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Seasonal patterns of spoilage of ice-stored cultured gilthead sea bream (Sparus aurata)

Seasonal patterns of spoilage of ice-stored cultured gilthead sea bream (Sparus aurata)

Food Chemistry 81 (2003) 263–268 Seasonal patterns of spoilage of ice-stored cultured gilthead sea bream (Sparus aur...

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Food Chemistry 81 (2003) 263–268

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

1. Introduction

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] (K. Grigorakis).

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

2.7. pH

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


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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 weight






Peritoneal fat

Perivisceral fat

Total deposit fat

August January

311 38 318 27

18.1 0.5 18.1 0.7

10.51.2 9.801.35

70.0 0.3 71.1 2.5

1.240.04 1.360.02

2.41 0.50 1.73 0.11

0.530.15 0.720.12

1.03 0.59 1.98 0.40

1.560.60 2.700.40

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 – 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


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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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