Journal of Food Engineering 115 (2013) 486–494

Contents lists available at SciVerse ScienceDirect

Journal of Food Engineering

journal homepage: www.elsevier .com/locate / j foodeng

Effects of chilled storage on quality of vacuum packed meagre fillets_Ismail Yüksel Genç a,⇑, Eduardo Esteves b, Jaime Aníbal c, Abdullah Diler a

a Department of Fishing and Processing Technology, Faculty of Fisheries, University of Suleyman Demirel, Turkeyb Departamento de Engenharia Alimentar, Instituto Superior de Engenharia, Universidade do Algarve and Centro de Ciências do Mar CCMAR – CIMAR Laboratório Associado, Portugalc CIMA – Centro de Investigação Marinha e Ambiental and Departamento de Engenharia Alimentar, Instituto Superior de Engenharia, Universidade do Algarve, Portugal

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

Article history:Available online 18 September 2012

Keywords:Meagre filletsQualityVacuum packagingShelf life prediction

0260-8774/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.jfoodeng.2012.09.007

⇑ Corresponding author. Address: Fishing and Proment, Faculty of Fisheries, University of Suleyman DeTel.: +90 246 313 34 47; fax: +90 246 313 34 52.

E-mail address: ismailygenc@gmail.com (_I.Y. Genç

The aim of this study was to experimentally assess several quality indices of meagre Argyrosomus regius(Asso, 1801) fillets packed in air (AP) and vacuum (VP) stored chilled (+4 �C) for up to 13 days. Consider-ing our experimental data on concentration of bacterial counts, shelf-life is estimated at ca. 6 days for APfillets and an additional 3–5 days for VP meagre fillets. Total volatile basic nitrogen (TVB-N) and trimeth-ylamine (TMA-N) did not reach the regulated limits (25–35 mg/100 g chilled fish). The models imple-mented in the software Seafood Spoilage and Safety Predictor predicted a relatively shorter shelf-lifeof 4.8–6.9 days for fish stored in air at +4 �C when compared to AP and VP fillets. Empirical data andthe models implemented in the software were used to predict the shelf-life of fillets if packaged underdifferent modified atmospheres (MAP). Chilled, MAP fillets are likely to have a longer shelf-life than APor VP samples if equilibrium CO2 concentration is substantially high.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Meagre, Argyrosomus regius (Asso, 1801), is a highly valuablescianid fish that is widely distributed in the Mediterranean, e.g.Portugal, Spain, France, Italy, and Turkey. The demand for this spe-cies is increasing daily (Naylor et al., 2000) since its nutritionalcomposition of about 20% proteins and 1.4% crude fat (Martinset al., 2006) favours its consumption as a ‘‘healthy food’’. Nonethe-less, the commercialised forms of processed seafood consist of por-tion-sized products, e.g. frozen skinned and/or breaded fillets andcubes, whole gutted and/or ungutted, sliced, smoked (FAO,2005-2011). Hence, the consumption of ready-to-eat minimallyprocessed seafood products, e.g. those that can be derived frommeagre, is preferable (Monfort, 2010).

Like other seafood (Frazier and Westhoff, 1988), fresh meagre ishighly perishable (Genç, 2012) due to microbial activity and/orspoilage-specific chemical reactions (viz. oxidation and rancidity)(Dalgaard, 2000, 2006), and thus has limited shelf-life. ‘‘Light’’preservation and/or proper packaging methods should be testedto help prolong the shelf-life (Huss et al., 2000; Poli et al., 2003).

Undoubtedly, vacuum packaging (VP) combined with chilledstorage, prolongs the shelf-life of seafood products by limitingthe availability of O2 that is necessary for the growth of aerobicbacteria. Moreover, other advantages of VP have been reported,

ll rights reserved.

cessing Technology Depart-mirel, 32500 Isparta, Turkey.

).

i.e. suitable moisture and gas permeability, correct assembly andprotection from the contamination with undesirable substancesfrom outer environment (Connel, 1995).

Modified atmosphere (MAP) and vacuum packaging (VP) of per-ishable (e.g. seafood) products maintains the hygienic characteris-tics (Özogul et al., 2004; Philips, 1996; Poli et al., 2006; Rotabakket al., 2008; Torrieri et al., 2011). Moreover, the combination ofMAP, VP and chilled storage has been found to extend the shelf lifeof fresh products (Pastoriza et al., 1996). There are several studiesabout VP fish, e.g. salmon (Salmo salar) (Dondero et al., 2004;Hansen et al., 2009), carp (Cyrinus carpio) (Krizek et al., 2004),rainbow trout (Salmo gairdneri) and Baltic herring (Clupea harengusmembras) (Randell et al., 1997) and Atlantic herring (Clupeaharengus) (Özogul et al., 2000), but no available data for VP ofmeagre fillets.

The objective of this study was to experimentally assess severalquality indices (microbiological and physical-chemical parame-ters) of meagre fillets packed in air and vacuum and stored chilledand by using empirical data and the models implemented in thesoftware Seafood Spoilage and Safety Predictor to predict theshelf-life of fillets if packaged under different modifiedatmospheres.

2. Materials and methods

2.1. Materials

Whole meagre, A. regius (Asso, 1801), were obtained from thecommercial circuit in Faro, Portugal. Specimens (mean

_I.Y. Genç et al. / Journal of Food Engineering 115 (2013) 486–494 487

weight ± SE: 862.71 ± 18.17 g) were kept in polystyrene boxescovered with ice during transportation to the laboratory. The fishwere washed with tap water and filleted under hygienic condi-tions. The average (±SE) weight of fillets was 124.5 g ± 4.51 g.

2.2. Packaging and storage conditions

Fillets were immediately packed in Combitherm� XX (WolffWalsrode AG, Germany) bags (200 � 200 mm) under atmosphericair (AP, sealing only) and vacuum (VP, at ca. 380 mm Hg) using amultipack vacuum packaging system (Interdibipack S.p.a., Italy).The packaging film was coextruded laminate composed of an exte-rior cast polyamide (PA) layer, a co-extruded interior barrier layercontaining ethylene vinylindene alcohol (EVOH) and a polyethyl-ene (PE) sealing layer. The oxygen transmission rate (OTR) is0.5 cm3/m2 d bar at 23 �C and 85% RH. Storage trials were carriedout for 13 days at +4 �C ± 0.5 �C.

2.3. Microbiological analysis

Samples (10 g) of fillets were aseptically placed into sterileStomacher� bags containing 90 ml of peptone water with NaCl(0.85% w/v) (Merck, Darmstadt, Germany) and homogenised for1 min (Stomacher� 400, Seward Ltd., London, UK). Aliquots of1 ml were poured in Petri dishes according to serial decimal dilu-tions before addition of appropriate media. For the enumerationof mesophilic aerobic and psychrophilic bacteria, PCA (Scharlau01-161, Germany) was incubated at 30 �C for 2 days (ISO, 2003)and at 6.5 �C for 10 days (ISO, 2001), respectively. Enterobacteri-ceae and lactic acid bacteria (LAB) were enumerated after inocula-tion of 1 ml aliquots into 10 ml of molten (at 45 �C) violet red bileglucose agar (VRBGA, Scharlau 01-295, Germany) and MRS agar(Scharlau 01-135, Germany), respectively. After settling, a 10 mloverlay of molten media was added and plates were incubated at37 �C for 48 h for VRBGA plates and 30 �C for 5 days for MRS plates.For hydrogen sulfide (H2S) producing bacteria, Iron Agar (IA) wasprepared and used according to NMKL (2006). Specifically, a thinoverlay of IA was poured on top of the IA to avoid fading of theblack colonies due to oxidation of iron sulfide (FeS). Petri disheswere then incubated at 25 �C for 72 h and black colonies werecounted as H2S-producing bacteria. All plates were examined visu-ally for typical colony types and morphological characteristicsassociated with each medium. Microbiological data, i.e. numberof colony forming units per unit mass were log-transformed priorto analysis, log (cfu g�1).

2.4. Chemical analysis

pH was determined directly from fish flesh using a digital meter(model Glp 21, Crison, Spain). Chemical spoilage was assessed bytwo indices: total volatile basic nitrogen (TVB-N) and trimethyla-mine (TMA-N). Both indexes were determined according to theConway method (Conway and Byrne, 1933); specifically, TMA-Nwas determined after the addition of formaldehyde. The flesh con-tent in TVB-N and TMA-N was expressed as mg TVB-N per 100 g ofsample and mg TMA-N per 100 g of sample, respectively.

2.5. Physical analysis

Colour measurements were carried out directly on fresh andpacked/chilled stored samples using a tristimilus colorimeter(model DR LANGE, Spectro-color, Spain) and examined accordingto the Hunter L,a,b colour scale (Hunter Associates LaboratoryInc., USA) where L refers to lightness (0 is black and 100 is white),a indicates greenness (a < 0) or redness (a > 0), and b measuresblueness (b < 0) or yellowness (b > 0) of samples. As summary

measure, total colour change (denoted DE) (Anon., 2008b) was cal-culated in accordance with:

DE ¼ ðDL2 þ Da2 þ Db2Þ12

where e.g. DL2 = (Lt � L0)2 and L refers to lightness at time t (Lt) andtime 0 (L0).

Hardness, i.e. the force required to attain a deformation of theproducts’ surface (Szczesniak, 2002), was determined via a com-pression test. This test was carried out using a texturometer (LFRATexture Analyzer, Brookfield Engineering Labs Inc., USA) equippedwith a 12.7 mm-diameter stainless steel spherical probe which ap-proached the sample at the speed of 1 mm�1 and compressed5 mm into the fillets. Measurements (in kgf, where 1 kgf = 9.806 N)were analysed using TexturePro Lite v1.1 software (BrookfieldEngineering Labs Inc., USA). Hardness was calculated as the peakforce of the first compression cycle.

2.6. Experimental design and statistical analysis

Analyses described above were carried out on days 0, 1, 3, 8 forair packed samples and days 0, 1, 3, 8 and 13 for vacuum packedfillets. On each occasion, two fillets were sampled. Several mea-surements were made on each fillet and averaged: six for pH, threefor colour, two for TVB-N and TMA-N and six for hardness. Resultsare reported as mean values ± standard errors. Two-way ANOVAwith factors storage time and package type (AP and VP) was per-formed for each of the quality parameters analysed. A significanteffect of the factors’ interaction term was further studied usingsimple effects test to compare package type at each storage time(Esteves, 2011). The analyses were done using IBM� SPSS� 19 forWindows (IBM Company, NY, USA).

2.7. Modelling microbial growth of empirical data and prediction ofshelf-life

The shelf-life of fillets packed under air and vacuum conditionswas estimated based on the chemical (i.e. TVB-N content) andmicrobiological criteria (Bremner, 2002), specifically counts ofmesophilic and psychrophilic aerobic and H2S-producing bacteria.The guidelines of ICMSF (1986) and the PHLS working group(Anon., 2000) and the regulated methods for the control of fisheryproducts (Anon., 2004, 2005, 2008a) were taken into consideration.

The microbial spoilage models implemented in the softwareSeafood Spoilage and Safety Predictor (SSSP v. 3.1, National Insti-tute of Aquatic Resources, Technical University of Denmark (DTUAqua), Denmark) were then used to model changes in the concen-tration of microorganisms over time, estimate kinetic parameters(e.g. maximum specific growth rate lmax) and predict productshelf-life under specific storage conditions.

Specifically, the storage temperature (T, �C, in the range 3–5 �C)(Eq. 1) and the initial counts of mesophilic, psychrophilic and H2S-producing bacteria (N0, logcfu/g, of ca. 3.2) were firstly enteredinto the models of Dalgaard et al. (1993)ffiffiffiffiffiffiffiffiffiffi

lmax

p¼ 0:0299 � ðT þ 7:08Þ ð1Þ

and

tSL ¼½logð107Þ � logðN0Þ� � lnð10Þ

lmax � 24ð2Þ

to obtain estimates of lmax (h�1) and shelf-life (tSL, in days). The pri-mary growth model (a log-transformed three-parameter logisticmodel; Eq. (3)) supporting the above equations has been validatedfor several species stored aerobic conditions, e.g. cod, haddock,sea bream or snapper (Dalgaard, 1999):

488 _I.Y. Genç et al. / Journal of Food Engineering 115 (2013) 486–494

logðNtÞ ¼ logNmax

1þ NmaxN0� 1

� �� expð�lmax � tÞ

0@

1A ð3Þ

where Nt (cfu/g) is the concentration of bacteria at time t, N0 (cfu/g)the initial concentration of bacteria, Nmax is the maximum concen-tration (cfu/g) and lmax the maximum specific growth rate (h�1).The observed (AP and VP fillets) and predicted growth of the mes-ophilic, psychrophilic and H2S-producing bacteria were comparedusing indices of models’ lack-of-fit (root mean square error, RMSE)and performance (bias and accuracy factor). The later were intro-duced by Ross (1996). Dalgaard (2002) proposed that the bias factorshould be in the range 0.75–1.25 for a model to be successfully val-idated. Values of the accuracy factor below 1.3 indicate reasonabledeviation between observed and predicted growth. Accessorily,product characteristics and storage conditions (i.e. initial celldensity, storage temperature and pH) were used as input into the(secondary) growth model of Ross and Dalgaard (2004):

ffiffiffiffiffiffiffiffiffiffilmax

p¼ b � ðT � TminÞ �

%CO2:max �%CO2

%CO2:max�ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiaW � aW:min

aW:ref � aw:min

s

�ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffipH � pHmin

pHref � pHmin

sð4Þ

where b is a constant and the model includes the cardinal parame-ters Tmin (theoretical minimum growth temperature), %CO2max (the-oretical maximum concentration of CO2 that allows growth), aw.min

(theoretical minimum water activity that allows growth) and pHmin

(theoretical minimum pH value that allows growth) – all these wereset at default values specifically b = 0.020, Tmin = –10 �C,%CO2max = 200, aw.min = 0.95, aw.ref = 0.997, pHref = 6.6 and pHmin = 5.The original model was developed for chilled product stored at aknown pH (pHref) and water activity (aw.ref) (Ross and Dalgaard,2004). The model (Eq. 4) allowed the prediction of shelf-life of mea-gre fillets under different storage conditions namely temperature(3–5 �C), equilibrium concentrations of CO2 in MAP (25–100%)and pH (6.5–8) (Dalgaard et al., 1997).

3. Results and discussion

3.1. Microbiological changes

The microbiological quality of fresh meagre fillets used in thisstudy was good as indicated by a low initial number of bacteria;mesophilic and psychrophilic aerobic, H2S-producing bacteria,Enterobactericeae and lactic acid bacteria counts were found tobe 3.41 ± 0.02 logcfu/g, 3.25 ± 0.60 logcfu/g, 3.18 ± 0.03 logcfu/g,1.86 ± 0.21 logcfu/g, and 2.82 ± 0.33 logcfu/g, respectively.

After fish fillets were exposed to the two different packagingatmospheres (AP and VP) and stored under chilled conditions,the microbiota increased significantly (p < 0.05). After 8 days ofstorage, mesophilic aerobic bacteria counts of AP fillets reached7 logcfu/g, the upper acceptability limit for freshwater and marinespecies by ICMSF (1986). On the other hand, even though the VPfillets would have been rejected in terms of organoleptic evalua-tion (intensity of off-odours and appearance), counts of mesophilicbacteria did not exceed 6.6 ± 0.1 logcfu/g (Fig. 1a) on day 13. Thedifferences between treatments were significant (p < 0.05) fromday 1 onwards. Similar to the results of VP meagre fillets’ meso-philic counts have been reported by Özogul et al. (2000) for VP her-ring (C. harengus) stored in ice.

Psychrophilic aerobic bacteria were naturally present in the ini-tial microbiota of the meagre fillets and their abundance increasedca. 3 log cfu/g with time of storage. VP fillets had already signifi-cant less bacteria than AP fillets on day 3. Similarly, on day 8,

significantly (p < 0.05) higher bacteria were recorded in AP fillets(6.93 ± 0.50 logcfu/g) compared to VP fillets (5.77 ± 0.27 logcfu/g). The later had 6.60 ± 0.29 logcfu/g after 13 days of chilled stor-age (Fig. 1b).

H2S-producing bacteria were also important in the spoilageprocess of meagre fillets (Fig. 1c). At the end of storage periodthe counts of H2S-producing bacteria were found to be 6.12 ±0.42 logcfu/g for AP fillets and 3.57 ± 0.61 logcfu/g for VP samples.The differences between treatments were statistically significant(p < 0.05) particularly on day 8. It is clear that VP significantly af-fected the growth of H2S-producing bacteria. Nonetheless, off-odours mentioned above could have developed as a result of anaer-obic H2S-producing bacteria growth favoured by VP conditions.

LAB that were initially present in the microbiota of meagre fil-lets at intermediate levels, ca. 3 logcfu/g, decreased first to about2 logcfu/g after 24 h of chilled storage and then increased to ca.5 logcfu/g on day 8 (Fig. 1d). The initial decrease in the counts ofLAB (and to a lesser extent in H2S-producing bacteria) could bedue to pH variations of fish species during storage (Huss, 1988,1995) and/or a ‘washing effect’ following the initial preparationof the fillets that involved rinsing with tap water. Similarly, Inacioet al. (2003) found a reduction in the counts of Pseudomonas sp.,H2S-producers and total number of bacteria in scad (Trachurus tra-churus) that lasted ca. 6 days after washing the specimens. Huido-bro et al. (2001) also found that washing delayed the limit ofmicrobiological acceptability of gilthead seabream (Sparus aurata).The final counts of LAB (day 13) for VP fillets were 6.21 ± 0.25 logc-fu/g. No significant differences (p > 0.05) were found between thetreatments at neither of times sampled, but the counts were signif-icantly different (p < 0.05) among the storage times. Stamatis andArkoudelos (2007b) reported lower results (<10 cfu/g) for the ini-tial LAB counts of AP/VP/MAP chub mackerel (Scomber colias japo-nicus). However, at the end of the storage period of 15 days LABcounts of chub mackerel were higher for AP and VP fillets (ca.7 logcfu/g and 6.8 logcfu/g, respectively).

Finally, Enterobacteriaceae (Fig. 1e) were the least abundantgroup among the microbiota studied, not exceeding 2.78 ±0.79 logcfu/g in AP fillets on day 8 and 3.45 ± 0.42 logcfu/g in VPsamples on day 13. The differences between treatments were notsignificant (p < 0.05). Higher results (6.70 ± 0.05 logcfu/g) havebeen reported for ice-stored meagre fillets (Hernández et al.,2009). The differences in Enterobacteriaceae counts between stud-ies could be the result of storage conditions; herein fillets werevacuum packaged whereas Hernández et al. (2009) studied thespoilage of meagre fillets stored under aerobic conditions.

Except for mesophilic aerobic bacteria, the microbiota of mea-gre fillets did not exceed 7 logcfu/g at the end of the storage peri-od. Notwithstanding, apart Enterobacteriaceae, the remaining taxaanalysed, reached unsatisfactorily high counts sensu Anon. (2000)– P106 cfu/mg.

3.2. Chemical changes

The initial TVB-N values of meagre fillets averaged14.63 ± 2.76 mg N/100 g sample. At the end of storage period,8 days for AP samples and 13 days for VP fillets, TVB-N valuesreached 18.84 ± 0.95 and 21.56 ± 2.64 mg N/100 g sample respec-tively (Fig. 2a). However, differences between treatments andtimes were not significant (p > 0.05). Similar results were reportedby Hernández et al. (2009) for fresh meagre fillets stored on ice,20.40 ± 0.97 mg N/100 g sample, but for a longer storage period,i.e. 18 days. The limits of TVB-N content reported as acceptablefor human consumption are in the range 25-35 mg N/100 g (Anon.,2005) but this is susceptible to variation among species. Despitethe relatively low levels of TVB-N after ca. 2-weeks of storage,the fillets would have been rejected for consumption due to the

Fig. 1. Changes in the counts of (a) mesophilic, (b) psychrophilic, (c) H2S-producing and (d) lactic acid bacteria and (e) Enterobacteriaceae during chilled storage of meagrefillets packed in air (AP) and under vacuum (VP). Means ± SE are plotted. Significant differences between treatments at specific time are noted with ⁄p < 0.05, ⁄⁄p < 0.01 and⁄⁄⁄p < 0.001.

_I.Y. Genç et al. / Journal of Food Engineering 115 (2013) 486–494 489

putrid off-odours. TVB-N, which is the standard, legal chemicalindicator of seafood spoilage, is appropriate for advanced spoilagebut is an insufficient sign of quality during the initial stages of sea-food spoilage (Clancy et al., 1995; Tejada and Huidobro, 2002). Infact, after 8 days samples were already unacceptable in terms ofoff-odours (a putrid smell was distinctly scented). In this study, de-spite the clearly spoiled degree of fillets, their TVB-N content waswithin regulated levels.

In marine fish, TMA-N is produced by decomposition of TMA-Ocaused by bacterial spoilage and enzymatic activity (Dalgaard,1995). At the beginning of the experiment the values of TMA-Nwere very low, 0.70 ± 0.49 mg N/100 g sample. In the end of stor-

age trials, concentrations of TMA-N in the meagre fillets were1.43 ± 1.70 mg N/100 g for AP samples and 2.00 ± 0.96 mgN/100 g for VP samples (Fig. 2c). Moreover, there were no signifi-cant (p > 0.05) differences between treatments and among storagetimes. The final concentration of TMA-N in the muscle tissues ofmeagre fillets indicates that the fillets were not spoiled in accor-dance with chemical criteria. Although the low initial values ob-served herein are in agreement with those published for Sparidaespecies, the final concentrations were substantially lower thanpublished, e.g. 8.87 mg N/100 g flesh of salted seabream (S. aurata)(Chouliara et al., 2004) and around 2 mg N/100 g (Tejada andHuidobro, 2002). Furthermore, Çakli et al. (2007) reported that

Fig. 2. Changes in (a) TVB-N content, (b) TMA-N content and (c) pH of meagre filletduring chilled storage. Means ± SE are plotted. Significant differences betweentreatments at specific time are noted with ⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001.

490 _I.Y. Genç et al. / Journal of Food Engineering 115 (2013) 486–494

the initial TMA-N values of whole ungutted seabass (Dicentrarchuslabrax) and seabream were 0.27 ± 0.01 mg N/100 g sample and0.44 ± 0.04 mg N/100 g sample, respectively. After 18 days ofice-storage, the TMA-N values increased several-fold to 5.08 ±0.02 mg N/100 g sample and 3.92 ± 0.07 mg N/100 g sample,respectively. Goulas and Kontominas, 2007) stated even lowerTMA-N values for MAP salted seabream with added oregano oilstored under chilled conditions, namely 0.31 ± 0.02 mg N/100 gsample. However, overall sensory assessments of the fillets wouldhave rendered the fillets unacceptable for consumption. The devel-opment of off-odours could be the result of sulfide compounds’

accumulation due to high H2S-producing bacteria counts ratherthan to the production of N-based compounds (i.e. TVB-N orTMA-N).

The pH of fresh fish flesh is close to neutrality. However, anaer-obic fermentation-based activity of glucose or glycogen causes adecrease in pH of fish flesh (Huss, 1995). In this study, the pH offresh meagre fillets averaged 6.82 ± 0.14, then increased to8.06 ± 0.04 for AP samples and 7.34 ± 0.59 for VP samples on day3 and, finally, decreased to 6.66 ± 0.02 at the end of the storageperiod. On day 3, AP fillets reached significantly higher (p < 0.05)pH than VP fillets (Fig. 2c). Lower results (6.2 ± 0.1 to 6.8 ± 0.1)were reported by Stamatis and Arkoudelos (2007a) for filletedVP/MAP sardines (Sardina pilchardus) stored at 3 �C for 15 days.The increase in pH after the first 3 days both for AP and VP meagrefillets may be the result of biogenic amines’ production that yieldshigher pH in fish meat (Jorgensen et al., 1988; Krizek et al., 2004).In contrast, the final levelling-out of pH could have been the resultof lactic acid production by LAB (Fernandes, 2009).

3.3. Physical changes

One of the most evident changes in quality of meagre fillets wasobserved in colour. Lightness of fillets, L, changed with time,becoming lighter just after 24 h of storage when compared to thefresh samples (Fig. 3a), but differences between treatments werenot significant (p > 0.05). Conversely, Hernández et al. (2009) re-ported that ice-stored meagre fillets were darker compared tofresh ones. The values of a, i.e. the green-red colour channel, wererelatively stable (at ca. �0.8) for AP fillets but displayed a fluctuat-ing pattern around �0.8 during the 13 days of the storage trials ofVP fillets (Fig. 3b). On the other hand, in the blue-yellow channel,the values of b showed an almost linear and significant (p = 0.004)increase for both AP and VP (Fig. 3c) throughout the storage periodbut the differences between treatments were again not statisticallysignificant (p > 0.05). Overall, the changes in DE values of fillets re-flected the variability described above for L, a and b. After the ini-tial 24 h of chilled storage, values of DE ranged from 3.12 to 4.02for AP fillets and varied between 2.52 and 4.42 for VP fillets(Fig. 3d) throughout the remainder of the sampling occasions. Asexpected, colour changed as a result of storage conditions andpackaging techniques seemingly influenced by microbiologicaland chemical loss of quality (Bonilla et al., 2007). The colour differ-ence values should be noticed by consumers since the values of DEfound herein exceed the just-noticeable-difference (JND) but‘unempirical’ threshold of 1.0 and even the ‘corrected’ limit of 1.9proposed by Mahy et al. (1994) to discriminate surface colours un-der the Hunter Lab system.

In terms of textural changes, the hardness of meagre fillets in-creased significantly (p < 0.05) with storage time; however therewere no significant (p > 0.05) differences between AP and VP fillets.The initial hardness values were found to be 2.44 ± 0.24 N for thefresh fillets and increased to 6.09 ± 0.95 N for the AP fillets onday 8 and 8.57 ± 1.28 N for the VP fillets on day 13. Sigurgisladottiret al. (2000, 2001) found that hardness increased in response toprocessing technique (i.e. freezing/thawing, dry salting, and smok-ing) and location of fillets. On the other hand, variations in the tex-tural properties of seafood also depend on the microstructure ofthe product, the storage conditions (frozen or chilled storage)and the species (Huriaux et al., 1999; Kim et al., 2005; Ladratet al., 2003). Commonly, seafood spoilage is associated with soften-ing of the tissues because of proteolysis and decreasing pH levels inthe range of 8.0–6.5. Unexpectedly, in this study, fillets’ hardeningis most probably the result of pH variations and bacterial activitythat can cause decrease in WHC and yield higher hardness values(Lonergan and Lonergan, 2005).

Fig. 3. Changes in (a–c) values of color parameters L, a and b, and (d) hardness during chilled storage of meagre fillets packed in air (AP) and under vacuum (VP). Means ± SEare plotted. Significant differences between treatments at specific time are noted with ⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001.

_I.Y. Genç et al. / Journal of Food Engineering 115 (2013) 486–494 491

3.4. Prediction of shelf life

Considering our data on concentration of mesophilic, psychro-philic and H2S-producing bacteria in AP and VP fillets and the morerestrictive guidelines of the PHLS working group (Anon., 2000), i.e.bacterial counts less than 6 logcfu/mg sample, shelf-life of meagrefillets is estimated at ca. 6 days for aerobically stored fish and 9–11 days for vacuum packed fillets – that is an additional 3–5 days.The 107 ufc/g limit proposed by ICMSF (1986) ‘‘extended’’ theshelf-life of AP fillets to 8 days and was not reached by VP fillets.On the other hand, TVB-N content of both AP and VP fillets didnot exceed the lower level determined by EU regulation (25–35 mg/100 g). Up to 2 days can/should be added to our findings

since this is the usual time elapsed between fishing/slaughterand market availability; the cumulative shelf-life, 8 days for AP fil-lets and 11–13 days for VP fillets, is probably excessive consideringthe putrid off-odours shown by fillets at the end of our storagetrials.

Estimates of shelf-life are in line with published results evenconsidering the different storage temperatures (i.e. 0 �C vs.+4 �C):It has been reported that the shelf-life of meagre fillets (Hernándezet al., 2003) and whole meagre (Poli et al., 2003) stored at 0 �C is9 days.

The microbial spoilage models implemented in the SSSP soft-ware, estimated the shelf-life of fillets stored in air (at 3–5 �C) tobe in the range 4.8–6.9 days for an initial cell count of

Table 1Bias and accuracy factors of microorganisms.

Microorganism RMSE Bias factor Accuracy factor

Mesophilic bacteria 2.99 1.20 1.20Psychrophilic bacteria 2.82 1.18 1.18H2S producing bacteria 4.18 1.44 1.44Lactic acid bacteria 5.51 1.64 1.64Enterobacteriaceae 6.36 1.98 1.98

492 _I.Y. Genç et al. / Journal of Food Engineering 115 (2013) 486–494

3.18–3.41 logcfu/g and a maximum specific growth rate of 0.09–0.13 h�1. The relatively shorter shelf-life is substantially influencedby the bias of the underlying models (see below).

The primary growth model used herein strongly overestimatedthe observed changes in concentration of H2S-producing over time(bias factor of 1.44, well over the 0.80–1.20 range) but provided(statistically) reasonable estimates for mesophilic and psychro-philic bacteria (RMSE of 2.99, bias factor of 1.20 and accuracy fac-tor less than 1.3) (Fig. 4). Models adjusted poorly to changes in theconcentration of LAB and Enterobacteriaceae: RMSE of 5.51 and6.36, respectively, clearly above 1.2. (Table 1).

Using the square root type (secondary) model of Ross andDalgaard (2004) we were able to compare the (estimated) growthand shelf-life of meagre fillets stored (at 3–5 �C) in aerobicconditions and in MAP (25–100% CO2) (Table 2).

The model predicts a shelf-life of 5.75 days for fish with pH of6.5 and stored in air at 3 �C; a much shorter period, 2.16 days, is

Fig. 4. Observed and predicted growth of (a) mesophilic, (b) psychrophilic,

predicted for pH of 8 and 5 �C. The former estimates are similarto the estimated shelf-life based on our empirical data, ca. 6 daysfor AP fillets; but the later are much briefer due to the increasedpH and storage temperature. Using increasingly higher concentra-tions of CO2 in MAP (from 25% to 100% at equilibrium) to store themeagre fillets would extend their shelf-life almost fourfold, i.e. to23 days using 100% CO2, in the more favourable conditions of lower

(c) H2S-producing, (d) lactic acid bacteria and (e) Enterobacteriaceae.

Table 2Predicted shelf-life (days) for meagre fillets stored under different conditions in air(AP) and in MAP using the square root type (secondary) model of Ross and Dalgaard(2004).

Storage condition pH Temperature (�C)

3 5

0% CO2 or AP 6.5 5.75 4.328 2.88 2.16

25% CO2 6.5 7.51 5.648 3.76 2.82

50% CO2 6.5 10.23 7.688 5.11 3.84

75% CO2 6.5 14.73 11.068 7.36 5.53

100% CO2 6.5 23.01 17.288 11.51 8.64

_I.Y. Genç et al. / Journal of Food Engineering 115 (2013) 486–494 493

temperature and pH, 3 �C and 6.5 respectively. Seemingly, at least75% of CO2 has to be added to the package’s atmosphere to notice-ably augment the product’s shelf-life when compared to VP fillets.Nonetheless, the validation of these predictions and a cost-benefitanalysis of applying this technology is advisable in a case-by-case,species-specific approach.

4. Conclusion

The quality of meagre fillets packed in air (AP) and vacuum (VP)and stored for up 13 days under chilled conditions was experimen-tally assessed. Vacuum packaging had a positive, significant effectover the shelf-life of the meagre fillets – VP fillets had an additional3–5 days of shelf-life to the period of ca. 6 days estimated for filletsstored in air. Microbiological indices (i.e. mesophilic and psychro-philic aerobic bacteria, H2S-producing bacteria and Enterobacteri-ceae counts) in VP fillets were significantly lower than those ofAP fillets. There were no significant changes in chemical attributesbetween AP and VP samples or storage periods. At the end of theexperiment, fillets were harder compared to fresh samples butno significant differences were found between treatments. Usingthe models implemented in the software Seafood Spoilage andSafety Predictor (SSSP), predicted shelf-life of aerobically storedfresh fillets at chill temperatures was 4.8-6.9 days for an initialcount of ca 3.2 logcfu/g which is in line with the experimentalfindings. Considering that MAP is increasingly used to extend theshelf-life of (sea)food products, existing predictive microbiologymodels are expected to help choose the storage conditions of alter-native seafood such as the meagre fillets studied herein. Using themodels implemented in the, software SSSP meagre MAP fillets arelikely to have a longer shelf-life than VP samples if the concentra-tion of CO2 at equilibrium is 75% or more CO2. Notwithstanding,these predictions should be tested experimentally.

Acknowledgements

Authors thank Engs. Neuza Rodrigues, Clarisse Ramalho, TeresaSoeiro, Teresa Cavaco, Vera Gonçalves, João Filipe Silva and Mrs.Sílvia Madeira at the Departmento de Engenharia Alimentar doInstituto Superior de Engenharia da Universidade do Algarve, andEng. Carla Mendes, at Continente Hipermercados S.A. for theirvaluable contribution to the work. _Ismail Yüksel Genç benefitedfrom a LLP Erasmus training program grant.

References

Anon., 2000. Guidelines for the microbiological quality of some ready-to-eat foodssampled at the point of sale. Communicable Disease and Public Health 3, 163–167.

Anon., 2004. Regulation (EC) No. 854/2004 of the European parliament and of thecouncil. Official Journal of the European Union L 226, 83–127.

Anon., 2005. Regulation (EC) No. 2074/2005 of the European parliament and of thecouncil. Official Journal of the European Union L 338, 27–59.

Anon., 2008a. Regulation (EC) No. 1022/2008 of the European parliament and of thecouncil. Official Journal of the European Union L 227, 18–20.

Anon., 2008b. Hunter L, A, b color scale. Applications Note 8 (9), 1–4.Bonilla, A.C., Sveinsdottir, K., Martinsdottir, E., 2007. Development of quality index

method (QIM) scheme for fresh cod (Gadus morhua) fillets and application inshelf life study. Food Control 18, 352–358.

Bremner, H.A., 2002. Safety and quality issues in fish processing. In: Modelling andPredicting the Shelf Life of Seafoods. Woodhead Publishing Ltd., Cambridge, UK,pp. 191–211.

Chouliara, I., Savvaidis, I.N., Panagiotakis, N., Kontaminas, M.G., 2004. Preservationof salted, vacuum-packaged, refrigerated seabream (Sparus aurata) fillets byirradiation: microbiological, chemical and sensory attributes. FoodMicrobiology 21, 351–359.

Clancy, G.S., Bearnes, D.A., Higgs, D.A., Dosanjh, B.S., 1995. Effect of methodology onthe determination of total volatile nitrogen and trimethylamine levels inpreviously frozen Pacific herring (Clupea harengus pallasi) stored at 2–5�C for upto 15 days. Canadian Technical Report of Fisheries and Aquatic Science 2047, 10.

Connel, J.J., 1995. Control of Fish Quality, Fishing New Books. Blackwell Science Ltd.,Cambridge, London, 241pp..

Conway, E.J., Byrne, A., 1933. An absorption apparatus for the micro-determinationof certain volatile substances. I. The micro-determination of ammonia.Biochemistry Journal 27, 419–429.

Çaklı, S�., Kılınç, B., Cadun, A., Dinçer, T., Tolasa, S., 2007. Quality differences of wholeungutted sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) whilestored in ice. Food Control 18, 391–397.

Dalgaard, P., Gram, L., Huss, H.H., 1993. Spoilage and shelf life of cod fillets packed invacuum or modified atmospheres. International Journal of Food Microbiology19, 283–294.

Dalgaard, P., 1995. Qualitative and quantitative characterization of spoilage bacteriafrom packed fish. International Journal of Food Microbiology 26, 319–333.

Dalgaard, P., Mejlholm, O., Huss, H.H., 1997. Application of an iterative approach fordevelopment of a microbial model predicting the shelf-life of packed fish.International Journal of Food Microbiology 38, 169–179.

Dalgaard, P., 1999. Modelling of seafood spoilage. In: Predictive MicrobiologyApplied to Chilled Food Preservation, Refrigeration Science and TechnologyProceedings. Refrigeration Science and Technology Proceedings, EUR 18816, pp.143–151.

Dalgaard, P., 2000. Fresh and lightly preserved seafood. In: Man, C.M.D., Jones, A.A.(Eds.), Shelf-Life Evaluation of Foods. Aspen Publishers, London, pp. 110–139.

Dalgaard, P., 2002. Modelling and predicting the shelf-life of food. In: Bremner, H.A.(Ed.), Safety and Quality Issues in Fish Processing. Woodhead Publishing Ltd.,pp. 191–219.

Dalgaard, P., 2006. Microbiology of marine muscle foods. In: Hui, Y.H. (Ed.),Handbook of Food Science, Technology and Engineering. CRC Press, Boca Raton,pp. 1–20.

Dondero, M., Cisternas, F., Carvajal, L., Simpson, R., 2004. Changes in quality ofvacuum-packed cold-smoked salmon (Salmo salar) as a function of storagetemperature. Food Chemistry 87, 543–550.

Esteves, E., 2011. Statistical analysis in food science. In: Cruz, R.M. (Ed.), PracticalFood and Research. Nova Science Publishers Inc., NY, USA, pp. 409–451.

FAO, 2005–2011. Cultured Aquatic Species Information Programme. Argyrosomusregius. Cultured Aquatic Species Information Programme. Text by Stipa, P.,Angelini, M. In: FAO Fisheries and Aquaculture Department (online). Rome.Updated 10 February 2005. (Cited 16 December 2011). <http://www.fao.org/fishery/culturedspecies/Argyrosomus_regius/en#tcNA00FE>.

Fernandes, R., 2009. Microbiology Handbook Fish and Seafood. Leatherheadpublishing, Cambridge, UK.

Frazier, W.C., Westhoff, D.C., 1988. Food Microbiology, Fourth ed. McGraw-HillBook Company, Singapore.

Genç, _I.Y., 2012. Microbiological, chemical and sensorial quality of Argyrosomus sp.(In Turkish with English abstract). M.Sc. Thesis, Graduate School of Natural andApplied Sciences, Isparta, 56pp.

Goulas, A.E., Kontominas, M.G., 2007. Combined effect of light salting, modifiedatmosphere packaging and oregano essential oil on the shelf-life of sea bream(Sparus aurata): biochemical and sensory attributes. Food Chemistry 100, 287–296.

Hansen, A.A., Mørkøre, T., Rudi, K., Rødbotten, M., Bjerke, F., Eie, T., 2009. Qualitychanges of prerigor filleted Atlantic salmon (Salmo salar L.) packaged inmodified atmosphere using CO2 emitter, traditional MAP, and vacuum. Journalof Food Science 74, 242–249.

Hernández, M.D., López, M.B., Álvarez, A., Ferrandini, E., García, B.G., Garrido, M.D.,2009. Sensory, physical, chemical and microbiological changes in aquaculturedmeagre (Argyrosomus regius) fillets during ice storage. Food Chemistry 114,237–245.

Huidobro, A., Pastor, A., López-Caballero, M.E., Tejada, M., 2001. Washing effect onthe quality index method (QIM) developed for raw gilthead seabream (Sparusaurata). European Food Research and Technology 212, 408–412.

Huriaux, F., Vandewalle, P., Baras, E., Legendre, M., Focant, B., 1999. Myofibrillarproteins in white muscle of the developing African catfish Heterobranchuslongifilis (Siluriforms, Clariidae). Fish Physiology and Biochemistry 21, 287–301.

Huss, H.H., 1988. Fresh fish-quality and quality changes. In: FAO Fisheries Series No.29. FAO, Rome, p. 40.

494 _I.Y. Genç et al. / Journal of Food Engineering 115 (2013) 486–494

Huss, H.H., 1995. Quality and quality changes in fresh fish, FAO Fisheries TechnicalPaper, 348, 195pp.

Huss, H.H., Jørgensen, L.V., Vogel, B.F., 2000. Control options for Listeriamonocytogenes in seafoods. International Journal of Food Microbiology 62,267–274.

ICMSF, 1986. Sampling plans for fish and shellfish. In: ICMSF (Ed.), Microorganismsin Foods. Sampling for Microbiological Analysis: Principles and ScientificApplications, second ed. University of Toronto Press, Toronto, Canada, pp.181–196.

Inacio, P., Bernardo, F., Vaz-Pirez, P., 2003. Effect of washing with tap water andtreated sea water on the quality of whole scad (Trachurus trachurus). EuropeanFood Research and Technology 217, 406–411.

ISO, 2001. ISO 17410: Microbiology of Food and Animal Feeding Stuffs – HorizontalMethod for the Enumeration of Psychrotrophic Microorganisms. InternationalOrganization for Standardization, Genève, Switzerland, 7pp..

ISO, 2003. ISO 4833: Microbiology of Food and Animal Feeding Stuffs – HorizontalMethod for the Enumeration of Microorganisms – Colony-Count Technique at30 �C. International Organization for Standardization, Genève, Switzerland,9pp..

Jorgensen, B.R., Gibson, D.M., Huss, H.H., 1988. Microbiological quality and shelf lifeprediction of chilled fish. International Journal of Food Microbiology 6, 295–307.

Kim, Y.S., Yongsawatdigul, J., Park, J.W., Thawornchinsombut, S., 2005.Characteristics of sarcoplasmic proteins and their interaction withmyofibrillar proteins. Journal of Food Biochemistry 29, 517–532.

Krizek, M., Vacha, F., Varlova, L., Lukasova, J., Cupakova, S., 2004. Biogenic amines invacuum-packed flesh of carp (Cyprinus carpio) stored at different temperatures.Food Chemistry 88, 185–191.

Ladrat, C., Verrez-Bagnis, V., Noe, J., Fleurence, J., 2003. In vitro proteolysis ofmyofibrillar and sarcoplasmic proteins of white muscle of sea bass(Dicentrarchus labrax L.): effects of cathepsins B, D and L. Food Chemistry 81,517–525.

Lonergan, E.H., Lonergan, S.M., 2005. Mechanisms of water-holding capacity ofmeat: the role of postmortem biochemical and structural changes. Meat Science71, 194–204.

Mahy, M., Van Eyckden, L., Oosterlinck, A., 1994. Evaluation of uniform color spacesdeveloped after the adoption of CIELAB and CIELUV. Color ResearchApplications 19, 105–121.

Martins, I., Porto, A., Oliveira, L., 2006. Tabela de composição de alimentos. InstitutoNacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal.

Monfort, M.C., 2010. Present market situation and prospects of meagre(Argyrosomus regius), as an emerging species in Mediterranean aquaculture.Studies and Reviews General Fisheries Commission for the Mediterranean, 89.FAO, Rome, 28pp..

Naylor, R.L., Goldburg, R.J., Primavera, J.H., Kautsky, N., Beveridge, M.C.M., Clay, J.,Folke, C., Lubshenco, J., Mooney, H., Troell, M., 2000. Effect of aquaculture onworld supplies. Nature 45, 1017–1024.

Özogul, F., Taylor, K.D.A., Quantick, P., Özogul, Y., 2000. Chemical, microbiologicaland sensory evaluation of Atlantic herring (Clupea harengus) stored in ice,modified atmosphere and vacuum pack. Food Chemistry 71, 267–273.

Özogul, F., Polat, A., Özogul, Y., 2004. The effects of modified atmosphere packagingand vacuum packaging on chemical, sensory and microbiological changes ofsardines (Sardina pilchardus). Food Chemistry 85, 49–57.

Pastoriza, L., Sampedro, G., Herrera, J.J., Cabo, M.L., 1996. Effect of modifiedatmosphere packaging on shelf-life of iced fresh hake slices. Journal of theScience of Food and Agriculture 71, 541–547.

Philips, C.A., 1996. Modified atmosphere packaging and its effects on themicrobiological quality and safety of produce. International Journal of FoodScience and Technology 31, 463–479.

Poli, B.M., Parisi, G., Zampacavallo, G., Iurzan, F., Mecatti, M., Lupi, P., Bonelli, A.,2003. Preliminary results on quality and quality changes in reared meagre(Argyrosomus regius): body and fillet traits and freshness changes in refrigeratedcommercial-size fish. Aquaculture International 11, 301–311.

Poli, B.M., Messini, A., Parisi, G., Scappini, F., Vigiani, V., Giorgi, G., Vincenzini, M.,2006. Sensory, physical, chemical and microbiological changes in European seabass (Dicentrarchus labrax) fillets packed under modified atmosphere/air orprepared from whole fish stored in ice. International Journal of Food Scienceand Technology 41, 444–454.

Randell, K., Hattula, T., Ahvenainen, R., 1997. Effect of packaging method on thequality of rainbow trout and Baltic herring fillets. Labensmittel-Wissenchaftund Technologie 30, 56–61.

Ross, T., 1996. Indices for performance evaluation of predictive models in foodmicrobiology. Journal of Applied Bacteriology 81, 501–508.

Ross, T., Dalgaard, P., 2004. Secondary models. In: McKeller, R.C., Lu, X. (Eds.),Modeling Microbial Responses in Foods. CRC Press, Boca Raton, USA, pp. 63–150.

Rotabakk, B.T., Birkeland, S., Lekang, O.I., Sivertsvik, M., 2008. Enhancement ofmodified atmosphere packaged farmed Atlantic halibut (Hippoglossushippoglossus) fillet quality by soluble gas stabilization. Food Science andTechnology International 14 (2), 179–186.

Sigurgisladottir, S., Ingvarsdottir, H., Torrissen, O.J., Cardinal, M., Hafsteinsson, H.,2000. Effects of freezing/thawing on the microstructure and the texture ofsmoked Atlantic salmon (Salmo salar). Food Research International 33, 857–865.

Sigurgisladottir, S., Sigurgardottir, M.S., Ingvarsdottir, H., Torrissen, O.J.,Hafsteinsson, H., 2001. Microstructure and texture of fresh and smokedAtlantic salmon, Salmo salar L., fillets from fish reared and slaughtered underdifferent conditions. Aquaculture Research 32, 1–10.

Stamatis, N., Arkoudelos, J.S., 2007a. Effect of modified atmosphere and vacuumpackaging on microbial, chemical and sensory quality indicators of fresh,filleted Sardina pilchardus at 3 �C. Journal of the Science of Food and Agriculture87, 1164–1171.

Stamatis, N., Arkoudelos, J., 2007b. Quality assessment of Scomber colias japonicusunder modified atmosphere and vacuum packaging. Food Control 18, 292–300.

Szczesniak, A.A., 2002. Texture is a sensory property. Food Quality and Preference13, 215–225.

Tejada, M., Huidobro, A., 2002. Quality of farmed gild head seabream (Sparus aurata)during ice storage related to slaughter method and gutting. European FoodResearch and Technology 215, 1–7.

Torrieri, E., Carlino, P.A., Cavella, S., Fogliano, V., Attianese, I., Buonocore, G.G., Masi,P., 2011. Effect of modified atmosphere and active packaging on the shelf-life offresh bluefin tuna fillets. Journal of Food Engineering 105, 429–435.