Bacterial histamine formation in mackerel and albacore was studied by inducing histamine in the muscles under controlled storage conditions. The optimum temperature for histamine formation was 25°C. The highest level of histamine detected was 283 mg/100 g in the 2-day stored mackerel; and 67.1 mg/100 g in the 6-day stored albacore. To identify the bacteria crucial to histamine formation, histamine formers were isolated using the conventional culture method. Enteric bacteria were most frequently isolated from the fish. Weak histamine formers were found in the gill and skin of fresh fish, and they required the enrichment step. Prolific histamine formers were mostly isolated from the decomposed muscles during storage at 25°C. Morganella morganii was the most prolific histamine former, producing >3,000 ppm in culture broth. M. morganii was the most prevalent histamine former in mackerel. In albacore, however, the most prevalent species was Hafnia alvei, a weak histamine former, resulting in less histamine accumulation than mackerel. Weak histamine formers, identified as natural bacteria in the marine environment, were found in mackerel during storage at 4°C after fish became unsuitable for human consumption. At 0°C, neither histamine-forming bacteria nor histamine was detected in fish. M. morganii formed significant amounts of histamine (>200 mg/100 g) in artificially contaminated fresh and frozen mackerel, albacore, and mahi-mahi when the fish were improperly stored at ambient temperatures (25°C). Growth of M. morganii was controlled by storage of fish at 4°C or below, but histamine formation was controlled only during frozen storage. For rapid detection of M. morganii, a PCR assay was developed by designing 16S rDNA targeted primers. Unique primers found for M. morganii were: the forward primer, 5'-CTCGCACCATCAGATGAACCCATAT-3'; and the reverse primer, 5'-CAAAGCATCTCTGCTAAGTTCTCTGGATG-3'. Nine CFU/ml of M. morganii inoculated in albacore homogenate were detected with a 6 h-enrichment of samples in TSB at 37°C. It would be necessary to monitor the presence of M. morganii in fish during handling and storage due to its high histamine-producing capability and prevent its contamination and proliferation after capture. The PCR assay developed in this study would be helpful to routinely monitor its presence in fish. / Graduation date: 2002

Changes in histamine, putrescine, and cadaverine concentrations in fresh and stored bluefish (Pomatomus saltatrix) were determined using a new HPLC method. The HPLC method utilized a 5.0% (w/v) trichloroacetic acid (TCA) extraction, pre-column fluorescamine derivitization, and fluorescence detection. The derivatives were stable over 24 h. The 5% TCA extraction produced percent recoveries of 98.6%, 98.7, and 100.0% for histamine, cadaverine, and putrescine respectively. The HPLC process including extraction, derivatization, and HPLC analyses was conducted in less than 45 minutes. Fresh bluefish was found to contain between <1 ppm and 99 ppm histamine, and no cadaverine or putrescine. Fresh bluefish fillets were stored at 5, 10, and 15 degrees C until sensory rejection. Fresh bluefish fillets inoculated with Morganella morganii were also stored at the same conditions. Histamine levels as high as 2200 ppm were observed in the inoculated fish stored at 15 degrees C. Overall, histamine achieved higher levels in the bluefish pieces inoculated with Morganella morganii. Histamine was present in greater amounts than putrescine and cadaverine in the bluefish samples. Histamine levels at each temperature exceeded the 50 ppm advisory level established by the FDA before 100% sensory rejection. Putrescine levels increased at each temperature during storage. Cadaverine was present only in uninoculated bluefish stored at 15 degrees C. Consumer risk from histamine poisoning seems to be the greatest in those fish stored at 5 degrees C where acceptance levels were higher and histamine levels above 100 ppm were observed. The presence of histamine-forming bacteria in fish-processing facilities was studied. Environmental sampling techniques were conducted in the Hampton Roads area of Virginia in fish-processing facilities that regularly handle scombroid fish or other fish which are known to accumulate histamine levels greater than 50 ppm. Surfaces that come into contact with the fish were swabbed and the histamine-forming bacteria from these areas were identified. One isolate each of Klebsiella ozaenae and Vibrio alginolyticus, and two isolates of Aeromonas sp. were found in the processing facilities. The study concluded that histamine-forming bacteria do not make up a large part of the microflora associated with fish-processing facilities. Fishing vessels were also sampled and no histamine-forming bacteria were identified. / Master of Science