398 Journal of Food Protection, Vol. 62, No. 4, 1999, Pages 398±402 Copyright Q, International Association of Milk, Food and Environmental Sanitarians Biogenic Amines and Sensory Changes Associated with the Microbial Flora of Mediterranean Gilt-head Sea Bream (Sparus aurata) Stored Aerobically at 0, 8, and 158C KONSTANTINOS KOUTSOUMANIS,* KYRIAKI LAMPROPOULOU, AND GEORGE-JOHN E. NYCHAS Agricultural University of Athens, Department of Food Science and Technology, Laboratory of Microbiology and Biotechnology of Foods, Iera Odos 75, Athens 11855, Greece MS 98-206: Received 11 August 1998/Accepted 18 November 1998 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/4/398/1670981/0362-028x-62_4_398.pdf by guest on 27 September 2021 ABSTRACT Changes in the concentrations of tyramine, agmatine, putrescine, cadaverine, spermidine, tryptamine, spermine, histamine, and trimethylamine were studied in parallel with the development of the microbial population during the storage of Mediter- ranean gilt-head sea bream (Sparus aurata) at three temperatures (0, 8, 158C). Changes in sensory scores were also recorded. Pseudomonads and H2S-producing bacteria were the dominant microorganisms. Enterobacteriaceae and lactic acid bacteria were also present in the ®sh micro¯ora. Among the biogenic amines, putrescine and cadaverine were detected when pseudo- monads exceeded 106 to 107 CFU/g. Histamine was produced only in samples stored at 158C. Tyramine, tryptamine, agmatine, and trimethylamine were absent regardless of the storage temperature. Sensory and microbial analyses are used to determine leagues (7) indicated that putrescine and cadaverine could ®sh quality. Because spoilage is a subjective evaluation and be used to assess the freshness of chilled, stored rainbow there is no general agreement on the early signs of ®sh trout (Salmon irideus). Okozumi and colleagues (29) spoilage, a number of chemical, physical, and microbiolog- showed that high levels of putrescine and cadaverine were ical methods have been proposed for detecting and mea- detected at the spoilage stage of horse mackerel meat when suring quality in ®sh (3, 13, 14, 15). Time-consuming mi- pseudomonads were the dominant bacterial ¯ora. Similar crobiological analyses may be replaced by analyses of results were reported by Suzuki and colleagues (31). These chemical changes associated with microbial growth on ®sh. two diamines have been proposed as freshness indicators Quantifying chemical changes could provide information for other ®sh species (11, 22, 39). Yamanaka and colleagues about the degree of spoilage. However, identifying the ideal (40) reported that in the fresh muscle of squid, agmatine metabolite for spoilage assessment is dif®cult (19) because was detected in small amounts, but its concentration in- (i) most metabolites are speci®c to certain organisms (e.g., creased with storage time and reached a very high level at gluconate to pseudomonads) and when these organisms are the stage of advanced decomposition. They concluded that not present or are inhibited by the food ecology, incorrect agmatine may be a useful freshness indicator for common spoilage information is provided; (ii) metabolites are the squid. In general, changes in the concentration of poly- result of the utilization of a speci®c substrate, but the ab- amines during the storage of ®sh were based on their re- sence of the given substrate or its presence in low quantities lation to bacteria that decarboxylate speci®c free amino ac- does not preclude spoilage; (iii) the rate of metabolite pro- ids (33, 34). duction and the metabolic pathways of bacteria are affected Although results concerning the formation of poly- by imposed environmental conditions (e.g., pH, oxygen amines have been reported for other ®sh species (18, 25, availability, temperature); (iv) accurate detection and mea- 29, 38, 39, 40), no data are available on changes of biogenic surements require sophisticated procedures, highly educated amines during spoilage of Mediterranean gilt-head sea personnel, time, and equipment; and (v) many metabolites bream (Sparus aurata). In addition, few studies have been give unsatisfactory retrospective information. made on the relation between bacterial counts and poly- Among chemical indicators, biogenic amines have amine production. Our aim, therefore, was to investigate been proposed for determining ®sh quality. These com- changes in biogenic amines during storage of Mediterra- pounds are found in very low levels in fresh ®sh, and their nean gilt-head sea bream at different temperatures and to formation is associated with bacterial spoilage (11). Mietz compare these with bacterial counts and sensory changes. and Karmas (26) proposed the biogenic amines index to evaluate the quality of canned tuna. The volatile compound MATERIALS AND METHODS trimethylamine (TMA) has been widely used as an indicator Microbiological analysis. Whole, fresh gilt-head sea bream of marine ®sh freshness (2, 5, 20, 23). Dawood and col- (Sparus aurata called tsipoura in Greek) were bought from Nireas s.a, a Chios island marine culture company, within 6 to 8 h after * Author for correspondence. Tel/fax: 30-1-5294693. they had been caught. The ®sh were transported to the laboratory J. Food Prot., Vol. 62, No. 4 BIOGENIC AMINES IN AEROBICALLY STORED GILT-HEAD SEA BREAM 399 TABLE 1. Changes in microbial ¯ora,a concentration of biogenic amine,b and sensory rating during the storage of Mediterranean sea bream at 08C Storage time (h) 0 48 96 144 192 240 336 Total viable counts 4.5 6 0.3 5.1 6 0.2 5.9 6 0.2 7.0 6 0.5 8.2 6 0.9 8.9 6 0.7 9.2 6 1.1 Pseudomonads 4.2 6 0.5 4.6 6 0.4 5.3 6 0.3 6.4 6 0.6 7.3 6 0.7 8.7 6 1.1 9.0 6 1.2 6 6 6 6 6 6 6 H2S-producing bacteria 2.9 0.3 3.2 0.3 4.0 0.3 5.7 0.5 6.4 0.5 7.1 0.3 7.4 0.5 Enterobacteriaceae 1.9 6 0.0 1.9 6 0.3 1.9 6 0.6 2.6 6 0.7 3.0 6 0.9 3.4 6 0.8 3.9 6 1.2 Lactic acid bacteria 1.5 6 0.3 2.1 6 0.5 1.9 6 0.8 2.3 6 0.5 2.9 6 0.5 3.5 6 0.7 4.5 6 0.6 Sensory rating 1 1 1 1.5 2.5 3 3 Biogenic aminesc Putrescine 0 0 0 0.12 6 0.16 0.34 6 0.16 0.68 6 0.08 1.33 6 0.31 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/62/4/398/1670981/0362-028x-62_4_398.pdf by guest on 27 September 2021 Cadaverine 0 0 0 0.26 6 0.15 0.39 6 0.22 0.71 6 0.06 1.27 6 0.32 Spermidine 1.22 6 0.39 0.95 6 0.07 0.93 6 0.08 0.83 6 0.17 0.70 6 0.02 0.60 6 0.18 0.49 6 0.03 Spermine 0.31 6 0.08 0.35 6 0.15 0.26 6 0.07 0.23 6 0.03 0.32 6 0.08 0.29 6 0.13 0.25 6 0.00 a log10 CFU/g. b mg/100 g. c The concentrations of tyramine, agmatine, tryptamine, histamine, and trimethylamine remained 0 mg/100 g during storage. in ice within 30 to 45 min after purchase. On arrival at the lab- 48C) and ®ltered through Whatman No. 2 ®lter paper. The ®ltrate oratory, they were gutted, ®lleted, and then divided into three was placed in a volumetric ¯ask and enough TCA 6% was added groups that were stored at 0, 8, or 158C for 336, 120, or 120 to make 20 ml. Each extract (2 ml) was derived with benzoyl hours, respectively. Two independent storage experiments were chloride according to the modi®ed method of Yen and Hsieh (41). conducted and two ®sh ®llets were analyzed on each occasion. To prepare standard amine solutions, we dissolved trypt- On each sampling occasion, a ®sh ®llet (25 g) was transferred amine-HCl (122.8 mg), putrescine-2HCl (182.9 mg), cadaverine- to a plastic bag (Seward Medical, London, UK) containing 225 2HCl (171.4 mg), spermidine-3HCl (175.3 mg), spermine-4HCl ml of 0.1% peptone water with 0.85% salt (wt/vol) and homog- (172.0 mg), histamine-2HCl (165.7 mg), TMA-HCl (161.7 mg), enized for 60 s using a Lab Blender 400, stomacher (Seward Med- and tyramine-HCl (126.7 mg) in 10 ml of deionized water and ical). used them as standard samples. The ®nal concentration of each For microbial enumeration, 0.1-ml samples of serial dilutions amine (free base) was 10 mg/ml solution. (1:10, diluent 0.1% peptone water with 0.85% wt/vol salt) of ®sh The presence of amines was determined using a Jasco (Japan) homogenates were spread on the surface of dry media. Research- Liquid Chromatograph consisting of a Model PU-980 Intelligent ers counted (i) total viable microbes on modi®ed Long and Ham- pump, a Model LG-980-02 ternary gradient unit, and a MD-910 mer's agar (mLHA; proteose peptone [Sigma, St. Louis, Mo., P multiwavelength detector. A LiChrospher 100 RP-18 reverse- 0431], 20 g/liter distilled water; gelatin [4070, Merck, West Point, phase column (5 mm, 125 3 3 mm interior diameter, E. Merck, Pa.], 40 g/liter; K2HPO4, 1 g/liter; NaCl, 10 g/liter; agar [L11, Germany) was used for separation. Oxoid, Basingstoke, UK], 15 g/liter; ammonium ferric citrate, The gradient elution program was at 1.1 ml/min, starting with 0.25 g/liter) (37), incubated at 108C for 7 d.; (ii) pseudomonads a 70:30 (vol/vol) water±acetonitrile mixture for 4 min.