Fishing for the Microbiome of Tropical Tuna.

Although tunas represent a significant part of the global fish economy and a major nutritional resource worldwide, their microbiome still remains poorly documented. Here, we conducted an analysis of the taxonomic composition of the bacterial communities inhabiting the gut, skin, and liver of two most consumed tropical tuna species (skipjack and yellowfin), from individuals caught in the Atlantic and Indian oceans. We hypothesized that each organ harbors a specific microbial assemblage whose composition might vary according to different biotic (sex, species) and/or abiotic (environmental) factors. Our results revealed that the composition of the tuna microbiome was totally independent of fish sex, regardless of the species and ocean considered. Instead, the main determinants of observed diversity were (i) tuna species for the gut and (ii) sampling site for the skin mucus layer and (iii) a combination of both parameters for the liver. Interestingly, 4.5% of all amplicon sequence variants (ASV) were shared by the three organs, highlighting the presence of a core-microbiota whose most abundant representatives belonged to the genera Mycoplasma, Cutibacterium, and Photobacterium. Our study also revealed the presence of a unique and diversified bacterial assemblage within the tuna liver, comprising a substantial proportion of potential histamine-producing bacteria, well known for their pathogenicity and their contribution to fish poisoning cases. These results indicate that this organ is an unexplored microbial niche whose role in the health of both the host and consumers remains to be elucidated.

Potential hazards associated with the consumption of Scombridae fish: Infection and toxicity from raw material and processing.

Scombridae fish (tuna, bonito and mackerel) have significant ecological and economic values. They are very appreciated by consumers worldwide for their high-quality flesh and for their high nutritional value. However, consumption of Scombridae fish is potentially hazardous. Indeed, several cases of infections and toxicity linked to the consumption of Scombridae fish as raw, or processed food products have been reported worldwide. In this review, we presented the most common health risks associated with Scombridae fish consumption. Diseases associated with the consumption of these fish are generally infectious or toxic and are caused by biological hazards, such as bacteria, viruses, parasites or chemicals hazards that enter the body through contaminated fish (polycyclic aromatic hydrocarbons, histamine) or by physical contaminants, such as heavy metals. The risks of contamination exist throughout the food chain, from primary production to the preparation of products for consumption.

Growth Kinetics and Spoilage Potential of Co-culturing Acinetobacter johnsonii and Pseudomonas fluorescens from Bigeye Tuna (Thunnus obesus) During Refrigerated Storage.

A. johnsonii and P. fluorescens are the well-known specific spoilage organisms in aquatic products and the study of the interactions between A. johnsonii and P. fluorescens are limited. This study aims to evaluate the growth kinetics, spoilage potential and interactions of A. johnsonii and P. fluorescens isolated from spoiled bigeye tuna (Thunnus obesus) by inoculating into sterile fish slices and stored at 4 °C for 6 days. The growth kinetics of A. johnsonii and P. fluorescens were fitted with Baranyi and Roberts model. The chemical indexes (total volatile base nitrogen (TVB-N), trimethylamine (TMA), pH, proteolytic activity and protein content) of each inoculated block of bigeye tuna were increased during refrigerated storage. Moreover, the higher contents of chemical indexes were observed in co-culture with A. johnsonii and P. fluorescens compared with single culture of A. johnsonii and P. fluorescens. In addition, atomic force microscopy (AFM) observation of co-culturing A. johnsonii and P. fluorescens inoculation into sterile fish slices revealed damage of myofibrillar protein structures and the protein degradation. Based on these parameters, a rapid method to evaluate spoilage potential of A. johnsonii and P. fluorescens was positively correlated with TVB-N value, TMA value and pH value (P < 0.05) by the correlation coefficient. Consequently, spoilage potential of microorganisms became stronger evaluated in a mixed culture than single culture. This paper provides insight for a detection method of interactions of A. johnsonii and P. fluorescens during refrigerated storage.

Histamine Production Behaviors of a Psychrotolerant Histamine-Producer, Morganella psychrotolerans, in Various Environmental Conditions.

Histamine food poisoning is a major safety concern related to seafood consumption worldwide. Morganella psychrotolerans is a novel psychrotolerant histamine-producer. In this study, the histamine production behaviors of M. psychrotolerans and two other major histamine-producers, mesophilic Morganella morganii and psychrotrophic Photobacterium phosphoreum, were compared in seafood products, and histamine accumulation by M. psychrotolerans was characterized at various pH and temperature levels in culture broth. The growth of M. psychrotolerans and P. phosphoreum increased similarly at 4 °C in canned tuna, but M. psychrotolerans produced much higher levels of histamine than P. phosphoreum. Histamine accumulation by M. psychrotolerans was induced at lower environmental pH condition at 4 and 20 °C. The optimal temperature and pH for producing histamine by crude histidine decarboxylase of M. psychrotolerans were 30 °C and pH 7, respectively. The activity of the crude HDC extracted from M. psychrotolerans cells at 10 °C retained 45% of the activity at 30 °C. Histidine decarboxylase gene expression of M. psychrotolerans was induced by low pH conditions. These results suggest that M. psychrotolerans are also a very important histamine-producer leading to histamine poisoning associated with seafood below the refrigeration temperature.

Histamine food poisoning: a sudden, large outbreak linked to fresh yellowfin tuna from Reunion Island, France, April 2017.

On 20 April 2017, an outbreak of histamine food poisoning occurred in a French military unit located near Paris. A total of 40 cases were identified (attack rate: 16.6%). We conducted a case-control study on 31 cases and 63 controls. Multivariate analysis pointed to cooked yellowfin tuna fillet as the very likely source of food poisoning (odds ratio = 156.8; 95% confidence interval: 18.4-1,338.4). The fresh yellowfin tuna was from Reunion Island and was supplied vacuum-sealed and packed with ice at the principal food market of Paris. No cold chain issues could be established in the upstream and downstream supply chains. Histamine concentration was found to be 1,720 mg/kg in leftover raw tuna, and 3,720 mg/kg in control cooked tuna, well above the threshold limit values defined by European regulations (200 mg/kg). The presence of Klebsiella variicola and Pantoea agglomerans, microorganisms of the Enterobacterales order that have been reported to produce histamine, was confirmed in the leftover raw tuna. This type of food poisoning is rarely recognised and confirmed. We describe the outbreak to highlight the specific key points of this type of investigation.

Multistate outbreak of Salmonella Paratyphi B variant L(+) tartrate(+) and Salmonella Weltevreden infections linked to imported frozen raw tuna: USA, March-July 2015.

Foodborne non-typhoidal salmonellosis causes approximately 1 million illnesses annually in the USA. In April 2015, we investigated a multistate outbreak of 65 Salmonella Paratyphi B variant L(+) tartrate(+) infections associated with frozen raw tuna imported from Indonesia, which was consumed raw in sushi. Forty-six (92%) of 50 case-patients interviewed ate sushi during the week before illness onset, and 44 (98%) of 45 who specified ate sushi containing raw tuna. Two outbreak strains were isolated from the samples of frozen raw tuna. Traceback identified a single importer as a common source of tuna consumed by case-patients; this importer issued three voluntary recalls of tuna sourced from one Indonesian processor. Four Salmonella Weltevreden infections were also linked to this outbreak. Whole-genome sequencing was useful in establishing a link between Salmonella isolated from ill people and tuna. This outbreak highlights the continuing foodborne illness risk associated with raw seafood consumption, the importance of processing seafood in a manner that minimises contamination with pathogenic microorganisms and the continuing need to ensure imported foods are safe to eat. People at higher risk for foodborne illness should not consume undercooked animal products, such as raw seafood.

Characterization of the spoilage potential of pure and mixed cultures of bacterial species isolated from tropical yellowfin tuna (Thunnus albacares).

AIM: The spoilage potential of 28 bacterial strains isolated from spoiled raw yellowfin tuna was evaluated. METHODS AND RESULTS: Bacterial species were inoculated in irradiated tuna matrix. Chemical changes, bacterial growth and sensory quality were monitored during aerobic storage at 8°C. Pseudomonas spp., Enterobacter spp. and Escherichia hermanii had no spoiling effect. Brochothrix thermosphacta and Carnobacterium divergens/maltaromaticum developed moderate unpleasant odours. Hafnia paralvei and Serratia spp. released strong off-odours (pyrrolidine, sulphur/cabbage). No bacterial group (except H. paralvei) combined with Pseudomonas spp. deteriorated the sensory quality of tuna. When C. divergens/maltaromaticum was associated with H. paralvei or B. thermosphacta, the odour is close to the naturally contaminated tuna stored on the same conditions. The pH, total volatile basic nitrogen (TVBN) and trimethylamine (TMA) were not correlated with the spoilage. CONCLUSIONS: The bacterial species had a different impact on the sensory quality of the fish. The bacterial interactions lead to an enhancement or an inhibition of the spoilage potential and the bacterial growth. SIGNIFICANCE AND IMPACT OF STUDY: The specific spoilage organism (SSO) appears to be an association of lactic acid bacteria (LAB) with Enterobacteriaceae or B. thermosphacta. Pseudomonas, often dominant at the sensory rejection time, is not a good quality indicator.

Tracing Origins of the Salmonella Bareilly Strain Causing a Food-borne Outbreak in the United States.

BACKGROUND: Using a novel combination of whole-genome sequencing (WGS) analysis and geographic metadata, we traced the origins of Salmonella Bareilly isolates collected in 2012 during a widespread food-borne outbreak in the United States associated with scraped tuna imported from India. METHODS: Using next-generation sequencing, we sequenced the complete genome of 100 Salmonella Bareilly isolates obtained from patients who consumed contaminated product, from natural sources, and from unrelated historically and geographically disparate foods. Pathogen genomes were linked to geography by projecting the phylogeny on a virtual globe and produced a transmission network. RESULTS: Phylogenetic analysis of WGS data revealed a common origin for outbreak strains, indicating that patients in Maryland and New York were infected from sources originating at a facility in India. CONCLUSIONS: These data represent the first report fully integrating WGS analysis with geographic mapping and a novel use of transmission networks. Results showed that WGS vastly improves our ability to delimit the scope and source of bacterial food-borne contamination events. Furthermore, these findings reinforce the extraordinary utility that WGS brings to global outbreak investigation as a greatly enhanced approach to protecting the human food supply chain as well as public health in general.

Photobacterium angustum and Photobacterium kishitanii, Psychrotrophic High-Level Histamine-Producing Bacteria Indigenous to Tuna.

Scombrotoxin fish poisoning (SFP) remains the main contributor of fish poisoning incidents in the United States, despite efforts to control its spread. Psychrotrophic histamine-producing bacteria (HPB) indigenous to scombrotoxin-forming fish may contribute to the incidence of SFP. We examined the gills, skin, and anal vents of yellowfin (n = 3), skipjack (n = 1), and albacore (n = 6) tuna for the presence of indigenous HPB. Thirteen HPB strains were isolated from the anal vent samples from albacore (n = 3) and yellowfin (n = 2) tuna. Four of these isolates were identified as Photobacterium kishitanii and nine isolates as Photobacterium angustum; these isolates produced 560 to 603 and 1,582 to 2,338 ppm histamine in marine broth containing 1% histidine (25°C for 48 h), respectively. The optimum growth temperatures and salt concentrations were 26 to 27°C and 1% salt for P. kishitanii and 30 to 32°C and 2% salt for P. angustum in Luria 70% seawater (LSW-70). The optimum activity of the HDC enzyme was at 15 to 30°C for both species. At 5°C, P. kishitanii and P. angustum had growth rates of 0.1 and 0.2 h(-1), respectively, and the activities of histidine decarboxylase (HDC) enzymes were 71% and 63%, respectively. These results show that indigenous HPB in tuna are capable of growing at elevated and refrigeration temperatures. These findings demonstrate the need to examine the relationships between the rate of histamine production at refrigeration temperatures, seafood shelf life, and regulatory limits.

Quality assessment of ice-stored tropical yellowfin tuna (Thunnus albacares) and influence of vacuum and modified atmosphere packaging.

Metagenomic, microbial, chemical and sensory analyses of Thunnus albacares from Martinique stored in ice (AIR - 0 °C), vacuum (VP - 4/8 °C) and modified atmosphere packaging (MAP - 4/8 °C) (70% CO2 - 30% O2) were carried out. The organoleptic rejection of AIR tuna was observed at day 13 when total bacterial counts equaled 10(6)-10(7) CFU g(-1). No extension of shelf-life was provided by VP and MAP. According to 16S rRNA gene sequence analyzed by Illumina MiSeq and PCR-TTGE, Rhodanobacter terrae was the main species of the freshly caught tuna. At the sensory rejection time, Brochothrix thermosphacta and Pseudomonas dominated the AIR products while B. thermosphacta alone or a mix of B. thermosphacta, Enterobacteriaceae and lactic acid bacteria (LAB) dominated the microbiota of MAP and VP products, respectively. The pH value remained stable in all trials, ranging from 5.77 to 5.97. Total volatile basic nitrogen (TVBN) and trimethylamine (TMA-N) concentrations were weak and not significantly different between batches. Lipid oxidation increased in the samples containing O2 (MAP > AIR). The initial concentration of histamine was high (75-78 mg kg(-1)) and stable up to 8 days but then significantly decreased in all trials to reach 25-30 mg kg(-1), probably due to the presence of histamine-decomposing bacteria.

Histamine fish poisoning in Australia, 2001 to 2013.

We report on human illness due to histamine fish poisoning outbreaks in Australia from 2001 to 2013. Histamine fish poisoning results from the ingestion of histamine contained within the flesh of certain fish species that naturally contain histidine, which has been converted to histamine by spoilage bacteria following poor handling or temperature control after harvesting. While symptoms vary, allergic symptoms such as facial flushing, headaches and rashes are frequently reported. Using the OzFoodNet outbreak register, published case reports and surveillance reports, we found data on 57 outbreaks of histamine fish poisoning, which affected 187 people, of whom 14% were hospitalised. There were no deaths reported. Outbreaks were generally small in size, with a median of 2 cases per outbreak (range 1 to 22 people), with 88% of outbreaks comprising less than 5 people. Tuna (in the family Scombridae) was the most frequently reported food vehicle, while 18 outbreaks involved non-scombridae fish. Median incubation periods among the outbreaks were short; being less than 1 hour for 22 outbreaks. The most frequently reported symptoms were diarrhoea and rash. Symptoms of facial/body flushing were reported for at least one case in 19 outbreaks and tingling, burning or swelling of the skin, especially around the lips for at least 1 case in 13 outbreaks. In 3 outbreaks, one or more cases were reported to have had respiratory distress or difficulty breathing. While the condition is often mild, improved recognition and appropriate treatment is important, as it will reduce the possibility of any severe health effects resulting from this condition. Key features of histamine fish poisoning outbreaks are the high attack rate, rapid onset, the typical symptoms and their short duration.

Microbiological safety of dry-cured fish from the raw material to the end of processing.

The commercialization of processed fish products is rising in restaurants and small to medium enterprises. However, there is a lack of data related to the microbiological safety of such products. In this study total aerobic colony count and Enterobacteriaceae, as proxy of process hygiene criteria, and detection of Listeria monocytogenes and concentration of histamine, as food safety criteria, were investigated in Salmo salar (salmon), Xiphias gladius (swordfish) and Thunnus albacares (yellowfin tuna), before, during, and at the end of a dry-curing process, performed in a dedicated cabinet, at controlled temperature, relative humidity and ventilation, up to 240 h. The microbiological parameters were investigated in the tested fish products by culture methods and shotgun metagenomic, while the presence of histamine, and other biogenic amines, was quantified by High Performance Liquid Chromatography. In the raw material, and up to the end of the dry curing process, the concentration of Enterobacteriaceae was always lower than 10 CFU/g, while total aerobic colony counts ranged between 3.9 and 5.4 Log CFU/g in salmon; 5.5 and 5.9 Log CFU/g in swordfish; 4.4 and 4.8 Log CFU/g in tuna. The pH values were significantly different between fish species, in the raw materials and during processing except for T4, occurring 70 h after the start of the process for salmon and after 114 h for swordfish and tuna. Water activity was different at specific sampling points and at the end of processing. Overall, 79 % of the sequences identified in the tested fish samples were assigned to y bacteria. The most abundant phyla were Pseudomonadota, Bacillota and Mycoplasmatota. The microbial populations identified by shotgun metagenomic in the tested fish species clustered well separated one from the other. Moreover, the microbial richness was significantly higher in salmon and tuna in comparison to swordfish. Listeria monocytogenes was not detected in the raw material by using the reference cultural method and very few reads (relative abundance <0.007) were detected in swordfish and tuna by shotgun metagenomic. Histamine producing bacteria, belonging to the genera Vibrio, Morganella, Photobacterium and Klebsiella, were identified primarily in swordfish. However, histamine and other biogenic amines were not detected in any sample. To the best of our knowledge this is the first paper reporting time point determinations of microbiological quality and safety parameters in salmon, swordfish and tuna, before, during and at the end of a dry-curing process. The data collected in this paper can help to predict the risk profile of ready to eat dry-cured fish products during storage before consumption.

Temperature-induced transcription of inflammatory mediators and the influence of Hsp70 following LPS stimulation of southern bluefin tuna peripheral blood leukocytes and kidney homogenates.

Temperature is known to influence inflammatory signalling in mammals, but far less understood in fish. The aim of the present study was to explore the potential effects of temperature on innate immune signalling in head kidney and leukocyte populations of the economically important southern bluefin tuna through the identification and utilization of gene expression targets in vitro. Here, we identified the mRNA sequences of five potential inflammatory mediators - TNFα (1 and 2), IL-1ß, IL-8, and Cox2 - and demonstrate induction of four - TNFα (2), IL-1ß, IL-8, and Cox2 - following LPS stimulation of both peripheral blood leukocytes and head kidney homogenates in vitro by real-time quantitative PCR. Comparison of transcriptional expression in cultures held at 18 and 25 °C (both within the presumed natural temperature range of this heterothermic species) showed accelerated transcription of cytokines TNFα, IL-1ß and IL-8 following LPS stimulation at 25 °C in both tissue types. Peak induction reached comparable levels for each transcript at both temperatures during the 24 h test period with only limited (if any) protraction in expression resulting from cold temperature (18 °C) incubation. Partial mRNA sequences were also identified for both the constitutively expressed and heat inducible chaperone proteins Hsc70 and Hsp70, and 24 h incubation at 25 °C was sufficient to induce Hsp70 transcription in leukocyte but not in head kidney cell populations. Taken together these findings suggest that temperature exerts influence in the timing but not the degree of an innate inflammatory response in bluefin tuna and that different cell populations have differential responsiveness to heat shock in this heterothermic species. Further, LPS stimulation failed to induce Hsp70 at either incubation temperature in leukocytes; whereas 25 °C incubation caused Hsp70 up-regulation in leukocytes with or without the presence of LPS. This suggests that Hsp70 does not play a direct role in immune responsiveness for this species and that an environmental temperature of 25 °C in excess of 24 h initiates a cellular stress response in blood cells of this organism. Lastly, a strong correlation between Hsp70 and IL-8 transcriptional expression was observed following LPS/heat shock stimulation of leukocytes and five potential heat shock response elements were subsequently identified on the gene promoter region of IL-8 indicating that heat shock co-activation of this chemokine previously identified in mammals is also likely present in fish.

Culturable microbiota of ranched southern bluefin tuna (Thunnus maccoyii Castelnau).

AIMS: The Australian tuna industry is based on the ranching of wild southern bluefin tuna (SBT, Thunnus maccoyii). Within this industry, only opportunistic pathogens have been reported infecting external wounds of fish. This study aimed to identify different culturable bacteria present in three cohorts of SBT and to determine normal bacteria and potential pathogens in isolates from harvest fish and moribund/dead fish. Post-mortem changes in the microbiota were also studied. METHODS AND RESULTS: Moribund/dead showed a greater proportion of members from the family Vibrionaceae than harvested fish; the latter presented mainly non-Vibrio species. In harvested fish spleens, Vibrio splendidus I complex was the most commonly identified group among Vibrio isolates, while most groups from the family Vibrionaceae were isolated from gills. For moribund/dead, Vibrio chagasii and Photobacterium damselae subsp. damselae were common in gill, spleen and kidney samples. Non-Vibrio isolates from gills were characterized using 16S rRNA sequencing as Flavobacteriaceae and classes Gammaproteobacteria and Alphaproteobacteria, mainly from the genera Winogradskyella and Tenacibaculum. Post-mortem changes showed dynamic shifts in bacterial dominance in gills, with Vibrionaceae and non-Vibrio spp. found in similar proportions initially and types related to Pseudoalteromonas ruthenica prevailing after 27 h. Spleen samples showed little bacterial growth until 5 h post-mortem, while various Vibrio-associated species were isolated 27 h post-mortem. CONCLUSIONS: Bacterial isolates found include a range of potentially pathogenic bacteria that should be monitored though most of them have yet to be associated with disease in tuna. SIGNIFICANCE AND IMPACT OF THE STUDY: This study forms a foundation for future research into the bacterial population dynamics under different culture conditions of SBT. An understanding of the bacterial compositions in SBT is necessary to evaluate the effects of some bacterial species on their health.

Detection, Identification, and Inactivation of Histamine-forming Bacteria in Seafood: A Mini-review.

Seafood is one of the essential sources of nutrients for the human diet. However, they can be subject to contamination and can cause foodborne illnesses, including scombroid fish poisoning caused by histamine. Many microorganisms can produce enzymes that eventually decompose endogenous histidine to histamine in postmortem fish muscles and tissues. One of these is histamine-forming bacteria (HFB), primarily found in the gills, gut, and skin of fishes. Previous studies linked a plethora of Gram-negative HFB including Morganella spp. and Photobacterium spp. to scombroid fish poisoning from many types of seafood, especially the Scombridae family. These bacteria possess the hdc gene to produce histidine decarboxylase enzyme. It was reported that Gram-negative HFB produced 6345 ppm in tuna and 1223 ppm in Spanish mackerel. Interestingly, Gram-positive HFB have been isolated in the seafood samples with lower histamine levels. It suggests that Gram-negative HFB are the major contributor to the accumulation of histamine in seafood. Several analytical methods are available to detect and identify HFB and their histamine metabolites from seafood substrates. Rapid test kits can be used in food production settings for early detection of histamine to avoid food intoxication. Furthermore, high hydrostatic pressure and irradiation treatment could prevent the proliferation of HFB and inactivate the existing histidine decarboxylase (HDC) activity. As demonstrated in different seafood model systems, the HDC activity was deactivated at a maximum high hydrostatic pressure level of 400 MPa. The complete inactivation of HFB was achieved by gamma irradiation at a dose of 4.0 kGy. Other postharvest treatments, like enzymatic degradation and electrolyzed oxidizing water, were studied as sustainable methods for bacterial growth prevention and enzyme inactivation. However, other HFB react differently to these treatment conditions, and further studies are recommended.

Species-specific DNA probe and development of a quantitative PCR assay for the detection of Morganella morganii.

AIMS: To develop a SYBR Green quantitative PCR assay (qPCR) for the specific detection of Morganella morganii, a fish pathogen responsible for the Histamine Fish Poisoning. METHODS AND RESULTS: A new primer set, amplifying a 179-bp fragment of the 16S rRNA gene, was selected for specificity, and 14 M. morganii strains and 32 non-Morganella strains were evaluated. The melting temperature of 84°C was consistently specific for the amplicon. Two standard curves were constructed: the minimum detection sensitivity was 0·563 pg of pure DNA, corresponding to DNA extracted from nine cells of M. morganii. The qPCR assay was evaluated in experiments with seeded fish samples, and the regression coefficient values were calculated. CONCLUSIONS: A highly specific and rapid assay was developed for the detection of M. morganii in tuna fish samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This method represents the first study about the quantification of pathogenic M. morganii in fish products. This approach can be utilized to prevent the presence of this undesirable species in the food chain.

Tracking spoilage bacteria in the tuna microbiome.

Like other seafood products, tuna is highly perishable and sensitive to microbial spoilage. Its consumption, whether fresh or canned, can lead to severe food poisoning due to the activity of specific microorganisms, including histamine-producing bacteria. Yet, many grey areas persist regarding their ecology, conditions of emergence, and proliferation in fish. In this study, we used 16S rRNA barcoding to investigate postmortem changes in the bacteriome of fresh and brine-frozen yellowfin tuna (Thunnus albacares), until late stages of decomposition (i.e. 120 h). The results revealed that despite standard refrigeration storage conditions (i.e. 4°C), a diverse and complex spoilage bacteriome developed in the gut and liver. The relative abundance of spoilage bacterial taxa increased rapidly in both organs, representing 82% of the bacterial communities in fresh yellowfin tuna, and less than 30% in brine-frozen tuna. Photobacterium was identified as one of the dominant bacterial genera, and its temporal dynamics were positively correlated with histamine concentration in both gut and liver samples, which ultimately exceeded the recommended sanitary threshold of 50 ppm in edible parts of tuna. The results from this study show that the sanitary risks associated with the consumption of this widely eaten fish are strongly influenced by postcapture storage conditions.

Modeling of pathogen survival during simulated gastric digestion.

The objective of the present study was to develop a mathematical model of pathogenic bacterial inactivation kinetics in a gastric environment in order to further understand a part of the infectious dose-response mechanism. The major bacterial pathogens Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. were examined by using simulated gastric fluid adjusted to various pH values. To correspond to the various pHs in a stomach during digestion, a modified logistic differential equation model and the Weibull differential equation model were examined. The specific inactivation rate for each pathogen was successfully described by a square-root model as a function of pH. The square-root models were combined with the modified logistic differential equation to obtain a complete inactivation curve. Both the modified logistic and Weibull models provided a highly accurate fitting of the static pH conditions for every pathogen. However, while the residuals plots of the modified logistic model indicated no systematic bias and/or regional prediction problems, the residuals plots of the Weibull model showed a systematic bias. The modified logistic model appropriately predicted the pathogen behavior in the simulated gastric digestion process with actual food, including cut lettuce, minced tuna, hamburger, and scrambled egg. Although the developed model enabled us to predict pathogen inactivation during gastric digestion, its results also suggested that the ingested bacteria in the stomach would barely be inactivated in the real digestion process. The results of this study will provide important information on a part of the dose-response mechanism of bacterial pathogens.

Emetic disease caused by Bacillus cereus after consumption of tuna fish in a beach club.

Several vomiting episodes were reported in December 2007 by the management of a beach club in Calvià. The subsequent case investigation confirmed tuna fish consumption a few hours before onset of emesis in all cases. Microbiological analyses detected high bacterial levels in ready-to-eat fish samples, indicating inappropriate cooking procedures. More important, elevated levels of Bacillus cereus were present both in raw and cooked fish. No other pathogens were detected, indicating B. cereus as the etiological agent. To our knowledge, this is the first case of emetic disease by B. cereus likely to be associated with fish consumption.

Identification and characterization of extracellular enzymes secreted by Aspergillus spp. involved in lipolysis and lipid-antioxidation during katsuobushi fermentation and ripening.

A mild-flavored soup stock made from katsuobushi is an important element of traditional Japanese cuisine and is the basic seasoning responsible for the taste. Fermented and ripened katsuobushi, known as karebushi, is manufactured by simmering skipjack tuna that is then smoke-dried, fermented, and ripened in a repeated molding process by five dominant Aspergillus species. Here, our aim was to characterize and identify the lipolytic enzymes secreted by the dominant Aspergillus species, especially A. chevalieri and A. pseudoglaucus, which are involved in hydrolyzing lipids during the molding process. The crude enzyme preparations from the five Aspergillus spp. cultivated on katsuobushi solid medium hydrolyzed triglycerides in fish oil, and more saturated and unsaturated fatty acids (C16:0, C16:1, C18:0, C18:1) were produced than major polyunsaturated fatty acids (C20:5, C22:6). On the basis of ion exchange chromatograms, the composition of the lipolytic enzymes was different in the five species. There was at least one active fraction with high hydrolytic activity toward fish oil in four of the Aspergillus spp., but not A. sydowii; the lipolytic enzyme secreted by A. sydowii had quite high activity toward the artificial substrate p-nitrophenyl butyrate, but low activity toward the natural oil. The lipolytic fractions from A. chevalieri and A. pseudoglaucus were further purified by hydrophobic interaction chromatography then gel-filtration chromatography; LC-MS-MS Mascot analysis identified a variety of lipolytic enzymes, including cutinase, esterase, phospholipase, and carboxyl esterase in the lipolytic fractions from these species. The identified enzymes had 30%-70% identity to previously reported or manually annotated lipases or esterases from taxa other than Aspergillus. The different lipolytic enzymes likely acted on triglycerides in the katsuobushi fish oil. Furthermore, catalase B and Cu/Zn superoxide dismutase, which limit oxidative damage of lipids, were also identified. These antioxidant enzymes may prevent lipid oxidation and rancidity as the lipolytic enzymes hydrolyze lipids during the long fermentation and ripening process. Umami and richness tastes tended to increase in extracts from culture of protease- and peptidase-producing A. sydowii. Our results will aid in the selection and application of desirable strains of Aspergillus species as starter cultures to improve the storage and quality of fermented and ripened karebushi.