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

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

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, Italyb 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, Italyc Department of Production Sciences and Innovation of Applied Agricultural Mediterranean Systems, University of Foggia, Via Napoli, 25, 71100 Foggia, Italy

⁎ Corresponding author. Department of Food ScienNapoli, 25, 71100 Foggia, Italy. Tel./fax: +39 881 589 2

E-mail address: ma.delnobile@unifg.it (M.A. Del Nob

0168-1605/$ – see front matter © 2009 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2009.07.024

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 May 2009Received in revised form 15 July 2009Accepted 20 July 2009

Keywords:Blue fish burgerEssential oilMAPShelf life

The combined effects of three essential oils [thymol, lemon extract and grapefruit seed extract (GFSE)] andmodified atmosphere packaging conditions (MAP) on quality retention of blue fish burgers was studied anddiscussed. In particular, samples were packaged in air and in three different gas mix compositions: 30:40:30O2: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 ofbiogenic amines was also evaluated.The obtained results highlight the possibility to improve the microbial quality of blue fish burgers by usingvery small amount of thymol (110 ppm), GFSE (100 ppm) and lemon extract (120 ppm) in combination withMAP. Based primarily on microbiological results, the combined use of the tested natural preservatives and apackaging system characterized by a high CO2-concentration, was able to guarantee the microbialacceptability of fish burgers until the 28th day of storage at 4 °C. On the other hand, results from sensoryanalyses 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 theproduct 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 humannutrition. Blue fish is characterized by protein composition of highbiological value and fatty acid composition especially rich inpolyunsaturated fatty acids, in particular omega-3. Although bluefish has a high nutritional value, the consumer choice shows apreference for medium-fine species that are scarce in the Mediterra-nean Sea and, therefore, mostly imported. As a consequence, blue fishare actually considered as fishing waste. The lack of consumption ofparticular fish species depends on the poor knowledge of theirnutritional characteristics, their optimal conditions to clean and cookand, above all, on the useful storage conditions to prolong the shelf lifeof these products. Fresh fish is, in fact, a highly perishable product dueto its biological composition (Haard, 1992; Gram and Dalgaard, 2002):during storage, the quality of fish quickly degrades as a result ofcomplex processes in which several forms of deterioration areimplicated (Amanatidou et al., 2000; González-Fandos et al., 2005).The main cause of deterioration is the microbial activity of typical

ce, University of Foggia, Via42.ile).

l rights reserved.

spoilage seafood microorganisms that limits the shelf life of packed aswell as unpacked fresh fish (Dalgaard, 1995; Fraser and Sumar, 1998;Gram and Dalgaard, 2002; Gram and Huss, 1996). Considerableresearch has been directed toward using various preservationstrategies to preserve or prolong the shelf life of fresh fish and themost suitable technology appears to be the modified atmospherepackaging (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 suggestthat MAP in combination with refrigeration could provide asubstantial shelf life extension of fresh fish. Nevertheless, it is worthnoting 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/ornatural preservatives (antimicrobials and antioxidants) have beenalso 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 preservationmethods 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

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 subsequenteffect 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 ourknowledge, on blue fish-based burgers.

In order to valorise fishing waste and move towards blue fishconsumption, in this study a new seafood product, represented by ablue fish-based burger, was developed and studied. In previousrelated works (Corbo et al., 2008, 2009a), among different EOs tested,thymol, lemon extract and grapefruit seed extract (GFSE) wereidentified as successful active natural preservatives to improve themicrobial stability of minimally processed fresh fish burgers. Inparticular, an optimal composition of these three antimicrobialcompounds (110 ppm of thymol, 100 ppm of GFSE and 120 ppm oflemon extract), able to increase (by about 40%) the microbiologicalacceptability limit of fish burgers stored under refrigeration andpackaged in air, was determined (Corbo et al., 2008).

This study was mainly initiated to evaluate the combined effect oftheMAP and the above-mentioned EOs (at their optimal composition)on the quality retention of blue fish burgers stored at 4 °C, in order toidentify a suitable preservation strategy. In particular, quality decay ofthe investigated products was assessed by monitoring three qualitysub-indices: nutritional, microbiological and sensorial.

2. Materials and methods

2.1. Raw material

Specimens of mackerel (Scomber japonicus) and hake (Merlucciusmerluccius), 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-coldwater (hypothermia) and packed in insulated polystyrene boxes withice. Then they were delivered to the laboratory within 2 h from themoment 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 containingthe three tested compounds was prepared with higher compoundconcentrations (2750 ppm of thymol, 2500 ppm of GFSE and3000 ppm of lemon extract), to enable greater subsequent dilutionof the samples. In order to enhance their water solubility, thecompounds were dissolved in ethyl alcohol (95%) and then dilutedwith distilled water (50%, v/v). The active solution was freshlyprepared 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 toobtain a mackerel-hake ratio of 70:30 (w/w). Mincing was performedby a domestic food processor (Multichef, Ariete, Firenze, Italy). Theobtained fish patty was homogenized in a bowl mixer with a spiraldough hook for 5 min and separated into two batches. An aliquot ofthe antimicrobial compounds solution was added to one batch inorder to obtain a final concentration of 110 ppm of thymol, 100 ppmof GFSE and 120 ppm of lemon extract (ACT samples), and thenmixedagain for 5 min, according to our previous work (Corbo et al., 2008a).As control samples, the same amount of 50% hydro-alcoholic solutionwas added to the second batch (CNTR samples). Mini fish burgers

were prepared by hand (25 g, 50–60 mm diameter) and packed inNylon/Polyethylene bags (95 µm, Tecnovac, San Paolo D'Argon,Bergamo, Italy) by means of S100-Tecnovac equipment. The bagswere 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, asspecified by the manufacturer. The samples were packaged in air andin three different gas mix compositions: 30:40:30 O2:CO2:N2, 50:50O2:CO2 and 5:95 O2:CO2. During storage at 4 °C, nutritional analysesand determination of biogenic amines were performed after 0, 14 and28 days. Microbiological, sensory analyses and determination of pHwere 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 wereanalyzed for nutritional analysis. The water content of burgers wasdetermined using a 5 g sample by heating at 105 °C to constant weight(AOAC, 2000). Fat was determined by the Soxhlet method, usingSoxtec 2055 (Foss, Denmark) according to AOAC procedures (AOAC,2000). Total nitrogen was determined by the Kjeldahl procedure inKjeltec (Foss, Denmark) and converted to crude protein by multi-plying by 6.25 (AOAC, 2000). Ash was determined as the remnantweight after calcination of an 8 g sample at 550 °C during 3 h (AOAC,2000). The fat and protein contents were expressed as percentage ondry matter (%). Lipid extraction followed the Bligh and Dyer method(Bligh and Dyer, 1959). Methyl esters were prepared by transmethy-lation using 2 MKOH inmethanol according to IUPACmethod (IUPAC,1987). Fatty acid methyl esters (FAME) were quantified by gas-chromatography. The fatty acids composition was analyzed by GC6890N (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 constantflow of 0.8 ml/min; the injector and the detector temperature wereset at 240 °C and 260 °C, respectively. The column temperature was60 °C, held for 5 min, raised to 180 °C at a rate of 25 °C/min andfurther up to 230 °C at a rate of 6 °C/min. The split usedwas 1:50. Fattyacids were identified by comparing the retention times of FAME witha standard 37 component FAME mixture (Supelco, Milan, Italy). Theresults 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 dilutedwith 225 ml of 0.1% peptone water with salt (0.85% NaCl) in aStomacher bag (Seward, London, England) and homogenized for1 min in a Stomacher Lab Blender 400 (Seward). Serial dilutions of fishhomogenates were plated on the surface of the appropriate media inPetri dishes. The media and the conditions used were: Plate CountAgar (PCA) incubated at 30 °C for 48 h for aerobic plate count (APC)(International Commission on Microbiological Specifications forFoods, ICMSF, 1986); Pseudomonas Agar Base (PAB), with addedCephaloridine Fusidin Cetrimide (CFC) supplement, incubated at25 °C for 48 h for Pseudomonas spp.; pour plated Iron Agar (IA),incubated at 25 °C for three days, for hydrogen sulphide-producingbacteria (HSPB); spread plated chilled IA, supplemented with 5 g/lNaCl and incubated at 15 °C for 7 days, for psychrotolerant and heatlabile aerobic bacteria (PHAB). The conditions used during the countsof HSPB and PHAB were those suggested by the Nordic Committee onFood Analyses, with regard to fish and fishery products (NCFA, 2006).All media were supplied from Oxoid (Milan, Italy). Microbiologicaldata were log-transformed and expressed as the average of tworeplicates. The variability coefficient, expressed as a percentage ratiobetween the standard deviation and the mean value was less than 7%.

The measurement of pH, conducted in duplicate, was performedon the first homogenized dilution of the fish samples with a pHmeter(Crison, Barcelona, Spain).

283M.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 HighPerformance Ion Chromatography system (HPIC), as described byCinquina et al. (2004). Two grams of homogenized fishwere extractedwith 5 ml of 20 mM methanesulfonic acid (MSA) for 10 min andcentrifuged 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 mMMSA. The obtained extractswere 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 suppressorcurrent was set at 74 mA. The eluent was MSA and the gradientconditions consisted of: 3 to 18 mmol/l from 0 to 10 min, 18 mmol/lfor 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 agingbetween 22 and 38 years (students and researchers of the Departmentof 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 qualityonly taking into account its odor, texture and drip loss. A score of 2 wasused as the threshold for product acceptability. During the evaluationsessions, the samples were coded by a letter and presented in randomorder.

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 andWilk, 1965). Then, data were processed by ANOVA, usingthe GLM procedure of Statistical Analysis System Version 8.1 (SASInst., Cary, USA) for repeated measures, using the treatment asrepeated factor. When significant effects were found (at Pb0.05), theStudent t-test was used to locate significant differences betweenmeans.

To assess the effectiveness of investigated packaging strategies onthe microbial stability of fish burgers, the viable cell concentration at28 days of storage (i.e., the extent of the period of observation) wascalculated for each microbial group, according to the Gompertzequation as re-parameterized by Corbo et al. (2006):

logðNðtÞÞ = logðN28Þ− A⋅exp −exp ðμmax⋅2:71Þ⋅λ − 28

A

� �+ 1

� �� �

+ A⋅exp −exp ðμmax⋅2:71Þ⋅λ − t

A

� �+ 1

� �� �ð1Þ

where: N(t) is the viable cell concentration (CFU/g) at time t; N28 isthe viable cell concentration at 28 days storage (CFU/g); A is related tothe difference between the decimal logarithm of bacterial loadattained at the stationary phase and the decimal logarithm of theinitial value of cell concentration; μmax is the maximal specific growthrate (Δlog CFU/g/day); λ is the lag time (day); t is the storage time

(day). The log N28 value was determined by fitting Eq. (1) to theexperimental data.

Regarding sensory analysis, the Sensory Acceptability Limit (SAL),defined as the time at which the fresh fish burger overall sensorialquality reaches its threshold value, was also calculated by using there-parameterized Gompertz equation (Corbo et al., 2006):

SAðtÞ = SAmin− AQ ⋅exp −exp ðμQmax⋅2:71Þ⋅

λQ− SALAQ

" #+ 1

( )( )

+ AQ ⋅exp −exp ðμQmax⋅2:71Þ⋅

λQ − tAQ

" #+ 1

( )( )ð2Þ

where: SA(t) is the fresh fish burgers sensory attribute at time t; AQ isrelated to the difference between the value of the sensory attributeattained at the stationary phase and the initial value of the sensoryattribute; µmax

Q is the maximal rate at which SA(t) changes; λQ is thelag time; SAmin is the threshold value of the sensory attribute; SAL isthe time at which SA(t) is equal to SAmin; and t is the storage time. Thevalue of SAmin is set up to 2.

3. Results and discussion

In this study the nutritional, microbiological and sensorial qualitysub-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 thehuman diet being rich inminerals, vitamins and polyunsaturated fattyacids (PUFA) (Karakoltsidis et al., 1995). Nutritional quality of fishburgers was unaffected by MAP and active compounds. As shown inTable 1, no differences emerged from analyses of moisture, fat, proteincontent 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 dominanceof two classes: saturated fatty acids (SFA) and PUFA. The last-mentioned are reported to improve the nutritional value and protectagainst diseases (Moreira et al., 2001). PUFA/SFA ratio found in theanalyzed fish burgers ranged from 1.01 to 1.34; the minimum value ofPUFA/SFA ratio recommended is 0.45 (Department of Health, 1994),which is lower than those obtained from all fish burgers speciesstudied in this work. The n6/n3 ratio was affected by treatments, evenif such difference may be ascribed to the variability among thesamples because neither packaging treatment nor time of treatmentmodified fatty acid composition of fish burgers. The UK Department ofHealth recommends an ideal ratio of n6/n3 of 4.0 at maximum(Department of Health, 1994). Values higher than themaximum valueare harmful to health and may promote cardiovascular diseases(Moreira et al., 2001). In the current study, the ratio of n6/n3 wasfound to range from 0.35 and 0.20 (Table 1), lower than themaximumthreshold (Department of Health, 1994).

3.2. Microbial quality

Fig. 1 shows the APC viable cell concentration plotted as a functionof storage time for all investigated samples. As it can be inferred, thehighest APC viable cell concentration was observed for the samplewithout antimicrobial compounds (control, CNTR), suggesting thatthe proposed packaging strategies were all effective in inhibiting theAPC growth. To quantitatively determine the efficiency of theproposed packaging strategies, Eq. (1) was fitted to the experimental

Table 1Values (Means±Standard Error) of chemical composition of CNTR samples (fish burgers without antimicrobial compounds) and ACT samples (fish burgers with antimicrobialcompounds), 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(control)

ACT-AIR

CNTR-30:40:30

ACT-30:40:30

CNTR-50:50

ACT-50:50

CNTR-5:95

ACT-5:95

SE Effects, P

Treatment

Moisture, % 0 74.97 73.60 74.97 73.60 74.97 73.60 74.97 73.60 2.30 NS28 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 NS28 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 NS28 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 NS28 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 NS28 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 NS28 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 NS28 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; ⁎⁎, Pb0.01.

Table 2Maximum cell load observed in CNTR samples (fish burgers without antimicrobial

284 M.A. Del Nobile et al. / International Journal of Food Microbiology 135 (2009) 281–287

data to determine the APC cell load at the end of the period ofobservation (i.e., log N28

APC). The log N28APC values obtained are listed in

Table 2. As it can be inferred from data shown in the table, there is asubstantial difference in the log N28

APC value between CNTR and ACT-AIR, which was about two orders of magnitude, thus suggesting thatthe tested active compounds inhibited the growth of APC. Data listedin 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 asynergistic way to inhibit the APC growth. In fact, the samplescontaining the antimicrobial agents showed the log N28

APC value thatwas at least one order of magnitude lower than the correspondingsample packed under MAP condition. For fresh water and marinespecies, the microbiological limit recommended by the ICMSF (1986)for APC at 30 °C is 7 log/g or log/cm2. Since in all samples (includingCNTR), APC never exceeded this value, the fish burgers could beconsidered microbiologically acceptable under all proposed condi-tions, even after 28 days of storage at 4 °C. However, it is worth notingthat spoiled marine fish is characterized by the development ofoffensive, 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 numberand types of bacteria isolated because of differences in nutrient andsalt requirements (as well as in terms of growth temperature) of the

Fig. 1. Evolution during storage at 4 °C of Aerobic Plate Count (APC) viable cellconcentration in the fish burgers. The curves are the fitting of Eq. (1) to theexperimental data. (○) Control in air; (▲) sample with active compounds in air; (◇)control under MAP 30:40:30 O2:CO2:N2; (●) sample with active compounds underMAP 30:40:30 O2:CO2:N2; (■) control under MAP 50:50 O2:CO2; (□) sample withactive compounds under MAP 50:50 O2:CO2; (◆) control under MAP 5:95 O2:CO2.

various SSOs (Nickelson and Finne, 1992). For this reason, counts ofPseudomonas spp., hydrogen sulphide-producing bacteria (HSPB)and psychrotolerant and heat labile aerobic bacteria (PHAB) werealso performed.

Fig. 2 shows the Pseudomonas spp. viable cell concentrationplotted as a function of storage time. Also in this case a substantialdifference between the CNTR and the other samples can be observed.In fact, the Pseudomonas spp. maximum population density of CNTRsample was always the highest one among the tested samples. Inorder to determine the efficacy of tested packaging strategies, thePseudomonas spp. cell load at the end of the storage time (i.e., logN28Pseudomonas) was also determined by fitting Eq. (1) to the experi-

mental data. The log N28Pseudomonas values are listed in Table 2. In this

case, results similar to those obtained for APC can be observed. It isworth 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 wasalways below the detection limit. These results allowed us to considerPseudomonas spp. of low importance tomicrobial quality evaluation ofblue fish burgers, since the calculated log N28

Pseudomonas values are very

compounds) and ACT samples (fish burgers with antimicrobial compounds), packed inAIR and in three differentMAP (30:40:30 O2:CO2:N2; 50:50 O2:CO2; 5:95 O2:CO2), at theend of the storage (28 days) at 4 °C, for each microbial group investigated.

Sample log N28APC log N28

Pseudomonas log N28HSPB log N28

PHAB

CNTR(control)

6.37±4.45·10−2 6.22±0.12 6.99±0.19 6.20±0.16

ACT-AIR 4.56±0.00 4.21±3.32·10−2 6.05±0.20 6.20±0.17CNTR-30:40:30

5.24±6.77·10−2 5.31±0.12 6.22±0.17 6.12±0.11

ACT-30:40:30

3.76±7.99·10−2 4.48±0.16 5.43±0.13 5.28±0.13

CNTR-50:50

4.92±0.22 4.84±0.13 5.25±0.14 6.39±0.11

ACT-50:50

3.65±5.36·10−2 –a 5.32±8.05·10−2 5.14±0.13

CNTR-5:95

4.25±8.51·10−2 –a 5.11±8.01·10−2 5.38±6.37·10−2

ACT-5:95

–a –a 4.91±0.16 4.95±0.11

Data are presented±standard deviation.APC, Aerobic Plate Count; HSPB, hydrogen sulphide-producing bacteria;PHAB, psychrotolerant and heat labile aerobic viable count.

a No growth.

Fig. 2. Evolution during storage at 4 °C of Pseudomonas spp. viable cell concentration inthe fish burgers. The curves are the fitting of Eq. (1) to the experimental data. (○)Control in air; (▲) sample with active compounds in air; (◇) control underMAP 30:40:30 O2:CO2:N2; (●) sample with active compounds under MAP 30:40:30O2:CO2:N2; (■) control under MAP 50:50 O2:CO2.

Fig. 4. Evolution during storage at 4 °C of psychrotolerant and heat labile aerobic viablecount (PHAB) viable cell concentration in the fish burgers. The curves are the fitting ofEq. (1) to the experimental data. (○) Control in air; (▲) sample with active compoundsin air; (◇) control under MAP 30:40:30 O2:CO2:N2; (●) sample with active compoundsunder MAP 30:40:30 O2:CO2:N2; (■) control under MAP 50:50 O2:CO2; (□) samplewith active compounds under MAP 50:50 O2:CO2; (◆) control underMAP 5:95 O2:CO2;(△) sample with active compounds under MAP 5:95 O2:CO2.

285M.A. Del Nobile et al. / International Journal of Food Microbiology 135 (2009) 281–287

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 numbersfor all investigated samples. The efficacy of the tested packagingsolutions was assessed by comparing the log N28

HSPB values, whichwere determined according to the procedure described above. Theobtained values are listed in Table 2. As for the two microbial groupsdiscussed beforehand, the antimicrobial compounds as well as the 3MAP conditions tested in this work inhibited the growth of HSPB.Moreover, MAP and active compounds acted in a synergistic wayagainst HSPB; the lowest cell loads, after 28 days of storage, wereobserved in all ACT samples packaged under MAP (log N28

HSPB wasabout 5 log CFU/g). These cell numbers indicated acceptable microbialquality for the blue fish burger investigated, given that HSPB (mainlyrepresented by Shewanella species) are considered the strongestspoilers of seafood from cold and temperate water and that significantamounts of sulfur compounds are being produced (and spoilage offish occurs) only when their numbers exceed 6 log CFU/g (Gram et al.,1987).

Fig. 4 shows the PHAB viable cell concentration plotted against thestorage time for all the investigated samples. Also in this case, resultshighlighted a synergistic effect of MAP and antimicrobial compoundson the growth of the above-mentioned microorganisms, as it can bemore easily inferred from data shown in Table 2. PHAB include Pho-tobacterium phosphoreumwhich is CO2-resistant and often dominatesthe 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 theexperimental data. (○) Control in air; (▲) sample with active compounds in air; (◇)control under MAP 30:40:30 O2:CO2:N2; (●) sample with active compounds underMAP 30:40:30 O2:CO2:N2; (■) control under MAP 50:50 O2:CO2; (□) sample withactive 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 andMAP (Sivertsvik et al., 2002). Several studieshave shown that 7 log CFU/g of this microorganism is required toproduce fish spoilage (Dalgaard et al., 1993; Dalgaard, 1995) and,therefore, the final cell numbers observed at the end of the storage inall samples (including CNTR) indicate an acceptable microbial quality.

Moreover, as it can be also observed in Figs. 1–4, all the testedpackaging strategies were able to severely affect both the lag phaseand the growth rate of each investigated microbial group. This aspectwas more evident for the pseudomonads count whose growth ratedecreased quite 10 times (from 2.28 of CNRT to about 0.20 during thetreatments) (data not shown).

Concerning the packaging treatments, results obtained in thisstudy are in good agreement with other studies reported in literature:MAP with 5:95 O2:CO2 resulted in the most effective for the inhibitionof all the investigated microbial groups. This fact may be attributed tothe inhibitory effect created by the presence of high CO2-concentra-tion on microbial growth. Actually, it is well known that in theabsence of O2 and in the presence of CO2, a bacteriostatic effect isexerted 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 andHuss, 1996; Sivertsvik et al., 2002). From the above results, it is alsoobvious to infer that the active compounds acted in a synergistic waywith MAP to delay microbial growth: blue fish burgers weremicrobiologically acceptable after 28 days of storage at 4 °C withcell loads considerably lower than control sample. Tassou et al. (1996)reported that the treatment of sea bream with olive oil, lemon juiceand oregano, followed by storage under MAP (40:30:30 CO2:O2:N2),showed bacteriostatic effect on the autochthonous flora. Mejlholmand Dalgaard (2002) reported that oregano oil (0.05% v/w) extendedthe shelf life of naturally contaminated MAP cod fillets until 21–26 days at 2 °C. Mahmoud et al. (2004) observed that the dippingtreatment of carp fillets in 1% (carvacrol and thymol) mixtureextended the shelf life of the product, packaged in air, for 12 days at5 °C. According to Harpaz et al. (2003), the addition of oregano andthyme at 0.05% v/v can considerably slow down the process ofspoilage of Asian sea bream. Goulas and Kontominas (2007), studyingthe combined effect of MAP (40:30:30 CO2:O2:N2) and oregano oil onthe shelf life of sea bream fillets stored under refrigeration, observed aconsiderable slowing down of fish spoilage. In a related studyconducted previously (Corbo et al., 2008), 110 ppm of thymol,100 ppm of GFSE and 120 ppm of lemon extract, guaranteed astatistically significant increase (of about 40% if compared to thecontrol sample) of the microbial acceptability limit of sea breamburgers, after 10 days of storage at 5 °C in air. Results obtained in this

Table 3Sensory Acceptability Limit (SAL) obtained in CNTR samples (fish burgers withoutantimicrobial compounds) and ACT samples (fish burgers with antimicrobialcompounds), packed in AIR and in three different MAP (30:40:30 O2:CO2:N2; 50:50O2:CO2; 5:95 O2:CO2), at the end of the storage (28 days) at 4 °C, for each sensorialattribute evaluated.

Sample SALO [day] SALT [day] SALDL [day] SALOQ [day]

CNTR (control) 7.82±0.28 N28 N28 8.21±0.57ACT-AIR 15.54±0.57 N28 N28 15.05±0.43CNTR-30:40:30 15.52±0.48 20.16±1.74 N28 17.67±0.62ACT-30:40:30 20.49±0.81 21.83±1.70 N28 19.47±1.06CNTR-50:50 17.73±0.48 22.30±1.30 26.51±0.58 17.67±0.62ACT-50:50 21.12±0.46 24.28±1.74 27.09±1.02 22.09±1.22CNTR-5:95 17.88±0.40 N28 23.74±1.62 20.96±0.48ACT-5:95 22.65±0.79 N28 N28 24.56±1.57

Data are presented±standard deviation.O, Odour; T, Texture; DL, Drip Loss; OQ, Overall Quality.

286 M.A. Del Nobile et al. / International Journal of Food Microbiology 135 (2009) 281–287

study highlight the possibility to improve themicrobial quality of bluefish burgers by using very small amount of thymol, GFSE and lemonextract in combination with MAP. In particular, the combined use ofthe tested natural preservatives and a packaging system characterizedby a high CO2-concentration, was able to guarantee the microbialacceptability of fish burgers until the 28th day of storage at 4 °C.

Concerning biogenic amines analyses, no amines were observedduring the entire observation period in all the investigated samples.

Finally, it is worth noting that in all samples pH value was about6.2 at the beginning of the storage and about 6.4 after 28 days; nosignificant differences were observed among the samples during theentire observation period (data not shown).

3.3. Sensorial quality

Active antimicrobial substances, used as preservatives in foods,often impart a certain flavor to products. On fish, carvacrol is said toproduce a “warmly pungent” aroma (Kim et al., 1995), thyme andoregano oils spread on whole Asian sea bass imparted herbal odor(Harpaz et al., 2003) and oregano oil on cod fillets produced adistinctive but pleasant flavor (Mejlholm and Dalgaard, 2002). On theother hand, it is well established that the use of MAP, especially whenCO2-concentration is high, could have an unpleasant effect on somesensorial parameters of the packed product (excessive exudate,softening of texture, discoloration, etc.) (Sivertsvik et al., 2002).Therefore, in order to evaluate the effects of the proposed preserva-tion strategies on the sensory acceptability of blue fish burgers, thesensory quality loss was also assessed. As an example, Fig. 5 shows thefresh fish burgers overall quality plotted against the storage time forall the investigated samples. As it can be inferred, all samples show asigmoidal trend. Data shown in Fig. 5 also highlight that there is amarked difference between CNTR sample and the other samplestested in this work. In particular, after the lag phase (about threedays), where all the samples show similar overall quality values, theoverall quality of CNTR sample decreased more rapidly than that ofthe other investigated samples, suggesting that all the proposedpackaging strategies are effective in minimizing the sensory qualityloss of the investigated food product. The sensory attributesmonitored in this work showed a similar trend (data not shown).To quantitatively determine the efficacy of the proposed packagingstrategies, the SAL value of the overall quality along with that of eachmonitored attribute, was determined according to the procedurereported above. Curves shown in Fig. 5 result from the fittingprocedure, whereas the SAL values obtained are listed in Table 3. Asit can be seen, the selected active compounds were effective in

Fig. 5. Blue fish burger overall quality plotted as a function of storage time for the fishburgers. The curves are the fitting of Eq. (2) 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 underMAP 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 5:95O2:CO2.

increasing the SAL values, even when they were used alone. In fact,the sample containing the active compounds and packed underordinary atmosphere showed an increase in the SALOQ (SAL of overallquality) value of about 100%, if compared to CNTR. As expected, theMAP conditions tested in this work were all effective in increasing theSALOQ value. Moreover, among the three tested MAP, 5:95 O2:CO2 ismost effective in slowing down the sensory quality loss of packedfresh fish burgers, as also observed for microbial quality. Also in thiscase, results show a synergistic effect of MAP and active compounds:in fact, the SALOQ values of samples whereMAP and active compoundsare used in combination are always higher than those of sampleswhere they are used alone. As also observed for the microbial qualityevaluation, the combined use of the tested natural preservatives and apackaging system characterized by a high CO2-concentration, resultedthe most effective preservation strategy in guaranteeing the sensoryquality of fish burgers.

It is worth noting that for all the tested samples, the odor is theattribute that limits the packed fresh fish burgers overall quality,suggesting that to further prolong the shelf life of the investigatedcommodity off-odors scavengers could be advantageously used.

4. Conclusions

Results obtained in this study highlight the possibility toimprove the quality of blue fish burgers by using very smallamount of thymol (110 ppm), GFSE (100 ppm) and lemon extract(120 ppm) in combination with MAP. From a microbiological pointof view, the combined use of the tested natural preservatives and apackaging system characterized by a high CO2-concentration, wasable to guarantee the microbial acceptability of fish burgers untilthe 28th day of storage at 4 °C. However, the sensorial quality losslimited the burgers shelf life to about 22–23 days. As compared toseveral 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 MAPis an inexpensive and uncomplicated method to extend shelf life ofpacked seafood. The investigated natural preservatives are relativelycheap and the addition of only 100 ppm of each compound is likelyto be cost-effective. On the other hand, the use of MAP is a practicaland economic technique, realized by small technical instruments.

Acknowledgment

This study was financially supported by Ministero dell'Economia edelle Finanze, Ministero dell'Istruzione, dell'Università e della RicercaScientifica e Tecnologica e l'Assessorato Bilancio e ProgrammazioneRegione Puglia by the programme “Accordo di Programma Quadroin Materia di Ricerca Scientifica della Regione Puglia – Progetto

287M.A. Del Nobile et al. / International Journal of Food Microbiology 135 (2009) 281–287

Esplorativo – Title: Valorizzazione di pescato di basso valorecommerciale attraverso trasformati ittici di IV gamma”.

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