Food Chemistry 81 (2003) 263–268 www.elsevier.com/locate/foodchem
Seasonal patterns of spoilage of ice-stored cultured gilthead sea bream (Sparus aurata) K. Grigorakisa,*, K.D.A. Taylorb, M.N. Alexisa a
National Centre for Marine Research, Laboratory of Nutrition, 16604 Agios Kosmas, Ellinikon, Athens, Greece b Food Research Centre, University of Lincoln, Brayford Pool, Lincoln LN6 7TS, UK Received 27 May 2002; received in revised form 9 September 2002; accepted 9 September 2002
Abstract Cage-cultured gilt-head sea bream (Sparus aurata) from Greek sea waters was sampled in January and in August for some chemical and microbial spoilage indicators during 15 days of ice storage. Winter ﬁsh reached higher levels of microbial populations (109 vs 107 in summer ﬁsh) at the end of the storage period. The pH showed an increase after 8 days of storage. TVBN showed a slow and uniform increase. The catabolism of adenine nucleotides showed a slow rate of inosine monophosphate decomposition and a small linear rate of hypoxanthine accumulation. The K value of summer ﬁsh was found to be initially higher than that of ﬁsh sampled in winter in the early stages of storage, but lower in the later stages when microbial spoilage occurred. These results indicate that summer ﬁsh have higher rates of autolytic activity but lower rates of microbial spoilage. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Gilt-head sea bream; Sparus aurata; Cultured; Ice-storage; Spoilage; Season; K value
2. Materials and methods
Aquaculture of gilthead sea bream (Sparus aurata) has major economic importance in the Mediterranean area. However, little literature exists on the changes in quality of this species when stored on ice as whole uneviscerated ﬁsh, which is one of the most common ways of storage (Alasalvar et al., 2001; Kyrana, Lougovois, & Valsamis, 1997; Santoro, Sarli, Sebastio, Sebastio, & Contin, 1996). The aim of this research was to examine the spoilage pattern of gilthead sea bream sampled in two diﬀerent seasons, winter and summer, and to evaluate the seasonal diﬀerences. This study included chemical (TBA, TVBN, ATP breakdown products, pH) and microbial (total and aerobial plate counts) indixes.
2.1. Culture and sampling conditions of ﬁsh
* Corresponding author. Tel.: +30-10-9820213; fax +30-109811713. E-mail address: [email protected]
Two samplings were carried out, one in winter (January), when the average water temperature was 14 C, and one in summer (August), when the average water temperature was 27 C. Fish were produced in a commercial cage culture unit (Poros Island, Argosaronicos gulf) according to standardproduction conditions, using an extruded diet (Europa Marine, TROUVIT S.A, Italy) containing 45% protein, 21% fat, and feeding ratios according to the diet manufacturer’s tables. Standard sampling methodology was used (i.e. ice killing of ﬁsh). The mean ﬁsh weights were 318 27 g (January) and 311 38 g (August). Both winter and summer ﬁsh were stored in ice for 15 days. Groups of three ﬁsh were removed from ice at days 0, 3, 8, 11 and 15 of storage time and analysed.
0308-8146/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0308-8146(02)00421-1
K. Grigorakis et al. / Food Chemistry 81 (2003) 263–268
2.2. Proximate composition
Proximate composition of the muscle was determined using a sample of ﬁsh on the ﬁrst day of their collection. The ﬁllets from ﬁve ﬁsh were removed and analysed individually for each ﬁsh, according to AOAC (1984). Hepatosomatic indices and deposit fat were also measured as described previously (Grigorakis, Alexis, Taylor, & Hole, 2002).
Dorsal white muscle (10 g) of was homogenised with 20 ml of distilled water and the pH measured using a pH meter (RADIOMETER COPENHAGEN, TYPE: MeterLab, PHM 210).
2.3. ATP breakdown products, K-value The HPLC methodology used for the ATP breakdown products determination was according to Ryder (1985). The instruments consisted of a Merck Hitachi L-6000 pump, a C18 HPLC column (5 mm, 100 RP, 4 250 mm), a PYE UNICAM PU 4020 UV detector set at a wavelength of 254 nm and chromatography computing integrator. The mobile phase was 0.04 M potassium dihydrogen orthophosphate (KH2PO4), 0.06 M dipotassium hydrogen orthophosphate (K2HPO4), and the ﬂow rate 2 ml/min with an injection volume 5 ml. The K values were calculated according to Saito, Arai, and Matsuyoshi (1959).
2.8. Statistical analysis For comparison of the means, one way analysis of variance (ANOVA) and the Tukey test were used. Parameters were correlated by two-tailed Pearson correlation. In all cases, conﬁdence levels were set at 95%.
3. Results and discussion 3.1. Composition There were only minor diﬀerences between winter and summer ﬁsh in the proximate composition of the muscle (Table 1). However, there were diﬀerences in total deposit fat, and in HSI. These could be due to nutritional factors (Grigorakis et al., 2002).
2.4. Total volatile basic nitrogen (TVBN) 3.2. Spoilage pattern of gilt-head sea bream TVBN determination took place in a Kjeltec unit by direct steam distillation over boric acid, following the extraction procedure of the Israeli Standard method (1976, IS 281) as described by Gelman, Pasteur, and Rave (1990). Titration was with 0.05 N HCl. The total volatile basic nitrogen content was expressed in mg / 100 g tissue. 2.5. Thiobarbituric acid reactive substances (TBARS) The TBARS were determined by a modiﬁcation of the method of Vyncke, (1978; Grigorakis, 1999). 2.6. Plate counts The skin from the dorsal anterior area was aseptically removed using sterilised scalpels and forceps. A quantity of about 1 g from the underneath ﬂesh was removed, chopped in pieces and aseptically homogenised with 10 ml of 1% peptone water. Further dilutions (from 10 2 up to 10 8), were prepared with peptone water. Pour plates were made for total plate count (TPC) as well as for aerobic plate count (APC) by using the proper dilution in the following media. Medium I: TPC: Standard plate count agar (OXOID), 2.35 g, NaCl, 2 g, in 100 ml of distilled water. Medium II: APC Tryptone soy agar (TSA, OXOID), 4 g, NaCl, 2 g, in 100 ml of distilled water. Incubation was for 2 days at 37 C.
The organoleptic shelf life of gilt-head sea bream varies according to diﬀerent studies from 15 (Santoro et al., 1996) to 18 days limit (Alasalvar et al., 2001; Kyrana et al., 1997) of ice storage. Thus, the period of 15 days of ice storage was near the sensory acceptability. Sampling within this period was selected in order to compare physicochemical and microbial indices of spoilage, of summer and winter ﬁsh, at stages before and at the limit of acceptability. For both seasons, gilt-head sea bream muscle was found to be sterile at least up to the third day (Fig. 1). Microbial development occurred, though at low levels of 102–104 cfu per g tissue, on the eighth day of ice storage. A much higher increase in microbial population was observed in later spoilage stages, with microbial population following a second order polynomial increase to 107 and 109 cfu/g in the 15th day for summer and winter samples respectively (Fig. 1). From the microbial measurements it can be concluded that aerobic spoilage is dominant (since the majority of the total plate count bacteria are aerobic, detected also by the APC). The pH of muscle remained stable (in both summer and winter samples) up to the third day of ice storage and started increasing thereafter, signiﬁcant diﬀerences from the initial values being apparent after day 11 for the summer samples and on day 15 for the winter samples (Table 2). In ﬁsh an initial pH decrease, soon after
K. Grigorakis et al. / Food Chemistry 81 (2003) 263–268
Table 1 Total ﬁsh weight in grammes, muscle proximate composition (%), hepatosomatic index (HSI), and fat deposition (% of total body weight) of cultured gilthead bream sampled in two seasons (August and January) (data are expressed as mean SD; n=5) Season
Total deposit fat
311 38 318 27
18.1 0.5 18.1 0.7
70.0 0.3 71.1 2.5
2.41 0.50 1.73 0.11
1.03 0.59 1.98 0.40
Fig. 1. Microbial development (total and aerobial plate count) in ice stored gilt-head sea bream sampled in winter and in summer. Regression values are 0.97 (winter), 0.97 (summer).
death, occurs during anaerobic metabolism of muscle glycogen and lactic acid concentration in the muscle. The lowest post mortem pH can vary from 5.4–7.2, depending on ﬁsh species, and this pH reduction is accompanied with the rigor mortis onset (Huss, 1988). In the present study no signiﬁcant initial decrease in pH was observed. This may be due to an equilibrium at the lowest pH even from the ﬁrst storage day, before measurement. In later spoilage stages, when microbial development occurs, the pH rises considerably, due to the metabolism of the microorganisms, which produces basic compounds. The pH change in the present study followed the same pattern as in a study of aquacultured gilt-head sea bream stored in ice (Kyrana et al., 1997). The lowest pH levels of the former study were, however, lower than those in the present study (reaching down to a value of
pH=6.10). These diﬀerences in pH possibly mirror different nutritional states of the ﬁsh, as lower pH levels could possibly be due to the higher initial levels of muscle glycogen (Love, 1980). However no nutritional information was available in that study to make comparisons in the two cases and conﬁrm such an hypothesis. Furthermore, another possible explanation for diﬀerences, at the lowest pH, is the size of the ﬁsh. In the former study ﬁsh were larger (average weight of 410 g) than the present ones, and muscle of larger ﬁsh tends to equilibrate after death to a lower pH than that of smaller ﬁsh (Love, 1992). This is also conﬁrmed by Sigholt et al. (1997) who found a strong covariance of post-mortem muscle pH with weight in a study on farmed Atlantic salmon. The cultured gilthead bream in the present study had a muscle fat level of 7–8%, which is between those of high-fat ﬁsh species and low-fat ones according to the study of Miyazawa et al. (1991) in six species. The levels of TBARs appear, however, to be close to those of highfat ﬁsh, reaching levels of 5 umol/kg tissue over a similar storage period. No seasonal diﬀerences were observed, indicating the same rate of lipid oxidation, regardless of the season (Table 2). The TVBN for gilt-head sea bream increased with the time of storage. However, the increase was small, from 15 mg N/100 g tissue to 25 mg N/100 g of tissue after 15 days of ice storage (Table 2). The last value is close to a roughly set acceptability limit of 30 mg N/100 g tissue for most ﬁsh species, such as sardine (Sardina pilchardus) (Ababouch et al., 1996), North-sea whiting (Merlangius merlangus) (Oehlenschlaeger, 1995) and many other cold-water ﬁsh species (Connell, 1975). Civera, Turi, Parisi, and Fazio (1995) has also observed a slow
Table 2 Changes of pH, Total Volatile Basic Nitrogen, and TBA-reactive substances in muscle of for gilt-head sea beam sampled in August and in January and during ice storage Storage time (day) 0 3 8 11 15
TVBN (mg/100 g tissue)
TBARS (umol/kg tissue)
6.15 0.03ax 6.14 0.04ax 6.17 0.03ax 6.25 0.03bx 6.34 0.02cx
6.40 0.05ay 6.35 0.04ay 6.49 0.05aby 6.49 0.04aby 6.53 0.06by
15.2 1.30ab 17.4 1.29ab 22.4 1.19bcy 20.5 1.37bc 25.2 0.89dy
14.43.16ax 17.272.13b 17.920.05bcx – 22.20.18cx
0.750.04ay 0.840.19ay 3.410.95ab 3.440.24b 4.630.48b
nd.ax nd.ax – 3.460.33b 4.330.47c
Diﬀerent letters (a–c) stand for statistically a signiﬁcant change in respect to the time of storage (P <0.05). Diﬀerent letters (x, y) denote statistically signiﬁcant diﬀerences of the parameter in respect to the season (P<0.05). nd, Not detectable; –, no result available.
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and regular increase of TVBN, of a similar pattern as the one presently found, in three Sparidae species during refrigerated storage. The slow, regular increase of TVBN found in this study for the period of 15 days of ice-storage, was slightly lower in winter ﬁsh than in summer ﬁsh (22 cf 25 mg N/100 g at the 15th day). The present ﬁndings indicate that gilthead sea bream, according to the EU categorisation (European Union, 1995), should rather be included in the group of ﬁsh for which the TVB-N limit for human consumption was set at 25 mg N / 100 g. Although the methodology that was adopted for the determination of TVB-N was diﬀerent from the reference procedure of the European Union (1995), a similarity of the results is expected, due to the similarity of the procedure used in this study to the direct distillation method used as a routine method in the former reference. A K value of around 25–35% for 15 days ice-stored gilthead sea bream (Fig. 2) seems to be indicative for ﬁsh near acceptability limit. These results agree with those found in a study on gilt-head sea bream storage (Santoro et al., 1996). Alasalvar et al. (2001) found that a K value of 39% correlated with the limit of acceptability for sea bream. ATP, ADP and AMP were found in very small and relatively stable quantities during ice storage, indicating rapid breakdown of ATP after death, and with ADP always present at highest concentrations of the three (Tables 3 and 4). Immediate depletion of ATP, down to negligible levels, within few hours of death was conﬁrmed for other ﬁsh species (Karube, Matsuoka, Suzuki, Watanabe, & Tokoyama, 1984; Ozogul, Taylor, Quantick, & Ozogul, 2000) and among them, for another Sparidae species, the snapper (Pagrus auratus) (Lowe, Ryder, Carragher, & Wells, 1993). A slow decomposition rate of IMP, on the other hand, was observed for gilt-head sea bream, with levels at day 15 of > 3 and > 5 umol/g for winter and summer, respectively (Tables 3 and 4). INO remained at low levels after 15 days of ice storage (20.72 and 22.60% of the total nucleotides in summer and winter, respectively). Similarly, small amounts of Hx in 15 days of ice storage were accumu-
Fig. 2. Seasonal K-values changes of ice-stored gilt-head sea bream. Best-ﬁt curves are third order polynomial (summer) and second order polynomial (winter) with regression values 0.90 and 0.99, respectively. The bars indicate the SD of the measured values.
lated. Thus, Hx consisted only 3.9 and 9.6% of the total nucleotides on the 15th day of summer and winter icestorage, respectively, which corresponded to concentrations of 0.27 and 0.49, umol/g tissue. Similar slow rates of Hx formation have been observed for several ﬁsh species (Ryder et al., 1984; Scott et al., 1992), and mostly tropical species (Bremner et al., 1988). The slow Hx build up in gilt-head bream suggests that the ATP complete degradation cycle proceeds at a slower pace than in most temperate-water species and agrees with results on the same species (Kyrana et al., 1997). This general pattern of ATP degradation with low formation of Hx, IMP preservation and low INO levels after 15 days of storage, as well as similar concentrations of these compounds, was also found in a previous study of the same species (Alasalvar et al., 2001). 3.3. Seasonal diﬀerences in spoilage The season in which the gilthead bream was sampled was found to aﬀect the spoilage pattern of the ﬁsh (as summer ﬁsh were found to keep slightly better than ﬁsh sampled in winter when stored on ice; this diﬀerence appears as higher microbial population, K-values, and pH in winter ﬁsh and at day 15 of ice storage). Some other studies have also reported a relationship between season and deterioration rate (Hattula, Kiesvaara, & Moran, 1993; Learson & Licciardelo, 2000; Nakayamu, Ooguchi, & Ooi, 1999). Seasonal composition changes have been found to aﬀect the spoilage pattern of ﬁsh (Huss, 1988). However, in the present results the compositional diﬀerences were found small (Table 1) and hence unlikely to aﬀect the spoilage pattern. Water temperature aﬀects shelf life (El Marakchi, Bennour, Bauchriti, Hamama, & Tagafait, 1990; Sumner, Orejana, & Cardial, 1986). Average water temperatures in Greece are about 27 C in summer, and about 14 C in winter, and so the summer ﬁsh surface microﬂora (highly responsible for bacterial spoilage) will receive a much more intensive thermal shock when the ﬁsh is placed on ice. Thus, bacteria would be expected to develop at higher rates in winter because they will need a shorter lag phase. This is conﬁrmed by microbiological data, where winter samples were found to have higher microbial loads after day 8 (Fig. 2). The observation corresponds with data showing that coldwater species spoil more quickly than tropical water species. This can be explained as due to Psychrotrophic Gram negative bacteria, which are present in temperate water species, and show a decreased lag phase, and thus spoilage proceeds more quickly (Ashie et al., 1996; Ward & Bai, 1988). Also, diﬀerent initial populations of surface bacteria may be a cause of diﬀerent deterioration rates formerly related to organoleptic shelf life (Shewan & Hobbs, 1967). There are indications, on the other hand, that higher autolytic activity occurs in
K. Grigorakis et al. / Food Chemistry 81 (2003) 263–268
Table 3 Concentrations (mmol/g tissue) of ATP breakdown products in cultured gilt-head sea bream sampled in winter (January), and their changes during storage on ice (data are expressed as meanSD) Storage time (day)
0 3 8 11 14 15
0.05 0.03 0.03 0.01 0.03 0.00 0.03 0.01 0.01 0.01 0.01 0.01
0.200.08 0.170.02 0.090.03 0.190.00 0.180.01 0.170.00
0.09 0.04 0.07 0.03 0.13 0.02 0.08 0.05 –a –a
8.210.23 6.080.27 5.870.10 6.620.83 3.450.25 3.260.02
0.02 0.04 0.04 0.06 0.38 0.03 1.23 0.07 1.04 0.05 1.15 0.01
0.060.01 0.150.04 0.280.02 0.350.03 0.400.03 0.490.01
Beyond detection limit.
Table 4 Concentrations (umol/g tissue) of ATP breakdown products in cultured gilt-head sea bream sampled in summer (August), and their changes during storage on ice (data are expressed as meanSD) Storage time (day)
0 3 8 11 15
0.140.13 0.130.06 0.0140.02 0.0130.02 0.030.02
0.030.02 0.140.09 0.270.01 0.250.01 0.120.13
0.010.02 0.160.03 0.010.02 –a –a
12.051.22 12.941.15 10.760.73 9.680.07 5.052.08
0.00.0 1.580.30 1.520.20 1.350.12 1.410.52
0.020.03 0.180.02 0.200.10 0.220.07 0.270.13
Beyond detection limit.
summer ﬁsh. This assumption is based on the seasonal diﬀerences of the K-value and its constituents. Although season and deterioration rate during ice storage have been previously correlated, as already mentioned, there are no reports of a correlation between season and K-value. Only Hattula et al. (1993) have mentioned a slight seasonal eﬀect in Finland-caught European whiteﬁsh, with August ﬁsh having a lower K-value increase rate. However, no possible explanation of this phenomenon was given. The seasonal diﬀerences in K-values of this study, could be explained by higher autolysis during summer and higher microbial spoilage during winter. Thus, in summer ﬁsh K-values were found to increase immediately at the ﬁrst 3 days of ice storage, reaching a plateau from the 3rd to 11th day. On the other hand, winter ﬁsh showed slower and more gradual K-value increases at initial stages of storage, while later (when microbial spoilage dominated) winter K-values increased much more rapidly and were found to be higher at the end of the storage period (Fig. 2). K-value correlation with bacterial development (TPC) was found to be signiﬁcant for both winter and summer experiments (0.981 and 0.952, respectively, P< 0.01), indicating a bacterial role in formation of the ATP breakdown products at late spoilage stages.
Acknowledgements This work was carried out as part of an EU INCO funded project.
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