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A study was carried out on the evolution of free amino acids and biogenic amines throughout the ripening process of fresh and frozen sardine. The effect of the level of fish gutting was also followed. During ripening a general increase of free amino
  CHANGES IN FREE AMINO ACIDS AND BIOGENIC AMINES DURING RIPENING OF FRESH AND FROZEN SARDINE R. MENDES , A. GONCALVES and M.L. NUNES Instituto de Investigapio das Pescas e do Mar Av. Brasilia 1400 Lisboa, Portugal Received for Publication January 23, 1998 Accepted for Publication July 7. 1998 ABSTRACT A study was carried out on the evolution o free amino acids and biogenic amines throughout the ripening process o resh and frozen sardine. The effect of the level offish gutting was also ollowed. During ripening a general increase of free amino acids content was observed in all samples. Histidine was the exception to this trend and decreased throughout the processing period. On the basis o the ratio of basidacidic amino acids, as an indicative o the ripened state, the fresh nobbed sardine ripened faster than the gutted fish. During the course of ripening a considerable increase in biogenic amines and particularly histamine occurred until the 20th day in products from fresh fish. The maximum histamine content allowed in ripened fish products in the European Union 400 mg/Kg) was never attained in this period. A general decrease in most amines was observed by the 240th day of ripening. Tyramine behaves differently and increased in the same period. The samples prepared with rozen fish consistently showed the lowest amine values throughout the ripening period. It seems that freezing has a good effect on the final quality of ripened products as it could decrease the bacterial activity responsible for biogenic amine formation. INTRODUCTION The ripening of sardine Sardina pilchardus) and anchovy Engraulis encrasicholus) is an ancient industry in Portugal that has remained almost unchanged for decades. At present, this industry faces great difficulty because of the lack of fresh fish, the only source of raw material used by the processors. On account of this, some projects dealing with the characterization of this process and the use of frozen fish are under development in this area. The hydrolysis of proteins is an essential phenomenon in the ripening process and depends primarily on the activity of the proteolytic enzymes present Author to whom correspondence should be sent. Journal of Food Biochemistry 23 (1999) 295-306. All Rights Reserved. Wbpyright 1999 by Food Nutrition Press, Inc., Trumbull, Connecticut. 295  296 R. MENDES, A. GONCALVES and M.L. NUNES in the fish (Kiesvaara 1975). During proteolysis, flavor and aroma substances are formed and a suitable consistency develops. Such substances are composed mainly of soluble nitrogenous compounds: amino acids, peptides and nucleo- tides, and their decomposition products (Kiesvaara 1975). The importance of free amino acids (FAA) as components responsible for the taste in fish is considerable in products where proteolysis is involved during their preparation. Furthermore, the proportion of basic and acidic amino acids of the total FAA (Basic FAAIAcidic FAA ratio) has been proposed as a criteria to follow the ripening degree of salted fish (Baldrati et al. 1977; Kiesvaara 1975). Although some studies have been already done with the ripening of sardine (Ayensa et al 1992), important aspects about the evolution of breakdown products, resulted from the proteolytic activity occurring during this process, are still not clear. Biogenic amines are among these products and result from the amino acid decarboxylase activity of some microorganisms on amino acids (Rice et al. 1976). A high incidence of toxic effects has been attributed to these compounds, in particular to histamine, which has been considered the primary causative agent responsible for the histaminic intoxication (Taylor et aZ 1984; Taylor 1985). In recent years the formation of cadaverine and putrescine has also been of interest because of the booster effect that these two amines produce on the histamine toxic action (Taylor 1986). Several authors found that histamine is more toxic in the presence of cadaverine and putrescine, since these may inhibit the in vivu mechanism of histamine detoxification by diamine oxidase and histamine N-methyltransferase (Taylor and Summer 1986). Semi-preserved fish products may contain large amounts of biogenic amines when prepared from raw material of poor quality. In these products it is also possible that amines arise throughout the production process, since it includes a ripening process involving microorganisms which can have amino acid decarboxylase activity (Veciana-Nogues et al. 1989). The large quantity of free histidine in the muscle that characterizes the Clupeidae (Yamanaka er ul 1986), may give rise to toxic levels of histamine. The present study evaluates the changes in biogenic amines and their correlation with the changes in the FAA level during the ripening of fresh and frozen sardine. The influence of the quality of the raw material and o the gutting process (partial or total) was also studied. MATERIALS AND METHODS Fish Samples Sardines Surdina pilchardus) were caught off the Portuguese coast by purse-seiners in November. After landing, the fish was transported to the  CHANGES IN FREE AMINO ACIDS 291 laboratory with ice and divided in two batches. The first batch was divided into two lots: Lot A: the fish was beheaded and partially gutted by hand (nobbed) leaving inside some viscera, washed in brine and then packed between salt layers in plastic containers to conform with commercial practice of ripening. Lot B: sardine was beheaded, thoroughly gutted (gutted) and further processed for ripening as in lot A. The second batch was immediately blast frozen at -5OC and stored for 20 days at -3OC. Frozen fish was thawed at room temperature and further processed in lots C and D, which received the same treatment as lots A and B, respective- ly. The salted fish was ripened for about 8 months at 22-24C. For analyses, 20 sardines were removed from each lot, filleted and used in the preparation of a composite sample. Chemical Analysis Trimethyiamine was determined by the Conway micro diffusion method described by Santos et al. (198 1 . Biogenic amines determination was carried out in 20 pl of 10 trichloroacetic acid extracts by the post-column method described by Ritchie (1991). The amines were separated in a SPHERI-5, C18 (220 x 4.6 mm) column from Brownlee Labs and detection was performed by fluorescence measurement (Ex. - 360 nm; Em. - 455 nm). Free amino acids were quantified in 10 TCA extracts after adjustment to pH 2.20. Free amino acids were fractionated by ion exchange chromatography according to Moore and Stein’s method (1951). The concentration of the amino acids was measured by reaction with ninhydrin reagent and spectrophotometric readings at 440nm and 5701x11. All determinations were performed in duplicate, obtaining the arithmetic means of these analyses. The results are expressed on a salt-free dry basis. RESULTS AND DISCUSSION Figure 1 shows the changes in TMA content during the ripening of sardine. A general increase was detected in all sardine lots from an initial value of approximately 6 mg/100 g to a final value of 25 mg/100 g. The highest values were consistently reported in lot A, produced with fresh nobbed fish, thus indicating a higher microbial activity in this lot. During ripening, notable changes were found in the total FAA content in the four sardine batches (Fig. 2). The total amount of FAA increased from 1.14 g/lOOg in the fresh sardine to 5.66 g/lOOg and 3.41 g/lOOg, respectively in the lots A (fresh nobbed) and B (fresh gutted). Increase in FAA was also noticed in the frozen fish which changed from an initial vdue of 1.58 g/IOOg to 4.83 g/lOOg and 3.89 g/lOOg in lots C (frozen nobbed) and D (frozen gutted),  298 R. MENDES, A. GONCALVES and M.L. NUNES respectively. Slight differences in day 0 between fresh and frozen sardine may result from enzymatic hydrolysis during the thawing process. Furthermore, during ripening the changes in the total content of FAA were similar in lots B and D as well as in A and C. On the other hand the total content of FAA was always higher in nobbed than in gutted fish lots. This behavior is in agreement with the higher proteolytic activity expected in the nobbed fish as a result of the remaining viscera. Thus it appears the gutting level was the main factor influencing amino acid accumulation during ripening. Trirnethylamine mgllOOg . ~~ 40.0 T - 30 0 20.0 10.0 0 50 100 150 200 250 FIG. 1. CHANGES OF TRIMETHYLAMINE CONTENTS DURING THE RIPENING OF SARDINE Data is expressed on a nonsalted dry basis. A - fresh nobbed, B - fresh gutted, C - frozen nobbed, D - frozen guned. FAA gll00 0.01 ' ' ' ' 50 100 150 200 250 Days FYB +-C--z] ____- FIG. 2. CHANGE OF FREE AMINO ACIDS (FAA) CONTENT DURING THE RIPENING OF SARDINE Legend text as in Fig. 1.  CHANGES IN FREE AMINO ACIDS 299 Figures 3 and 4 show the changes of the individual FAA acid during ripening in each sardine lot. In general, in the first stages of ripening, the contents of most FAA were very low, excluding histidine which was the major component, about 81.5 and 83.2 of the total (in day 0 in the frozen and fresh fish, respectively. This has been referred as a common feature of the composition of muscle FAA in dark-fleshed fish (Sakaguchi el al. 1982; Murai et al 1982). As time passed, the FAA contents increased and the changes were more evident in the nobbed fish. Histidine was an exception and in the first 20 days of ripening decreased from 1.29 C and D) and 0.95 g/lOOg (A and B) in day 0 to 0.56 (C), 0.46 (D), 0.54 (A) and 0.46 (B) g/lOOg. Judging from the results, a significant part of the histidine in fresh samples (A and B) was converted to histamine (Him) in the first 20 days of ripening, since the degree of Him production was proportional to the loss of histidine. After 20 days ripening, histidine contents were almost constant probably as a result of the equilibrium between the processes of formation, catabolism and leaching of this FAA to the brine. During the ripening of the gutted batches (lots B and D), histidine was the major component but after approximately 150 days of ripening, glutamic acid, lysine, leucine and alanine were also present in high concentration. The FAA profile was approximately the same in the nobbed fish (A and C) and glutamic acid was the predominant amino acid. Figure 5 shows the ratio of basic FAA/acidic FAA during ripening. This ratio changed after 150 days, from its initial value of 26, to a value of approximately 1 in the nobbed batches: respectively, 1 OO and 0.99 in the frozen and fresh lots. These values are in accordance with the data reported by Kiesvaara (1975) and Baldrati et al. (1977) for other fish species, who concluded this rate is indicative of the ripened state. Lots B and D did not attain these values after 240 days of ripening and also, did not ripen completely. On the other hand, lots A and C attained an index of respectively, 0.79 and 0.89, at the end of the experimental period, which seems to correspond to a degree of over ripening, as it was assessed by sensorial analysis. On the basis of this index, which has been used as an indication of the ripened state, the fresh nobbed sardine was considered to have ripened faster than the gutted fish. The changes in biogenic amines during the ripening of fresh and frozen sardine are shown in Fig. 6. At the start of the ripening the fish was judged to be fresh on the basis of the sensory properties, TMA value (< 8 mg/lOOg) and biogenic amine content ( C 0. lmg/kg). In the lot prepared with fresh, nobbed sardine (A) a very rapid increase of the Him content occurred in the initial 20 days, attaining in this period the highest value detected in all the lots (349 mg/kg). The level of Him decreased rapidly thereafter to a value of 150 mglkg and remained almost constant until the end of the experiment. This behavior may
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