FLAVOUR CHANGES DURING THE STORAGE OF MILKFISH (CHANOS CHANOS FORSKAL) IN ICE AND AT -12.2OC
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1 FLAVOUR CHANGES DURING THE STORAGE OF MILKFISH (CHANOS CHANOS FORSKAL) IN ICE AND AT -12.2OC C.G. Beza and E.C. Sison Department of Food Science and Technology, College of Agriculture University of the Philippines at LOS Baiios Laguna 372, Philippines Sensory evaluation, chemical and microbiological tests were carried out on brackishwater and freshwater milkfish during storage in ice and at -12.2OC in order to monitor flavour changes. The results indicated that freshwater milkfish deteriorated faster than brackish water milkfish characteristic flavour in freshwater milkfish comlated well with IMP levels. However, undesirable flavours were detected at day 12 due to the accumulation of volatile bases and rancidity by-products. The formation of volatile bases and the rise in ph were attributed to bacterial activity and development of rancidity was due to lipolytic activities. In brackishwater milkfish, flavour scores correlated well with hypoxanthine levels. Undesirable flavours detected at days 2 1 and low TVN levels were found. The characteristic flavour of milkfish was maintained during storage at -12.2OC, and after 6 months of storage the fish were still edible. The predominant microflora found in milkfish were Alcaligenes Achromobacter, Citrobacter, Flavobacter, Pseudomonas, Bacillus, Micrococcus and Vibrio. Microbiological counts increased during storage in ice but the counts did not change significantly during storage at -12.2OC. The chemical and bacteriological changes accompanying flavour development in fish from temperate waters have been the subject of much research (Jones, 1961), but in the case of tropical fish little information is available. Several reports have suggested that fish from tropical waters, whether freshwater or marine, have different flavour characteristics from those of cold-water species. The relationship between autolytic and bacterial changes in temperate fish is also fairly well documented. For tropical fish, information on bacterial changes is limited; on autolytic changes and their control, the information is almost non-existent. The rate of degradation of flavour compounds has not been extensively studied on Pacific Coast species (ACTI, 1974). This study follows up the changes in flavour components and microbiological counts, of brackishwater and freshwater mi1k;ish (Chanos chanos Forskal) commonly known in the Philippines as bangus. As such, it contributes to the knowledge on the microbiology and chemistry of tropical fish. MATERlALS AND METHODS Materials Freshwater milkfish (Chanos chanos Forskal), weighing 250 to 300 g and measuring 25 to 30 cm long were obtained from the fishpens of Laguna de Bay in the Los Barios area. were obtained from fishponds in the vicinity of Dagupan City, Pangasinan.
2 Methods Freshwater and brackishwater milkfish were held in melting ice inside a styrofoam thermo-chest. Fish and ice were placed in alternate layers and the top layer of ice was replenished as necessary. The thermo-chest was placed in a chill-room, maintained at 15.6~~ throughout the storage period of 30 days. Further fish were individually wrapped in polyethylene bags and stored in a freezer at -12.2'~. Sample were taken every three days during the iced storage trial and monthly during the frozen storage trial. The samples were subjected to chemical and microbiological analysis and the results were related to changes in flavour. Sensory evaluation Taste panel assessment was used to detect flavour changes during the storage period. Fish were descaled, eviscerated, wrapped in aluminium foil and steamed for 15 min. The odour and flavour characteristics of the anterio-dorsal portion of the cooked fish were evaluated by 8 semi-trained panelists. Chemical analysis For all the tests, except TBA, a sample of muscle was taken from the anterio-dorsal portion after removing the skin. ph - a ph measurement was made on 15 g of muscle macerated in 15 ml of distilled water. Total volatile nitrogen (TVN) - TVN values were determined by the modified Lucke and Geidel macrodistillation method of Pearson (1970). 2-Thiobarbituric acid value (TBA) - TBA values were measured using the extraction method of CSIRO (1975) which was modified from the distillation and 2-TBA reaction method of Tarladgis et al. (1960). Inosine S'monophosphate (IMP) - IMP was determined on a perchloric acid extraction by ion-exchange separation and absorbancy measurements using the method of Spinelli and Kemp (1966). Hypoxanthine (Hx) - Hx was determined by the xanthine oxidase method as adapted from Jones and Murray (1964). Microbiological analysis fish. Microbiological analyses were carried out on samples put aseptically from the anterio-dorsal area of each Eleven g of muscle were weighed aseptically and homogenized in 99 ml of sterile distilled water in a Waring blend r. Appropriate serial dilutions were made and aliquots were plated in duplicate on Tryptone Glucose Yeast Extract Agar (TGYA). The plates were incubated for 48 h at 3 2O~. Plates containing 30 to 300 colonies were counted. Twenty colonies were selected at random and isolated into TGYA slants for the determination of morphological and cultural characteristics. Representative isolates were further cultured into specific media for biochemical tests to establish their identity at the generic level. Changes in total bacterial levels were mon~tored throughout the storage period. Visual assessment During the storage period, changes in the general appearance and texture were recorded based upon a scoresheet prepared for snapper (Surnner, 1974, as cited by Olley, 1977).
3 RESULTS AND DISCUSSION Sensory evaluation Changes in odour scores Odour scores decreased during storage in ice. Strong off-odours were detected at day 15 in the freshwater fish and after 24 days in the brackishwater fish. The fresh fish odour was lost by day 12 in the freshwater fish and by day 21 in the brackishwater fish. During frozen storage no off-odours were detected but, after 6 months, there was a slight loss of the fresh odour. Changes in flavour scores Flavour scores decreased during storage in ice (Table 1) The freshwater fish lost their flavour more rapidly than did the brackishwater fish. Taste panelists detected the loss of the fish flavour at day 12 for the freshwater fish and after 21 days for the,brackishwater fish. Table 1 Flavour scores of milkfish during storage in ice Flavour scores Multiple regression analysis of flavour scores against other factors indicated that hypoxanthine determined the characteristic flavour of brackishwater milkfish whilst IMP influenced the flavour of freshwater fish. TVN and TBA values also affected the flavour scores. The bases formed by bacterial and autolytic action and the byproducts of rancidity may have been volatilized during cooking. Flavour quality loss was detected earlier in the freshwater fish because, although IMP values were still high enough to impart a meaty flavour, the level of the volatile bases and rancidity by-products were sufficiently high to dominate the flavour profile. In brackishwater fish Hx was the predominant flavour; IMP levels were lower. Initially, the Hx values of brackishwater fish were higher than those of freshwater fish but lower flavour scores were observed in the former. It has been reported that Hx in fish imparts a bitter flavour and it is possible that the panelists detected this since Hx was the dominant flavour component. The relatively low levels of the nitrogenous bases and rancidity by-products had no detectable effect on flavour.
4 In brackishwater fish, the fall in flavour scores accelerated from the third week due to the accumulation of rancidity by-products and Hx. In freshwater fish, the flavour scores decreased at a faster rate than in the brackishwater fish but the rate slowed probably because of the masking effect of IMP. Regression analysis of flavour scores with psychrophilic counts indicated that flavour change in freshwater fish was influenced predominantly by the psychrophilic count, while in brackishwater fish, the influence of the psychrophiles was complemented by autolytic changes. Flavour loss was detected when the psychrophilic count was 104fg in freshwater fish, and 105fg in brackishwater fish. Flavour scores decreased with time during storage at -12.Z C. However, milkfish may be frozen and stored longer than 5 months at constant low temperature. Conditions in this experiment were not constant since intermittent power failures occurred and the quality of the frozen fish was affected by the repeated thawing and freezing. Regression analyses between flavour scores and chemical parameters for the milkfish stored at -12.2~~ showed that the changes were mainly due to autolytic rather than bacteria1 activity. Chemical changes The ph of milkfish during storage in ice and at -12.2'~ increased significantly with time of storage. This rise in ph may have been due to the accumulation of basic compounds as a result of proteolytic and putrefactive action. TVN As shown in Table 2, the TVN value of freshwater fish during storage in ice increased sharply at day 12 and, after day 21, the values declined. The rapid increase in TVN value coincided with the onset of quality deterioration as detected by sensory evaluation. The de-arnination of peptides and amino acids could have caused the rise in TVN value. Despite the rise in the microbial population during the latter part of storage TVN values fell. Possibly the ' nitrogenous bases were being metabolized by bacteria faster than they were being produced.
5 Table 2 TBA values of milkfish during storage in ice TVN (wet basis) (mg/100 g muscle) TVN values also decreased during storage at -12.2~~. The TBA values of brackishwater and freshwater fish in ice rose and fell throughout the storage period but, in general, an upward trend was observed (Table 3). Rancidity was detected at day 15 in freshwater fish and at day 21 in brackishwater fish but the results did not permit any conclusions to be drawn. Table 3 TBA values of milkfish during storage in ice TBA values (Ag 32) TBA values in milkfish during storage at '~ increased with time, indicating that even in frozen storage, oxidative rancidity occurred.
6 IMP IMP was degraded during storage in ice (Table 4). Freshwater milkfish had higher initial IMP levels than brackishwater milkfish but in both types of fish IMP degradation occurred at the same rate. IMP concentration decreased as the number of psychrophiles increased indicating that they were involved in the degradation of IMP IMP was similarly degraded during storage at '~ (Table 5). In the freshwater fish IMP declined at a rate of 0.69 umoleslglday and, in the brackishwater fish, at a rate of 0.52 umoleslglday. Hypoxanthine Hypoxanthine increased during storage in ice and at -12.2~~(Table 7). The Hx values of brackishwater fish were higher than those of freshwater fish during storage in ice. The relatively slow increase in Hx concentration of freshwater fish may indjcate that the ribose group of inosine was cleaved very slcwly (Botta and Shaw, 1976). The formation of Hx correlated with the increase in the number of psychrophiles in freshwater fish. Even in frozen storage the autolytic enzymes brought about increases in Hx values as storage progressed. Table 4 IMP values of milkfish during storage in ice IMP content (umolelg)
7 Table 5 (months) IMP values of milkfish during storage at -12.2~~ r IMP content (umolelg) Table 6 Hx values of milkfish during storage in ice Hx content (pmolelg) t (months) Table 7 Hx values of milkfish during storage at -12.2'~ Hx content (pmolelg)
8 Microbiological changes Quantitative changes Microbiological counts in milkfish increased during -storage in ice (Table 8). The initial counts in brackishwater and freshwater milkfish were of the same order of magnitude but the predominant flora differed. Table 8 Total microbiological counts of milkfish during storage in ice Total plate count/g muscle During storage in ice the total microbiological counts, as well as psychrophilic counts were higher on freshwater fish than on brackishwater fish causing the former to spoil faster. The higher counts on freshwater fish may be due to bacteria in the gut content. The freshwater fish were not fasted prior to harvesting whilst the brackishwater fish were fasted for at least a day before harvest and the gut content may have had low microbial counts. Microbiological counts in milkfish stored at -12.2'~ did not change significantly during the 6 months of storage. Qualitative changes Qualitatively, the flora did not differ very much between freshwater and brackishwater fish; the difference lay in the relative proportions of the predominant genera. There was a predominance of the gram-negative nonsporeforming rods, mainly Alcaligenes-Achromobacter, Citrobacter (Escherichlh. fratndii), Vibrio, Pseudomonas and Flavobacter, and the grampositive cocci, Micrococcus and Pediococcus. Bacillus and Lactobacillus were found to a certain extent. Mesophilic organisms predominated since milkfish came from relatively warm. tropical water. The bacterial found in the milkfish are similar to those reported by Moorjani (1976) in tropicai fish from Indian waters.
9 Citrobacter predominated over the Alcdigenes-Achromobacter group in the brackishwater fish whereas the opposite was observed in freshwater fish. Since the Alcaligenes-Achromobacter group produce basic metaboiic by-products their predominance may partly explain the higher ph of the freshwater milkfish. The considerable extensionof the shelf-life of milkfish when compared to temperate fish may be due partly to the lower population of psychrophiles in these tropical fish. Visual appearance The eyes of the brackishwater fish stored in ice began to lose translucency and started to become cloudy at day 18. Similar changes were observed at day 12 in the freshwater fish. The flesh started to become waxy white at day 21 in the former and at day 12 in the latter. The texture was firm and elastic up to the third day, after which time the flesh became progressively soft and inelastic. Milkfish stored at -12.2~~ were of optimum appearance up to the second month. By the sixth month, the eyes had become cloudy but the gills were still pink. The flesh was slightly translucent, and the scales had loosened. ACKNOWLEDGEMENT The authors wish to express their gratitude to the IRR Chemistry Department for allowing the use of the W spectrophotometer. REFERENCES Advisory Committee on Technology Innovation (ACTI). Food science in developing countries: a selection of 1974 unsolved problems. National Academy of Sciences, Washington D.C., p.62 Botta, J. and D. Shaw, Chemical and sensory analysis of roundnose grenadier stored in ice. J.Foo~ Sci 41: CSIRO, Quantitative determination of malonaldehyde in rancid comminuted fish. Hobart, Tasmania, Division of 1975 Food Research. (Unpubl. MS.) Jones, N., Fish flavours. In Proceedings of flavour chemistry symposium. Campbell Soup Co., New Jersey, p.61' Jones, N. and J. Murray, Rapid estimations of hypoxanthine concentrations as indices of the freshness of chill stored fish. JSci. Food Agric., 15(10):763 Moorjani, M., Some notes on the microbiology of tropical fish in Indian waters. Report of the Second Session of 1976 of the IPFC working party on fish technology and marketing. IPFC, 17th Session, Colombo, Sri Lanka, 27 Oct.4 Nov OIley, J., Temperamre and seafood spoilage. Paper given at the 1977 School of seafood processors. Hobart, 1977 Tasmania, CSIRO Pearson, D., The chemical analysis of foods. Chemical Publishing Co. Inc. New York, p SpineHi, J. and Jemp. Rapid measurements of IMP and total adenosine nucleotide in fish tissue. J. Agric. Food 1966 Chem., l4(2): 176 Sumner, J., Scoresheet for snapper.h Proceedings of fish quality seminar, New Zealand (as cited by Olley, 1977) 1974 Tarladgis, B.B. et al., A distillation method for the quantitative determination or malonaldehyde in rancid Gods J.ArnOil Chem Soc., 1960: 37-44
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