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Application of the Freshness Quality Index (K Value) for Fresh Fish to Canned Sardines from Northwestern Mexico
JOURNAL OF FOOD COMPOSITION AND ANALYSIS ARTICLE NO.
10, 158–165 (1997)
FC970529
Application of the Freshness Quality Index (K Value) for Fresh Fish to Canned Sardines from Northwestern Mexico F. A. Va´zquez-Ortiz, R. Pacheco-Aguilar,1 M. E. Lugo-Sanchez, and R. E. Villegas-Ozuna Centro de Investigacio´n en Alimentacio´n y Desarrollo, A. C. Apartado Postal 1735, Hermosillo, Sonora 83000, Mexico Received July 10, 1996, and in revised form December 17, 1996 Adenosine triphosphate (ATP) and its breakdown products (ADP, AMP, IMP, HxR, Hx) contents in three commercial brands of canned sardine from Northwest Mexico were determined by HPLC. From this data the so called ‘‘K’’ value (a freshness quality index) was calculated. A homogenate of sardine extract in 0.6 M perchloric was used for the separation in a simple run on a mBondapak RP-C18 commercial column. The six compounds were separated in 12 min. The retention time of nucleotides could be measured within /0.2 relative standard deviation (CV õ 2.2%). The coefficient of variation of the relative peak areas and calibration factors, based on the external standard calculation methodology were within 6% or less. Canned sardines from the different commercial brands showed a K value in the range from 34.7 to 56.3%. Even though processors claimed that sardines are canned prior to 36 h postcatch, results suggested that the fish processed had an equivalent biochemical age at 07C, from 9 to 15 days postcapture at the time of processing. The methodology should be useful for measuring the original freshness of canned fish and as indicator of postcapture handling operations. q 1997 Academic Press
INTRODUCTION
Canning of Monterey sardine (Sardinops sagax caerulea) is the second most important economical activity of the fishing industry in the Gulf of California. Several processing plants have been operating for the last 30 years mainly in the State of Sonora, Mexico. Regardless of the impact of this fishery, little is known about the effect of the domestic postcatch handling techniques on the quality of the processed fish. Domestic canners make the statement that their sardine is processed no longer that 36 h after catch; nevertheless, it is well known that postcatch handling techniques still show some deficiencies that could affect the quality of fish before it is processed. Postcatch handling and processing of fish affect the quality of the finished product. After harvest, breakdown of proteins, lipids, and nucleotides occurs by enzymatic action of naturally present enzymes, producing undesirable flavors, odors, and texture problems. Methods to inhibit this enzyme activity such as maintaining the product at low temperature or in salt brines are used to preserve the freshness of fish products. Many methods have been used for assessing quality in fresh fish and shellfish. Most methods are based on measuring products or by-products of protein breakdown, degradation of trimethylamine oxide (TMAO), or lipid oxidation. More recently, spectrofluorophotometry and biosensor methodologies have been developed (Kaminishi 1 To whom correspondence and reprints request should be addressed. Fax: (62) 80-04-21. E-mail: rpacheco @cascabel.ciad.mx.
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et al., 1992; Morin, 1990; Matsumura and Hirota, 1989). In the last decade, biochemical indices of quality based on nucleotide degradation (hypoxanthine and ‘‘K’’ value) have received special attention for monitoring fish freshness during handling and processing (Uchiyama, 1988; Ryder, 1985). When a fresh fish deteriorates, it passes from rigor mortis to dissolution of rigor mortis, autolysis, and finally to bacterial spoilage. Therefore, methods for estimating the freshness of fish must estimate the ‘‘degree of freshness’’ in the autolysis stage. Loss of freshness is caused by endogenous biochemical changes in muscle not by bacterial action, as fish will spoil under aseptic conditions through natural enzyme degradation (Saito et al., 1959; Ehyra and Uchiyama, 1986). Fish freshness must be distinguished from fish deterioration estimated using bacterial spoilage. The former is called biochemical or enzymatic freshness and the latter bacterial freshness or spoilage. The level of major adenine nucleotides and their related compounds have been utilized extensively as an index of freshness of fish muscle before bacterial spoilage commences (Murata and Sakaguchi, 1986). From the quantification of adenosine5*-triphosphate (ATP) in muscle and its breakdown products, namely adenosine-5*diphosphate (ADP), adenosine-5*- and inosine-5*-monophosphate (AMP, IMP), inosine (HxR), and hypoxhantine (Hx), a practical biochemical index for estimating freshness, the so-called K value has evolved. It is documented (Hughes and Jones, 1966) that nucleotides are fairly stable during heat processing. On the other hand, endogenous enzymes are denatured and inactivated during the heating in the canning process. Thus, K values can be calculated from the concentration of ATP and its breakdown products in the processed sardines to define the degree of fish freshness before processing. The objective of this study was to determine the K value in three commercial brands of canned sardines by reverse phase high performance liquid chromatography (HPLC), to obtain information reflecting the degree of freshness or biochemical age of the raw fish at the moment of processing. Since ATP and other nucleotides are resistant to the thermal process applied during canning the calculation of the K value would reflect the adequacy of postcatch handling techniques. HPLC by ion exchange or reverse phase has been reported for the determination of K value (Currie et al., 1982; Ryder, 1985). This analysis has proved to be a useful technique with many applications in marine food and related fields (Greene et al., 1990; Yokoyama et al., 1992). The present paper reports a rapid, accurate, single injection procedure for determination of ATP, ADP, AMP, IMP, HxR, and Hx in canned sardines. MATERIALS AND METHODS
Commercial Canned Sardines Samples from three different commercial brands of canned sardine (Sardinops sagax caerulea) were obtained from local markets in 1994. Three different lots of 15 cans (450 g/can) for each brand were sampled. Brands were identified as B1, B2, and B3. Sample and Extract Preparation The cans were drained and a homogenate was prepared with the solid content of three cans from each lot using a food processor Cusinart 8 Plus (Cusinart Inc., Green-
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wich, CT). No separation of white (fast) and dark (slow) muscles was done on the cooked sardines before making the homogenate. Extracts for analyses were prepared by blending 5 g of sardine homogenate with 25 ml of 0.6 M perchloric acid at 07C for 1 min in a waring blender homogenizer according to Ryder (1985). The homogenate was centrifuged at 3000g for 10 min in a refrigerated centrifuge (0–27C) Beckman Model J2-21 (Beckman Inst. Inc., Palo Alto, CA), and 10 ml of the supernatant was immediately neutralized to pH 6.5–6.8 with 1 M potassium hydroxide. After standing at 07C for 30 min, formed potassium perchlorate was removed by filtration through Whatman paper No. 1, and the filtrate was diluted to 20 ml prior to storage at 0207C for subsequent analysis. High-Pressure Liquid Chromatography (HPLC) Analysis A Varian 9010 solvent delivery system pump equipped with a model 9050 UVVis absorbance detector, a GC-STAR Workstation Automation System (Varian Assoc. Inc., Walnut Creek), and a Rheodyne 7125 injector of 10-ml loop (Rheodyne Inc., Cotati, CA), was used for all analyses. Separations were achieved on a 30 cm 1 3.9 mm ID reverse-phase mBondapak RP-C18 stainless steel column (Whatman Inc., Clifton, NJ) equilibrated at 307C. The mobile phase of 0.04 M potassium dihydrogen orthophosphate and 0.06 M dipotassium hydrogen orthophosphate dissolved in purified distilled water was used at a flow rate of 2 ml/min. The eluant was monitored at 254 nm with full-scale deflection set at 0.2 absorbance units. The detector response for each of the six nucleotides, nucleosides, and bases found in samples was calibrated daily by injecting varying amounts of a solution containing 0.166 mM of each different standard compound (Fig. 1). Quantification was done by use of external standard (Sigma Chemical Co., St. Louis, MO). Calibration factors were calculated from this data. All standard solutions were filtered through a 0.22-mm aqueous filter prior to injecting onto the column as recommended by Ryder (1985). Identification and Quantification The identification of nucleotides, nuclosides, and bases peaks was made by using retention time comparison with standards and by standard addition or ‘‘spiking’’ (Johnson and Stevenson, 1978). Precision was examined by analyzing five replicate standard mixtures and comparing calibration factors for the respective mixtures (Table 1). Analyses were carried out in duplicate. Endogenous concentration were obtained from unspiked aliquots of the homogenate. Statistical Analysis Descriptive statistics (mean, standard deviation, and variation coefficient), one-way ANOVA, and multiple comparison by the Tukey test were applied. A significance level of 5% was used. RESULTS AND DISCUSSION
The chromatographic run took about 13 min and allowed the simultaneous determination of the above listed compounds (Fig. 1). The six purines were sufficiently well resolved to allow their accurate quantitation. At injection levels of 0.166 mM/10 ml the coefficients of variation (CV) ranged from 5.32% for ADP to 0.84% for IMP (Table
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FIG. 1. Separation of standard ATP and its breakdown products. Operating conditions: mBondapak Column; flow rate, 1.0 ml/min; movile phase, 0.04 M potassium dihydrogen orthophosphate and 0.06 M dipotassium hydrogen orthophosphate. Peaks: (1) IMP, (2) ATP, (3) ADP, (4) AMP, (5) Hx, (6) HxR.
1). The retention time could be measured within /0.2 relative standard deviation (CV Å 2.14%). The relative peaks area, based on the external standard calculation methodology, were within 5.01% CV or less. The total peak area had a variation coefficient of 2.96%. The elution times for the ATP and ADP standard peaks were very close and the peak areas overlapped (Fig. 1), which caused the higher coefficient of variation for ADP (Table 1). An almost identical pattern for ATP and its breakdown products has been reported by Ryder (1985). TABLE 1 PRECISION OF HPLC METHOD: COEFFICIENT OF VARIATION QUANTITATION OF FIVE REPLICATE STANDARD INJECTIONS
OF
a b
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FIG. 2. Typical chromatogram of ATP and its breakdown products in a sample of canned Monterey sardine from Northwest Mexico. Operating conditions same as described in the legend to Fig. 1. Peaks: (1) IMP, (4) AMP, (5) Hx, and (6) HxR.
The typical chromatogram of ATP and its breakdown products in a sample of canned sardines is shown in Fig. 2; data analysis for the three brands is described in Table 2. ATP and ADP were not detected in high concentrations. Due to this fact, the lack of resolution between ATP and ADP was not an analytical problem and it was not necessary to modify the mobile phase composition for separating those peaks. It is well documented in the literature that rates and patterns of changes in the level
TABLE 2 ATP
AND ITS
BREAKDOWN PRODUCTS IN CANNED SARDINES NORTHWEST MEXICO (mM/10 ml)
FROM
Note. Data is the result of duplicates. (— — —) Not detected.
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of adenine nucleotides and their related compounds during storage differ according to fish species (Dingle and Hines, 1970), storage conditions (Uchiyama et al., 1970), and muscle types (Murata and Sakaguchi, 1986). With regard to muscle types the change of the K value in dark muscle increases more rapidly than in white muscle, because catabolic rate of ATP, and therefore the increase in K value, is faster in dark than in white muscle. In the present study no separation of dark and white muscles in the cooked sardine body was done before getting the extracts, thus the results reflect the combination of ATP catabolism rates from both muscles and could underestimate the sardine freshness at the time of processing because of the high K values obtained. Nevertheless, since the entire edible portion of Monterey sardine muscle (dark and white) is canned and consumed in Mexico, not like tuna where muscles are separated, it was decided that working with the muscle homogenate will give a more precise data about sardine freshness at the moment of processing in this specific ready-to-eat food item. Further studies are required like those in other pelagic fish species such as Sardinops melanosticta (Watabe et al., 1991) and Seriola quinqueradiata (Murata and Sajaguchi, 1986), to determine if there is a significant difference in the ATP catabolism rates between both muscles in Monterey sardine (Sardinops sagax caerulea). Watabe et al. (1991) had reported that ATP levels in white (fast) muscle of sardine (Sardinops melanosticta) almost disappeared after 12 h of iced storage; similar results for Monterey sardine (Sardinops sagax caerulea), same species as in this study, were reported by Lugo-Sanchez and Pacheco-Aguilar (1996) after a storage period at 07C/ 24 h. Based on those results, the low ATP and ADP concentrations in samples indicated that canned sardines analyzed in this study were older than 24 h postcatch at the moment of processing. The results showed an average high nucleotide concentration and variation among samples for IMP, within a range from 0.170 to 0.562 mM/10 ml (Table 2). This result was the expected, since, as mentioned before, ATP is rapidly degraded to AMP and subsequently to IMP shortly after death by partial dephosphorylation and deamination mediated by endogenous enzymes. The disappearance of IMP has been correlated with a loss of freshness and flavor in some fish species. On the other hand, Hx accumulation in fish tissue reflects the initial phase of autolytic deterioration as well as subsequent contribution through bacterial spoilage as mentioned by Woyewoda et al. (1986). Ehira and Uchiyama (1986) pointed out that spoilage odor in fish kept at 07C did not appear even after 17 days, where the viable bacteria (incubated at 207C) was on the order of 105 g, the minimum of the range considered to be the threshold of the initial spoilage. The organoleptically perceived quality was lost after 14 to 15 days, which correlated well with the 45% K value. This statement indicates that fish freshness is lost before bacteria count increases significantly. Likewise, Wakao and PalmaEstrada (1983) evaluated the freshness changes of sardine (Sardinops sagax) stored at 27C in boxes with ice, ice and water, water and salt; their results showed that at that temperature sardine muscle has an ATP low autolytic rate with a 20% K value after 6 days of ice storage. Similarly, Lugo-Sanchez and Pacheco-Aguilar (1996) in their study on Monterey sardine reported the relationship between freshness and postcatch storing time at 07C for the spring catching season with the linear equation %K Å 3.28 (days) / 6.2, (r Å 0.9864; P õ 0.0001). That equation gave K values of 25.9, 39, and 52.1% after 6, 10, and 14 days of storage at 07C, respectively. In the present study, canned sardine from the three commercial brands showed a
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TABLE 3 ‘‘K’’ VALUES IN COMMERCIAL BRANDS OF CANNED SARDINES FROM NORTHWEST MEXICO
a Values are in days and calculated from the equation of Lugo-Sanchez and Pacheco-Aguilar (1996). Data are the result of duplicates. No difference was detected among means (P ú 0.05).
K value in the range from 34.7 to 56.3%; no statistical difference (P § 0.05) was detected among the brands (Table 3). With regard to data reported by Wakao and Palma-Estrada (1983), the analyzed canned sardine clearly exceeded 6 days postcapture at 27C before it was processed. Based on the freshness behavior of Monterey sardine as reported by Lugo-Sanchez and Pacheco-Aguilar (1996), the equivalent biochemical age (storage maintained at 07C) of the raw fish at the moment of canning fluctuated between 9 and 15 days postcatch. Domestic fishermen and processors assured that canning and thermal processing is carried out no more than 36 h postcapture, a period that represents the real or chronological postcatch age. If this statement is correct, results of this study suggested a far from optimum postcatch handling of sardine before canning. In order to assure a higher freshness and a better overall quality of the canned product, it is necessary to apply more strict temperature controls during postcatch handling. The results of the study showed the usefulness of using the K value methodology for measuring the original freshness of canned fish. REFERENCES Currie, R. W., Sporns, P., and Wolfe, F. H. (1982). A Method for the analysis of ATP metabolites in beef skeletal muscle by HPLC. J. Food Sci. 47, 1226–1228, 1234. Dingle, J. N., and Hines, J. A. (1971). Degradation of inosine 5*-monophosphate in skeletal muscle of several north Atlantic fishes. J. Fish. Res. Bd. Canada. 28, 1125. Ehira, S., and Uchiyama, H. (1986). Determination of fish freshness using the K value and comments on some other biochemical changes in relation to freshness. In Seafoods Quality Determination (D. E. Kramer and J. Liston, Eds.), pp. 185–207. Elsevier Science, Amsterdam. Kaminishi, Y., Matsumo, T., Shindo, J., Miki, H., and Nishimoto, J. I. (1992). A rapid determination of ATP in fish muscle using luciferase of American firefly (Photinus pyrarlis). Bull. Jpn. Soc. Sci. Fish. 58, 1551–1555.
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Greene, D. H., Babbitt, J. K., and Reppond, K. D. (1990). Patterns of nucleotide catabolism as freshness indicators in flatfish from the Gulf of Alaska. J. Food Sci. 55, 1236–1238. Hughes, R. B., and Jones, N. R. (1966). Measurement of hypoxanthine concentration in canned herring as an index of the freshness of the raw material with a comment on flavor relations. J. Sci. Food Agric. 17, 434–438. Johnson, E., and Stevenson, R. (1978). Quantitative Analysis in Basic Liquid Chromatography. (Varian Associates, Eds.), Chap. IX, p. 223. Hansen Way, Palo Alto, CA. Lugo-Sanchez, and Pacheco-Aguilar (1996). Pattern of Nucleotide Catabolism as Freshness Indicator in Chilled Monterey Sardine (Sardinops sagax caerulea) Muscle from the Gulf of California. J. Food Biochem., in preparation. Matsumura, T., and Hirota, I. (1989). Determination of Adenosine Triphosphate in Seawater Using a Spectofluorophotometer. J. Fac. Mar. Sci. Technol. Tokai Univ. Tokaidai Kiyo Kaiyogaku Bull. 28, 29– 41. Morin, J. F. (1990). Determination of K Values by Biosensor. In Advances in Fisheries Technology for Increased Profitability (M. N. Voigt and J. R. Botta, Eds.), pp. 481–485. Technomomic Publishing, Lancaster, PA. Ryder, J. M. (1985). Determination of adenosine triphosphate and its breakdown products in fish muscle by High-Performance Liquid Chromatography. J. Agric. Food Chem. 33, 678–680. Saito, T., Arai, K., and Matsuyoshi, M. (1959). A new method for estimating the freshness of fish. Bull. Japan. Soc. Sci. Fish. 24, 749–750. Uchiyama, H., Ehira, S., Kawahara, T., and Shimidu, W. (1970). Significance in measuring volatile base and trimethylamine nitrogen and nucleotides in fish muscle as indices of freshness. Bull. Japan. Soc. Sci. Fish. 36, 177. Uchimaya, H. (1988). Biochemical determination of fish freshness and partial freezing as a means of keeping freshness of fish and its products. Aquaculture International Congress and Exposition, p. 14. Vancouver, Canada. Wakao, A. W., and Palma-Estrada, J. (1983). Freshness evaluation in sardine and jack mackerel using K value and nitrogenated volatile bases. Aquaculture 76(1–2), 135–143. Watabe, S., Kamal, M., and Hashimoto, K. (1991). Postmortem changes in ATP, creatinine phosphate, and lactate in sardine muscle. J. Food Sci. 56, 151–153, 171. Woyewoda, A. D., Shaw, S. J., Ke, P. J., and Burns, B. G. (1986). Recommended Laboratories Methods for Assessment of Fish Quality. Canadian Technical Report of Fisheries and Aquatic Science No. 1448, pp. 106–108. Fisheries and Oceans, Canada. Yokoyama, Y., Sakaguchi, M., Kawai, F., and Kanamori, M. (1992). Changes in concentration of ATPrelated compounds in various tissues of oyster during ice storage. Bull. Jpn. Soc. Sci. Fish. 58, 2125– 2136.
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10, 158–165 (1997)
FC970529
Application of the Freshness Quality Index (K Value) for Fresh Fish to Canned Sardines from Northwestern Mexico F. A. Va´zquez-Ortiz, R. Pacheco-Aguilar,1 M. E. Lugo-Sanchez, and R. E. Villegas-Ozuna Centro de Investigacio´n en Alimentacio´n y Desarrollo, A. C. Apartado Postal 1735, Hermosillo, Sonora 83000, Mexico Received July 10, 1996, and in revised form December 17, 1996 Adenosine triphosphate (ATP) and its breakdown products (ADP, AMP, IMP, HxR, Hx) contents in three commercial brands of canned sardine from Northwest Mexico were determined by HPLC. From this data the so called ‘‘K’’ value (a freshness quality index) was calculated. A homogenate of sardine extract in 0.6 M perchloric was used for the separation in a simple run on a mBondapak RP-C18 commercial column. The six compounds were separated in 12 min. The retention time of nucleotides could be measured within /0.2 relative standard deviation (CV õ 2.2%). The coefficient of variation of the relative peak areas and calibration factors, based on the external standard calculation methodology were within 6% or less. Canned sardines from the different commercial brands showed a K value in the range from 34.7 to 56.3%. Even though processors claimed that sardines are canned prior to 36 h postcatch, results suggested that the fish processed had an equivalent biochemical age at 07C, from 9 to 15 days postcapture at the time of processing. The methodology should be useful for measuring the original freshness of canned fish and as indicator of postcapture handling operations. q 1997 Academic Press
INTRODUCTION
Canning of Monterey sardine (Sardinops sagax caerulea) is the second most important economical activity of the fishing industry in the Gulf of California. Several processing plants have been operating for the last 30 years mainly in the State of Sonora, Mexico. Regardless of the impact of this fishery, little is known about the effect of the domestic postcatch handling techniques on the quality of the processed fish. Domestic canners make the statement that their sardine is processed no longer that 36 h after catch; nevertheless, it is well known that postcatch handling techniques still show some deficiencies that could affect the quality of fish before it is processed. Postcatch handling and processing of fish affect the quality of the finished product. After harvest, breakdown of proteins, lipids, and nucleotides occurs by enzymatic action of naturally present enzymes, producing undesirable flavors, odors, and texture problems. Methods to inhibit this enzyme activity such as maintaining the product at low temperature or in salt brines are used to preserve the freshness of fish products. Many methods have been used for assessing quality in fresh fish and shellfish. Most methods are based on measuring products or by-products of protein breakdown, degradation of trimethylamine oxide (TMAO), or lipid oxidation. More recently, spectrofluorophotometry and biosensor methodologies have been developed (Kaminishi 1 To whom correspondence and reprints request should be addressed. Fax: (62) 80-04-21. E-mail: rpacheco @cascabel.ciad.mx.
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et al., 1992; Morin, 1990; Matsumura and Hirota, 1989). In the last decade, biochemical indices of quality based on nucleotide degradation (hypoxanthine and ‘‘K’’ value) have received special attention for monitoring fish freshness during handling and processing (Uchiyama, 1988; Ryder, 1985). When a fresh fish deteriorates, it passes from rigor mortis to dissolution of rigor mortis, autolysis, and finally to bacterial spoilage. Therefore, methods for estimating the freshness of fish must estimate the ‘‘degree of freshness’’ in the autolysis stage. Loss of freshness is caused by endogenous biochemical changes in muscle not by bacterial action, as fish will spoil under aseptic conditions through natural enzyme degradation (Saito et al., 1959; Ehyra and Uchiyama, 1986). Fish freshness must be distinguished from fish deterioration estimated using bacterial spoilage. The former is called biochemical or enzymatic freshness and the latter bacterial freshness or spoilage. The level of major adenine nucleotides and their related compounds have been utilized extensively as an index of freshness of fish muscle before bacterial spoilage commences (Murata and Sakaguchi, 1986). From the quantification of adenosine5*-triphosphate (ATP) in muscle and its breakdown products, namely adenosine-5*diphosphate (ADP), adenosine-5*- and inosine-5*-monophosphate (AMP, IMP), inosine (HxR), and hypoxhantine (Hx), a practical biochemical index for estimating freshness, the so-called K value has evolved. It is documented (Hughes and Jones, 1966) that nucleotides are fairly stable during heat processing. On the other hand, endogenous enzymes are denatured and inactivated during the heating in the canning process. Thus, K values can be calculated from the concentration of ATP and its breakdown products in the processed sardines to define the degree of fish freshness before processing. The objective of this study was to determine the K value in three commercial brands of canned sardines by reverse phase high performance liquid chromatography (HPLC), to obtain information reflecting the degree of freshness or biochemical age of the raw fish at the moment of processing. Since ATP and other nucleotides are resistant to the thermal process applied during canning the calculation of the K value would reflect the adequacy of postcatch handling techniques. HPLC by ion exchange or reverse phase has been reported for the determination of K value (Currie et al., 1982; Ryder, 1985). This analysis has proved to be a useful technique with many applications in marine food and related fields (Greene et al., 1990; Yokoyama et al., 1992). The present paper reports a rapid, accurate, single injection procedure for determination of ATP, ADP, AMP, IMP, HxR, and Hx in canned sardines. MATERIALS AND METHODS
Commercial Canned Sardines Samples from three different commercial brands of canned sardine (Sardinops sagax caerulea) were obtained from local markets in 1994. Three different lots of 15 cans (450 g/can) for each brand were sampled. Brands were identified as B1, B2, and B3. Sample and Extract Preparation The cans were drained and a homogenate was prepared with the solid content of three cans from each lot using a food processor Cusinart 8 Plus (Cusinart Inc., Green-
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wich, CT). No separation of white (fast) and dark (slow) muscles was done on the cooked sardines before making the homogenate. Extracts for analyses were prepared by blending 5 g of sardine homogenate with 25 ml of 0.6 M perchloric acid at 07C for 1 min in a waring blender homogenizer according to Ryder (1985). The homogenate was centrifuged at 3000g for 10 min in a refrigerated centrifuge (0–27C) Beckman Model J2-21 (Beckman Inst. Inc., Palo Alto, CA), and 10 ml of the supernatant was immediately neutralized to pH 6.5–6.8 with 1 M potassium hydroxide. After standing at 07C for 30 min, formed potassium perchlorate was removed by filtration through Whatman paper No. 1, and the filtrate was diluted to 20 ml prior to storage at 0207C for subsequent analysis. High-Pressure Liquid Chromatography (HPLC) Analysis A Varian 9010 solvent delivery system pump equipped with a model 9050 UVVis absorbance detector, a GC-STAR Workstation Automation System (Varian Assoc. Inc., Walnut Creek), and a Rheodyne 7125 injector of 10-ml loop (Rheodyne Inc., Cotati, CA), was used for all analyses. Separations were achieved on a 30 cm 1 3.9 mm ID reverse-phase mBondapak RP-C18 stainless steel column (Whatman Inc., Clifton, NJ) equilibrated at 307C. The mobile phase of 0.04 M potassium dihydrogen orthophosphate and 0.06 M dipotassium hydrogen orthophosphate dissolved in purified distilled water was used at a flow rate of 2 ml/min. The eluant was monitored at 254 nm with full-scale deflection set at 0.2 absorbance units. The detector response for each of the six nucleotides, nucleosides, and bases found in samples was calibrated daily by injecting varying amounts of a solution containing 0.166 mM of each different standard compound (Fig. 1). Quantification was done by use of external standard (Sigma Chemical Co., St. Louis, MO). Calibration factors were calculated from this data. All standard solutions were filtered through a 0.22-mm aqueous filter prior to injecting onto the column as recommended by Ryder (1985). Identification and Quantification The identification of nucleotides, nuclosides, and bases peaks was made by using retention time comparison with standards and by standard addition or ‘‘spiking’’ (Johnson and Stevenson, 1978). Precision was examined by analyzing five replicate standard mixtures and comparing calibration factors for the respective mixtures (Table 1). Analyses were carried out in duplicate. Endogenous concentration were obtained from unspiked aliquots of the homogenate. Statistical Analysis Descriptive statistics (mean, standard deviation, and variation coefficient), one-way ANOVA, and multiple comparison by the Tukey test were applied. A significance level of 5% was used. RESULTS AND DISCUSSION
The chromatographic run took about 13 min and allowed the simultaneous determination of the above listed compounds (Fig. 1). The six purines were sufficiently well resolved to allow their accurate quantitation. At injection levels of 0.166 mM/10 ml the coefficients of variation (CV) ranged from 5.32% for ADP to 0.84% for IMP (Table
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FIG. 1. Separation of standard ATP and its breakdown products. Operating conditions: mBondapak Column; flow rate, 1.0 ml/min; movile phase, 0.04 M potassium dihydrogen orthophosphate and 0.06 M dipotassium hydrogen orthophosphate. Peaks: (1) IMP, (2) ATP, (3) ADP, (4) AMP, (5) Hx, (6) HxR.
1). The retention time could be measured within /0.2 relative standard deviation (CV Å 2.14%). The relative peaks area, based on the external standard calculation methodology, were within 5.01% CV or less. The total peak area had a variation coefficient of 2.96%. The elution times for the ATP and ADP standard peaks were very close and the peak areas overlapped (Fig. 1), which caused the higher coefficient of variation for ADP (Table 1). An almost identical pattern for ATP and its breakdown products has been reported by Ryder (1985). TABLE 1 PRECISION OF HPLC METHOD: COEFFICIENT OF VARIATION QUANTITATION OF FIVE REPLICATE STANDARD INJECTIONS
OF
a b
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Standard deviation. Coefficient of variation.
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FIG. 2. Typical chromatogram of ATP and its breakdown products in a sample of canned Monterey sardine from Northwest Mexico. Operating conditions same as described in the legend to Fig. 1. Peaks: (1) IMP, (4) AMP, (5) Hx, and (6) HxR.
The typical chromatogram of ATP and its breakdown products in a sample of canned sardines is shown in Fig. 2; data analysis for the three brands is described in Table 2. ATP and ADP were not detected in high concentrations. Due to this fact, the lack of resolution between ATP and ADP was not an analytical problem and it was not necessary to modify the mobile phase composition for separating those peaks. It is well documented in the literature that rates and patterns of changes in the level
TABLE 2 ATP
AND ITS
BREAKDOWN PRODUCTS IN CANNED SARDINES NORTHWEST MEXICO (mM/10 ml)
FROM
Note. Data is the result of duplicates. (— — —) Not detected.
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of adenine nucleotides and their related compounds during storage differ according to fish species (Dingle and Hines, 1970), storage conditions (Uchiyama et al., 1970), and muscle types (Murata and Sakaguchi, 1986). With regard to muscle types the change of the K value in dark muscle increases more rapidly than in white muscle, because catabolic rate of ATP, and therefore the increase in K value, is faster in dark than in white muscle. In the present study no separation of dark and white muscles in the cooked sardine body was done before getting the extracts, thus the results reflect the combination of ATP catabolism rates from both muscles and could underestimate the sardine freshness at the time of processing because of the high K values obtained. Nevertheless, since the entire edible portion of Monterey sardine muscle (dark and white) is canned and consumed in Mexico, not like tuna where muscles are separated, it was decided that working with the muscle homogenate will give a more precise data about sardine freshness at the moment of processing in this specific ready-to-eat food item. Further studies are required like those in other pelagic fish species such as Sardinops melanosticta (Watabe et al., 1991) and Seriola quinqueradiata (Murata and Sajaguchi, 1986), to determine if there is a significant difference in the ATP catabolism rates between both muscles in Monterey sardine (Sardinops sagax caerulea). Watabe et al. (1991) had reported that ATP levels in white (fast) muscle of sardine (Sardinops melanosticta) almost disappeared after 12 h of iced storage; similar results for Monterey sardine (Sardinops sagax caerulea), same species as in this study, were reported by Lugo-Sanchez and Pacheco-Aguilar (1996) after a storage period at 07C/ 24 h. Based on those results, the low ATP and ADP concentrations in samples indicated that canned sardines analyzed in this study were older than 24 h postcatch at the moment of processing. The results showed an average high nucleotide concentration and variation among samples for IMP, within a range from 0.170 to 0.562 mM/10 ml (Table 2). This result was the expected, since, as mentioned before, ATP is rapidly degraded to AMP and subsequently to IMP shortly after death by partial dephosphorylation and deamination mediated by endogenous enzymes. The disappearance of IMP has been correlated with a loss of freshness and flavor in some fish species. On the other hand, Hx accumulation in fish tissue reflects the initial phase of autolytic deterioration as well as subsequent contribution through bacterial spoilage as mentioned by Woyewoda et al. (1986). Ehira and Uchiyama (1986) pointed out that spoilage odor in fish kept at 07C did not appear even after 17 days, where the viable bacteria (incubated at 207C) was on the order of 105 g, the minimum of the range considered to be the threshold of the initial spoilage. The organoleptically perceived quality was lost after 14 to 15 days, which correlated well with the 45% K value. This statement indicates that fish freshness is lost before bacteria count increases significantly. Likewise, Wakao and PalmaEstrada (1983) evaluated the freshness changes of sardine (Sardinops sagax) stored at 27C in boxes with ice, ice and water, water and salt; their results showed that at that temperature sardine muscle has an ATP low autolytic rate with a 20% K value after 6 days of ice storage. Similarly, Lugo-Sanchez and Pacheco-Aguilar (1996) in their study on Monterey sardine reported the relationship between freshness and postcatch storing time at 07C for the spring catching season with the linear equation %K Å 3.28 (days) / 6.2, (r Å 0.9864; P õ 0.0001). That equation gave K values of 25.9, 39, and 52.1% after 6, 10, and 14 days of storage at 07C, respectively. In the present study, canned sardine from the three commercial brands showed a
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TABLE 3 ‘‘K’’ VALUES IN COMMERCIAL BRANDS OF CANNED SARDINES FROM NORTHWEST MEXICO
a Values are in days and calculated from the equation of Lugo-Sanchez and Pacheco-Aguilar (1996). Data are the result of duplicates. No difference was detected among means (P ú 0.05).
K value in the range from 34.7 to 56.3%; no statistical difference (P § 0.05) was detected among the brands (Table 3). With regard to data reported by Wakao and Palma-Estrada (1983), the analyzed canned sardine clearly exceeded 6 days postcapture at 27C before it was processed. Based on the freshness behavior of Monterey sardine as reported by Lugo-Sanchez and Pacheco-Aguilar (1996), the equivalent biochemical age (storage maintained at 07C) of the raw fish at the moment of canning fluctuated between 9 and 15 days postcatch. Domestic fishermen and processors assured that canning and thermal processing is carried out no more than 36 h postcapture, a period that represents the real or chronological postcatch age. If this statement is correct, results of this study suggested a far from optimum postcatch handling of sardine before canning. In order to assure a higher freshness and a better overall quality of the canned product, it is necessary to apply more strict temperature controls during postcatch handling. The results of the study showed the usefulness of using the K value methodology for measuring the original freshness of canned fish. REFERENCES Currie, R. W., Sporns, P., and Wolfe, F. H. (1982). A Method for the analysis of ATP metabolites in beef skeletal muscle by HPLC. J. Food Sci. 47, 1226–1228, 1234. Dingle, J. N., and Hines, J. A. (1971). Degradation of inosine 5*-monophosphate in skeletal muscle of several north Atlantic fishes. J. Fish. Res. Bd. Canada. 28, 1125. Ehira, S., and Uchiyama, H. (1986). Determination of fish freshness using the K value and comments on some other biochemical changes in relation to freshness. In Seafoods Quality Determination (D. E. Kramer and J. Liston, Eds.), pp. 185–207. Elsevier Science, Amsterdam. Kaminishi, Y., Matsumo, T., Shindo, J., Miki, H., and Nishimoto, J. I. (1992). A rapid determination of ATP in fish muscle using luciferase of American firefly (Photinus pyrarlis). Bull. Jpn. Soc. Sci. Fish. 58, 1551–1555.
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Greene, D. H., Babbitt, J. K., and Reppond, K. D. (1990). Patterns of nucleotide catabolism as freshness indicators in flatfish from the Gulf of Alaska. J. Food Sci. 55, 1236–1238. Hughes, R. B., and Jones, N. R. (1966). Measurement of hypoxanthine concentration in canned herring as an index of the freshness of the raw material with a comment on flavor relations. J. Sci. Food Agric. 17, 434–438. Johnson, E., and Stevenson, R. (1978). Quantitative Analysis in Basic Liquid Chromatography. (Varian Associates, Eds.), Chap. IX, p. 223. Hansen Way, Palo Alto, CA. Lugo-Sanchez, and Pacheco-Aguilar (1996). Pattern of Nucleotide Catabolism as Freshness Indicator in Chilled Monterey Sardine (Sardinops sagax caerulea) Muscle from the Gulf of California. J. Food Biochem., in preparation. Matsumura, T., and Hirota, I. (1989). Determination of Adenosine Triphosphate in Seawater Using a Spectofluorophotometer. J. Fac. Mar. Sci. Technol. Tokai Univ. Tokaidai Kiyo Kaiyogaku Bull. 28, 29– 41. Morin, J. F. (1990). Determination of K Values by Biosensor. In Advances in Fisheries Technology for Increased Profitability (M. N. Voigt and J. R. Botta, Eds.), pp. 481–485. Technomomic Publishing, Lancaster, PA. Ryder, J. M. (1985). Determination of adenosine triphosphate and its breakdown products in fish muscle by High-Performance Liquid Chromatography. J. Agric. Food Chem. 33, 678–680. Saito, T., Arai, K., and Matsuyoshi, M. (1959). A new method for estimating the freshness of fish. Bull. Japan. Soc. Sci. Fish. 24, 749–750. Uchiyama, H., Ehira, S., Kawahara, T., and Shimidu, W. (1970). Significance in measuring volatile base and trimethylamine nitrogen and nucleotides in fish muscle as indices of freshness. Bull. Japan. Soc. Sci. Fish. 36, 177. Uchimaya, H. (1988). Biochemical determination of fish freshness and partial freezing as a means of keeping freshness of fish and its products. Aquaculture International Congress and Exposition, p. 14. Vancouver, Canada. Wakao, A. W., and Palma-Estrada, J. (1983). Freshness evaluation in sardine and jack mackerel using K value and nitrogenated volatile bases. Aquaculture 76(1–2), 135–143. Watabe, S., Kamal, M., and Hashimoto, K. (1991). Postmortem changes in ATP, creatinine phosphate, and lactate in sardine muscle. J. Food Sci. 56, 151–153, 171. Woyewoda, A. D., Shaw, S. J., Ke, P. J., and Burns, B. G. (1986). Recommended Laboratories Methods for Assessment of Fish Quality. Canadian Technical Report of Fisheries and Aquatic Science No. 1448, pp. 106–108. Fisheries and Oceans, Canada. Yokoyama, Y., Sakaguchi, M., Kawai, F., and Kanamori, M. (1992). Changes in concentration of ATPrelated compounds in various tissues of oyster during ice storage. Bull. Jpn. Soc. Sci. Fish. 58, 2125– 2136.
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