Biogenic Amines and Sensory Changes Associated with the Microbial Flora of Mediterranean Gilt-Head Sea Bream Sparus Aurata) Stored Aerobically at 0, 8, and 15؇C)

398

Journal of Food Protection, Vol. 62, No. 4, 1999, Pages 398±402 Copyright ᮊ, 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 15؇C)

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, 15ЊC). 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 pseudomonads exceeded 106 to 107 CFU/g. Histamine was produced only in samples stored at 15ЊC. 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 0ЊC Storage time (h)

0 48 96 144 192 240 336

Total viable counts 4.5 Ϯ 0.3 5.1 Ϯ 0.2 5.9 Ϯ 0.2 7.0 Ϯ 0.5 8.2 Ϯ 0.9 8.9 Ϯ 0.7 9.2 Ϯ 1.1 Pseudomonads 4.2 Ϯ 0.5 4.6 Ϯ 0.4 5.3 Ϯ 0.3 6.4 Ϯ 0.6 7.3 Ϯ 0.7 8.7 Ϯ 1.1 9.0 Ϯ 1.2 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ 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 Ϯ 0.0 1.9 Ϯ 0.3 1.9 Ϯ 0.6 2.6 Ϯ 0.7 3.0 Ϯ 0.9 3.4 Ϯ 0.8 3.9 Ϯ 1.2 Lactic acid bacteria 1.5 Ϯ 0.3 2.1 Ϯ 0.5 1.9 Ϯ 0.8 2.3 Ϯ 0.5 2.9 Ϯ 0.5 3.5 Ϯ 0.7 4.5 Ϯ 0.6 Sensory rating 1 1 1 1.5 2.5 3 3 Biogenic aminesc

Putrescine 0 0 0 0.12 Ϯ 0.16 0.34 Ϯ 0.16 0.68 Ϯ 0.08 1.33 Ϯ 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 Ϯ 0.15 0.39 Ϯ 0.22 0.71 Ϯ 0.06 1.27 Ϯ 0.32 Spermidine 1.22 Ϯ 0.39 0.95 Ϯ 0.07 0.93 Ϯ 0.08 0.83 Ϯ 0.17 0.70 Ϯ 0.02 0.60 Ϯ 0.18 0.49 Ϯ 0.03 Spermine 0.31 Ϯ 0.08 0.35 Ϯ 0.15 0.26 Ϯ 0.07 0.23 Ϯ 0.03 0.32 Ϯ 0.08 0.29 Ϯ 0.13 0.25 Ϯ 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- 4ЊC) 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 15ЊC 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 ϫ 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 10ЊC for 7 d.; (ii) pseudomonads a 70:30 (vol/vol) water±acetonitrile mixture for 4 min. The pro- on cetrimide fusidin cephaloridine agar (CFC; Oxoid, CM 559 gram proceeded linearly to 50:50 water±acetonitrile with the same supplemented with SR 103), incubated at 20ЊC for 2 days (24), ¯ow rate over 2 min. This composition and ¯ow rate were main- and (iii) lactic acid bacteria on MRS medium, pH 6.2 (Oxoid, CM tained for 3 min, then changed to 70:30 water±acetonitrile at 1.1 361), incubated at 25ЊC for 5 days. For Enterobacteriaceae and ml/min for 2 min. The whole spectra (190±800 nm) of the chro- hydrogen sulphide±producing bacteria, a 1.0-ml sample was in- matograms were analyzed. The solvents were HPLC grade. To oculated into 10 ml of molten (45ЊC) violet-red bile glucose agar identify peaks, solutions of reference substances were analyzed (VRBGA; Oxoid, CM 485) and iron agar (IA; Oxoid, CM 867), using the same program, and their retention times and spectra respectively. After they had set, a 10-ml overlay of molten me- were compared. The precision of the results was always better dium was added. VRBGA plates were incubated at 30ЊC for 24 than Ϯ5%. h. The large colonies with purple halos were counted (27). IA Sensory evaluation. The dorsal half of each sample ®llet plates were incubated at 20ЊC. Black colonies formed by the pro- was heated (80ЊC, 15 min) in unsealed plastic bags. The quality duction of H2S were enumerated after 2 to 3 days (12). was assessed by 5 to 7 laboratory-trained panelists. A scoring Three replicates of at least three appropriate dilutions were scale with three categories was used (5). Class 1 corresponded to enumerated. All plates were examined visually for typical colony high-quality ®llets without any off-odors or off-¯avors; Class 2, types and morphologic characteristics associated with each growth to slight off-odors or -¯avors but acceptable quality (initial de- medium. In addition, selectivity of each medium was checked by composition stage); Class 3, to unacceptable quality (advanced Gram staining and microscopic examination of smears prepared decomposition stage). from randomly selected colonies. RESULTS AND DISCUSSION High-performance liquid chromatography (HPLC) analysis of biogenic amines. Five grams of each ®sh sample were Changes in microbial ¯ora. The changes in the mi- homogenized with 10 ml of 6% (wt/vol) trichloroacetic acid crobial ¯ora of sea bream during storage under aerobic con- (TCA). The homogenate was centrifuged (12,000 rpm, 20 min, ditions at 0, 8, and 15ЊC are shown in Tables 1, 2, and 3, 400 KOUTSOUMANIS ET AL. J. Food Prot., Vol. 62, No. 4

TABLE 2. Changes in microbial ¯ora,a concentration of biogenic amines,b and sensory rating during the storage of Mediterranean sea bream at 8ЊC Storage time (h)

0 20324872108120

Total viable counts 4.5 Ϯ 0.3 5.1 Ϯ 0.2 5.3 Ϯ 0.3 6.1 Ϯ 0.4 7.1 Ϯ 0.9 8.0 Ϯ 0.4 8.6 Ϯ 0.3 Pseudomonads 4.1 Ϯ 0.5 4.9 Ϯ 0.4 5.1 Ϯ 0.7 5.6 Ϯ 0.7 6.9 Ϯ 1.2 7.6 Ϯ 0.6 8.1 Ϯ 0.3 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ H2S-producing bacteria 2.9 0.3 3.7 0.3 4.2 0.8 5.0 0.4 6.2 1.1 7.0 0.1 7.2 0.2 Enterobacteriaceae 1.9 Ϯ 0.0 2.5 Ϯ 0.1 2.8 Ϯ 0.4 3.5 Ϯ 0.6 4.2 Ϯ 0.5 5.1 Ϯ 1.0 5.0 Ϯ 1.0 Lactic acid bacteria 1.5 Ϯ 0.3 2.4 Ϯ 0.6 2.7 Ϯ 0.4 3.4 Ϯ 0.5 4.1 Ϯ 0.5 4.6 Ϯ 0.2 4.6 Ϯ 0.5 Sensory rating 1 1 1 1.5 2.5 3 3 Biogenic aminesc

Putrescine 0 0 0.12 Ϯ 0.16 0.11 Ϯ 0.15 0.24 Ϯ 0.08 0.65 Ϯ 0.02 1.25 Ϯ 0.51 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 0.16 Ϯ 0.21 0.35 Ϯ 0.17 0.96 Ϯ 0.02 1.46 Ϯ 0.47 Spermidine 0.88 Ϯ 0.02 0.96 Ϯ 0.44 0.49 Ϯ 0.01 0.67 Ϯ 0.01 0.64 Ϯ 0.20 0.46 Ϯ 0.32 0.46 Ϯ 0.15 Spermine 0.46 Ϯ 0.10 0.48 Ϯ 0.05 0.34 Ϯ 0.01 0.40 Ϯ 0.10 0.31 Ϯ 0.01 0.36 Ϯ 0.03 0.30 Ϯ 0.01 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. respectively. Total viable counts reached approximately 8 storage of ®sh at 0 and 8ЊC (Tables 1 and 2). Changes in to 9.2 log10 cycles (CFU/g) by the end of the storage pe- the sensory score were evident only when the pseudomo- riods, regardless of temperature. Pseudomonads were the nads and H2S-producing bacteria reached approximately dominant population of ®sh stored at all temperatures, fol- 106 to 107 CFU/g. Taoukis and colleagues (32) reported that lowed by H2S-producing bacteria. These ®ndings agree the end of the shelf-life of Mediterranean boque (Boops with those of Gram and colleagues (17) and Drosinos and boops) stored aerobically at 0, 3, 7, and 10ЊC was reached Nychas (9, 10), who reported that the microbial population when pseudomonads and H2S-producing bacteria reached of ®sh stored aerobically consists almost exclusively of 107 CFU/g. pseudomonads and H2S-producing bacteria. The counts of the H2S-producing bacteria were always lower than those Production of biogenic amines. The potential use of of pseudomonads (0.5 to 1.6 log CFU/g) at the end of the biogenic amine concentration as a criterion for evaluating storage period. Other studies that focused on ice storage of freshness and spoilage in the ®sh industry has been recently various Mediterranean and tropical ®sh had similar ®ndings reviewed (3, 13). The most commonly used amine is TMA, (9, 10, 15). Enterobacteriaceae and lactic acid bacteria since it is produced during chilled storage of ®sh. TMA is were also members of the microbial population, but these formed from bacterial utilization of trimethylamine oxide Ͻ groups remained at low numbers ( log10 5 CFU/g) during (TMAO), a naturally occurring osmoregulatory substance

TABLE 3. Changes in microbial ¯ora,a concentration of biogenic amine,b and sensory rating during the storage of Mediterranean sea bream at 15ЊC Storage time (h)

0 20 32 48 72 108 120

Total viable counts 4.5 Ϯ 0.3 5.7 Ϯ 0.6 6.1 Ϯ 0.5 7.8 Ϯ 1.3 8.7 Ϯ 1.1 9.0 Ϯ 0.8 9.1 Ϯ 1.0 Pseudomonads 4.1 Ϯ 0.5 5.2 Ϯ 0.8 5.7 Ϯ 0.8 7.4 Ϯ 1.2 8.5 Ϯ 1.2 8.8 Ϯ 1.0 8.9 Ϯ 1.1 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ H2S-producing bacteria 2.9 0.3 4.1 0.7 5.1 1.2 7.2 0.8 7.8 0.7 8.4 0.8 8.3 0.8 Enterobacteriaceae 1.9 Ϯ 0.0 3.1 Ϯ 0.5 4.7 Ϯ 0.3 5.7 Ϯ 0.3 6.3 Ϯ 0.3 6.7 Ϯ 0.2 6.9 Ϯ 0.1 Lactic acid bacteria 1.5 Ϯ 0.3 1.4 Ϯ 0.2 2.3 Ϯ 0.4 3.3 Ϯ 0.5 4.4 Ϯ 0.6 6.2 Ϯ 0.5 6.3 Ϯ 0.4 Sensory rating 1 1 1.5 2.5 3 3 3 Biogenic aminesc Putrescine 0 0.19 Ϯ 0.27 0.24 Ϯ 0.31 0.42 Ϯ 0.21 2.02 Ϯ 0.94 5.31 Ϯ 2.48 6.58 Ϯ 3.20 Cadaverine 0 0 2.63 Ϯ 3.71 3.42 Ϯ 3.54 14.51 Ϯ 8.49 26.66 Ϯ 14.4 28.28 Ϯ 9.66 Spermidine 0.74 Ϯ 0.18 0.56 Ϯ 0.31 0.35 Ϯ 0.10 0.41 Ϯ 0.11 0.42 Ϯ 0.26 0.46 Ϯ 0.29 0.37 Ϯ 0.23 Spermine 0.49 Ϯ 0.06 0.45 Ϯ 0.12 0.40 Ϯ 0.05 0.31 Ϯ 0.04 0.28 Ϯ 0.13 0.25 Ϯ 0.04 0.16 Ϯ 0.02 Histamine 0 0 0 5.37 Ϯ 4.64 10.07 Ϯ 1.73 15.26 Ϯ 8.72 17.59 Ϯ 8.89 a log10 CFU/g. b mg/100 g. c The concentrations of tyramine, agmatine, tryptamine, and trimethylamine remained 0 mg/100 g during storage. J. Food Prot., Vol. 62, No. 4 BIOGENIC AMINES IN AEROBICALLY STORED GILT-HEAD SEA BREAM 401 found in most ®sh species. It is well established that only of the bacteria that decarboxylate histidine belong to En- Shewanella putrefaciens and Photobacterium phosphoreum terobacteriaceae (1, 34, 35). produce TMA (4, 5), and pseudomonads cannot use TMAO Among the polyamines detected in this study, putres- (15). The contradictory results reported (2, 20, 21, 23, 28, cine and cadaverine could be used as freshness indicators 30, 38) concerning the utility of this compound as an in- of Mediterranean sea bream. These were the only amines dicator of ®sh freshness could be due to the fact that mi- detected before initial decomposition, and they increased crobial ¯ora were not taken into account. In our study, the markedly during storage at all temperatures. Similar results absence of TMA in ®sh samples was evident (Tables 1, 2, indicating that the determination of putrescine and cadav- and 3). These ®ndings agree with results obtained for other erine can be used to assess freshness in other ®sh species Mediterranean ®sh (6, 8, 10), where no or extremely low have been reported (7, 11, 39). The absence of histamine quantities of TMA were produced. TMA may not be pre- in samples stored at 0 and 8ЊC con®rmed the observation sent because S. putrefaciens never reached levels of 108 to of other researchers that histamine occurrence is extremely 9 variable and its production is a function of time, tempera- 10 CFU/g, a population considered crucial for TMA pro- 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 duction (5). Another possible reason is the varying content ture, and the micro¯ora present (36). To understand the re- of TMAO in different ®sh species and seasons (3). The lationship between formation of biogenic amines and bac- inhibitory effect of pseudomonads against S. putrefaciens terial counts, research on polyamine production by each (16) in ®sh extract and on ®sh tissue could also explain the bacterial group of the Mediterranean sea bream micro¯ora absence of TMA. is required. Among the other biogenic amines, only spermidine and ACKNOWLEDGMENTS spermine were present initially. These two amines remained at the same levels or decreased slightly during storage in This study was carried out as a part of EU-FAIR (FAIR-1090) and all samples. Tyramine, tryptamine, and agmatine were not Greek National (EPET-II, proposal 21) projects. detected after storage at any temperatures. REFERENCES

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