Food Research International 42 (2009) 1147–1152

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Food Research International

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Role of biogenic amines as index of freshness in beef meat packed with differentbiopolymeric materials

Fernanda Galgano *, Fabio Favati, Malvina Bonadio, Vitina Lorusso, Patrizia RomanoDipartimento di Biologia, DBAF, Università degli Studi della Basilicata, Viale dell’Ateneo Lucano, 10, 85100 Potenza, Italy

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

Article history:Received 2 March 2009Accepted 22 May 2009

Keywords:Biogenic aminesBiopolymersCadaverineFresh beef meatPackagingShelf-lifeTyramine

0963-9969/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodres.2009.05.012

* Corresponding author. Tel.: +39 0971 205570; faxE-mail address: fernanda.galgano@unibas.it (F. Ga

The main objectives of this work were to evaluate the chemical and microbiological fresh beef meat qual-ity packed in aerobic atmosphere with biopolymers, to investigate the possible role of biogenic amines(BAs) as indicators of spoilage in fresh beef meat stored at 4 �C for 8 days. The results of this researchhighlighted that for fresh meat packaging it could be possible to replace the PS tray/PVC film system, withan expanded PLA biopolymeric tray heat-sealed with a biopolymeric film, characterized by a negligibleenvironmental impact in comparison with the use of synthetic plastic materials. The storage time differ-entiated the meat samples on the basis of pH and microbiological characteristics. With regard to BAs,tyramine and cadaverine resulted strongly influenced by the storage time, and to a less extent putrescineand spermidine. Tyramine and cadaverine could be used as spoilage indexes of fresh beef meat chilledand packed in aerobic atmosphere with biopolymers.

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1. Introduction

The utilization in food packaging of materials compostable andbiodegradable, such as biopolymers, may represent a good techno-logical opportunity for reducing the amount of plastic wastes, withpositive consequences on the environment (Alves et al., 2006; Cut-ter, 2006). Biopolymers are obtained from renewable resources andare generally characterized by a relatively high permeability to O2

and H2O; therefore, their utilization in food packaging is of interestmainly for those foodstuffs, such as meat, having few gas andwater vapor barrier requirements, with a shelf-life limited to afew days of storage (Avella et al., 2005).

Packaging of fresh beef meat can be realized in several ways:under vacuum, under air, in very permeable packages, usuallymade using expanded polystyrene (PS) trays wrapped with PVCfilm or in an O2 enriched atmosphere in a barrier packaging mate-rials. Interesting results have been recently also reported regardingthe use of biopolymeric materials for meat packaging in combina-tion with the use of PS trays or protective atmosphere (Cannarsi,Baiano, Marino, Sinigaglia, & Del Nobile, 2005; Cornini, Puglisi,Vannini, & Fava, 2005).

The shelf-life of fresh meat is generally evaluated by monitoringthe microbiological and sensorial changes of the product duringstorage time. In addition to these traditional methods, chemicalmetabolites produced in the microbiological deterioration of the

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food products, such as biogenic amines (BAs), have been also pro-posed as quality indicators of meat (Rokka, Eerola, Smolander,Alakomi, & Ahvenainen, 2004).

The formation and accumulation of BAs in foods is the result ofthe enzymatic amino acids decarboxylation due to microbial en-zymes and to tissue activity; therefore, the determination of thesecompounds is of a great interest, not only for their potential risk onhuman health, namely for histamine and tyramine, but also be-cause they could be considered indicators of food quality andfreshness, being the BAs associated to the degree of food fermenta-tion or degradation (Favaro, Pastore, Saccani, & Cavalli, 2007). TheBAs principally found in fresh and processed meat are putrescine(PUT), cadaverine (CAD), histamine (HIS) and tyramine (TYR),while natural polyamines levels, such as spermidine (SPD) andspermine (SPM) slightly change during storage or processing(Favaro et al., 2007).

The different concentration of these compounds in fresh andmeat products can be explained by the varying properties of meatsubstrates from different sources and by microbial floras with dif-ferent biochemical potentialities for metabolizing amino acids.Many Enterobacteriaceae, Pseudomonas spp. and certain Lactobacilli,Enterococci and Staphylococci are particularly involved in BAs for-mation; these amine-positive microorganisms may be naturallypresent in meat products or may be introduced by contaminationbefore, during or after processing (Rokka et al., 2004).

In addition, also the packaging system can affect the qualitativeand quantitative formation of BAs (Hernández-Jover, Izquierdo-Pulido, Veciana-Nogués, Marinè-Font, & Vidal-Carou, 1997;

1148 F. Galgano et al. / Food Research International 42 (2009) 1147–1152

Kaniou, Samouris, Mouratidou, Eleftheriadou, & Zantopoulos,2001; Krizek, Scott Smith, & Phebus, 1995; Lee & Yoon, 2001;Patsias, Chouliara, Paleologos, Savvaidis, & Kontominas, 2006;Rokka et al., 2004).

Several studies were carried out dealing with the possible use ofBAs as indicators of meat quality during storage time (Balamatsia,Paleologos, Kontominas, & Savvaidis, 2006; Hernández-Jover,Izquierdo-Pulido, Veciana-Nogués, Marinè-Font, & Vidal-Carou,1996; Patsias et al., 2006; Rokka et al., 2004; Silva & Glória,2002; Vinci & Antonelli, 2002; Kaniou et al., 2001), but to ourknowledge, at present no data are available in the scientificliterature dealing with the levels of BAs in fresh beef meat duringchilling storage and packaged with biopolymeric materials.

The aims of this work were to evaluate the fresh beef meatquality packed in aerobic atmosphere with biopolymers, from achemical and microbiological point of view, to investigate thepossible role of the BAs as indicators of spoilage in fresh beefmeat stored at 4 �C for 8 days under aerobic conditions andpacked with different biopolymeric materials and to correlatemicrobial changes in meat under the same storage conditionswith BAs formation.

2. Materials and methods

2.1. Meat sampling and packaging

Meat was obtained from six young Podolian bulls, all fed withthe same diet, and slaughtered at 18 months of age, with a meanweight of 444 ± 21 kg. Dressed carcasses were divided into twosides and chilled for 24 h at 4 �C. Afterwards, the Longissimus lum-borum muscle was chosen as representing muscles of greatestmass from the left side of each animal, and all the removed sec-tions were vacuum-packaged and aged at 4 �C until 8 days post-mortem. The muscles were then cut and for each thesis two steaksof similar size (100 g, thickness 1 cm) were packed using an ex-panded PLA tray (23 cm � 14.5 cm � 3.5 cm) having a biodegrad-able pad (Coopbox Europe SpA, Italy), and either overwrappedwith a PVC film, or heat-sealed with three different biopolymericfilms: PLA (Treophan, Germany), Mater-Bi-1, a monolayer film,and Mater-Bi-2, a coextruded tri-layer film having a bettermechanical performances. Both Mater-Bi films (Novamont, Italy)were compostable and based on polyesters partially derived fromrenewable sources. In Table 1 are reported the main technical char-acteristics of the films as reported in the technical data sheet sup-plied by the producers. For control, meat samples were packedusing PS trays with a polyolefinic pad (Coopbox Europe), wrappedwith a PVC film, hence replicating a commercially available pack-aging system for fresh meat. All samples were stored at 4 �C for8 days and the meat quality was assessed throughout the wholestorage period by using chemico-physical and microbiologicalindexes.

Table 1Principal technical characteristics of the packaging films as reported in the technicaldata sheets supplied by the film producers.

Characteristics Packaging film

PLA Mater-Bi-1 Mater-Bi-2 PVC

Thickness (lm) 25 35 35 12WVTR (g/m2 24 h)(38 �C; 100% R.H.) 175 408 842 34.1OTR (cm3/m2 24 h)(23 �C; 50% R.H.) 610 1246 2491 2940Tensile strength MD (MPa) >70 35 32 44.5

WVTR = water vapor transmission rate, OTR = oxygen transmission rate, MD = ma-chine direction.

2.2. Chemical analyses

2.2.1. Determination of pHThe determination of pH was assessed using a pH meter (Model

SA720, Orion, Milano, Italy) equipped with a probe for solids(FC200B, Hanna Instruments, Milano, Italy).

2.2.2. Analysis of biogenic aminesSamples were prepared and derivatized by modifying the meth-

od proposed by Vinci and Antonelli (2002).

2.2.2.1. Derivatization and concentration. A precisely weighed 5 gsample was homogenized with 20 mL of 6% TCA, centrifuged at12,000 g for 20 min at 4 �C and filtered through Whatman No.1 fil-ter paper. Afterwards, the supernatant was brought up to 50 mLwith distilled water. An aliquot of the extract acid (1.5 mL) wasmixed with 300 lL of a saturated NaHCO3 solution, 200 lL of NaOHand 2 mL of dansyl chloride (10 mg/mL acetone). The dansylationreaction was conducted in the dark at room temperature for15 min. Afterwards, the residual dansyl chloride was removed byadding 200 lL of 30% NH3; after 15 min at 20 �C, each samplewas brought up to 5 mL with acetonitrile and filtered through0.22 lm PTFE filter (Alltech, Milano, Italy).

2.2.2.2. HPLC analysis. The chromatographic system consisted of aVarian (Leini, Italy) 9012 pump, a Varian 9050 UV/VIS detectorand a personal computer running the chromatographic softwareSTAR 4.5 (Varian). The sample (10 lL) was injected onto a C18Gemini column (5 lm, 250 � 4.6 mm, Phenomenex, Torrance, CA)equipped with a Gemini C18 guard column (Phenomenex). Thepeaks were detected at 254 nm and the elution program consistedof the gradient system shown in Table 2, with a flow rate of 0.8 mL/min. The limits of detection for BAs were 2 mg/kg for TRYPT,0.8 mg/kg for PUT, CAD and HIS and 0.5 mg/kg for TYR, SPD andSPM.

2.3. Reagents and standard solutions

All the chemicals used were of suitable analytical grade andpurchased from Carlo Erba (Milano, Italy). Cadaverine (CAD), hista-mine (HIS), putrescine (PUT), spermidine (SPD), spermine (SPM),tryptamine (TRYPT), tyramine (TYR), dansyl chloride were ob-tained from Fluka (Milano, Italy). Amines were purchased ashydrochloride salts of the highest available purity, and the analyt-ical results were referred to the free base. A stock standard solutionwas prepared by accurately weighing about 25 mg of each aminein a 25 mL volumetric flask and bringing up to volume with 6%TCA. Five working standard solutions, containing all the aminesat concentrations ranging from 5 to 40 mg/L, were then preparedfrom the stock solution.

2.4. Microbiological analyses

Samples of 10 g of meat were dispersed in 90 mL of a 0.85%(w/v) sterile NaCl solution in a stomacher Lab-Blender (mod. 400,

Table 2HPLC elution program for the biogenic amines analysis in meat.

Time (min) A (%) B (%)

0.01 30 701.00 30 70

11.00 0 10019.00 0 10020.00 30 7025.00 30 70

A = acetonitrile, B = H2O.

F. Galgano et al. / Food Research International 42 (2009) 1147–1152 1149

PBI International, Milano, Italia). Decimals dilutions were preparedand plated on specific media. The following microbial groups wereevaluated:

� Brochotrix thermosphacta on Streptomycin Thallous Acetate Acti-dione (STAA) Agar (Oxoid, Basingstoke, England) supplementedwith streptomycin sulphate and thallous acetate, incubated at22 �C for 48 h.

� Enterobacteriaceae on Violet Red Bile Glucose Agar (VRBGA)(Oxoid) inoculated by pour plate and incubated at 30 �C for24 h in aerobic conditions.

� Pseudomonas spp. on Pseudomonas Agar Base (PAB) Oxoid) withselective supplement CFC, incubated at 25 �C for 48 h.

� Psychrotrophic aerobic bacteria, on Plate Count Agar (PCA)(Oxoid) incubated at 7 �C for 10 days.

2.5. Statistical data treatment

In order to study the effect of the storage time and packagingsystem on the meat composition, data were processed by analysisof variance (ANOVA) and the Least Significant Difference (LSD) testwas performed for comparison of means (P < 0.05). Correlationanalysis was also applied to the data. All statistical procedureswere computed using the statistical package SAS (ver. 9.1., 2005)(SAS Institute Inc., Cary, NC, USA).

3. Results and discussion

3.1. Effect of the storage time on meat quality

All the chemical and microbiological parameters considered formeat quality evaluation were influenced by the storage time (Table3), and in particular the pH value significantly increased over thestorage period (P < 0.05). A similar result has been reported by Ny-chas, Drosinos, and Board (1998), which ascribed this trend to anincrease in the production of proteases by psychrotrophic bacteria.However, in the present study no significant correlation betweenpH value and psychrotrophic bacteria count could be highlighted(Table 5).

As far as the biogenic amines, these moieties were initiallyfound in the meat samples at a concentration of about 40.6 mg/kg, and after 8 days of storage their level showed a 12% increase,reaching a value of about 46.0 mg/kg. Furthermore, this increase

Table 3Chemico-physical and microbiological parameters of fresh beef meat stored at 4 �C as a fu

Chemico-physical and microbiological parameters Storage time (days)

0 3

pH 5.68a ± 0.13 5.CAD (mg/kg fresh weight) 1.56a ± 0.03 1HIS (mg/kg fresh weight) 1.92a ± 0.18 1PUT (mg/kg fresh weight) 1.77a ± 0.08 1SPD (mg/kg fresh weight) 2.07a ± 0.15 2SPM (mg/kg fresh weight) 9.67a ± 0.58 11TRYPT (mg/kg fresh weight) 21.68a ± 2.51 22TYR (mg/kg fresh weight) 1.97a ± 0.16 3B. thermosphacta (log cfu/g) 2.05d ± 0.18 3Enterobacteriaceae (log cfu/g) 2.85d ± 0.22 4Pseudomonas spp. (log cfu/g) 2.83d ± 0.21 4Psychrotrophics (log cfu/g) 4.83d ± 0.27 6

Values are the mean of six determinations (n = 6) ± SD.Data in the same row with different superscripts are significantly different (LSD test atn.s. not significant.* Significant for P < 0.05.*** Significant for P < 0.001.

resulted to be positively correlated to the pH trend during the meatstorage (r = 0.67).

Working with cow and chicken meat, after 5 days of storage at4 �C Vinci and Antonelli (2002) have reported a total BAs content of52 and 105 mg/kg in the red and in the white meat, respectively. Inthe latter BAs concentration increased earlier and more rapidlythan in the former, and this could be explained taking into accountthe presence of shorter muscular fibers in chicken, with respect tothe muscular fibers present in cow meat. This fact could facilitatethe attack of proteins by proteolytic enzymes, with consequentmajor availability of amino acid precursors for the BAs production(Vinci & Antonelli, 2002).

As reported in Table 3, the most abundant amines in fresh beefmeat samples were TRYPT and SPM and their concentration wasnot affected by the storage time, in agreement to the data reportedby Vinci and Antonelli (2002). Conversely, in chicken meat PUT andCAD have been found to represent the most abundant amines (Na-don, Ismond, & Holley, 2001), while TRYPT is commonly found infermented meat products, being principally related to the activityof fermentative lactic acid bacteria (Durlu-Özkaya, Ayhan, & Vural2001; Favaro et al., 2007; Hernández-Jover et al., 1997).

In the meat samples analyzed, the assessed initial concentrationof PUT was about 1.8 mg/kg, a value higher than that reported inliterature for fresh beef meat (Kaniou et al., 2001). Only after8 days of storage the PUT level showed a significant increase ofabout 19% with respect to the initial value (Table 3, P < 0.05), whilefor an analogous storage period Kaniou et al. (2001) have recordeda more marked increase, equal to 65% with respect to the initialvalue.

With regard to CAD, the level of this amine in the meat samplesstudied increased over time, being the data already significant afteronly 6 days of storage and reaching a maximum of 1.9 mg/kg after8 days, with a final 23% increase with respect to the initial value(Table 3, P < 0.001). Working on fresh unpacked beef meat samplesstored at 4 �C, Kaniou et al. (2001) have reported that CAD, whosedetection limit was 0.84 mg/kg, could be detected only after 8 daysof storage, when the assessed concentration of this amine reachedthe value of about 3 mg/kg.

It should be pointed out that CAD increased earlier and morerapidly than PUT, as also confirmed in literature (Edwards, Dainty,Hibbard, & Ramantanis, 1987). Changes in PUT and CAD levels havebeen shown to be generally correlated with microbial spoilage,storage temperature and storage time of meat (Dainty, 1986; Dur-lu-Özkaya et al., 2001; Favaro et al., 2007; Min et al., 2007; Rokka

nction of the storage time.

Significance

6 8

75ab ± 0.13 5.71a ± 0.14 5.82b ± 0.13 *

.58a ± 0.03 1.78b ± 0.04 1.92b ± 0.05 ***

.77a ± 0.22 1.79a ± 0.15 1.85a ± 0.20 n.s.

.84a ± 0.10 1.91ab ± 0.20 2.11b ± 0.17 *

.11a ± 0.14 2.31b ± 0.17 2.22ab ± 0.18 *

.49a ± 0.56 11.51a ± 0.65 10.17a ± 0.54 n.s.

.20a ± 1.88 23.38a ± 2.45 20.75a ± 1.92 n.s.

.81b ± 0.24 6.21c ± 0.19 7.02cd ± 0.21 ***

.99c ± 0.24 5.30b ± 0.26 6.48a ± 0.32 ***

.20c ± 0.34 5.59b ± 0.32 6.52 a ± 0.41 ***

.55c ± 0.22 6.92b ± 0.24 8.17a ± 0.41 ***

.86c ± 0.35 7.96b ± 0.27 8.71a ± 0.24 ***

P < 0.05).

1150 F. Galgano et al. / Food Research International 42 (2009) 1147–1152

et al., 2004), as in the present study; conversely Edwards et al.(1987) have reported a correlation only among these two aminesand microbial spoilage and not with storage time of meat.

With reference to TYR, the initial amount recorded in the freshmeat was about 2 mg/kg, being this value similar to that reportedin literature for fresh beef meat by Saccani, Tanzi, Pastore, Cavalli,and Ray (2005) and higher than that found in fresh chicken meatby Balamatsia et al. (2006) (0.3 mg/kg). In the present study TYRand CAD were the amines whose increase showed the highestsignificance (Table 3, P < 0.001), according to the data reported byVinci and Antonelli (2002), who have suggested to use these twoamines as spoilage indicators of fresh meat during storage. Otherauthors (Krizek et al., 1995; Rokka et al., 2004) have consideredTYR, PUT and CAD as spoilage indicators of meat in function of stor-age time and temperature, while in other studies only TYR showedto be the best freshness index in meat packed under vacuum (Ed-wards et al., 1987; Smith, Kenney, Kastner, & Moore, 1993).

In the meat samples analyzed in this study, the level of HIS didnot significantly change during storage (Table 3), similarly to whatreported in the literature by several authors investigating the HISlevels in various meat products during storage (Bauer, Seuss, Paul-sen, & Vali, 1994; Krizek et al., 1995; Min et al., 2007; Rokka et al.,2004; Smith, Kenney, Kastner, & Moore, 1993). Moreover, HIS wasdetected only on the 5th day of storage by Kaniou et al. (2001).Some factors have been found to affect the HIS level; for example,a low storage temperature (4 �C) does not favor HIS formation,being this process linked to the activity of mesophilic bacteria thatrequire temperatures higher than 15 �C. Furthermore, in the HISformation process the optimum temperature for the histidinedecarboxylase enzyme activity is 30 �C (Kaniou et al., 2001).

In the meat samples studied the assessed SPD concentrationsshowed a slight tendency to increase over time, and this trendwas substantiated by the statistical analysis of the data. Con-versely, the SPM levels did not significantly change over the wholestorage period, similarly to what reported in the literature (Bala-matsia et al., 2006; Patsias et al., 2006; Vinci & Antonelli, 2002).In meat SPM and SPD are naturally occurring polyamines, whileother BAs, such as PUT, CAD, TYR and TRYPT, may be formed duringstorage or even during processing of meat products (Patsias et al.,2006). Generally the SPD content in fresh meat is lower than 5 mg/kg, as in the present study, while for SPM, whose level is usuallycomprised between 20 and 40 mg/kg, the concentrations assessedin the samples ranged between 9.7 and 11.5 mg/kg.

The obtained data for SPD, SPM, as well as those for HIS andTRYPT, indicated that these amines cannot be used as freshnessindicators for refrigerated beef meat, because their concentrationsremained nearly constant throughout the entire storage period.Conversely, all the microbiological indices considered in this studyincreased significantly (P < 0.001) over time, especially during thefirst 3 days of storage. Similar results have been reported in a workon Podolian meat by Cannarsi et al. (2005), even if the initial valuesof psychrotrophics, B. thermosphacta and Pseudomonas spp. werehigher than those recorded in the present study.

The permissible maximum level of total bacteria in refrigeratedmeat ranges from 107 to 109 cfu/g; however, the correlation be-tween bacterial numbers and sensorial spoilage is imprecise, thusmaking difficult to use bacterial levels as a spoilage index (Borch,Kant-Muermans, & Blixt, 1996). In fact, Young, Reviere, and Cole(1998) have reported that the bacterial count at which meat couldbe considered spoiled (107 cfu/g) did not coincide with the panel-ists rejection of meat on the basis of color, while Insausti et al.(2001) have reported that meat samples with bacterial counts low-er than 107 cfu/g were unacceptable from a sensory point of view.Therefore, it is necessary, but not sufficient, to use microbiologicalindices for quality evaluation of meat; however, rather than the to-tal count of bacteria, it is preferable to use as a spoilage indicator

the growth of a specific spoilage bacteria, such as Pseudomonasspp. or B. thermosphacta.

With regard to the relation of BAs production to microbial countin meat, there are controversial opinions. Enterobacteriaceae arecommon contaminants of meat and meat products (Durlu-Özkayaet al., 2001), and in agreement to the results of the present study,other research on BAs formation in meat found Enterobacteriaceaeto be principally responsible for CAD production (Guerrero-Legar-reta & Chavez-Gallardo, 1991; Katikou, Georgantelis, Paleologos,Ambrosiadis, & Kontominas, 2006; Ruiz-Capillas, & Jiménez-Col-menero, 2004) (Table 5, P < 0.001). Furthermore, large amountsof CAD present in beef have been associated with heavy contami-nation by Enterobacteriaceae (Halász, Barath, Simon-Sarkadi, & Hol-zapfel, 1994). In the present research, a significant correlationresulted also between TYR and Enterobacteriaceae (P < 0.05), evenif this amine has been mostly associated with the growth of lacticacid bacteria strains (Smith et al., 1993). Conversely, no significantcorrelation was found among PUT, TRYPT, HIS and Enterobacteria-ceae (Table 5) and no correlation was also found among SPM andSPD and the all types of microorganisms tested, confirming the factthat these two amines are naturally occurring amines in fresh andbeef meat (Patsias et al., 2006).

On the other hand, some authors (Durlu-Özkaya et al., 2001;Kaniou et al., 2001) have associated not only CAD and TYR, but alsoHIS and PUT with the growth of Enterobacteriaceae in chilled freshmeat. Balamatsia et al. (2006) have reported that HIS formation infresh chicken meat was noted only when Enterobacteriaceaereached a population higher than 107 cfu/g after 11 days of storageat 4 �C.

In the literature, PUT and CAD have been shown to be alsocorrelated to psychrotrophic bacteria, in particular to some Pseudo-monas strains (Balamatsia et al., 2006; Guerrero-Legarreta, &Chavez-Gallardo, 1991; Halász et al., 1994; Patsias et al., 2006).In the present study, the pychrotrophics, Pseudomonas spp. and B.thermosphacta resulted strongly correlated to TYR formation(P < 0.001) and to a lesser extent to PUT, CAD and HIS, while onlyB. thermosphacta resulted to be correlated to TRYPT formation(P < 0.05) (Table 5). It should be pointed out that B. thermosphactais the predominant psychrotrophic microorganism associated withthe spoilage of fresh meat stored in refrigeration conditions (Borchet al., 1996).

3.2. Effect of packaging system on meat quality

Shelf-life evaluation of the fresh beef steaks stored at 4 �C for8 days and packed with biopolymeric materials showed that nosignificant differences existed among the various packaging sys-tems on the basis of all the microbiological and chemical parame-ters considered (Table 4).

The meat is generally packed in air, under vacuum or in protec-tive atmosphere, and the packaging system can contribute to dis-criminate the type of microflora and the type of BAs found in theproduct. Under air, Pseudomonas and Enterobacteriaceae becomethe dominant spoilage bacteria, while under vacuum the lactic acidbacteria contribute significantly to the meat microflora (Balama-tsia et al., 2006).

Krizek et al. (1995) have reported that the packaging under vac-uum can influence the quantitative and qualitative formation ofBAs; in particular, TRYPT, HIS and 2-phenylethylamine were notdetected in meat samples during the whole storage period atrefrigeration temperature, while TYR was the most abundant bio-genic amine, with the major increase during storage either in meatsamples packed in air or in under vacuum. Significantly higher con-centrations of PUT were recorded by Patsias et al. (2006) in aerobi-cally-packaged meat samples as compared to samples packedunder protective atmosphere. Kaniou et al. (2001) reported a BAs

Table 4Chemico-physical and microbiological parameters of fresh beef meat stored at 4 �C as a function of the packaging system.

Chemico-physical and microbiological parameters Packaging system Significance

A B C D E

pH 5.75a ± 0.13 5.74a ± 0.15 5.73a ± 0.16 5.72a ± 0.15 5.75a ± 0.14 n.s.CAD (mg/kg fresh weight) 1.70a ± 0.33 1.71a ± 0.35 1.77a ± 0.38 1.66a ± 0.37 1.71a ± 0.34 n.s.HIS (mg/kg fresh weight) 1.73a ± 0.29 1.72a ± 0.18 1.92a ± 0.25 1.94a ± 0.28 1.85 a ± 0.32 n.s.PUT (mg/kg fresh weight) 1.96a ± 0.21 1.95a ± 0.22 1.87a ± 0.20 1.89a ± 0.25 1.84a ± 0.23 n.s.SPD (mg/kg fresh weight) 2.21a ± 0.17 2.17a ± 0.22 2.16a ± 0.23 2.13a ± 0.18 2.18a ± 0.19 n.s.SPM (mg/kg fresh weight) 10.05a ± 0.72 11.21a ± 0.64 10.70a ± 0.61 11.27a ± 0.65 10.33a ± 0.70 n.s.TRYPT (mg/kg fresh weight) 24.99a ± 1.28 19.96a ± 2.40 21.10a ± 2.22 23.20a ± 2.19 20.75a ± 2.26 n.s.TYR (mg/kg fresh weight) 4.55a ± 0.62 4.68a ± 0.54 5.01a ± 0.54 4.64a ± 0.45 4.88a ± 0.38 n.s.B. thermosphacta (log cfu/g) 3.79a ± 0.26 3.75a ± 0.31 3.82a ± 0.41 3.75a ± 0.26 3.70a ± 0.18 n.s.Enterobacteriaceae (log cfu/g) 4.29a ± 0.41 4.24a ± 0.27 4.10a ± 0.24 4.23a ± 0.17 4.20a ± 0.16 n.s.Pseudomonas spp. (log cfu/g) 4.80a ± 0.31 4.84a ± 0.42 4.76a ± 0.35 4.61a ± 0.32 4.82a ± 0.29 n.s.Psychrotrophics (log cfu/g) 6.53a ± 0.35 6.46a ± 0.34 6.51a ± 0.51 6.60a ± 0.42 6.64a ± 0.48 n.s.

A: control (PS tray wrapped with PVC film); B: PLA tray wrapped with PVC film; C: PLA tray heat-sealed with PLA film; D: PLA tray heat-sealed with Mater-Bi-1 film; E: PLAtray heat-sealed with Mater-Bi-2 film.n.s. not significant.Values are the mean of six determinations (n = 6) ± SD.Data in the same row with different superscripts are significantly different (LSD test at P < 0.05).

Table 5Correlation analysis: Pearson coefficients.

pH TRYPT PUT CAD HIS TYR SPD SPM Psychrotrophics Pseudomonasspp.

Brochotrixthermosphacta

Enterobacteriaceae

pH 1.00TRYPT �0.21 1.00PUT �0.18 0.21 1.00CAD 0.01 0.24* �0.12 1.00HIS �0.08 0.23* �0.08 0.08 1.00TYR �0.35** 0.19 0.64*** 0.33** 0.26* 1.00SPD �0.07 �0.08 0.14 0.01 0.59*** 0.28* 1.00SPM �0.16 �0.28* 0.19 �0.19 0.45*** 0.32** 0.49*** 1.00Psychrotrophics �0.06 0.16 0.33** 0.28* 0.27* 0.53*** 0.21 0.16 1.00Pseudomonas spp. �0.15 0.04 0.28* 0.29* 0.28* 0.59*** 0.12 0.16 0.91*** 1.00B. thermosphacta 0.18 0.25* 0.32** 0.30** 0.32** 0.47*** 0.14 0.07 0.86*** 0.84*** 1.00Enterobacteriaceae 0.20 �0.06 �0.02 0.44*** 0.19 0.25* 0.18 0.16 0.74*** 0.77*** 0.69*** 1.00

* Significant for P < 0.05.** Significant for P < 0.01.*** Significant for P < 0.001.

F. Galgano et al. / Food Research International 42 (2009) 1147–1152 1151

content, derived by the sum of PUT, CAD and HIS, of 5 mg/kg after8 days of storage of meat samples in air, while this level wasreached in meat samples packed under vacuum after 12 days ofstorage.

4. Conclusions

The results of this research have highlighted that for fresh meatpackaging it could be possible to replace the PS tray/PVC film sys-tem, with a commercially viable solution, represented by an ex-panded PLA biopolymeric tray heat-sealed with a biopolymericfilm, characterized by a negligible environmental impact in com-parison with the use of synthetic plastic materials.

Shelf-life evaluation of the fresh beef steaks stored at 4 �C for8 days showed that no significant differences existed among thevarious packaging systems on the basis of the microbiologicaland chemical parameters considered.

The storage time differentiated the meat samples on the basis ofpH and microbiological characteristics. With regard to BAs, TYRand CAD resulted to be strongly influenced by the storage time,and to a lesser extent PUT and SPD. TYR and CAD could be usedas spoilage indexes in fresh beef meat chilled and packed in aerobicatmosphere with biopolymers.

The correlations found among microbial changes in meat duringstorage and BAs formation, highlighted that CAD was principally

associated with Enterobacteriaceae counts, while the pychrotroph-ics, Pseudomonas spp. and B. thermosphacta resulted to bestrongly correlated to TYR formation and to a lesser extent toPUT, CAD and HIS.

Acknowledgements

The authors would like to thank Novamont Italy for supplyingthe Mater-Bi films, Treophan Germany for the PLA film, and Coop-box SpA. Italy for supplying the trays and PVC film. This researchwas financially supported by MIUR – PRIN 2005 research project‘‘Improvement of meat production and quality in order to revaluethe native cattle”.

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