.03 1.05 1.03 1.05 1.03 1.05 0.17 NS
28 1.03 1.02 1.02 1.01 1.08 1.05 1.03 1.34
n6/n3 0 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.02 ⁎⁎
28 0.25 0.23 0.27 0.22 0.24 0.22 0.24 0.20

SE, standard error; NS, not significant; ⁎⁎, P b 0.01.

data to determine the APC cell load at the end of the period of
observation (i.e., log NAPC APC
28 ). The log N28 values obtained are listed in
Table 2. As it can be inferred from data shown in the table, there is a
substantial difference in the log NAPC
28 value between CNTR and ACT-
AIR, which was about two orders of magnitude, thus suggesting that
the tested active compounds inhibited the growth of APC. Data listed
in Table 2 also highlight that among the MAP samples, 5:95 O2:CO2
was the most efficient gas mixture in inhibiting this microbial group.
As it can also be seen, MAP and tested active compounds acted in a
synergistic way to inhibit the APC growth. In fact, the samples
containing the antimicrobial agents showed the log NAPC 28 value that
was at least one order of magnitude lower than the corresponding
sample packed under MAP condition. For fresh water and marine
species, the microbiological limit recommended by the ICMSF (1986)
for APC at 30 °C is 7 log/g or log/cm2. Since in all samples (including
CNTR), APC never exceeded this value, the fish burgers could be
considered microbiologically acceptable under all proposed condi-
tions, even after 28 days of storage at 4 °C. However, it is worth noting
that spoiled marine fish is characterized by the development of
offensive, fishy, rotten H2S off-odors and flavours (Gram and Huss,
1996) which are mainly related to specific spoilage organisms (SSOs).
The plating medium used in a standard APC could affect the number
and types of bacteria isolated because of differences in nutrient and
salt requirements (as well as in terms of growth temperature) of the
various SSOs (Nickelson and Finne, 1992). For this reason, counts of
Pseudomonas spp., hydrogen sulphide-producing bacteria (HSPB)
and psychrotolerant and heat labile aerobic bacteria (PHAB) were
also performed.
Fig. 2 shows the Pseudomonas spp. viable cell concentration
plotted as a function of storage time. Also in this case a substantial
difference between the CNTR and the other samples can be observed.
In fact, the Pseudomonas spp. maximum population density of CNTR
sample was always the highest one among the tested samples. In
order to determine the efficacy of tested packaging strategies, the
Pseudomonas spp. cell load at the end of the storage time (i.e., log
NPseudomonas
28 ) was also determined by fitting Eq. (1) to the experi-
mental data. The log NPseudomonas
28 values are listed in Table 2. In this
case, results similar to those obtained for APC can be observed. It is
worth noting that Pseudomonas spp. viable cell concentration of ACT-
50:50 O2:CO2, CNTR-5:95 O2:CO2, and ACT-5:95 O2:CO2 samples was
always below the detection limit. These results allowed us to consider
Pseudomonas spp. of low importance to microbial quality evaluation of
blue fish burgers, since the calculated log NPseudomonas
28 values are very
Table 2
Maximum cell load observed in CNTR samples (fish burgers without antimicrobial
compounds) and ACT samples (fish burgers with antimicrobial compounds), packed in
AIR and in three different MAP (30:40:30 O2:CO2:N2; 50:50 O2:CO2; 5:95 O2:CO2), at the
end of the storage (28 days) at 4 °C, for each microbial group investigated.

Sample log NAPC

28 log NPseudomonas
28 log NHSPB
28 log NPHAB
28
−2
CNTR 6.37 ±4.45 · 10 6.22 ± 0.12 6.99 ± 0.19 6.20 ± 0.16
(control)
−2
ACT-AIR 4.56 ± 0.00 4.21 ±3.32·10 6.05 ± 0.20 6.20 ± 0.17
CNTR- 5.24 ±6.77 · 10− 2 5.31 ± 0.12 6.22 ± 0.17 6.12 ± 0.11
30:40:30
ACT- 3.76 ±7.99 · 10− 2 4.48 ± 0.16 5.43 ± 0.13 5.28 ± 0.13
30:40:30
CNTR- 4.92 ± 0.22 4.84 ± 0.13 5.25 ± 0.14 6.39 ± 0.11
50:50
−2 a −2
ACT- 3.65 ±5.36 · 10 – 5.32 ±8.05·10 5.14 ± 0.13
50:50
CNTR- 4.25±8.51·10− 2 –a 5.11 ±8.01·10− 2 5.38 ±6.37·10− 2
5:95
ACT- –a –a 4.91 ± 0.16 4.95 ± 0.11
Fig. 1. Evolution during storage at 4 °C of Aerobic Plate Count (APC) viable cell
5:95
concentration in the fish burgers. The curves are the fitting of Eq. (1) to the
experimental data. (○) Control in air; (▲) sample with active compounds in air; ( ) ◇ Data are presented ± standard deviation.
control under MAP 30:40:30 O2:CO2:N2; (●) sample with active compounds under APC, Aerobic Plate Count; HSPB, hydrogen sulphide-producing bacteria;
MAP 30:40:30 O2:CO2:N2; (■) control under MAP 50:50 O2:CO2; (□) sample with PHAB, psychrotolerant and heat labile aerobic viable count.
◆
active compounds under MAP 50:50 O2:CO2; ( ) control under MAP 5:95 O2:CO2. a
No growth.
 M.A. Del Nobile et al. / International Journal of Food Microbiology 135 (2009) 281–287
Fig. 2. Evolution during storage at 4 °C of Pseudomonas spp. viable cell concentration in
the fish burgers. The curves are the fitting of Eq. (1) to the experimental data. (○)
◇
Control in air; (▲) sample with active compounds in air; ( ) control under
MAP 30:40:30 O2:CO2:N2; (●) sample with active compounds under MAP 30:40:30
O2:CO2:N2; (■) control under MAP 50:50 O2:CO2.
low compared to the 8–9 log CFU/g required to spoil chilled fish
(Gram et al., 1989).
Fig. 3 shows the evolution during storage of the HSPB cell numbers
for all investigated samples. The efficacy of the tested packaging
solutions was assessed by comparing the log NHSPB 28 values, which
were determined according to the procedure described above. The
obtained values are listed in Table 2. As for the two microbial groups
discussed beforehand, the antimicrobial compounds as well as the 3
MAP conditions tested in this work inhibited the growth of HSPB.
Moreover, MAP and active compounds acted in a synergistic way
against HSPB; the lowest cell loads, after 28 days of storage, were
observed in all ACT samples packaged under MAP (log NHSPB 28 was
about 5 log CFU/g). These cell numbers indicated acceptable microbial
quality for the blue fish burger investigated, given that HSPB (mainly
represented by Shewanella species) are considered the strongest
spoilers of seafood from cold and temperate water and that significant
amounts of sulfur compounds are being produced (and spoilage of
fish occurs) only when their numbers exceed 6 log CFU/g (Gram et al.,
1987).
Fig. 4 shows the PHAB viable cell concentration plotted against the
storage time for all the investigated samples. Also in this case, results
highlighted a synergistic effect of MAP and antimicrobial compounds
on the growth of the above-mentioned microorganisms, as it can be
more easily inferred from data shown in Table 2. PHAB include Pho-
tobacterium phosphoreum which is CO2-resistant and often dominates
the spoilage microflora of fresh marine fish, particularly for products
Fig. 3. Evolution during storage at 4 °C of hydrogen sulphide-producing bacteria (HSPB)
viable cell concentration in the fish burgers. The curves are the fitting of Eq. (1) to the
experimental data. (○) Control in air; (▲) sample with active compounds in air; ( ) ◇
control under MAP 30:40:30 O2:CO2:N2; (●) sample with active compounds under
MAP 30:40:30 O2:CO2:N2; (■) control under MAP 50:50 O2:CO2; (□) sample with
◆
active compounds under MAP 50:50 O2:CO2; ( ) control under MAP 5:95 O2:CO2; (△)
sample with active compounds under MAP 5:95 O2:CO2.
285
Fig. 4. Evolution during storage at 4 °C of psychrotolerant and heat labile aerobic viable
count (PHAB) viable cell concentration in the fish burgers. The curves are the fitting of
Eq. (1) to the experimental data. (○) Control in air; (▲) sample with active compounds
◇
in air; ( ) control under MAP 30:40:30 O2:CO2:N2; (●) sample with active compounds
under MAP 30:40:30 O2:CO2:N2; (■) control under MAP 50:50 O2:CO2; (□) sample
◆
with active compounds under MAP 50:50 O2:CO2; ( ) control under MAP 5:95 O2:CO2;
(△) sample with active compounds under MAP 5:95 O2:CO2.
packaged in vacuum and MAP (Sivertsvik et al., 2002). Several studies
have shown that 7 log CFU/g of this microorganism is required to
produce fish spoilage (Dalgaard et al., 1993; Dalgaard, 1995) and,
therefore, the final cell numbers observed at the end of the storage in
all samples (including CNTR) indicate an acceptable microbial quality.
Moreover, as it can be also observed in Figs. 1–4, all the tested
packaging strategies were able to severely affect both the lag phase
and the growth rate of each investigated microbial group. This aspect
was more evident for the pseudomonads count whose growth rate
decreased quite 10 times (from 2.28 of CNRT to about 0.20 during the
treatments) (data not shown).
Concerning the packaging treatments, results obtained in this
study are in good agreement with other studies reported in literature:
MAP with 5:95 O2:CO2 resulted in the most effective for the inhibition
of all the investigated microbial groups. This fact may be attributed to
the inhibitory effect created by the presence of high CO2-concentra-
tion on microbial growth. Actually, it is well known that in the
absence of O2 and in the presence of CO2, a bacteriostatic effect is
exerted on aerobic microflora, thus inhibiting Gram-negative bacteria,
such as Pseudomonas spp. and hydrogen sulphide-producing bacteria
(i.e. Shewanella species) (Debevere and Boskou, 1996; Gram and
Huss, 1996; Sivertsvik et al., 2002). From the above results, it is also
obvious to infer that the active compounds acted in a synergistic way
with MAP to delay microbial growth: blue fish burgers were
microbiologically acceptable after 28 days of storage at 4 °C with
cell loads considerably lower than control sample. Tassou et al. (1996)
reported that the treatment of sea bream with olive oil, lemon juice
and oregano, followed by storage under MAP (40:30:30 CO2:O2:N2),
showed bacteriostatic effect on the autochthonous flora. Mejlholm
and Dalgaard (2002) reported that oregano oil (0.05% v/w) extended
the shelf life of naturally contaminated MAP cod fillets until 21–
26 days at 2 °C. Mahmoud et al. (2004) observed that the dipping
treatment of carp fillets in 1% (carvacrol and thymol) mixture
extended the shelf life of the product, packaged in air, for 12 days at
5 °C. According to Harpaz et al. (2003), the addition of oregano and
thyme at 0.05% v/v can considerably slow down the process of
spoilage of Asian sea bream. Goulas and Kontominas (2007), studying
the combined effect of MAP (40:30:30 CO2:O2:N2) and oregano oil on
the shelf life of sea bream fillets stored under refrigeration, observed a
considerable slowing down of fish spoilage. In a related study
conducted previously (Corbo et al., 2008), 110 ppm of thymol,
100 ppm of GFSE and 120 ppm of lemon extract, guaranteed a
statistically significant increase (of about 40% if compared to the
control sample) of the microbial acceptability limit of sea bream
burgers, after 10 days of storage at 5 °C in air. Results obtained in this
286 M.A. Del Nobile et al. / International Journal of Food Microbiology 135 (2009) 281–287

study highlight the possibility to improve the microbial quality of blue Table 3
fish burgers by using very small amount of thymol, GFSE and lemon Sensory Acceptability Limit (SAL) obtained in CNTR samples (fish burgers without
antimicrobial compounds) and ACT samples (fish burgers with antimicrobial
extract in combination with MAP. In particular, the combined use of compounds), packed in AIR and in three different MAP (30:40:30 O2:CO2:N2; 50:50
the tested natural preservatives and a packaging system characterized O2:CO2; 5:95 O2:CO2), at the end of the storage (28 days) at 4 °C, for each sensorial
by a high CO2-concentration, was able to guarantee the microbial attribute evaluated.
acceptability of fish burgers until the 28th day of storage at 4 °C.
Sample SALO [day] SALT [day] SALDL [day] SALOQ [day]
Concerning biogenic amines analyses, no amines were observed
CNTR (control) 7.82 ± 0.28 N 28 N28 8.21 ± 0.57
during the entire observation period in all the investigated samples. ACT-AIR 15.54 ± 0.57 N 28 N28 15.05 ± 0.43
Finally, it is worth noting that in all samples pH value was about CNTR-30:40:30 15.52 ± 0.48 20.16 ± 1.74 N28 17.67 ± 0.62
6.2 at the beginning of the storage and about 6.4 after 28 days; no ACT-30:40:30 20.49 ± 0.81 21.83 ± 1.70 N28 19.47 ± 1.06
significant differences were observed among the samples during the CNTR-50:50 17.73 ± 0.48 22.30 ± 1.30 26.51 ± 0.58 17.67 ± 0.62
ACT-50:50 21.12 ± 0.46 24.28 ± 1.74 27.09 ± 1.02 22.09 ± 1.22
entire observation period (data not shown). CNTR-5:95 17.88 ± 0.40 N 28 23.74 ± 1.62 20.96 ± 0.48
ACT-5:95 22.65 ± 0.79 N 28 N28 24.56 ± 1.57
3.3. Sensorial quality
Data are presented ± standard deviation.
O, Odour; T, Texture; DL, Drip Loss; OQ, Overall Quality.
Active antimicrobial substances, used as preservatives in foods,
often impart a certain flavor to products. On fish, carvacrol is said to
produce a “warmly pungent” aroma (Kim et al., 1995), thyme and increasing the SAL values, even when they were used alone. In fact,
oregano oils spread on whole Asian sea bass imparted herbal odor the sample containing the active compounds and packed under
(Harpaz et al., 2003) and oregano oil on cod fillets produced a ordinary atmosphere showed an increase in the SALOQ (SAL of overall
distinctive but pleasant flavor (Mejlholm and Dalgaard, 2002). On the quality) value of about 100%, if compared to CNTR. As expected, the
other hand, it is well established that the use of MAP, especially when MAP conditions tested in this work were all effective in increasing the
CO2-concentration is high, could have an unpleasant effect on some SALOQ value. Moreover, among the three tested MAP, 5:95 O2:CO2 is
sensorial parameters of the packed product (excessive exudate, most effective in slowing down the sensory quality loss of packed
softening of texture, discoloration, etc.) (Sivertsvik et al., 2002). fresh fish burgers, as also observed for microbial quality. Also in this
Therefore, in order to evaluate the effects of the proposed preserva- case, results show a synergistic effect of MAP and active compounds:
tion strategies on the sensory acceptability of blue fish burgers, the in fact, the SALOQ values of samples where MAP and active compounds
sensory quality loss was also assessed. As an example, Fig. 5 shows the are used in combination are always higher than those of samples
fresh fish burgers overall quality plotted against the storage time for where they are used alone. As also observed for the microbial quality
all the investigated samples. As it can be inferred, all samples show a evaluation, the combined use of the tested natural preservatives and a
sigmoidal trend. Data shown in Fig. 5 also highlight that there is a packaging system characterized by a high CO2-concentration, resulted
marked difference between CNTR sample and the other samples the most effective preservation strategy in guaranteeing the sensory
tested in this work. In particular, after the lag phase (about three quality of fish burgers.
days), where all the samples show similar overall quality values, the It is worth noting that for all the tested samples, the odor is the
overall quality of CNTR sample decreased more rapidly than that of attribute that limits the packed fresh fish burgers overall quality,
the other investigated samples, suggesting that all the proposed suggesting that to further prolong the shelf life of the investigated
packaging strategies are effective in minimizing the sensory quality commodity off-odors scavengers could be advantageously used.
loss of the investigated food product. The sensory attributes
monitored in this work showed a similar trend (data not shown).
4. Conclusions
To quantitatively determine the efficacy of the proposed packaging
strategies, the SAL value of the overall quality along with that of each
Results obtained in this study highlight the possibility to
monitored attribute, was determined according to the procedure
improve the quality of blue fish burgers by using very small
reported above. Curves shown in Fig. 5 result from the fitting
amount of thymol (110 ppm), GFSE (100 ppm) and lemon extract
procedure, whereas the SAL values obtained are listed in Table 3. As
(120 ppm) in combination with MAP. From a microbiological point
it can be seen, the selected active compounds were effective in
of view, the combined use of the tested natural preservatives and a
packaging system characterized by a high CO2-concentration, was
able to guarantee the microbial acceptability of fish burgers until
the 28th day of storage at 4 °C. However, the sensorial quality loss
limited the burgers shelf life to about 22–23 days. As compared to
several other mild preservation procedures like low dose irradia-
tion, addition of protective cultures, or high-pressure treatments,
the addition of natural compounds used in combination with MAP
is an inexpensive and uncomplicated method to extend shelf life of
packed seafood. The investigated natural preservatives are relatively
cheap and the addition of only 100 ppm of each compound is likely
to be cost-effective. On the other hand, the use of MAP is a practical
and economic technique, realized by small technical instruments.

Acknowledgment
Fig. 5. Blue fish burger overall quality plotted as a function of storage time for the fish
burgers. The curves are the fitting of Eq. (2) to the experimental data. (○) Control in air; This study was financially supported by Ministero dell'Economia e
◇
(▲) sample with active compounds in air; ( ) control under MAP 30:40:30 O2:CO2:N2; delle Finanze, Ministero dell'Istruzione, dell'Università e della Ricerca
(●) sample with active compounds under MAP 30:40:30 O2:CO2:N2; (■) control under
Scientifica e Tecnologica e l'Assessorato Bilancio e Programmazione
MAP 50:50 O2:CO2; (□) sample with active compounds under MAP 50:50 O2:CO2; ( ) ◆ Regione Puglia by the programme “Accordo di Programma Quadro
control under MAP 5:95 O2:CO2; (△) sample with active compounds under MAP 5:95
O2:CO2. in Materia di Ricerca Scientifica della Regione Puglia – Progetto
 M.A. Del Nobile et al. / International Journal of Food Microbiology 135 (2009) 281–287
Esplorativo – Title: Valorizzazione di pescato di basso valore
commerciale attraverso trasformati ittici di IV gamma”.
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