Freshness Quality of Harp Seal (Phoca Groenlandica) Meat Fereidoon Shahidi, Xin Chong, and Edward Dunajski J

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Freshness Quality of Harp Seal (Phoca groenlandica) Meat Fereidoon Shahidi, Xin Chong, and Edward Dunajski J. Agric. Food Chem., 1994, 42 (4), 868-872• DOI: 10.1021/jf00040a005 • Publication Date (Web): 01 May 2002 Downloaded from http://pubs.acs.org on April 19, 2009

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 060 J. Agric. Food Chem. 1994, 42, 868-872

Freshness Quality of Harp Seal (Phoca groenlandica) Meat

Fereidoon Shahidi,’ Xin Chong, and Edward Dunajski Department of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada A1B 3x9

Freshness quality of seal meat during the post-mortem storage period was determined by monitoring the profile of nucleotides and their breakdown products. Analytical conditions for quantitative determination of adenosine 5’-triphosphate, adenosine 5’-diphosphate, adenosine 5’-monophosphate, inosine 5’-monophosphate, hypoxanthine, xanthine, and inosine in harp seal meat extracts were examined by reversed-phase high-performance liquid chromatography (HPLC). Freshness indicator K, KO,Ki, G, and Pvalues were calculated. Linear increases in K,Ki, and KOvalues with increasing storage periods of up to 19 days were observed. Comparison of KOvalues of seal with those from beef indicated that seal meat behaves similarly to beef. Comparison of the Ki values of seal meat with those of cod and pollock indicated that seal meat has a better keeping quality than fish species. Comparison of the G and P values of seal meat with those of cod indicated a similar trend. Thus, HPLC for nucleotide determination lends itself to the evaluation of seal freshness quality. The K,Ki, KO,and P values offered a better indication for seal meat freshness during the early stages of storage than the G value.

INTRODUCTION these nucleotides are advantageous. Thus, the concept of the K value (eq 2) was first introduced by Saito et al. Seal meat has a high protein content with a well-balanced (1959). amino acid composition (Shahidi et al., 1990). It is low in cholesterol (approximately 66 mg/100 g) and nucleic K (7%) = acids (approximately 2 mg/g) and is a rich source of B [INOI + [Hxl vitamins, iron, and long-chain w-3 fatty acids. Interest in seal meat as a highly nutritional source of muscle food is [ATP] + [ADP] + [AMP] + [IMP] + UNO1 + [Hxl well documented (Shahidi et al., 1990). Freshness quality 100 (2) of muscle foods is related to biochemical changes taking ATP, ADP, and AMP disappear approximately 24 h place during the post-mortem period. In particular, post-mortem. In practice, muscle foods obtained from autolytic and biochemical deteriorations become more the market are at least 24 h after death. Hence, the K important when proper chilling and handling procedures value can be reduced to are not used. [IN01 + [Hxl Immediately after death, adenosinetriphosphate (ATP) Ki (5%) = x 100 (3) begins to degrade to uric acid through the pathway [IMP] + FINO1 + [Hxl (Terasaki et al., 1965) Assays monitoring IMP, INO, and Hx are therefore 1 2 3 4 5 67 sufficient for determination of fish freshness. However,

ATP -+ ADP -, AMP -+ IMP -+ IN0 -+ Hx -+ X -+ U when nucleotides are degraded fully, the usefulness of these (1) indicators is questionable (Jones and Murray, 1964; Kassemsarn where ADP is adenosine diphosphate, AMP is adenosine et al., 1973). Burns et al. (1985) have proposed another freshness monophosphate, IMP is inosine monophosphate, IN0 is indicator for fish, namely the G value (eq 4), which is inosine, Hx is hypoxanthine, X is xanthine, and U is uric acid. In most cases, the rate-determining step is (5) or (6), [Hxl + UNO1 depending on the species of animal being examined G= (4) (Karube et al., 1984; Karube and Sode, 1988). Conse- UNO1 + [IMP] + [AMPI quently, the concentration of IN0 and Hx increases with the length of the storage period. Contents of IN0 or Hx based on accumulation of Hx and disappearance of IMP, in fish muscles have been used as indicators of freshness AMP, and IN0 in the muscle after slaughter. The index (Jones and Murray, 1964; Burt, 1977). is useful over the entire iced shelf life of lean fish. Molecules of ATP are rapidly degraded after death to A second quality indicator, namely the P value (eq 51, AMP and subsequently to IMP by partial dephosphor- serves as an indicator of spoilage during the early stages ylation. The dephosphorylation of IMP is primarily of chilled storage: autolytic and occurs during the early stages of chilled [Hxl + [INOI storage. The disappearance of IMP has been correlated P= (5) with the loss of fresh fish flavor in some species (Jones IINOI + [IMP] + [Hxl + [AMPI and Murray, 1961; Fraser, 1965; Spinelli et al., 1969).The Values of G and P can provide a basis for the establish- effect of Hx accumulation in fish tissues reflects the initial ment of a grading system in fish, and these have been phase of autolytic deterioration and later includes con- compared with other fish freshness tests and physical tributions through bacterial spoilage. evaluations for several fish species. These values are While useful indices of freshness have been based on applicable to most fish samples,although species variations individual nucleotides or their breakdown products, can be expected. Values of G and P are most useful with indicators that incorporate the measurement of several of lean fish. In fatty fish, however, factors such as devel-

0021-8561/94/1442-0868$04.50/0 0 1994 American Chemical Society Freshness Quallty of Harp Seal Meat J. Agric. Food Chem., Vol. 42, No. 4, 1994 860 opment of rancidity may render the product undesirable before meaningful G and P values can be obtained. As an indication of freshness of meat, Nakatani et al. ox A HX (1986) proposed the use of the KOvalue (eq 6) in which v IN0 v IMP KO(76) = [([IN01 + [Hxl)/([ATPl + [ADPI + AMP [AMPI + [AD] + [IMP] + [IN01 + [Hxl + [XI)] X ADP 100 (6) ,A ATP 0241 adenosine (AD) and X were also included. A well- Time (hours) established specific and fast ion-pair reversed-phase high- I performance liquid chromatographic (HPLC) method may be employed. This method allows most of the nucleotides I Y-4 to be simultaneously determined on the basis of the property of the molecular electrical charges (Murray and Thomson, 1983;Stocchi et al., 1987; Langand Rizzi, 1986; Watanabe et al., 1989). The objective of this study was to evaluate the freshness quality of harp seal meat using nucleotide analyses. Conditions suitable for separation of ATP-related compounds in seal meat were examined. Results of nucleotide analyses were used to calculate relevant indicators of seal meat freshness. MATERIALS AND METHODS 0 5 10 15 20 Time (days) Materials. Nucleotides and nucleoside standards were ob- Figure 1. Changes in the content of nucleotides and nucleosides tained from Sigma Chemical Co. (St. Louis, MO). HPLC grade of seal meat during post-mortem storage at 0 "C. Each point methanol was supplied by Fisher Scientific Ltd. (Dartmouth, represents the mean value of triplicate determinations of samples NS). Other chemicals used were of commercialACS grade. A 0.1 from seven seals standard deviation. M potassium phosphate buffer, pH 6.0, containing 8 mM * tetrabutylammonium hydrogen sulfate (buffer B for pump B) Stocchi method was used for the analytical separation conditions and a 0.1 M potassium phosphate buffer, pH 6.0, containing 8 (Stocchi et al., 1987): 0.01-3.8 min at 100% buffer B, 7.8 min mM tetrabutylammonium hydrogen sulfate and 30 % (v/v) at up to 20% buffer A, 15 min at up to 40% buffer A, 19.5 min methanol (buffer A for pump A) were used. at up to 100% buffer A, and hold until 27 min. The gradient was Instruments. A Shimadzu HPLC system consisting of two then immediately returned to 100% buffer B and was held there Model LC-6A pumps with a mixing chamber, a Model SPD-6AV until 31 min, when the analysis was terminated. The flow rate UV-visible detector, a Model SIL-6B autoinjector, a Model SCL- was 1 mL/min, and detection was measured at 254 nm. The 6B system controller, and a Model CR501 Chromatopac (data analyses were performed at room temperature, but the samples processor) were used. A 10-rm particle size LC-18-T reversed- were kept at 0 "C until the time of the injection. Quantification phase analytical column (4.5 mm X 24 cm) from Supelco (Oakville, of the endogenous nucleotide and nucleoside concentrations was ON) and a guard column (4.5 mm X 5 cm) which was coupled carried out using the datamodule in the external standardization with the analytical column were used. mode. Results are expressed as micromoles per gram of muscle. Sample Preparation. Seven harp seal carcasses hunted in TheK,KO, Ki, G, and Pvalues were then calculated. Mean values the waters adjacent to St. Anthony, NF, during April 1991 were of, at least, triplicate determinations * standard determinations used in this study. All seals were bled well and eviscerated, 30 are reported in each case. min post-mortem, at the collection site. A known amount of longissimusdorsi muscle was cut from each seal at different time RESULTS AND DISCUSSION intervals, as indicated in the figures, and nucleotides were extracted into perchloric acid as described below. The remaining Figure 1 shows changes in the concentration of nucle- carcasses were placed inside plastic bags and stored on ice in a otides and nucleosides during a 3-week storage of seal meat refrigerated room at 0-4 "C during the course of the experiments in a refrigerated room at 0-4 "C. Concentrations of ATP, which lasted 23 days. ADP, and AMP decreased and concentrations of IMP, Nucleotide Extraction. Ten grams of finely chopped seal INO, Hx, and X increased during the first 48 h. After 48 muscle was weighed and homogenized in 20 mL of 0.6 N perchloric h, ATP was no longer detected, but the concentrations of acid for 30 s using a Brinkmann Polytron homogenizer (Brink- ADP and AMP remained unchanged at a very low level. mann Polytron, Westbury, NY) at maximum speed. The IMP concentration increased extremely quickly, reaching homogenate was filtered through a Whatman No. 1filter paper, and the resultant filtrate containing the nucleotides extract was a maximum after 48 h, and then started a decline to almost kept for up to 3 weeks at -60 "C until use. zero at day 23. However, the level of IN0 increased during HPLC Analyses. Before HPLC analyses, the nucleotide the first 12 days before starting to decline. The steady extracts were thawed at 0-4 "C and the pH was adjusted to 6.5 increase in the concentration of Hx was noted up to 19 by diluting it 1/10 with 0.1 M KzHP04 to avoid crystallization days. During the 23-day post-mortem storage, there was of perchloric acid during the HPLC analyses. The pH-adjusted a steady increase in the concentration of X with time. sample was then filtered through a 0.45-pm nylon filter (Cameo These observations are in line with the reported results 11, MSI, Westboro, MA) into the HPLC sampling vial and was on fish freshness (Burt, 1977; Jones and Murray, 1964). then used directly for analysis. Nucleotides in the samples were The disappearance of IMP has been correlated with the determined by a reversed-phase HPLC. Twenty microliters of a standard or filtered solution was injected into the column loss of fresh fish flavor in some species (Fraser, 1965; automatically using the autoinjector. The detector response for Spinelli et al., 1969; Jones and Murray, 1961). In addition, each nucleotide and nucleoside was calibrated daily by injecting Hx has been reported to be a good indicator of freshness a known mixture of the reference compounds. Initially, a four- (Jahns et al., 1976); the present data on seal agree with level calibration run was performed, giving a linear response this suggestion and show that xanthine may serve as a (standard curve) for each reference compound. A modified good indicator for seal meat freshness. 87Q J. Agric. Food Chem., Vol. 42, No. 4, 1994 ShahMi et al.

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- 10 I Time (hours) Lc 0 5 10 15 20 25 0 5 10 15 20 25 Time (days) Time (days) Figure 2. Changes in freshness indicator K values of seal meat Figure 4. Changes in freshness indicator Ki values of seal meat during storage at 0 "C. Each point represents the mean value of during storage at 0 OC. Each point represents the mean value of triplicate determinations of samples from seven seals f standard triplicate determinations of samples from seven seals i standard deviation. deviation.

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0 10 20 30 40 ! 0.1 Time (hours) lot I I 0 5 10 15 XI 25 :: Y Time (days) Figure 3. Changes in freshness indicator KOvalues of seal meat during storage at 0 OC. Each point represents the mean value of triplicate determinations of samples from seven seals f standard deviation. 19. However, no clear trend during the early storage period The commonly used indicators of freshness quality, (upto 13 days) was noticed when G values were considered namely K, G, P, KO,and Ki values, of seal meat were (Figure 6). However, a linear increase in the G values was calculated from the concentrations of the nucleotides. noticed during prolonged storage of the meat. Compared Figures 2-4 show changes of K, KO,and Ki values of seal with other freshness indicators, the G value is the only meat during a 23-day storage period. During the first 6 parameter without Hx in the denominator. Thus, it is h, no clear trends in changes of K, KO,and Ki values were clear that Hx plays an important role in the freshness evident. However, K, KO,and Ki increased linearly with quality of seal meat and may serve as a good freshness storage time (19 days). Figure 5 shows a linear increase quality indicator by itself. in P values during the storage period between days 2 and Comparison of K and KOvalues of seal meat with KO Freshness Quality of Harp Seal Meat J. Agric. Food Chem., Vol. 42, No. 4, 1994 871

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0 5 io 15 20 25 0 5 10 15 20 25 Time (days) Time (days) Figure 6. Changes in freshness indicator G values of seal meat Figure 8. Changes in freshness indicator Ki values of seal meat as compared with wild Atlantic cod and cod fillets from Halifax as compared with Pacific cod and pollock (Green and Bernatt- Laboratory Aquaria (HLA) (Burns et al., 1985) during storage Byme, 1990) during storage at 0 OC. at 0 OC. in I 1 1

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0 5 10 15 20 25 0 5 io 15 20 25 Time (days) Time (days) Figure 9. Changes in freshness indicator P values of seal meat Figure 7. Changes in freshness indicators Ki and KOvalues of as compared with wild Atlantic cod and cod from Halifax seal meat as compared with beef (Watanabe et al., 1989) during Laboratory Aquaria (HLA) (Burns et al., 1985) during storage storage at 0 "C. at 0 "C. values of beef (Watanabe et al., 1989) is shown in Figure of Atlantic wild cod and the cod from the Halifax 7. It is apparent that seal meat behaves similarly to beef. Laboratory Aquaria (HLA) (Burns et al., 1985) indicated Both seal meat and beef are from warm-blooded animals, a similar trend (Figures 6-9). and this might explain the similarity in their post-mortem In summary, the present study shows that the HPLC behavior. Comparison of the Ki values of seal meat with method provides an adequate procedure for evaluating those of the Pacific cod and pollock (Greene and Bernatt- freshness quality of harp seal meat. Data presented also Byme, 1990)indicated that seal meat has a better keeping indicated that harp seal meat behaves similarly to beef in quality than either species of fish considered (Figure 8). terms of its storage freshness quality. The K,Ki, KO, and Comparison of the G and P values of seal meat with those Pvalues offered a better indication for seal meat freshness 872 J. Agric. Food Chem., Vol. 42, No. 4, 1994 ShahMi et al. than the G value, as a better linear relationship was aegkefinus),lemon sole (Pleuronecetes microcephalus) and obtained between these indicators and the storage of the plaice (Pleuronecetes platessu). J. Food Sci. 1973,28,28-30. meat for up to 19 days. Lang, H. R. M.; Rizzi, A. Separation of purine bases, nucleosides and nucleotides by a column-switch technique combining ACKNOWLEDGMENT reversed-phase and anion-exchange high-performance liquid chromatography. J. Chromatogr. 1986,355, 115-120. This work was financially supported by a NIFDA grant Luong, J. H. T.; Male, K. B.; Huynh, M. D. Applications of from the Department of Fisheries of Newfoundland and polarography for assessment of fish freshness. J. Food Sci. Labrador. We are grateful to Mr. Michael Keats for 1991,56, 335-337, 340. technical support and assistance during post-mortem Murray, J.; Thomson, A. B. Reversed-phase ion pair separation, period studies on board a long-liner. of nucleotide and related products in fish muscle. J. High Resolut. Chromatogr. Chromatogr. Commun. 1983, 6, 209- LITERATURE CITED 210. Nakatami, Y.; Fujita, T.; Sawa, S. Changes in ATP-related Burns, B. G.; Ke, P. J.; Irvine, B. B. "Objective procedure for fish compounds of beef and rabit muscles and a new index of freshness evaluation based on nucleotide changes usinga HPLC freshness of muscle. Agric. Biol. Chem. 1986, 50, 1751. system". Canadian technical report of fisheries and aquatic Saito, J.; Arai, K.; Matsuyoshi, M. A new method for estimating sciences, No. 1373; 1985. the freshness of fish. Bull. Jpn. SOC.Sci. Fish. 1959,24,749- Burt, J. R. Hypoxanthine: a biochemical index of fish quality. 752. Process Biochem. 1977,12, 32-35. Shahidi, F.; Synowiecki, J.; Naczk, M. Seal meat-A potential Fraser, D. I.; Weistein, H. M.; Dyer, W. J. Post-mortem,glycolytic source of muscle food: Chemical composition, essential amino and associated changes in the muscle of trap- and trawl-caught acids and colour characteristics. Can. Znst. Food Sci. Technol. cod. J. Fish. Res. Board Can. 1965,22, 83-100. J. 1990,23, 137-139. Green, D. H.; Bernatt-Byrne, E. I. Adenosine triphosphate Spinelli, J.; Pelroy, G.; Miyouchi, D. Quality indicies that can be catabolites as flavor compounds and freshness indicators in used to assess irradiated seafoods. InFreezing and Irradiation Pacific cod (Gadus macrocephalus) and pollock (Theragra ofFish;Kreuzer, R., Ed.; Fishing News Books: London, 1969; chalcogranama). J. Food Sci. 1990,55, 257-258. p 425. Jahns, F. D.; Howe, J.; Coduri,R. J.;Rand,A. G. Aviaualenzyme Stocchi, V.; Cucchiarini, L.; Canestrari, F.; Piacentini, M. P.; test to assess fish freshness. Food Technol. 1976, 30, 27-30. Fornaini, G. A very fast ion-pair reversed-phase HPLC method Jones, N. R.; Murray, J. Nucleotide concentration in codling for the separation of the most significant nucleotides and their (Gadus Callarias) muscle passing through rigor mortis at 0 degradation products in Human red blood cells. Anal. Biochem. "C. 2. Vgl. Physiol. 1961, 44, 174-177. 1987,167, 181-190. Jones, N. R.; Murray, J. Rapid measures of nucleotide dephos- Watanabe, A.; Tsuneishi, E.; Takimoto, Y. Analysis of ATP and phorylation in iced fish muscle. Their value as indices of its breakdown products in beef by reversed-phased HPLC. J. freshness and of inosine 5'-monophosphate concentration. J. Food Sci. 1989,54, 1169-1172. Sci. Food Agric. 1964, 15, 684-690. Karube, I.; Sode, K. Enzyme and microbial sensor. In Analytical Uses of Immobilized Biological Compounds for Detection, Medical and Zndustrial Uses;Guilbault, Mascini, Eds.; Reidel Received for review December 13, 1993. Accepted January 17, Publishing: Boston, 1988; pp 115-130. 1994.' Karube, I.; Matsuka, H.; Suzuki, S.; Watanabe, E.; Toyama, K. Determination of fish freshness with an enzyme sensor.J. Agric. Food Chem. 1984,32, 314-319. Kassemsarn, B.; Sanz Perez, B.; Murray, J.; Jones, N. R. @ Abstract published in Advance ACS Abstracts, March Nucleotide degradation in the muscle of iced haddock (Gadus 1, 1994.