Department of Seafood Research, Danish Institute for Fisheries Research (DIFRES), Lyngby, Denmark

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1 Journal of Applied Microbiology ISSN ORIGINAL ARTICLE Biogenic amine formation and microbial spoilage in chilled garfish (Belone belone belone) effect of modified atmosphere packaging and previous frozen storage P. Dalgaard, H.L. Madsen, N. Samieian and J. Emborg Department of Seafood Research, Danish Institute for Fisheries Research (DIFRES), Lyngby, Denmark Keywords biogenic amines, garfish, histamine, histamine fish poisoning, Photobacterium phosphoreum. Correspondence Paw Dalgaard, Department of Seafood Research, Danish Institute for Fisheries Research, c/o Technical University of Denmark, Søltofts Plads Building 221, DK-28, Kgs. Lyngby, Denmark. 25/768: received 7 July 25, revised 4 December 25 and accepted 5 December 25 doi:1.1111/j x Abstract Aims: To evaluate biogenic amine formation and microbial spoilage in fresh and thawed chilled garfish. Methods and Results: Storage trials were carried out with fresh and thawed garfish fillets at or 5 C in air or in modified atmosphere packaging (MAP: 4% CO 2 and 6% N 2 ). During storage, sensory, chemical and microbial changes were recorded and histamine formation by isolates from the spoilage microflora was evaluated at 5 C. Photobacterium phosphoreum was responsible for histamine formation (>1 ppm) in chilled fresh garfish. The use of MAP did not reduce the histamine formation. Strongly histamine-producing P. phosphoreum isolates formed ppm at 5 C, whereas below 6 ppm was formed by other P. phosphoreum isolates. Frozen storage inactivated P. phosphoreum and consequently reduced histamine formation in thawed garfish at 5 C markedly. Conclusions: Photobacterium phosphoreum can produce above 1 ppm of histamine in chilled fresh garfish stored both in air and in MAP. Freezing inactivates P. phosphoreum, extends shelf life and markedly reduces histamine formation in thawed MAP garfish during chilled storage. Significance and Impact of the Study: At 5 C, more than 1 ppm of histamine was formed in garfish; thus even when it is chilled this product represents a histamine fish-poisoning risk. Introduction Every spring, schools of garfish (Belone belone belone) migrate through Danish waters into the Baltic Sea for spawning and feeding in shallow waters. During August to October, they return to the eastern North Atlantic Ocean (Dorman 1991). In Denmark, there is a tradition for both commercial and recreational fishing of garfish and landings are of 5 1 tons per year. This is relatively high compared with Estonia, Greece, Italy, Spain and Turkey where recorded garfish landings vary from 1 to >1 tons per year ( fisoft/fishplus.asp, assessed May 25). In commercial fishing in Denmark, garfish are typically caught in pond nets and transported to a harbour where they are iced and later gutted and filleted in fish shops prior to sale. In households or canteen kitchens, fillets are likely to be stored overnight, and sometimes longer, in refrigerators at about 5 C. Garfish are mainly sold as chilled fresh fillets although its properties allow processing by freezing or smoking (Bruun 1988). Modified atmosphere packaging (MAP) is used extensively for retail distribution of fresh fish, but to our knowledge this technology has not been used commercially or studied experimentally for garfish. In Denmark, garfish has caused outbreaks of histamine fish poisoning (HFP). In 1997, two HFP outbreaks involving 25 children and 4 adults were caused by grilled garfish containing 75 9 and 12 ppm of histamine, ª 26 The Authors 8 Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26) 8 95

2 P. Dalgaard et al. Histamine formation in chilled garfish respectively. In 21, 13 children became ill after consumption of grilled garfish with 1 12 ppm of histamine. Symptoms occurred within 6 min after the intake of garfish and included strong headache, rash, oral burning, a tingling sensation in fingers and diarrhoea. Related to these three outbreaks, about 5% of those consuming garfish fell ill and 25 5% of the ill persons were treated in a hospital. All the three recorded outbreaks occurred during autumn (Hess Thaysen and Sloth 1997; A. Kjølby and A. Perge, personal communication). Histamine fish poisoning is the most common reason for finfish-borne human disease, and although finfish like mackerel, mahimahi and tuna have been extensively studied HFP remains an important problem for consumers and the seafood sector. In fact, HFP has caused about half of all reported finfish-borne outbreaks of disease in both the United States and the United Kingdom (Lehane and Olley 2; Flick et al. 21; Gillespie et al. 21; CSPI 24). Histamine in seafood is formed by the decarboxylation of free amino acid histidine, and products that cause HFP must contain sufficient concentrations of free histidine to allow formation of at least 5 1 ppm of histamine. In addition, histidine decarboxylase producing micro-organisms must be present and able to grow to 1 7 CFU g )1 or more. If micro-organisms produce other biogenic amines in significant amounts, these may increase the toxicity of histamine (Lehane and Olley 2; Flick et al. 21). Relying on concentrations of biogenic amines, different indices of seafood quality have been suggested but it seems disputable that decomposition protects consumers from hazardous concentrations of biogenic amines (Lehane and Olley 2; Flick et al. 21). Off-flavours is used by some experts to detect histamine in seafood, but at the same time a large number of HFP outbreaks have documented that many consumers eat seafood with high concentrations of histamine despite possible off-flavours. Many studies only found significant histamine formation in fresh fish, when products were stored at temperatures above 7 1 C and under these conditions mesophilic Enterobacteriaceae were primarily responsible for histamine formation (Taylor 1986; Lehane and Olley 2; Flick et al. 21; Kim et al. 24). However, above 5 1 ppm of histamine can be formed in various species of naturally contaminated fresh fish when stored at 5 C (Okuzumi et al. 1981, 1982; Yamanaka et al. 1984; Ababouch et al. 1991, 1996; Silva et al. 1998; Emborg et al. 25). For garfish, the content of free histidine, the microflora that develops during refrigerated storage and the profile of biogenic amines that they may produce have not been described. It is also unclear if toxic concentrations of histamine can be formed in properly chilled garfish or if storage at high temperature is required. The objectives of the present study were to evaluate biogenic amines formation, shelf life, chemical changes and microbial changes in chilled garfish. Fillets of fresh garfish were stored in air and in MAP at both and 5 C and the effect of frozen storage at )22 C on histamine and biogenic amines formation in thawed garfish at 5 C was studied. Materials and methods Storage trials with naturally contaminated garfish fillets Two series of storage trials (during spring and autumn) were carried out with fresh and thawed garfish (B. belone belone). Garfish were caught in pond nets during the last week of April (Spring) in Musholm bugt, Storebælt (55Æ4 N, 11Æ1 E) and during the first week of October (Autumn) in Køge bugt (55Æ5 N, 12Æ5 E). Water temperature was 1 12 C both in spring and in autumn. When taken live from pond nets, the garfish were landed within 1 h, iced and directly transported to DIFRES. The average weights of the garfish specimens were 415 g in the spring (n ¼ 5) and 64 g in the autumn (n ¼ 25). For each of the two series of experiments, garfish specimens were divided into six sub-batches and submitted to six different treatments. In brief, the experimental design included four treatments of fresh garfish fillets stored either in air or in MAP at and 5 C. Two sub-batches of garfish were frozen and later after thawing, fillets were stored in air and in MAP at 5 C. The fresh garfish were gutted and filleted using a dedicated garfish-filleting device. Skin-on fillets were cut into pieces of appropriate size for packaging. One hundred and twenty grams of fillet pieces were placed in plastic trays and packed with 2Æ5 3Æ vols of gas (3 36 ml) using a Multivac C5 packaging machine (Multivac Ltd, Vejle, Denmark). Garfish fillets from two sub-batches were packed in air using packs of 7 lm thick polyethylene film (H92, Topiplast Ltd, Greve, Denmark) with high permeability of >6 g m )2 d )1 for water vapour, >3 cm 3 m )2 d )1 atm )1 for O 2 and >14 cm 3 m )2 d )1 atm )1 for CO 2. Fillets from two different subbatches were packed in a modified atmosphere with 4% CO 2 and 6% N 2 (AGA, Copenhagen, Denmark). The gas mixture was chosen as preliminary experiments showed that it reduced oxidation of garfish flesh and previously resulted in relatively long shelf life of fresh and thawed MAP salmon (Emborg et al. 22). A 117 ± 6 lm packaging film (NEN 4 HOB/LLPDE 75, Amcore Flexibles, Horsens, Denmark) with low gas permeability of 3Æ9 gm )2 d )1 for water vapour, Æ45 ± Æ15 cm 3 m )2 d )1 atm )1 for O 2 and 1Æ8 ±Æ6 cm 3 m )2 d )1 atm )1 for CO 2 was used. This film consisted of polyamide, ethylenevinyl, ª 26 The Authors Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26)

3 Histamine formation in chilled garfish P. Dalgaard et al. polyamide, polyurethane adhesive and polyethylene layers. Packs of fresh garfish in air and in MAP were stored both at and 5 C. Whole garfish from two sub-batches were glazed and frozen in a blast freezer at )45 C until the core temperature was below )2 C. Whole frozen garfish were stored at )22 C during 5 6 weeks and then thawed overnight at 5 C. The thawed garfish were gutted, filleted and packed as described above for fresh garfish. Packs of thawed garfish in air and in MAP were stored at 5 C. The temperature of all sub-batches of garfish was followed during frozen and chilled storage by data loggers (Tinytag, Gemini Data Loggers Ltd, Chichester, UK). Sampling and analyses Packs from all the studied sub-batches of skin-on garfish fillets were removed for analyses at regular intervals during chilled storage at or 5 C. At each sampling time, three packages were evaluated. The composition of the atmosphere in packs was determined by a gas analyser (Combi Check 98-1, PBI Dansensor, Ringsted, Denmark), drip loss was measured as previously described (Dalgaard et al. 1993) and sensory, microbiological and chemical analyses were carried out as indicated below. Sensory analyses At each sampling time, off-odour development for each sub-batch of chilled garfish was evaluated by two persons with years of experience in sensory evaluation of quality changes in seafood. Off-odours were graded as absent, weak or strong. In addition, cooked samples from each sub-batch of chilled garfish were evaluated by 5 8 trained judges. This evaluation was carried out two or three times around the expected time of sensory spoilage. All panelists evaluated two portions of garfish from each sub-batch every time analyses were carried out. Portions of 25 g were heated in coded porcelain bowls in a convection oven (1 C, 14 min) and then served to the panelists. In addition to garfish stored at or 5 C, freshly thawed garfish samples, previously kept at )4 C, were included in each sensory evaluation as controls. Appearance, off-flavours and texture of samples were evaluated by using a simple scale with three classes, where 3 corresponds to a spoiled sample (Dalgaard 2). The shelf life of a sub-batch of garfish was defined as the time when off-odours were present (weak or strong) and 5% or more of the panelists determined the cooked samples to be in class 3. Training of panelists in this shelf life determination procedure was carried out with garfish fillets stored at 5 C. Microbiological analyses All sub-batches of skin-on garfish fillets were analysed for concentrations of aerobic plate counts (APC), Photobacterium phosphoreum and H 2 S-producing organisms. Concentrations of Enterobacteriaceae, fluorescent pseudomonads and lactic acid bacteria (LAB) were determined on the first day of chilled storage and at the time of sensory spoilage. In addition, the concentrations of these groups of bacteria were determined during chilled storage for selected sub-batches. Twenty grams of garfish flesh with skin were diluted tenfold in chilled physiological saline (PS) (Æ85% NaCl and Æ1% Bacto-Peptone ; Becton Dickinson, Sparks, MD, USA) and homogenized for 6 s in a Stomacher (Seward Laboratory Blender, London, UK). From this homogenate, a series of tenfold dilutions was prepared with chilled PS. APC were determined by spread plating on prechilled plates of Long and Hammer agar (van Spreekens 1974) with 1% NaCl (LH). LH plates were incubated at 15 C for 7 days. Photobacterium phosphoreum was enumerated at 15 C by a conductance-based incubation method using three vials for each garfish sample (Dalgaard et al. 1996). For samples of garfish with very low concentrations of P. phosphoreum, a change in conductance was not observed for all vials, and thus a simple most probable number method was used to calculate the concentration of the bacteria (Dalgaard et al. 1997a). H 2 S-producing organisms were enumerated by pour plating in Iron agar Lyngby (IA; CM964, Oxoid) (25 C, 3 days). Enterobacteriaceae were quantified by spreading 1 ml of diluted sample on plates with 5 ml of Tryptone Soya Agar (TSA; CM 131, Oxoid), then after 2 h at room temperature Violet Red Bile Glucose agar (VRBG; CM4585, Oxoid) was added. These TSA/VRBG plates were incubated (25 C, 48 h) and red and/or purple colonies counted as Enterobacteriaceae. Fluorescent pseudomonads were enumerated by spread plating on King agar B (Merck, 1Æ1991) with 1Æ% glycerol. Plates were incubated 48 h at 25 C and fluorescent colonies counted under UV light. Concentrations of LAB were determined by pour plating in Nitrate Actidion Polymyxin agar with ph 6Æ2 (25 C, 3 days) (Davidson and Cronin 1973). To identify the dominating spoilage microflora in garfish fillets, a total of 12 colonies were isolated from LH agar at the time of sensory spoilage. Colonies on highest dilutions of LH agar were isolated, pure cultured and characterized by colony morphology, Gram reaction, motility, catalase and cytochrome oxidase tests, fermentation of glucose and sensitivity to vibriostaticum (15 lg). Gram-negative, oxidase-negative (or late positive) and fermentative coccobacilli with sensitivity to vibriostaticum, i.e. Photobacterium-like isolates, were tested for metabolism of arginine and ornithine, reduction of trimethylamine-n-oxide (TMAO), production of gas from glucose, growth with % NaCl and at and 35 C, assimilation of d-mannitol, bioluminescence and hydrolysis of gelatine ª 26 The Authors 82 Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26) 8 95

4 P. Dalgaard et al. Histamine formation in chilled garfish (Dalgaard et al. 1997b). Gram-negative, catalase and oxidase-positive, motile and nonfermentative rods were tested for the metabolism of arginine and ornithine and for the reduction of TMAO and H 2 S production. Pseudomonas-like isolates unable to reduce TMAO were tested for fluorescence on King agar B, fermentation of maltose, production of indole and hydrolysis of gelatine (Stenström and Molin 199). Shewanella-like TMAO reducing isolates were further tested for assimilation of citrate, d-gluconate, sucrose, growth with and 6% NaCl (w/v) and at, 4 and 37 C (Ziemke et al. 1998). Gramnegative, catalase-positive, oxidase-negative and vibriostaticum-resistant isolates (Enterobacteriaceae-like) were characterized using API 2 E (BioMérieux, Marcy-l Etoile, France) and tested for fermentation of lactose, xylose and trehalose, phenylalanine deaminase, reduction of nitrate and TMAO and growth at 2, 3 and 37 C. Gram-positive isolates were further tested for growth on acetate agar, final ph after growth in La broth (Reuter 197), production of gas from glucose and gluconate, ammonia production from arginine and production of acetoin from glucose (Voges Proskauer). Carnobacterium-like isolates were tested for fermentation of inulin, d-lactose, d-mannitol, methyl-a-d-glucoside, methyl-a-d-mannoside and d-xylose (Collins et al. 1987). The biochemical identification tests were carried out as previously described (Dalgaard et al. 1997b, 23). Isolates were identified by general keys (Barrow and Feltham 1999), the keys indicated above for specific species or by the APILAB plus software (BioMérieux) for Enterobacteriaceae-like isolates. To support the identification, the following type and reference strains were included in the study: Carnobacterium piscicola (DSM 273 T ), Morganella morganii subsp. morganii (LMG 7874 T ), P. phosphoreum (ATCC 114 T ), Pseudomonas fluorescens (ATCC 1325 T ) and Shewanella baltica (NCTC 1735 T ). Chemical analyses To characterize garfish fillets, samples were homogenized using a blender and dry matter, free amino acids, lipid content, NaCl content, organic acids, ph, trimethylamine (TMA), TMAO and total volatile nitrogen (TVN) were determined in triplicate. These analyses were carried out prior to chilled storage and as previously described (Barkholt and Jensen 1989; Dalgaard et al. 1993; Emborg et al. 22). During chilled storage of fresh and thawed garfish, ph, concentration of TMAO, TVN, TMA and biogenic amines were determined. Biogenic amines were extracted from 15 g of garfish with 7Æ5% trichloroacetic acid and quantified by HPLC where postcolumn derivatization with o-phthadialdehyde (OPA) allowed agmatine, b-phenylethylamine, cadaverine, histamine, putrescine, spermidine, spermine, tryptamine and tyramine to be determined by fluorescence detection at 43 nm (Jørgensen et al. 2a). For sub-batches of garfish studied during spring, the concentration of organic acids was also determined at the time of sensory spoilage. Histamine and biogenic amine formation by isolates from the dominating microflora Forty-six selected isolates from spoilage microflora were screened for the production of histamine and other biogenic amines. Isolates were selected to represent all identified groups of bacteria from all sub-batches of chilled garfish. Biogenic amine formation was studied using a liquid media (growth medium broth, GMB; Dalgaard et al. 1994), added TMAO (441 mg-n l )1 ), arginine (1 ppm), lysine (5 ppm), histidine (75 ppm), phenylalanine (5 ppm) and tyrosine (9 ppm) (GMB with amino acids, GMB-AA) to match the concentrations of these compounds in garfish fillets (Table 1). Sterile broth with Æ5% NaCl was incubated overnight in air or in 25% CO 2 /75% N 2 followed by adjustment of ph to 6Æ25. From the broth in air, 8Æ5 ml portions were transferred to sterile test tubes with normal test tube caps and stored in air. From the broth in 25% CO 2 /75% N 2, 8Æ5 ml portions were transferred to sterile Hungate tubes and added a headspace gas of 25% CO 2 /75% N 2. All isolates were pre- Table 1 Characteristics of garfish fillets Average ± SD (n ¼ 3) Spring Autumn Dry matter (%)* 21Æ8 ± Æ84 28Æ9 ± Æ27 Lipid (%)* 2Æ1 ±Æ6 5Æ3 ±1Æ1 Lactic acid (ppm)* 3284 ± ± 393 ph* 6Æ45 ± Æ4 6Æ2 ± Æ1 Trimethylamine-N-oxide (mg-n kg )1 )* 66 ± ± 9 Total volatile nitrogen (mg-n kg )1 )* 14 ± 5 14 ± 4 Free amino acids (ppm) Alanine* 786 ± ± 45 Arginine 13 ± ± 8 Aspartic acid 25 ± 13 1 ± 2 Cystine 199 ± ± 6 Glutamic acid 461 ± ± 15 Glycine* 538 ± ± 28 Histidine 684 ± ± 644 Isoleucine* 87 ± ± 3 Leucine 137 ± ± 5 Lysine 559 ± ± 26 Methionine* 63 ± ± 1 Phenylalanine* 69 ± ± 3 Proline* 122 ± 24 3 ± 1 Serine* 325 ± ± 5 Tyrosine 117 ± ± 5 Valine* 124 ± 18 7 ± 4 *Concentrations differed significantly (P < Æ5) between spring and autumn. ª 26 The Authors Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26)

5 Histamine formation in chilled garfish P. Dalgaard et al. cultured at 5 C in the same medium as used to evaluate histamine and biogenic amine formation. From these precultures, broth in the two atmospheres were inoculated with 1 4 CFU ml )1 and stored at 5 C. Each isolate was analysed in duplicate. Bacterial isolates from garfish stored in air were studied using test tubes in air, and isolates from MAP garfish were studied using broth saturated with 25% CO 2 /75% N 2 and stored in Hungate tubes. All cultures were incubated until the absorbance (54 nm) no longer increased more than Æ5 U during 24 h. After filter sterilization and appropriate dilution of the broth, the concentrations of biogenic amines were determined by HPLC as described above. Furthermore, histamine and biogenic amine formation by selected isolates were evaluated in the same broth but with 2Æ% NaCl, ph 5Æ5 and stored in air at 2 C. Effect of ph and MAP on biogenic amine formation by Photobacterium phosphoreum at 5 C To evaluate how differences in ph between spring and autumn garfish (Table 1) influence growth and biogenic amine formation by P. phosphoreum, a mixture of six isolates from fresh garfish was studied during storage in air and in MAP. Sterile GMB-AA, prepared as described above, was divided into four portions. Two portions were incubated overnight in air and two other portions in 25% CO 2 /75% N 2. For broth portions from each of the two atmospheres, ph values were adjusted to 6Æ45 and 6Æ, respectively, to match the ph of spring and autumn garfish. The six P. phosphoreum isolates were precultured individually in GMB-AA at 5 C and then mixed (Pp mix). The four portions of broth were inoculated with Pp mix to a concentration of 1 2 CFU ml )1. From the broth in air, 8Æ5 ml portions were transferred to sterile test tubes with normal test tube caps and stored in air. From the broth in 25% CO 2 /75% N 2, 8Æ5 ml portions were transferred to sterile Hungate tubes and added a headspace gas of 25% CO 2 /75% N 2. All cultures were stored at 5 C. At regular intervals during storage at 5 C, P. phosphoreum was enumerated by spread plating on prechilled LH plates, ph was measured and the concentrations of biogenic amines were determined by HPLC as described above. Statistical analyses The degree of garfish spoilage, determined as the percentage of samples in class three (per cent-class 3, Y variable) was related to chemical and microbiological changes in garfish (X variables) by partial least squares regression (PLSR). unscrambler Ò (version 9Æ1, CAMO A/S, Trondheim, Norway) was used for this multivariate data analysis. Prior to PLSR analysis, each variable was centred and scaled by subtracting the mean value and dividing by the SD across all samples. Full crossvalidation of models was used. anova and the t-test were used to evaluate differences between samples and treatments. The Wilcoxon signed rank test was used to evaluate the effect of season, atmosphere and previous freezing on histamine formation and growth of P. phosphoreum during chilled storage of garfish. A multifactor anova with storage time as covariate was used to evaluate the effect of treatments on drip loss from garfish fillets as well as the effect of ph and atmosphere on biogenic amine formation by P. phosphoreum in broth. Statistical analyses were carried out using statgraphics Ò plus v. 5Æ1 (Manugistics Inc. Rockville, MD, USA). The four-parameter log-transformed logistic model was used to estimate maximum specific growth rates (l max ) from microbial growth curves (Dalgaard et al. 1994). Results Storage trials Product characteristics and storage conditions Garfish caught during immigration to the Baltic Sea differed in several ways from those caught in October when leaving this area (Table 1). In the spring, the fish flesh contained significantly less dry matter, lipid and lactic acid (P Æ1) and had significantly higher ph and concentration of TMAO (P Æ1). In both cases, the flesh contained little NaCl (Æ1 Æ3%). The concentration of free amino acids in garfish fillets seemed higher during the spring, but the difference was not statistically significant for all amino acids (Table 1). The free histidine concentration of c. 64 ppm indicates that about 45 ppm of histamine may be formed in garfish fillets. For newly caught autumn garfish concentrations of APC and P. phosphoreum were, respectively, 4Æ2 ±Æ2 and 1Æ ±Æ7 log CFU cm )2 of skin, 4Æ4 ±Æ3 and 3Æ1 ±Æ1 log CFU g )1 of gills and 7Æ7 ±1Æ3 and 5Æ1 ±3Æ5 log CFU g )1 of intestinal content (n ¼ 3, results not shown). Just after packaging the modified atmospheres contained CO 2 concentrations from 37Æ4 to39æ8%. CO 2 dissolved in the fish flesh and equilibrium concentrations of CO 2 in the modified atmosphere ranged from 23Æ to29æ% with an overall average of 26Æ3%. The average storage temperatures varied from )Æ3 Cto)Æ1 C for a target value of C and from 4Æ9 C to5æ3 Cfor a target value of 5 C. For the target value of C during autumn, the storage temperature was )Æ34 ± 1Æ15 C from day to day 17 and +Æ1 ±Æ6 C from day 18 to ª 26 The Authors 84 Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26) 8 95

6 P. Dalgaard et al. Histamine formation in chilled garfish day 38. The average frozen storage temperatures were )22Æ ± 1Æ7 C and )21Æ5 ± Æ7 C in spring and autumn, respectively. Shelf life and sensory changes In general, shelf life of garfish was similar in spring and autumn (Table 2). The exception was a very long shelf life of 38 days for fresh MAP autumn garfish at C. A temperature of )1 C to)2 C during the early part of the storage period for that specific sub-batch of autumn garfish may have contributed to this unexpectedly long shelf life. MAP extended the shelf life of fresh spring garfish at C and of fresh garfish at 5 C by 43% when compared with storage in air, whereas the same modified atmosphere extended the shelf life of frozen and thawed garfish at 5 C by more than 7% (Table 2). The sensory spoilage characteristics of garfish were similar during spring and autumn and results for fresh and thawed sub-batches are shown in Table 2. A PLSR-model with two PLS-components explained 76% of the observed variation in the per cent-class 3 data (Y-variable) and a good correlation (r 2 ¼ Æ86) between observed and predicted per cent-class 3 data was observed. This PLSR-model relied on changes in 17 measured microbial and chemical parameters, but the model explained only 56% of the variation in these parameters. Thus, a subset of microbiological and chemical data may be sufficient to explain changes in the Y-variable. In fact, the per cent-class 3 data from all subbatches of garfish from spring and autumn were equally well described by a simple PLSR-model with one PLScomponent, relying on only four X-variables [Eqn (1)], where concentrations are in Log CFU g )1 for APC and H 2 S-producing bacteria, ppm for histamine and mg-n kg )1 for TVN. The standard error of prediction was 21%-class 3 for eqn (1) and it should be used accordingly and not for detailed prediction of per centclass 3 values. %-class 3 ¼ 4 þ 53½APCŠþ43½H 2 SŠ þ 16½HistŠþ64½TVNŠ: ð1þ Chemical changes Histamine formation in chilled garfish was observed only when APC were about 1 7 CFU g )1 or above (Fig. 1). The formation of detectable concentrations of histamine started approximately at the time of sensory product rejection but thereafter the amounts produced strongly depended on storage temperature and previous freezing of the fish (Fig. 1). For fresh garfish, MAP had no clear effect on the time of histamine formation (Fig. 1). However, the combined effect of previous freezing and MAP resulted in both a considerable shelf life extension (Table 2) and a pronounced reduction (P <Æ1) in the formation of histamine (Fig. 1). Between spring and autumn, the formation of histamine during storage only differed significantly for fresh MAP garfish at 5 C (P <Æ1), where histamine formation was more pronounced in spring garfish (Table 2). Very little (<1 ppm) b-phenylethylamine, spermine and tryptamine were formed in garfish and concentrations of agmatine, cadaverine, putrescine and tyramine were lower than the formation of histamine. As for histamine (Fig. 1), the concentrations of other biogenic amines mainly increased at and after the time of sensory product rejection (Fig. 2). Substantial amounts of TMA were formed during storage of garfish (Fig. 3a; Table 2). The TMA and TVN concentrations increased at the same time during storage, indicating that little if any ammonia was produced (Fig. 3b). Therefore, the ammonia-like sensory spoilage characteristics (Table 2) probably resulted from TMA. The TMA formation in garfish corresponded closely to the consumption of TMAO. In fact the average yield of mg-n TMA from mg-n TMAO was 1Æ in the spring and Æ93 in the autumn. Other chemical changes included the formation of acetic acid ( 1 ppm) and pyruvic acid (c. 5 ppm) in fresh but not in thawed garfish. ph and concentrations of CO 2 increased, respectively, by as much as Æ5 Æ7 U and 1% in sub-batches where substantial formation of TMA and biogenic amines was observed at the end of the storage period (Table 2; Figs 1 3). The drip loss from garfish fillets increased with storage time (P <Æ1) and for some treatments a maximum level of about 6% (w/w) drip loss was reached (Table 2). During storage, the percentage (w/w) of drip loss increased more rapidly in thawed than in fresh garfish (P < Æ1) whereas season, storage atmosphere and chill storage temperature had no significant effect on the increase of percentage (w/w) of drip loss. Microbiological changes The initial APC of fresh garfish fillets in air were 3Æ6 ±Æ6 and 4Æ1 ±Æ2 log CFU g )1, respectively, in spring and autumn. The initial concentrations of P. phosphoreum and H 2 S-producing micro-organisms were much lower but after some days of aerobic storage at both C and 5 C they made up an important part of APC (Fig. 1). Only in fresh autumn garfish did MAP significantly (P <Æ5) reduced the growth of P. phosphoreum but MAP markedly inhibited the growth of H 2 S-producing micro-organisms in all sub-batches of garfish (Fig. 1, Tables 3 and 4). The spoilage microflora of fresh garfish fillets in air was dominated by Ps. fluorescens/putida, P. phosphoreum and S. baltica (H 2 S-producing) with 2% Aeromonas/Vibrio present in spring garfish. Photobacterium phosphoreum to a much larger ª 26 The Authors Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26)

7 Histamine formation in chilled garfish P. Dalgaard et al. Table 2 Shelf life and spoilage characteristics of fresh and thawed garfish fillets Fresh garfish Frozen/thawed garfish C 5 C 5 C Air MAP Air MAP Air MAP Shelf life (d) Spring Autumn >16* Sensory spoilage characteristics Aerobic plate counts, log (CFU g )1 ) Histamine at sensory spoilage, ppm Histamine 1 3 days after spoilage, ppm Trimethylamine (mg-n kg )1 ) Total volatile nitrogen (mg-n kg )1 ) Dark colour; ammonia-like, oxidized and nauseous flavour; dry and soft texture Dark colour; sour and oxidized flavour; dry texture Dark colour; ammonia-like, oxidized, sour and sharp flavour; dry and soft texture Dark colour; ammonia-like, sour and sharp flavour; dry and soft texture Dark colour:; ammonia-like, oxidized, musty and putrid flavour; dry and soft texture Dark colour; ammonia-like, sour and musty flavour; dry and tough texture Spring 7Æ8 ±Æ2 7Æ4 ±Æ3 8Æ6 ±Æ1 7Æ4 ±Æ1 9Æ3 ±Æ1 8Æ2 ±Æ2 Autumn 8Æ ±Æ1 5Æ9 ±Æ4 7Æ8 ±Æ6 6Æ7 ±Æ1 8Æ1 ±Æ4 6Æ8 ±Æ5* Spring Æ ±Æ 13±12 149±59 57±95 2±2 32±16 Autumn 11 ± 19 1 ± 2 28 ± ± 91 ± 19 ± 3* Spring 21 ± ± ± ± ± ± 38 Autumn 46 ± 585à 127 ± ± 339 Spring 22 ± ± ± ± ± ± 5 Autumn 26 ± 6 27 ± ± ± 6 12 ± 2 1 ± 2* Spring 155 ± ± ± ± ± ± 79 Autumn 23 ± ± ± ± ± 28 2 ± 11* ph Spring 6Æ53 ± Æ14 6Æ7 ± Æ12 6Æ68 ± Æ22 6Æ81 ± Æ4 6Æ43 ± Æ7 6Æ44 ± Æ13 Autumn 6Æ4 ± Æ4 6Æ6 ± Æ3 6Æ12 ± Æ5 6Æ19 ± Æ2 6Æ1 ± Æ5 6Æ4 ± Æ1* Drip loss (%, w/w) Spring 5Æ6 ± 2Æ9 4Æ9 ± Æ6 2Æ5 ± 1Æ8 3Æ3 ± 1Æ9 2Æ5 ± 1Æ5 5Æ9 ± Æ9 Autumn 4Æ4 ±2Æ5 5Æ3 ±1Æ6 2Æ2 ±Æ5 3Æ7 ±1Æ5 4Æ3 ±1Æ7 4Æ6 ±Æ7* *Experiment was stopped before the thawed MAP garfish became sensory spoiled. Samples of spring garfish also had ammonia-like and nauseous flavours and soft texture, whereas samples of autumn garfish had putrid flavour and a tough texture. àconcentrations in the three packs were 32, 11 and 188 ppm of histamine, respectively. Not determined. ª 26 The Authors 86 Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26) 8 95

8 P. Dalgaard et al. Histamine formation in chilled garfish Garfish in air (a) Fresh at C MAP garfish (b) Fresh at C Log 1 (CFU g 1 ) (c) Fresh at 5 C (d) Fresh at 5 C Histamine (ppm) (e) Thawed at 5 C Storage period (days) 9 (f) Thawed at 5 C Figure 1 Histamine formation ( ) and microbiological changes in fresh and frozen and thawed garfish. Aerobic plate counts determined on Long and Hammer agar (h), H 2 S-producing micro-organisms detected in iron agar (d) and Photobacterium phosphoreum (s). fl indicates the time of sensory spoilage. extend dominated the spoilage microflora of fresh MAP garfish (Fig. 1; Table 3). Initial APC in thawed garfish fillets were 2Æ5 ±Æ4 and 3Æ9 ±Æ1 log CFU g )1 in spring and autumn, respectively. Photobacterium phosphoreum was not detected in the thawed product (Fig. 1; Table 3) but H 2 S-producing micro-organisms, Enterobacteriaceae and fluorescent pseudomonas grew and reached high concentrations (Table 4). The spoilage microflora of thawed garfish in air was dominated by Ps. fluorescens/putida and S. baltica, whereas for thawed MAP garfish S. baltica, Carnobacterium maltaromaticum (8% in autumn), Aeromonas/Vibrio (27% in spring) and a psychrotolerant M. morganii-like bacteria (9 1%) dominated the spoilage microflora (Table 3). ª 26 The Authors Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26)

9 Histamine formation in chilled garfish P. Dalgaard et al. Garfish in air MAP garfish 175 (a) Fresh at C (b) Fresh at C Biogenic amines (ppm) 45 (c) Fresh at 5 C 45 (d) Fresh at 5 C (e) Thawed at 5 C (f) Thawed at 5 C Storage period (days) ), putres- Figure 2 Biogenic amine formation in fresh and frozen and thawed spring garfish during chilled storage. Agmatine (h), cadaverine ( cine (s) and tyramine (d). fl indicates the time of sensory spoilage. Histamine and biogenic amine formation by isolates from the dominating microflora Forty-six isolates were evaluated but only five P. phosphoreum isolates from fresh garfish and two M. morganii-like isolates from thawed garfish formed high concentrations of histamine at 5 C (Table 5). The five P. phosphoreum isolates formed ppm of histamine, whereas the remaining 19 isolates of P. phosphoreum studied formed ª 26 The Authors 88 Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26) 8 95

10 P. Dalgaard et al. Histamine formation in chilled garfish mg-n kg 1 7 (a) (b) Table 3 Composition of the dominating spoilage microflora in chilled garfish Percentage of isolates* Fresh garfish C 5 C 5 C Frozen/thawed garfish Air MAP Air MAP Air MAP Photobacterium phosphoreum Spring Autumn Pseudomonas fluorescens/putida Spring Autumn 7 1 Shewanella baltica Spring Autumn Other bacteria as explained in the text Spring Autumn 45 1 *9 11 isolates were characterized for each of the 12 sub-batches of garfish. Four of these 1 isolates were bioluminescent whereas all other isolates of P. phosphoreum were nonbioluminescent Storage period (days) Figure 3 (a) Trimethylamine (TMA) formation in fresh garfish stored in air at C (h), in MAP at C ( ), in air at 5 C (s), in MAP at 5 C (d) as well as in frozen and thawed garfish stored in air at 5 C (n) and in MAP at 5 C ( ). (b) Changes in concentrations of trimethylamine-n-oxide (.), TMA ( ) and total volatile nitrogen (*) during storage of fresh MAP garfish at C. less than 6 ppm of histamine (Table 5). Interestingly, all the weak histamine-producing P. phosphoreum isolates were nonbioluminescent, whereas two of the five strongly histamine-producing P. phosphoreum isolates were bioluminescent. It was confirmed that for seven weak histamineproducing isolates of P. phosphoreum they formed <6 ppm of histamine even at optimal conditions for histamine formation (2 C, ph 5Æ5 and with 2% NaCl). Both histamine-producing variants of P. phosphoreum formed agmatine (<5 3 ppm), cadaverine (<5 126 ppm), putrescine (<5 311 ppm), b-phenylethylamine (<5 53 ppm) and tyramine (<5 418 ppm). Of these biogenic amines only the formation of agmatine differed significantly (P ¼ Æ4) between the two variants of P. phosphoreum, with the strong histamine producers forming least agmatine. The psychrotolerant M. morganii-like isolates formed putrescine (<5 17 ppm) and tyramine (<5 16 ppm) but no agmatine, cadaverine and b-phenylethylamine. Shewanella baltica formed cadaverine (9 66 ppm) and putrescine (16 86 ppm) and one of the Table 4 Growth of groups of bacteria in frozen and thawed garfish Maximum specific growth rate (h )1 ) Concentration at sensory spoilage (log CFU g )1 ) Air MAP Air MAP H 2 S-producing bacteria Spring 1Æ95 ± Æ54 1Æ23 ± Æ23 7Æ6 ± 1Æ5 7Æ3 ± 1Æ3 Autumn 1Æ79 ± Æ6 1Æ6 ± Æ5 6Æ5 ± Æ5 6Æ7 ± Æ5 Enterobacteriaceae Spring 1Æ9 ± Æ7 1Æ31 ± Æ22 8Æ3 ± Æ4 8Æ1 ± Æ4 Autumn * 5Æ4 ±Æ6 5Æ7 ±Æ9 Fluorescent pseudomonas Spring 1Æ73 ± Æ236 7Æ9 ±Æ2 Autumn *Not measured. eight isolates studied formed tyramine (117 ppm). Aeromonas produced putrescine (<5 8 ppm) and tyramine (<5 62 ppm), whereas C. maltaromaticum produced only tyramine ( ppm). Isolates of Ps. fluorescens/putida and Vibrio formed <5 ppm of all the studied biogenic amines. Effect of ph and MAP on biogenic amine formation by Photobacterium phosphoreum at 5 C ph (6Æ7 ± Æ9 or 6Æ48 ± Æ9) and atmosphere (air or 25% CO 2 /75% N 2 ) had little effect on the growth of P. ª 26 The Authors Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26)

11 Histamine formation in chilled garfish P. Dalgaard et al. Table 5 Histamine production by individual isolates from the dominating spoilage microflora of chilled garfish Histamine production at 5 C Isolates from garfish Test tubes stored in air Hungate tubes with 25% CO 2 /75% N 2 No. of isolates Origin of isolates Avg. histamine production (mg l )1 )* No. of isolates Origin of isolates Average histamine production (mg l )1 )* Photobacterium phosphoreum 45 9 A, C < B, D <5 53 (n ¼ 1), (n ¼ 5) Pseudomonas fluorescens/putida 29 6 A,C,E <5 à à à Shewanella baltica 21 5 A, C, F < B, D, F <5 5 Carnobacterium maltaromaticum 12 2 B, F <5 5 Aeromonas and Vibrio 9 2 C <5 2 F <5 Morganella morganii-like 2 2 F *Each strain was analysed in duplicate. A, fresh garfish in air at C; B, fresh MAP garfish at C; C, fresh garfish in air at 5 C; D, fresh MAP garfish at 5 C; E, thawed garfish in air at 5 C; F, thawed MAP garfish at 5 C. àno isolates obtained from MAP garfish. No isolates obtained from garfish in air. Log CFU ml Storage period at 5 C (days) phosphoreum (Fig. 4). Nevertheless, ph 6Æ7 as compared with ph 6Æ48 significantly increased the formation of histamine (Fig. 4) as well as the formation of cadaverine, putrescine and tyramine (P <Æ5). Storage in MAP when compared with storage in air did not significantly influence the formation of cadaverine, histamine, putrescine and tyramine by P. phosphoreum (P > Æ5). Discussion Figure 4 Growth (open symbols) and histamine formation (solid symbols) by Photobacterium phosphoreum at 5 C. Experiments were carried out at ph 6Æ48 ± Æ9 (h,, s, d) orph6æ7 ± Æ9 (n,,,,.), in air (h,, n, ) or under a modified atmosphere with 25% CO 2 and 75% N 2 (s, d,,,.). Several previous studies evaluated occurrence, growth and other biological aspects of B. belone belone and other species and subspecies of garfish (Dorman 1991; Collette 23; Uckun et al. 24). The present study showed that Histamine (ppm) biogenic amines and above 1 ppm of histamine can be formed in fresh garfish at 5 C and that this was primarily due to the growth and activity of P. phosphoreum. In addition, we documented changes in quality attributes of chilled garfish (Figs 1 3; Tables 2 4). Similar data are not available for garfish, and our results will be compared with the studies of other fish with a high concentration of free histidine including sauries (Scomberesocidae) that are closely related to garfish (Lovejoy 2). Garfish with 7 12 ppm of histamine have caused HFP outbreaks and this concentration of histamine can be formed in fresh garfish fillets if these are stored under refrigeration 1 3 days beyond the end of shelf life as determined by trained sensory evaluators (Table 2; Fig. 1). At C histamine formation was much lower than that at 5 C but even at C 18 ppm of histamine were determined in one sample of autumn garfish (Table 2; Fig. 1). MAP did not reduce the formation of histamine in fresh garfish (Fig. 1). For tuna with ph 5Æ8, MAP (4% CO 2 and 6% O 2 ) strongly inhibited growth and histamine formation by both P. phosphoreum and psychrotolerant M. morganii-like bacteria (Emborg et al. 25). To reduce the formation of histamine and other biogenic amines modified atmospheres with 4 6% CO 2 and 4 6% O 2 were also efficient for naturally contaminated hake at C but only 15 ppm of histamine was formed in this product when stored in air (Ruiz-Capillas and Moral 21). In preliminary experiments with fresh garfish fillets, MAP with 4% CO 2 and 6% O 2 resulted in grey/brown colouration and oxidized off-flavours after only 7 days at 5 C. Therefore, MAP with 4% CO 2 and 6% O 2 was not applicable with garfish. For herring, MAP with 6% CO 2 and 4% N 2 reduced the concentration of ª 26 The Authors 9 Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26) 8 95

12 P. Dalgaard et al. Histamine formation in chilled garfish histamine from 396 to 197 ppm after 16 days at 2 C and for sardines the same atmosphere after 15 days at 5 C resulted in c. 1 ppm histamine compared with c. 2 ppm for storage in air (Özogul et al. 22, 24). As compared with garfish (Fig. 1), the low concentrations of histamine in herring and sardines suggest different spoilage microflora and this may also explain the different effect of MAP on histamine formation. Similar to fresh garfish (Fig. 2), a modified atmosphere with 6% CO 2, 15% O 2 and 25% N 2 did not reduced the formation of agmatine, cadaverine and histamine in hake at C compared with storage in air (Ruiz-Capillas and Moral 21). Of the biogenic amines only the formation of putrescine in garfish was inhibited by MAP with 4% CO 2 and 6% N 2 when compared with storage in air (Fig. 2). For pink shrimps at 1Æ6 C, MAP with 45% CO 2,5%O 2 and 5% N 2 stimulated agmatine and cadaverine formation when compared with storage in air (López-Caballero et al. 22). Some stimulation of agmatine and cadaverine formation was also observed in MAP garfish (Fig. 2). López- Caballero et al. (22) indicated P. phosphoreum as an important part of the spoilage microflora in pink shrimps. This was also observed for fresh garfish (Fig. 1; Table 3), and a similar effect of MAP on agmatine and cadaverine formation therefore is not surprising. In contrast, a modified atmosphere with CO 2 and N 2 reduced cadaverine formation in herring (Özogul et al. 22). Again this suggests that the microflora in herring in that study differed from the microflora observed in garfish in the present study (Table 3). It is well documented that the rate of histamine formation in chilled fresh fish increase with the storage temperature (Kimata 1961; Frank et al. 1983; Yamanaka et al. 1984; Frank and Yoshinaga 1987; Veciana-Nogués et al. 1997; Silva et al. 1998). In fact, many studies and several reviews have concluded that histamine formation in seafood by psychrotolerant bacteria is insignificant and that storage of fresh fish at to 4Æ4 C is efficient to prevent critical histamine formation (Taylor 1986; FDA 21; Kim et al. 24). However, this conclusion is not in agreement with our garfish data (Table 2; Fig. 1) or with data for naturally contaminated fresh Pacific saury, sardine, mackerel, skipjack and yellowfin tuna, where more than 1 ppm of histamine developed during storage at 5 C (Okuzumi et al. 1981, 1982;, Yamanaka et al. 1984; Yamanaka and Matsumoto 1989; Yamanaka 199; Ababouch et al. 1991, 1996; Silva et al. 1998; Emborg et al. 25). Histamine formation in, e.g. Pacific saury, mackerel and yellowtail, can be much higher at 17 2 C than at 35 C (Kimata 1961; Yamanaka et al. 1984) and this further indicates that histamine formation in naturally contaminated seafood can be caused by psychrotolerant and heat-labile bacteria like P. phosphoreum. To reduce the unacceptably high occurrence HFP, it therefore seems important to understand and limit the growth and activity of P. phosphoreum and maybe other psychrotolerant histamine-producing bacteria in chilled seafood. The occurrence and growth of P. phosphoreum as observed in fresh garfish fillets is not surprising (Fig. 1; Table 3). As previously observed for different marine fish high concentrations of P. phosphoreum were found in the intestinal content of garfish and contamination of fillets during preparation is most probable. Photobacterium phosphoreum is both psychrotolerant and remarkably resistant to CO 2 and the observed growth to high concentrations in fresh garfish stored in air and MAP (Fig. 1) corresponds to observations for many other fresh marine fish (van Spreekens 1974; Okuzumi et al. 1982; Okuzumi and Awano 1983; Dalgaard et al. 1997a; Dalgaard 26). Although the growth response of P. phosphoreum is relatively well understood, highly variable histamine formation has been reported with some isolates being negative and others producing more than 8 ppm (Table 6). Differences in test media, incubation conditions and histamine detection methods most likely caused some of the observed variability (Table 6). However, the occurrence of weakly and strongly histamine-producing variants of P. phosphoreum, as observed in the present study (Table 5), seems essential to explain the importance of this bacteria in seafood and why some studies found P. phosphoreum to form only low concentrations of histamine (Morii et al. 1988; Takahashi et al. 23). In broth, MAP did not stimulate histamine formation by P. phosphoreum isolates when compared with storage in air (Fig. 4), but the pronounced histamine formation in fresh MAP garfish at 5 C may be explained by selection of the strongly histamine-producing variant of P. phosphoreum under these storage conditions (Tables 3 and 5). However, inhibition of P. phosphoreum by the high concentrations of other bacteria in fresh garfish in air may also explain the tendency to higher histamine concentrations in fresh MAP garfish (Fig. 1). Plasmids with histidine decarboxylase genes have been observed for Vibrio anguillarum (Barancin et al. 1998) and Lactobacillus hilgardii (Lucas et al. 25). Such plasmids have not been reported for P. phosphoreum but if they exist this could explain why P. phosphoreum isolates with such plasmids produce much more histamine than other isolates. For one P. phosphoreum isolate (NUFM 262), both a constitutive and an inducible histidine decarboxylase has been observed (Morii and Kasama 1995, 24). Thus, it is a possibility that weak histamine-producing isolates of P. phosphoreum form only one of these enzymes but further research is needed to understand and control the histamine formation by P. phosphoreum. ª 26 The Authors Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26)

13 Histamine formation in chilled garfish P. Dalgaard et al. Table 6 Histamine formation by isolates of Photobacterium phosphoreum Origin of isolates Common mackerel Mackerel, Pacific saury, Sardine NCIMB 844, 1275 and 3 other isolates Horse mackerel Horse mackerel Horse mackerel Dried sardine and squid Dried sardine and squid Fresh VP Yellowfin tuna Fresh VP Yellowfin tuna Substrate Mackerel infusion broth, 2.5% NaCl Mackerel infusion broth, 2.5% NaCl Fatty herring with 2% NaCl Broth, 1 ppm histidine, 2.5% NaCl Broth, 1 ppm histidine, 2Æ5% NaCl Broth with added histidine Broth, 1 ppm histidine, 2.% NaCl Broth, 1 ppm histidine, 2.% NaCl Broth, 15 ppm histidine, 1.% NaCl Yellowfin tuna, vacuum-packed Temperature, time ph Isolates Histamine formation References 5 C, 1 d isolates positive, 5 formed 3 4 ppm (Okuzumi et al. 1981) 5 C, 1 d isolates positive, only (Okuzumi et al. 1982) one formed > 1 ppm 4 C, 8 d * Mix of ppm (van Spreekens 1987) 2 C, h 6Æ ppm (Morii et al. 1986) C, 16 d 6Æ * ppm (Morii et al. 1988) 25 C, 24 h * ppm (Takahashi et al. 23) 12 C, 7 d 5Æ8 2 3 ppm (Kanki et al. 24) 4 C, 14 d 5Æ8 2 3 ppm (non-luminous), (Kanki et al. 24) 3 ppm (luminous) 1 C, 6 d 5Æ ppm (Emborg et al. 25) 2 C, 14 d 5Æ8 Mix of ppm (Emborg et al. 25) *Not indicated in manuscript. Vacuum-packed. We found P. phosphoreum to produce agmatine, cadaverine, putrescine, b-phenylethylamine and tyramine in broth and this correspond to its formation of these biogenic amines in cold-smoked salmon (Jørgensen et al. 2b) and fresh garfish fillets (Fig. 2). It remains to be determined whether concentrations of biogenic amines produced by P. phosphoreum potentiate the toxic effect of histamine in seafood (Lehane and Olley 2). Autumn garfish have caused more recorded outbreaks of HFP than spring garfish. The autumn garfish had lower ph (6Æ2 ± Æ1) than spring garfish (6Æ45 ± Æ4) (Table 1) and histamine formation by P. phosphoreum in broth with ph 6Æ7 ± Æ9 was more pronounced than at ph 6Æ48 ± Æ9 (Fig. 4). Thus, more histamine may be formed in autumn garfish due to their lower ph but this was not clearly observed in our studies with naturally contaminated garfish (Table 2). Photobacterium phosphoreum is inactivated by freezing at about )2 C (Guldager et al. 1998; Emborg et al. 22) and its absence in frozen and thawed garfish was therefore expected (Fig. 1; Table 3). Photobacterium phosphoreum is responsible for histamine formation in fresh garfish as discussed above and its inactivation by freezing explains the marked delay of histamine formation in thawed garfish when compared with fresh fish (Fig. 1). In the absence of P. phosphoreum, psychrotolerant Enterobacteriaceae including M. morganii-like bacteria grew to high concentrations in thawed garfish at 5 C (Table 4). The M. morganii-like bacteria isolated from thawed garfish were able to grow at 2 C (results not shown) and they most likely formed the detected concentration of histamine in thawed garfish at 5 C (Fig. 1; Table 5). The two studied isolates of M. morganii-like bacteria produced putrescine (<5 17 ppm) and tyramine (<5 16 ppm) and most likely formed the observed concentrations of these biogenic amines in thawed garfish at 5 C (Fig. 2). However, psychrotolerant M. morganii-like bacteria never dominated the microflora in thawed MAP garfish and their activity may have been limited by the remaining microflora. It is noteworthy that the psychrotolerant M. morganii-like bacteria from thawed garfish resembled the recently reported psychrotolerant and strongly histamine-producing M. morganii-like bacteria from fresh vacuum-packed tuna (Emborg et al. 25). To understand and prevent histamine formation in chilled seafood, our data support that both P. phosphoreum and psychrotolerant M. morganii-like bacteria should be taken into account as suggested by Emborg et al. (25). The shelf life of small and dark fleshed fish is often 4 9 days at C when stored in air (Dalgaard 2). For Pacific saury, however, obvious spoilage has been observed after 1 12 days at 2Æ5 C (Okuzumi et al. 1982) and this corresponds to the shelf life of garfish (Table 2). Five to six weeks of frozen storage at )22 C extended the ª 26 The Authors 92 Journal compilation ª 26 The Society for Applied Microbiology, Journal of Applied Microbiology 11 (26) 8 95