A chemical assessment of freshness in stored adductor muscle from scallops

 

 

A.E.Massa^I; M.E.Paredi^II; M.Crupkin^{III, *}

^IComisión de Investigaciones Científicas de la Pcia. de Buenos Aires (CIC)
^IIComisión de Investigaciones Científicas de la Pcia. de Buenos Aires (CIC), Universidad Nacional de Mar del Plata
^IIIUniversidad Nacional de Mar del Plata, CEMSUR-CITEP, Marcelo T de Alvear 1168 - 7600, Mar del Plata – Argentina. E-mail: mcrupkin@mdp.edu.ar

 

 

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ABSTRACT

The postmortem catabolism of adenosine triphosphate (ATP) in cold-stored adductor muscles from scallops (Zygochlamys patagónica) was studied. Changes in the pH of stored muscles were also studied. The ATP content increased for a short time after death and afterwards decreased up to 24 hr of storage. Thereafter, the nucleotide level remained unchanged up to the end of storage. The ADP content slightly decreased up to 48 hr and after that remained unchanged. The AMP slowly accumulated to around 15% of the total nucleotide concentration when the ATP decreased. Small amounts of IMP were detected in all samples. Conversely, adenosine (Ado) was not detected. Inosine (HxR) increased slightly after 48 hr of storage and hypoxanthine (Hx) increased significantly after 24 hr. The 260/250 absorbance ratio of muscle extracts and the pH of stored muscles fell sharply up to 24 hr and then decreased slowly up to the end of storage. The hypoxanthine concentration and the 260/250 absorbance ratio could be reliable indicators of storage age in adductor muscles from scallops.

Keywords: scallop, nucleotides, cold storage.

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INTRODUCTION

The patagonian scallop, Zygochlamys patagónica (King and Broderip, 1832), is a new fishery in the Argentine Continental Shelf. The marine bivalve is distributed around the southern tip of South America from 42º S in the Pacific to 35º S in the Atlantic, between 40 and 200 m deep (Waloszek and Waloszek, 1986). Consumption of fresh and frozen scallop adductor muscles has increased remarkably during the past few years.

Consequently, it is very important to investigate the biochemical postmortem changes in scallop adductor muscles, since those changes are closely related to the eating quality of the flesh. In postmortem muscles the degradation of ATP proceeds as follows: ATP ® ADP ® AMP ® IMP ® Inosine (HxR) ® Hypoxanthine (Hx) (Kassemsarn et al., 1963; Ehira and Uchiyama, 1987). The rate and pattern of changes in nucleotides and their related compounds differ considerably between fish species (Ryder, 1985), muscle type (Obatake et al., 1988), and factors related to handling and storage conditions (Surette et al., 1988). In marine invertebrates a major route for conversion of adenosine monophosphate (AMP) to inosine (HxR) via adenosine (Ado) rather than inosine monophosphate (IMP) was proposed (Saito et al., 1958; Arai, 1960; Hiltz and Dyer, 1970). This idea was supported by the finding that the muscle tissue of bivalves has a much lower level of AMP deaminase activity than that of fish and mammals (Fujisawa and Yoshino, 1985). However, several reports on the accumulation of IMP in postmortem molluscan muscle are available (Nakamura et al., 1976; Suwetja et al., 1989). Contrary to the results of Saito et al. (1958) and Arai (1960), a small amount of IMP was detected in the same species of cephalopods and bivalves (Suwetja et al., 1989). IMP but not Ado was detected in squid (Illex argentinus) mantle (Sagedhal et al., 1997). These studies suggest that depending on the different biological conditions of the specimens, different mechanisms of AMP degradation could be operating in the muscles of marine invertebrates (Suwetja et al., 1989; Sakaguchi et al., 1990; De Vido de Mattio et al., 1992).

It is widely accepted that nucleotides influence the taste and flavor of fish and mollusc meat (Matsumoto and Yamanaka, 1990). IMP contributes to the pleasant, delicious taste and fresh flavor of the meat and its degradation to hypoxanthine is responsible for the progressive loss of desirable flavor (Fletcher and Statham, 1988). Procedures for studying nucleotide degradation give a basis for valid and useful indices of fish quality. The chemical assessment of freshness in postmortem storage of shellfish muscle hasn't been as well established as that for fish. Because of that the purpose of this work was to study the nucleotide catabolism pattern and the 260/250 absorbance ratio in extracts of cold-stored adductor muscles of scallop. The changes in the pH of stored muscles were also studied.

 

MATERIALS AND METHODS

Live scallops (Zygochlamys patagonica) were collected in the area from 39º24' S to 55º 56' W and 104 m deep in the Argentine Sea. Mature specimens with shell of heights 55-65 mm were selected. Immediately after death, the shells were cleaned and the adductor muscles were dissected and carefully freed from adhering pancreatic and liver tissue. Adductor muscles were stored at 2-4ºC for up to 120 hr. At zero time and at different periods of storage the muscles were cut into small pieces and thoroughly mixed to ensure homogeneity. Aliquots were taken for the following studies.

Nucleotide Analysis

Five grams of muscle was homogenized in an Omni-mixer with 20 mL of 7% cold perchloric acid for 1 min. The suspension was centrifuged for 10 min at 10,000 x g at 2-4ºC. The supernatant was immediately neutralized to pH 6.5-6.8 with 30% KOH. The extract was stored on ice for 30 min. The precipitate was removed by filtration through Whatman 1 filter paper. The filtrate was diluted to 50 ml with distilled water and stored at – 30ºC until analysis. Prior to HPLC analysis each sample was filtered through a 0.2 µm filter.

HPLC Analysis

Nucleotides and related compounds were determined by high performance liquid chromatography (HPLC) according to Ryder´s procedure (1985). The HPLC system used in this study was a 2350 ISCO (Pro-Tean LC INERT) pump, 2360 ISCO (Pro Tean LC INERT) gradient programmer, UV/VIS V₄ ISCO detector and CHEM-RESEARCH 150 integrator. Separations were performed on a reverse-phase Lichrosper (5 µm) stainless steel column (2.5 x30 cm) maintained at 30ºC. The mobile phase consisted of 0.04 M K₂HPO₄ + 0.06 M KH₂PO₄, pH 6.5 (Solution A) and methanol (Solution B) as follows: isocratic elution 100% A, 0-12 min; lineal gradient from 100% A to 85%A/15%B, 12-15 min; isocratic elution 85%A/15%B, 15-27 min; lineal gradient from 85%A/15%B to 100% A, 27-30 min; post time 8 min before next injection.

The eluate was monitored by UV absorption at 254 nm. The chromatographic peak of each ATP breakdown product was calibrated by injecting known concentrations of the following standards: Adenosine-5-triphosphate (ATP), Adenosine-5-diphosphate (ADP), Adenosine-5-monophosphate (AMP), Inosine-5-monophosphate (IMP), Adenosine (Ado), Inosine (HxR), Hypoxanthine (Hx), Xanthine (Xt) and Adenine (Ad) (Sigma Chemical Co. St. Louis, MO).

260/250 Absorbance Ratio

The 260/250 ratio was determined as the ratio of the absorbance of perchloric acid muscle extracts at 260 nm to that at 250 nm (Korhonen et al., 1990). One gram of tissue was homogenized in an Omni-mixer with 25 ml of 20% cold perchloric acid for 1 min and then filtered through Whatman 1 filter paper. The filtrate was diluted with 3 volumes of distilled water. Absorbance was measured immediately using a Metrolab 1700 spectrophotometer at 250 and 260nm.

Measurement of the pH Value

Two grams of pooled muscles were homogenized in five volumes of distilled water and then the pH values were determined with a pH meter (Hanna-Instrument HI 9321).

 

RESULTS AND DISCUSSION

Figure 1 shows the HPLC chromatogram of the standard solution containing nine authentic compounds: ATP, ADP, AMP, IMP, Ado, HxR, Hx, Xt and Ad. The ATP-related compounds were effectively separated within 30 min with good reproducibility. The HPLC chromatograms of ATP and catabolites related to ATP breakdown from scallop adductor muscles at zero and 120 hr after death are shown in Figure 2. ATP and ADP accounted for about 85% of the total nucleotide content in scallop adductor muscle immediately after death (Fig. 2A). At 120 hr of storage, these high-energy metabolites had been degraded with concomitant increases in AMP, HxR, and Hx (Fig. 2B).

 

 

 

Figure 3 shows the changes in the contents of ATP and its related compounds in adductor muscles stored at 2-4ºC. After death the ATP level was 4.45 ± 0.96 µmol/g. The ATP content increases for a short time after death because the nucleotide is regenerated by degradation of phosphagen prior to destruction of ATP (Iwamoto et al., 1988). A maximum ATP concentration (5.63 ± 0.41 µmol/g) occurred 2 hr after death, and then the ATP content fell sharply up to 48 hr of storage. Thereafter, the nucleotide level remained unchanged up to the end of storage.

The ADP level slightly decreased up to 48 hr and then remained unchanged. AMP slowly accumulated up to around 15% of the total nucleotide concentration when ATP decreased. A small amount of IMP was detected during cold storage. Conversely, adenosine was not detected. In marine invertebrates, several authors (Saito et al., 1958; Arai, 1960; Hilz and Dyer, 1970) proposed a major route for conversion of AMP to HxR via adenosine (Ado) rather than IMP. However, different reports are available on the accumulation of IMP in postmortem molluscan muscles (Sakamoto et al., 1973; Nakamura et al., 1976; Suwetja et al., 1989; Sagedhal et al., 1997). These apparently contradictory reports could be due to different biological conditions of the specimens (season, sex, physiological condition, etc.) (Sakaguchi et al., 1990; De Vido de Mattio et al., 1992).

HxR slightly increased and reached 0.86 ± 0.27 µmol/g after 48 hr of cold storage. Hx significantly increased after 24 hr and reached 2.65 ± 0.57 µmol/g at the end of storage.

Differential UV spectroscopy of perchloric acid extracts has been used to study nucleotide breakdown in beef and poultry muscle (Davidek and Velisek, 1973; Honikel and Fisher, 1977), based on the measurement of postmortem conversion of adenosine nucleotides to derivatives IMP, HxR and Hx. Adenosine nucleotides show an absorption maximum at 259-260 nm, IMP and inosine at 248.5-249 nm and hypoxanthine at 249-250 nm. The R-value was defined as the 250/260 absorbance ratio. The inverse of this value, the 260/250 absorbance ratio was successfully used to determine the effects of antemortem stress on the rate of onset of rigor mortis and associated biochemical changes in fish muscle (Korhonen et al., 1990). As can be seen in Fig. 4, at zero time of storage the 260/250 absorbance ratio was 1.13 ± 0.05. Korhonen et al. (1990) reported initial values of 1.07 and 0.97 for muscle from unstressed and stressed fish, respectively. Initial values of 1.26 and 1.22 were reported for adductor muscle from the scallop Aequipecten tehuelchus in good and poor biological condition, respectively (De Vido de Mattio et al., 2001). During storage of adductor muscle from Zygochlamys, the ratio showed a rapid decrease between zero and 24 hr (0.0063 260/250 absorbance units per hour), and then gradually declined to 0.93 ± 0.02 up to 96 hr of cold storage (Fig. 4.).

Figure 2 also shows the changes in pH in scallop adductor muscles during storage at 2-4ºC. The initial pH in scallop adductor muscles was 7.27 ± 0.12. During the first 24 hr of cold storage, the pH value fell sharply (0.021 pH units per hour) and then decreased gradually. The last pH was 6.63 ± 0.14. Changes in the postmortem pH in scallop adductor muscles are related to the increase in lactic and octopine contents, end products of glycolysis (Hiltz and Dyer, 1971; Kawashima and Yamanaka, 1995). The amount of lactic acid and octopine produced is related to the amount of stored carbohydrate (glycogen) in the living tissue. In addition, the nutritional status of the fish and the amount of stress and exercise encountered before death will have a dramatic effect on the levels of stored glycogen and consequently on the last postmortem pH.

 

CONCLUSIONS

Hx content increased significantly after 24 hr. This change was accompanied by a decrease in both the 260/250 absorbance ratio and the pH. Hypoxanthine concentration and the 260/250 absorbance ratio could be reliable indicators of storage age in adductor muscle from scallops.

 

ACKNOWLEDGMENTS

This work was supported by FONCYT grants (PICT O374/98).

 

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Received: March 5, 2002
Accepted: February 11, 2003

 

 

* To whom correspondence should be addressed