Dissertations / Theses on the topic 'Fish spoilage'

Assessment of freshness and quality of meat and fish is a major activity of both food regulatory agencies and the food industry. Various methods are used for measuring fish and meat quality, each with its particular advantages and limitations. However, methods based on monitoring the products of proteolysis have received relatively little attention. The objective of the present study was to identify specific protein and peptide products of proteolysis as indicators of freshness and quality during chilled storage of fresh fish and meat.
Samples of meat and fish were subjected to chilled storage; at intervals of 0, 2, 4, 8, 12 and 16 days, samples were subjected to protein and peptide extraction, and separation of individual sarcoplasmic and myofibrillar proteins by SDS and native electrophoresis. These extracted proteins along with acid soluble nitrogen (ASN) were separated by RP-HPLC, fractions were collected and identified by electrospray ionization mass spectrometry (ESI-MS).
RP-HPLC separated at least thirty fractions from the ASN extract of fresh fish. ESI-MS revealed the presence of at least twenty-five polypeptides with molecular weights (MW) ranging from 2 to 32 kDa. The relative area % of the polypeptides with MW 32.8 kDa and 42.8 kDa decreased during the storage while polypeptides of MW of 10.9 kDa and 16.7 kDa increased during storage. Changes in polypeptides of MW 12, 34.2 and 42.8 kDa was also observed. The sarcoplasmic protein extracted from ground and whole meat contained at least 12 polypeptides with MW ranging from 11 to 42 kDa. The relative area % of polypeptide of MW of 35.7 kDa decreased during storage. The results suggest that changes in proteins and polypeptides of MW 10.9, 12, 16.7, 32.8, 34.2 and 42.88 kDa in fish and 35.7 kDa in meat could serve as indicators of spoilage.

This study investigates the growth and metabolite production of microorganisms causing spoilage of Atlantic cod (Gadus morhua) fillets packaged under air and modified atmosphere (60 % CO2, 40 % O2). Samples were provided by two different retailers (A and B). Storage of packaged fillets occurred at 4 °C and 8 °C. Microbiological quality and metabolite production of cod fillets stored in MAP 4 °C, MAP 8 °C and air were monitored during 13 days, 7 days and 3 days of storage, respectively. Volatile compounds concentration in the headspace were quantified by Selective ion flow tube mass spectrometry and a correlation with microbiological spoilage was studied. The onset of volatile compounds detection was observed to be mostly around 7 log cfu/g of total psychrotrophic count. Trimethylamine and dimethyl sulfide were found to be the dominant volatiles in all of the tested storage conditions, nevertheless there was no close correlation between concentrations of each main VOC and percentages of rejection based on sensory evaluation. According to results it was concluded that they cannot be considered as only indicators of the quality of cod fillets stored in modified atmosphere and air.

Eight samples of varying qualities and ages of surimi were used in the development of a new method for testing the quality of surimi. The effects of salt, pH, concentration, heating temperatures and times, and cooling times were observed. These results were used in the development of the McRae-Manning Test. By employing this method, it was possible to determine the difference between fresh high quality surimi, fresh low quality surimi, and old surimi. For this test, surimi was mixed at a 15% concentration, heated at 90 C for 20 minutes in plastic syringes, cooled and evaluated. The surimi was evaluated by emptying the samples onto prepared transparent sheets and measuring how far the sample spreads with time. The transparent sheets had circular measurements which indicated the amount of spread. Best results were obtained when the sheets were elevated at one end.

This thesis purposes and develops inductively coupled LC (inductive-capacitive) pH sensors based on pH-sensitive electrode pair. The LC resonator circuit is based on a varactor and measures the low frequency potential difference. For wireless pH monitoring, the resonator circuit is integrated with a pH-sensitive electrode pair. This sensor demonstrates a linear response over 2 to 12 pH dynamic range, 0.1 pH accuracy and long-term stability. Accurate measurement of pH using electrode-based sensors is affected by temperature variation. A technique of simultaneously measuring two parameters, pH and temperature, with a single RLC resonator based sensor is presented. An algorithm is developed, which applies both pH and temperature measurement to incorporate temperature compensation in pH measurement. For in-fluid applications, an encapsulation method is applied to the LC resonator based sensor to reduce the influence of medium permittivity and conductivity on the sensor measurement. Non-invasive way to obtain reliable pH information from bacterial culture bioprocesses is demonstrated with the fluid embeddable sensor. The pH sensor is remodeled to an acidic and basic volatile sensor by embedding the electrodes in a hydrogel host electrolyte. Tests demonstrate that the volatile sensor has a detection limit of 1.5 ppm and 2 ppm for ammonia and acetic acid vapor, respectively. Application of the volatile sensor to fish spoilage monitoring shows that the sensor is capable of detecting the product rejection level with good sensitivity in real-time. It is important to develop low cost wireless passive pH sensor technologies for embedded applications such as bioprocess and food spoilage monitoring. The electrode-based passive LC sensor approach employed in this thesis overcomes drawbacks of some of the early developed passive pH sensors and can lead to an inexpensive implementation using printed electronics technology.

Listeria monocytogenes is a common post-processing contaminant in ready-to-eat vacuum packaged (VP) cold smoked salmon. Since this psychrotrophic pathogen can grow at refrigerated temperatures (~4°C), other safety barriers in addition to temperature are needed to ensure the continued safety of VP cold smoked salmon. One such novel barrier could be the pulsed light (PL) treatment of the product prior to packaging or treating the product through a transparent package.
Pulsed light destruction kinetics of L. monocytogenes were evaluated while dispensed into a liquid media, on the surface of a general purpose agar and on the surface of cold smoked salmon. Results showed that PL technology was an effective surface sanitation method (a decimal reduction time or D-value of 0.91, 1.37 and 2.25 s exposure of PL at 800, 700 and 600 V, respectively, and a resulting z value of 500 V) on the agar plate. However, it had only a limited success when applied to liquid samples as well as directly on the surface of cold smoked salmon (D-value ranged from 93 s to 24 min).
Sensory quality of VP cold smoked salmon subjected to selected PL treatments was monitored during storage for 14 days at 4°C. Both color and odor scores remained within acceptable limits over the 14 day storage period. Subsequent challenge studies were carried out with L. monocytogenes applied on VP cold smoked salmon. An overall reduction in counts was observed in samples stored at 4°C over 28 days; however, after PL treatment (day 0), there was no significant reduction in counts. Color and odor scores maintained acceptable values over 14 days. Additional experiments were carried out to determine the effects of (1) 1.5% salt, (2) 6% oil, (3) a representative salmon media and (4) background microflora (lactic acid bacteria) on the PL inactivation of L. monocytogenes. All of these factors significantly affected the destruction of L. monocytogenes by increasing the D-value (adding resistance to pulsed light destruction).
Overall, these studies have shown that PL treatment in combination with low temperature storage (4°C) has the potential to extend the shelf-life of VP cold smoked salmon products without compromising sensory quality. However further investigation into higher treatment voltages is necessary in order to achieve a higher target kill of L. monocytogenes.

Seafood permits the transmission of many bacterial pathogens. In order to reconcile consumer demands with important safety standards, traditional means of regulating microbial spoilage and safety hazards in foods are combined with novel technologies. These include biological antimicrobial systems, such as the use of lactic acid bacteria (LAB) and/or their bacteriocins, such as Carnobacterium maltaromaticum CS526 and its bacteriocin piscicocin CS526. The aims of this study were to investigate the presence of Listeria monocytogenes in temperate seafood, namely fresh and smoked salmon, fresh and smoked haddock, and fresh mussels and oysters. Additionally, there was an aim to recover, characterise and use bacteriocin-like-substance to control Listeria monocytogenes in cold smoked haddock. Vibrio spp., Enterobacteriaceae representatives, total aerobic heterotrophic counts and Listeria monocytogenes were isolated from commercially prepared smoked and fresh Atlantic salmon, smoked and fresh haddock, live mussels and oysters using selective media and tryptone soya agar (TSA). Vibrio spp. occurred in high densities (>106 CFU gˉ1) in mussels and Enterobacteriaceae representatives were recorded at >106 CFU gˉ1 in fresh salmon. Total aerobic heterotrophic counts in fresh salmon, live mussels and oysters reached 107, > 107, and > 106 CFU gˉ1, respectively. Listeria monocytogenes was recorded at 5.0 x 104 CFU gˉ1 in mussels. In total sixty one bacterial isolates were recovered from the seafood examined. The results revealed 19 genera of bacteria, i.e. Acinetobacter, Aerococcus, Aeromonas, Bacillus, Brochothrix, Carnobacterium, Citrobacter, Corynebacterium, Enterobacter, Escherichia coli, Moraxella, Micrococcus, Pseudomonas, Psychrobacter, Serratia, Shewanella, Staphylococcus, Vibrio and Listeria. The prominent characteristics of fish spoilage isolates were demonstrated by the ability of the isolates to reduce trimethylamine oxide (TMAO) to trimethylamine, and to produce H₂S. Sh. baltica OS185, Aeromonas spp. HB-6, Sh. baltica, Sh. putrefaciens, A. hydrophila HX201006-3, A. salmonicida subsp. achromogenes, A. hydrophila, C. freundii, Enterobacter cloacae were strong producers of TMA and H₂S. The spoilage microorganisms were tested for potential pathogenicity. The result revealed that 6/15 of the spoilage microorganisms produced proteolytic, lecithinase, blood (β and α haemolysin) and elastinase activity, respectively, whereas 7/15 of the spoilage microorganisms showed lipolytic activity. Cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances of Carnobacterium maltaromaticum MMF-32 and KOPRI 25789 producing strains isolated from commercially prepared smoked salmon were investigated for their potential antimicrobial activity against potentially pathogenic and food spoilage microorganisms. Generally, a broad spectrum of activity was revealed against potentially pathogenic and food spoilage microorganisms in vitro. Cold-smoked haddock treated with bacteriocin producing C. maltaromaticum MMF-32, C. piscicola A9b bacˉ phenotype nonbacteriocin producing strain a mutant of C. piscicola A9b bac+, cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances was challenged with L. monocytogenes ATCC 19114 up to 103 CFU gˉ1, respectively. Samples were stored at 4 °C for 10 days. L. monocytogenes and total bacterial counts were determined along with changes in total volatile base nitrogen (TVBN) and biogenic amines production as well as texture, colour and odour. Although the study on anti-listerial effects of C. maltaromaticum MMF-32 was not successful, this organism did have a positive effect on retention of firmness and sensory perception in cold smoked haddock.

Thesis (D. Tech. Environmental health) -- Central University of technology, Free State, 2011
Apart from being the primary food source of many cultures around the world, fish contains notable amounts of essential fatty acids that are required by the human body, thus making fish a vital part of the human diet. In South Africa Cape hake is a well-known and highly consumed local fish species, which is transported from coastal areas countrywide where the fresh fish are displayed on ice in various retail stores. Fish is known to be highly susceptible to spoilage and, as a result, the maintenance of the cold-chain in related products is of particular importance. Additionally, recent trends showing a decline in natural fish resources have instigated growing concerns about the sustainability and optimal utilisation of fish as a food source. Against this backdrop, this study aimed at determining the influence of storage parameters on selected triglycerides and their possible metabolic pathways. Also applying prediction modelling of fatty acids and volatiles as instruments to assess exposure of Cape hake fillets to excessive microbial contamination and, in effect, be indicative of the environmental parameters (for example temperature) that may influence such contamination. Randomly selected juvenile hakes were filleted and stored under various simulated retail storage conditions, under either controlled or uncontrolled environmental conditions. For each hake filleted, one fillet was inoculated with an increased load of autochthonous microbiota, and the corresponding fillet was kept at similar temperature conditions. All fillets were monitored over a ten day period, during which fatty acid and volatile samples were collected and analysed. From the resulting triglycerides a selection of fatty acids were profiled and their possible metabolic pathways investigated. Fish maturity, the distribution of the fatty acids and the implication thereof in the nutritional value were also assessed. Conventional chemometric methods utilising mathematical expressions were subsequently utilised in order to predict contamination and whether the cold chain was sustained, while an artificial neural network (ANNs) were designed to predict excessive microbial contamination in the fillets. The results showed that the nutritional value of fish differs notably with its maturity and size. Mathematical equations were furthermore found to be effective assessment instruments to indicate the percentage differences in storage temperature, as well as consequent microbial influences. Thus, this approach may introduce mathematical prediction modelling as a promising mechanism to assess Cape hake spoilage. An artificial neural network (ANN) was successfully designed, that succeeded in distinguishing between Cape hake fillets displayed and stored on ice that have been exposed to excessive contamination and those that have not been exposed. In the latter case, the selected variable was a fatty acid, hexadecanoic acid, used as biochemical indicator. This modulating approach may provide a platform for future shelf-life studies on related muscle tissue. Ultimately, the study endeavoured to add to the body of knowledge regarding the biochemical and microbiological changes related to Cape hake storage, the prediction thereof via contemporary methods and contributing to the safety and effective utilization of this unique and declining South African nutritional resource.

Die Bedingungen für die Vermarktung von Fischereierzeugnissen sind streng geregelt. Die Deutsche Fisch-Hygiene-Verordnung (VO) schreibt in den §§ 4 (1) und 6 vor, dass alle Fische unverzüglich nach dem Fang und der Tötung ausgenommen werden müssen. In diesem Punkt ist die deutsche VO strenger als das europäische Gemeinschaftsrecht (RL 91/493/EWG). Sowohl durch die in einzelnen Bundesländern unterschiedliche Auslegung des Begriffes "unverzüglich" als auch durch die in anderen EU-Ländern erlassenen Vorschriften ergibt sich eine gewisse Rechtsunsicherheit bei der Vermarktung von Ganzfischen. Im Gegensatz zu Seefischen gibt es bei Süßwasserfischen nur wenige Untersuchungen zum Vergleich der sensorischen und hygienischen Parameter von ausgenommen und unausgenommen gelagerten Fischen. Ziel der Untersuchungen war es daher, an den zwei Modellfischarten Regenbogenforelle (Oncorhynchus mykiss) und Ostsee-Zander (Sander lucioperca) gesundheitlich-hygienische als auch qualitative Gründe für bzw. gegen das Ausnehmen von Süßwasserfischen zu ermitteln. Zum einen wurde während der Lagerung bis zum Verderb wiederholt der Keimstatus der Fische bestimmt, wobei sowohl die Verderbsorganismen als auch potentiell humanpathogene Keime (Vibrio spp., Aeromonas spp., Clostridium spp., Salmonella spp., Listeria spp.) im Fischgewebe erfasst wurden. Zum anderen wurden fischartspezifische Frischegrad- und Kochprobenschemata entwickelt, die den Verderbsprozessen der beiden Süßwasserfischarten während der Lagerung angepasst sind und für eine Bestimmung der sensorischen Eigenschaften angewendet werden können. Darüber hinaus erfolgte bei den Zandern eine Untersuchung der Muskulatur und der Eingeweide auf einen Befall mit humapathogenen Nematoden und eine Bestimmung von flüchtigen Basenstickstoff (TVB-N) in der Filetmuskulatur. Die vorliegenden Ergebnisse lassen den Schluss zu, dass unausgenommene Fische, sofern eine optimale Lagerung gewährleistet ist, hygienisch unbedenklich sind und für einen Zeitraum von einigen Tagen qualitativ gleichwertig mit ausgenommenen Fischen bleiben, bevor autolytische Bauchhöhlenprozesse die Qualität des Ganzfisches beeinträchtigen. Dabei hängt der Zeitraum unter anderem von der Fischart und dem Nüchterungsgrad ab. Mikrobiologische Gründe sprechen eher gegen ein frühzeitiges Ausnehmen, da die bakterielle Belastung der Bauchhöhle durch die Schlachtung höher ausfällt als bei unausgenommen gelagerten Fischen. Demnach kann durch ein spätes Ausnehmen zwar ein schnellerer Qualitätsverlust bei Ganzfischen auftreten, aber aus hygienischer Sicht, auch im Zusammenhang mit humanpathogenen Keimen, ist eine erhöhte Gesundheitsgefährdung des Verbrauchers nicht zu erwarten.
Conditions for the marketing of fishery products are strictly regulated. In Germany, the fish hygiene regulations (§§ 4(1) and 6) demand the gutting of all fishes immediately after the catch. This regulation is more restrictive than the corresponding EU-legislation (RL 91/493/EWG). But several German counties interprete the term "immediately" differently, and most other EU-countries have more permitting regulations, thus creating legal ambiguities in the marketing of whole fishes. Unlike the situation in seafish there are only few surveys in fresh water fish comparing possible sensoric and hygienic effects of gutted and ungutted storage. The aim of this study therefore was to examine health and hygiene related as well as qualitative reasons for or against an immediate gutting of freshwater fish, using aquaculture rainbow trout (Oncorhynchus mykiss) and pike-perch (Sander lucioperca) from the Baltic Sea as model fish species. The bacterial load in several tissues of gutted and ungutted fish, spoilage organisms as well as potentially pathogenic bacteria (Vibrio spp., Aeromonas spp., Clostridium spp., Salmonella spp., Listeria spp), were determined repeatedly throughout the storage period and statistically compared. Furthermore, species-specific grading schemes for the whole fish as well as for steamed fillets were developed that allowed the assessment of spoilage and of the sensoric characteristics of gutted and ungutted fishes. In pike-perch, muscle tissue and guts were examined for pathogenic nematode larvae and the fillet content of total volatile basic nitrogen was determined. The results of this study lead to the conclusion that ungutted fish, as long as optimal storage conditions are guaranteed, bear no special hygienic risks and keep a quality comparable to gutted fish for at least some days before autolytic processes in the body cavity compromise the quality of the whole fish. The length of this storage period depends mainly on the fish species and the filling of the digestive tract. From a microbiological point of view early gutting is not advisable because the gutting process itself results in a higher bacterial contamination of the body cavity than that found in fish stored ungutted. Therefore, a later gutting may lead to a faster loss of quality in whole fish but a higher hygienic risk for the consumer, even in connection with bacteria pathogenic for humans, can not be stated.

En la presente Tesis Doctoral se estudió la capacidad de conservación y la calidad sensorial de diversos platos preparados a base de carne o pescado cocinados mediante la tecnología sous vide y conservados en refrigeración. El objetivo fue estudiar el deterioro y determinar la vida comercial con el fin de introducir mejoras tecnológicas. Se determinaron diversos parámetros microbiológicos (enterobacterias totales, aerobios y anaerobios psicrófilos, bacterias ácido lácticas y mohos y levaduras), físico-químicos (pH, aw, acidez, textura instrumental y color CIELab) y sensoriales (análisis descriptivo cuantitativo del aspecto, olor, olor, sabor y textura). Los resultados obtenidos indican que los tiempos y temperaturas recomendados para cocinar la carne y/o el pescado sous vide aseguraron una correcta pasteurización. No se detectaron cambios apreciables de humedad, acidez, color o textura. Sólo el análisis sensorial permitió evaluar el deterioro de la carne y/o pescado sous vide durante su almacenamiento. La tecnología sous vide proporcionó platos refrigerados a base de carne y/o pescado con una alta aceptación y una vida comercial adecuada para su distribución en sistemas de catering.
The present PhD thesis analyses the preserving capacity and the sensory quality of different meat-or-fish-based dishes which are cooked by using the sous vide method and recreating the conditions of catering industry.The aim was to study the spoilage and to determine the shelf-life of these products to introduce technological improvements. For this purpose, several parameters were established: microbiological (aerobic and anaerobic psychrotrophs, lactic acid bacteria, Enterobacteriaceae, mould and yeasts), physical-chemical (pH, aw, acidity, instrumental texture and colour CIELab) and sensory (QDA of the appearance, colour, odour, flavour and texture). The results indicated that the periods of time and temperatures which are recommended to cook sous vide meat or fish guaranteed a correct pasteurization which prevented the proliferation, at 2 ºC, of Enterobacteriaceae and altering microorganisms. Only the sensory analysis permitted the evaluation of the spoilage of the sous vide cooked meat or fish during their storage. All this would leave a considerable safety margin in the cooking process so that the sensory quality could be adjusted. Besides, no noticeable variations of humidity, acidity, colour or texture were detected by the physical-chemical techniques, which were employed.

Le poisson est un produit très périssable dont l’altération résulte essentiellement de la croissance bactérienne. Comparé aux régions tempérées, peu d’études portent sur le microbiote d’altération des poissons tropicaux. En Martinique, le thon jaune (Thunnus albacares) et l’ombrine ocellée (Sciaenops ocellatus) représentent des poissons d’intérêt pour les filières pêche et aquaculture. Dans le but de mieux connaître le microbiote d’altération de ces poissons, des analyses culturales et aculturales (séquençage de nouvelle génération des amplicons d’ARNr 16S, Illumina MiSeq) ont été réalisées.Une grande diversité d’espèces bactériennes a été retrouvée dans le thon et l’ombrine fraîchement pêchés (104 et 887 OTUs, respectivement) et la plupart d’entre elles sont communément isolées des poissons (Chryseobacterium, Burkholderia, Flavobacterium, Psychrobacter, Arthrobacter, Staphylococcus). Certaines, comme Ralstonia sp. et Rhodanobacter terrae, en quantité importante dans le thon frais, sont plus atypiques. Au cours de l’entreposage du thon sous-glace, Pseudomonas et Brochothrix deviennent dominants. L’emballage sous atmosphère modifiée (MAP) ou sous vide (VP) entraine clairement la sélection de Brochothrix dans un cas et d’un mélange de Brochothrix, bactéries lactiques (Lactococcus piscium, Carnobacterium maltaromaticum) et d’entérobactéries (Hafnia paralvei) dans l’autre, et ne permet pas une augmentation significative de la durée de conservation. Pour les filets d’ombrine, peu de différences sont observées entre MAP et VP dont le microbiote se compose essentiellement de bactéries lactiques (Carnobacterium spp., Vagococcus spp., Lactococcus spp., Leuconostoc spp.). La durée de conservation est étendue de 15 jours par rapport au poisson entier sous air.L’inoculation de différentes espèces bactériennes dans de la chair pauci-microbienne de thon ou d’ombrine a montré que Hafnia paralvei et Serratia spp. sont les espèces les plus altérantes. Brochothrix thermosphacta et Carnobacterium spp. produisent aussi des odeurs indésirables mais de façon plus modérée. Chez Pseudomonas, les espèces ne sont pas toutes altérantes et présentent même parfois des capacités à empêcher le développement des mauvaises odeurs induites par d’autres bactéries (Pseudomonas psychrophila/fragi) et à dégrader l’histamine (Pseudomonas cedrina, Pseudomonas plecoglossicida/monteilii). En parallèle, des tests sensoriels et des dosages physico-chimiques ont également été réalisés pour comprendre les conséquences de la croissance bactérienne et identifier des indicateurs fiables pour l’évaluation du degré d’altération des produits
Fish is a highly perishable product and spoilage is mainly due to the bacterial growth. Compared to temperate regions, few studies examined the spoilage microbiota of tropical fish. In Martinique, yellowfin tuna (Thunnus albacares) and red drum (Sciaenops ocellatus) are essential fish of fisheries and aquaculture sectors. For a better characterization of the microbial ecosystem, culture-dependent and culture-independent (next-generation sequencing of 16S rRNA amplicons, Illumina MiSeq) methods were carried out.A wide diversity of species was found in freshly caught tuna and red drum (104 and 887 OTUs, respectively) and most of them are commonly isolated from fish (Chryseobacterium, Burkholderia, Flavobacterium, Psychrobacter, Arthrobacter, Staphylococcus). Others, such as Ralstonia sp. and Rhodanobacter terrae, largely present in fresh tuna, are less familiar. During the ice-storage of tuna, Pseudomonas and Brochothrix became dominant. The modified atmosphere packaging (MAP) and vacuum packaging (VP) clearly leaded to the selection of Brochothrix in one case and to a mixture of Brochothrix, lactic acid bacteria (Lactococcus piscium, Carnobacterium maltaromaticum) and enterobacteria (Hafnia paralvei) in the other case, and not conduct to a significant increase of the shelf-life. For red drum fillets, few differences were observed between MAP and VP with a microbiota essentially composed by lactic acid bacteria (Carnobacterium spp., Vagococcus spp., Lactococcus spp., Leuconostoc spp.). The shelf-life was extended by 15 days compared to the whole fish ice-stored.The inoculation of different bacterial species into the pauci-microbial flesh of tuna or red drum showed that Hafnia paralvei and Serratia spp. were the most spoiling bacteria. Brochothrix thermosphacta and Carnobacterium spp. produced more moderate undesirable odors. Among the Pseudomonas genus, not all species induced spoiling effects and some of them are even able to prevent the development of unpleasant odors from other bacteria (Pseudomonas psychrophila/fragi) and to degrade histamine (Pseudomonas cedrina, Pseudomonas plecoglossicida/monteilii).At the same time, sensory tests and physico-chemical assays were performed to understand the consequences of the bacterial growth and to identify reliable indices for the evaluation of the spoilage degree of the products

Smoked fish products are currently part of the Portuguese diet, being accessible to a large group of people. They are presented on Portuguese commercial point of sale packed in a vacuum, in small sliced pieces or in the form of a fillet. Fishes such as salmon, salmon-trout and swordfish, with European provenance, are used as raw material for the cold smoking production. The shelf-life depends on the type of cold smoking applied, ranging from 2 to 6 weeks at refrigeration temperatures ≤5ºC. Microbiological and physicochemical characteristics have demonstrated the dominance of Lactic Acid Bacteria, Enterobacteriaceae, and vibrios, among others, with the possibility of other groups of microorganisms being present, such as the pathogen Listeria monocytogenes. Attemptsto create a quality index ofcold-smoked fish products havebeen the subject of several studies in order to establish a correlation between the useful shelf-life of the product and the physicochemical, sensorial and microbiological characteristics. The main objective of this research was to characterize the microbial ecology of vacuum packed cold-smoked fish available in a Portuguese commercial point of sale and at a pilot-scale production by the identification of determinant variables, which influence the microbiological and physicochemical quality of the cold smoked fish. The investigation was conducted to characterize commercial cold-smoked fish products available in the Portuguese market, mainly salmon (Salmo salar) and salmon trout (Oncorhynchus mykiss), considering microbiological and physicochemical studies. From microbiological characterization, the ability of different bacteria isolated from cold-smoked fish to produce biogenic amines was evaluated, using different descarboxylation agar growth medium. A pilot-scale cold smoking controlled experiments using salmon trout were conducted to study: a) the effect of the application of ozone as a disinfected agent on whole fresh fish and fillets on reduction of microorganisms, including L. innocua; b) the effect of a previous freezing step (-20°C) of individual samples of vacuum packed cold-smoked salmon trout before product commercialized at chilled storage on microbial ecology c) the effect of combined treatments of salting/drying/smoking (wet or dry salting, addition of sugar in the salting mixture and long and short smoking) on microbiological and physicochemical properties followed by the use of vacuum and modified atmospheres packaging chilled storage conditions at 5ºC. Results from samples obtained at the Portuguese point of saleevidenced differences on shelf-life products, as well as on microbiological numbers of the cold-smoked samples. Some of the cold-smoked samples would be at the limit of the allowed microbial load for RTE products, even before reaching shelf-life ́s limit. The results also suggested the decrease in coefficient of variation of samples for aerobic plate counts and for numbers of Enterobateriaceaein controlled time and temperature laboratory conditions. The results demonstrated the suitability of growth culture medium on selection of bacteria producing biogenic amines. Some bacterial strains belonging to the group of LAB and Enterobacteriaceaewere positive for tyramine production and less for histamine production. Complementary results obtained from HPLC determinations, showed higher concentration of tyramine production by Carnobacterium divergensand by Lactoccocus lactis lactis.From gaseous ozone treatments applied to whole and fresh fillets salmon trout, a decrease of less than 1Log10in L. innocua(as a surrogate for the pathogen L. monocytogenes) numbers occurred on ozone treated samples in all sampling occasions. Aerobic Plate Count was slightly lower on fresh fillets after treatment and during three weeks of storage. From ozone treatments applied to whole fresh fish, a reduction greater than 1 Log10/g of L. innocuaoccurred on smoked samples at the end of the storage period. These results were more pronounced when the slime present on the whole fish surface was removed. From a previous freezing step (-20ºC) the results showed an general effect on microbial load throughout storage of previously frozen samples at initial stage of chill storage (1stweek) for total aerobic plate count, LAB and H2S producing bacteria, as compared to non-frozen cold-smoked samples only chilled. Asignificant increase in numbers of H2S-producing bacteria was observed in previously frozen samples, independently of the type of salting applied. Enterobacteriaceaegroup wasless affected by the previously freezingstep. DSC thermograms showed changes in muscle structure after salting/cold-smoking process. The stability of myofibrillar proteins were affected by salting/smoking treatment and the additional freezing step can result in decrease in product quality. Results from combined treatments of salting/drying/smoking on production of cold-smoked salmon trout revealed that wet salting treatment (especially for shorter salting times) did not produce the same results on microbiological and physicochemical characteristics on the end product, compared with dry salting. Overall, dry salting is preferable to brining for reducing microbial growth in cold-smoked salmon trout stored in VP.Higher sugar content in the salting mixture (salt:sugar|3:1) induced an increase in microbiological numbers. The smoking process characterized by long drying and short smoking times (Group II –Dry 6h and Smoke 2h) encouraged a general increase in microbiological numbers of cold-smoked samples, with asignificant increase in LAB counts, but a negative effect on the samples regarding microbiological quality, with significant increase in Enterobacteriaceae and H2S-producing bacteria. After 3 week storage the average of samples presented levels of trimethylamine(TMA) (up to 30 mg in 100 g of fish). A positive effect of short dry and long smoke exposure on microbial ecology was observed (Group I –Dry 2h and Smoke 6h) in cold-smoked fish, dry salted and packaged either in VP or MAP. MAP represents an alternative of packaging to VP, reducing the microbiological activity of some spoilage bacteria.The present research highlights how essential is the improvement of careful control in three areas of work that compose the production of cold smoked fish: (1) the control of quality of the raw material; (2) the design of technological procedures applied during the curing/smoking preservation process and (3) type of packaging and chill storage conditions. Advances in the integrated production of cold smoked fish using the preservative combined effects as the 'hurdle concept' should be applied defining technical procedures to product stability and safety commercialization.
Os produtos da pesca fumados fazem atualmente parte da alimentação dos portugueses, estando acessível a um grande grupo de pessoas. Apresentam-se nas superfícies comerciais portuguesas embaladas a vácuo, em peças pequenas laminados, e ou em forma de filete. Peixes como o salmão, a truta salmonada e o espadarte, com proveniência europeia, são utilizados como matéria-prima para a fumagem a frio do pescado. A vida de prateleira depende do tipo de fumagem a frio aplicado, variando entre 2 a 6 semanas, armazenado a temperaturas de refrigeração <5ºC. As características microbiológicas e físico-químicas têm demonstrado a dominância de Bactérias Ácido-Lácticas, Enterobacteriaceae, e vibrios, havendo a possibilidade de outros microrganismos estarem presentes como é o caso de Listeria monocytogenes, afetando a qualidade e a segurança destes produtos. Nos últimos anos, a tentativa de criar um índice de qualidade de peixe fumado a frio foi alvo de vários estudos, com o objetivo de estabelecer uma correlação entre a vida útil do produto e as características físico-químicas, sensoriais e microbiológicas. A presente investigaçãoteve como principal objetivoa caracterização da ecologia microbiana de peixe fumado a frio disponível no mercado português e à escala piloto, através da produção de peixe fumado a frio, identificando determinadas variáveis com influência na qualidade microbiológica e química destesprodutos. Numa primeira faseda investigação foi efetuada a caracterização microbiológicados produtos de pescado fumado a friodisponíveis no mercado português, essencialmente salmão (Salmo salar) e truta salmonada (Oncorhynchus mykiss). Relativamente à caracterização microbiológica, bactérias isoladas de salmão e truta salmonada foram testadas para a produção de aminas biogénicas, tiramina e de histamina, utilizando meios de cultura específicos de crescimento. Estudos à escala piloto sobre o processo de fumagem a frio de truta salmonada foram conduzidos, com objetivode estudar: a) o efeito da aplicação do ozono, enquanto agente desinfetante em filete e peixe inteiro de truta salmonada fresca na redução de microrganismos viáveis totais e L. innocua; b) a aplicação de um passo prévio de congelação (-20ºC) em amostras individuais de truta salmonada embalada a vácuo na ecologia microbiana do produto; c) o efeito de tratamentos combinados de salga/secagem/fumagem (salga seca e húmida, adição de açúcar na mistura da salga, duração curta e longa de fumagem) e embalamento a vácuo ou em atmosferas modificadas nas características físico-químicas e microbiológicas do produto final.Resultados sobre a classificação das amostras comerciais de peixe fumado a frio embalado a vácuo, demonstraram variabilidade das amostras, pelas diferenças nos períodos de vidas de prateleira e características microbiológicas. Algumas amostras apresentavam já estar muito próximo dos limites de rejeição estabelecidos para produtos prontos a comer, antes de terminar a vida de prateleira. Os resultados evidenciaram que em condições controladas de tempo e temperatura, houve uma diminuição do coeficiente de variação nas amostras, e para o número de microrganismos aeróbicos totais e grupo Enterobacteriaceae. Sobre o resultado da pesquisa de estirpes produtores de aminas biogénicas em condições específicas em meio de cultura, os resultados indicaram a habilidade de algumas bactérias LAB e Enterobacteriaceaeproduzirem tiramina e menos a histamina. Resultados complementares utilizando HPLC para a quantificação das aminas, mostraram níveis elevados de tiramina produzidos pelas bactérias Carnobacterium divergense Lactoccocus lactis lactis. Os resultados envolvendotratamento com ozono gasoso em filetes e em peixe inteiro fresco, mostrou um decréscimo inferior a 1Log10/g de L. innocuaem amostras tratadas com ozono em todas as experiências. Contagens totais de microrganismos viáveis foram baixas no peixe fresco e durante a armazenagem a frio ao final de três semanas. Uma redução superior a 1Log10/g de L. innocuafoi observada em peixe tratadono final do período da armazenagem. Quando retirado o slimeda superfície do peixe, o efeito de redução foi mais pronunciado. Relativamente ao tratamento prévio da congelação (-20ºC) aplicado em amostras individuais fumadas a frio de truta salmonada embalada a vácuo, os resultados evidenciaram um aumento da carga microbiana nas amostras previamente congeladas na primeira semana de armazenamento em refrigeração,essencialmente para as bactérias aeróbias totais, LAB e bactérias produtoras de H2S. Um aumento significativo para bactérias produtoras de H2S foi observado, independentemente do tipo de salga a que foram sujeitos. O processo prévio do passo da congelação pareceu ter menos efeito no grupo Enterobacteriaceae. Alterações na estrutura das proteínas musculares da truta-salmonada após o processo de salga/fumagem, foram evidenciadas nos termogramas obtidos por Differencial Scanning Calorimetry (DSC).A estabilidade das proteínas miofibrilares foram afetadas pelo processo da salga/fumagem e o processo adicional de congelação poderá afetara qualidade microbiológica da truta-salmonada fumada a frio.Os resultados obtidos sobre os tratamentos combinados da salga/secagem/fumagem revelaram que a salga húmida e salga seca apresentaram diferentes efeitos nas características físico-químicas e microbiológicas do produto final. Na generalidade, a salga seca apresentou efeito maior na perda de peso (menor rendimento do processo), e melhor desempenho na obtenção de teores de sal em fase aquosa, havendo efeito no controlo/redução do crescimento microbiano. A presença de maior teor de açúcarna mistura com sal (sal:açucar|3:1) induziu um incremento no crescimento microbiano nas amostras em geral, quando comparado com a mistura (sal:açucar|5:1).Relativamente ao efeito da fumagem, os resultados indicaram que a combinação 6h de secagem e 2h de fumagem (Grupo II) induziu um crescimento significativo de bactérias ácido lácticasno produto final, com efeitos similares em outros microrganismos, como o grupo Enterobacteriaceaee bactérias produtoras de H2S. Ao final de três semanas de armazenamento em refrigeração, foram registadas um aumento do teor médio de trimetilamina (TMA) (superior a 30 mg. em 100 g de peixe). Comparativamente, o tratamento combinado de 2h de secagem e 6h de fumagem (Grupo I) mostrou ser mais eficaz no controlo do crescimento microbiano, em amostras tratadas por salda seca (8h) e embaladas a vácuo ou em atmosferas modificadas.O embalamento em atmosferas modificadas representa uma alternativa reduzindo a actividade microbiana de alguns microorganismos degradativos.Genericamente o presente estudo evidencia a necessidade do controlo e implementação de procedimentos controlados no processo de produção de fumagem a frio de pescado, considerando (1) A qualidade microbiológica e química da matéria-prima; (2) A definição do processo tecnológico a aplicar (descrição, objetivose características produto final) e (3) Tipo de embalamento e controlo das condições de armazenagem. A criação de processos de fumagem a frio de peixe baseados em ‘'hurdle concept technology’ poderão através da sinergia dos agentes de preservação constituir uma solução à estabilidade e comercialização em segurança destes produtos.

The biogenic amines content in fish products has been widely studied due to their potential toxicity and possible indication of the spoilage degree of food. One particular amine, histamine, is behind several outbreaks of food poisoning, particularly in fish products. However, histamine alone appears to be insufficient to cause intoxications, being putrescine and cadaverine, potentiators agents that contribute to its toxicity. Several methodologies have been studied and developed for determination of bi-ogenic amines in food products. Ion Mobility Spectrometry coupled with Multi Capil-lary Columns or Gas Chromatography offers a higher sensitivity and selectivity on com-plex biological matrices analysis, providing the monitoring of trace levels of volatile compounds. The aim of this thesis is to evaluate Gas Chromatography coupled with Ion Mobil-ity Spectrometry as a tool for monitoring non-volatile amines emission from fish tissues matrices, allowing the detection and establishment of specific patterns of biogenic amines. Samples of histamine dihydrochloride, putrescine, cadaverine, tyramine, trypta-mine, spermine, spermidine and phenethylamine were analysed. Samples of atlantic bo-nito, atlantic horse mackerel and sardine were collected and analysed over time of four days to allow an assessment of fish spoilage. With the exception of histamine, it was possible to obtain an identifier pattern for all analysed amines. The presence of some amines was also observed in the fish samples spectra.

The main objective of this study was the characterization of surface associated microbial communities in breweries. In addition, the beer-spoiling potential of isolated strains and biofilm samples was investigated. Some studies reported the identity of cultivatable organisms from industrial plants. However, there were no data available about the composition of biofilm communities from these habitats for cultivation-independent techniques. Consequently, the fatty acid methyl esters (FAMEs) analysis, the fluorescence in situ hybridization (FISH) and the construction and investigation of 16S rRNA gene clone libraries were applied to reveal the structure of these communities. All of these methods have different advantages and therefore, they complement each other to get a more reliable picture of the biofilm communities. The cultivation method was included in this study because it enables a verification of results from other studies. Furthermore, the obtained strains are genuine brewery isolates and can be used for physiological tests. Isolates were obtained from seven different sample sites (Chapter 1 and 5). They were identified and affiliated to 25 different genera. Some of these strains were inoculated in beer but none of them was able to grow in it (Chapter 1 and 5). However, these strains can still be harmful for the industry, e.g. if they are able to form biofilms. This aspect was investigated by analyzing the potential of the isolates to produce acyl-homoserine lactones (AHLs) (Chapter 6). These quorum sensing mediating molecules are involved in the maturation process of biofilms. Indeed, some strains were found to secrete these autoinducer molecules, they mainly belonged to the genus Pseudomonas. An abundant proportion among the isolates was constituted by members of the Enterobacteriaceae (Chapter 7). In the beginning of this study, there was a minor suspicion concerning their beer-spoiling potential. Indeed, all isolated Enterobacteriaceae were found to be able to multiply in non-alcoholic beer under access of oxygen but they represented no risk for filled beer. The beer-spoiling potential of biofilm communities was investigated by inoculating them in beer (Chapter 3). These enrichments allowed the detection of minor proportions of beer-spoiling organisms. About 25% of the biofilms contained microorganisms which were able to multiply in beer with 4.8% of ethanol (v/v). The absence of anaerobic beer-spoiling bacteria in most of the biofilms was confirmed by using specific FISH probes for Pectinatus and Megasphaera cells (Chapter 9). However, Pectinatus cells constituted one of the most abundant groups in two biofilm communities. These samples clearly demonstrated that brewery biofilms can become hazardous for the quality of the product. The acetic acid bacteria were supposed to be abundant brewery biofilm organisms. This was not confirmed by any method used (Chapter 8). Instead, FISH signals were found for many other taxa in considerable proportions, e.g. communities from the conveyors consisted of members of the Eukarya, Archaea, Alpha-, Beta-, Gammaproteobacteria, Cytophaga-Flavobacteria, Planctomycetales, Actinobacteria and Firmicutes (Chapter 1). Such diverse communities were also evidenced for three other biofilms analyzed by FISH (Chapter 2 and 9). Whereas the FISH technique allows the specific detection of single cells, the FAME analysis targets all organisms present, except the Archaea. The fatty acid profiles of 78 biofilms indicated significant differences between the communities, even between those which were exposed to similar conditions. In addition, repeated sampling of identical sites revealed a temporal variability of the microbial communities (Chapter 3). Characteristical fatty acids of beer-spoiling bacteria were almost absent. Typical fatty acids of Eukarya dominated nearly half of all biofilms. The high proportions of Eukarya in some biofilms was not confirmed, as these samples were also investigated by FISH. This divergence was found to be due to the higher biomass of eukaryotic cells compared to bacterial cells (Chapter 3). As some wild yeast strains were isolated and characterized, they are a potential source of these fatty acids. In contrast to the revealed bacterial diversity, most of the isolated yeasts were assigned to Saccharomyces or Candida spp. (Chapter 4). The Saccharomyces spp. showed a high beer-spoiling potential and many Candida species were able to form biofilms. The construction of 16S rRNA gene clone libraries and the analysis of the clones with amplified ribosomal DNA restriction analysis (ARDRA) was performed with two biofilm communities (Chapter 2). Clones with identical ARDRA patterns were grouped and some representatives were identified by sequencing. These clone sequences were affiliated to 30 different genera, most of which were members of the Alpha- and Gammaproteobacteria and the Bacteroidetes. In addition, some clone sequences were assigned to uncultured organisms. Despite of the presence of 53 and 59 different ARDRA patterns in the two clone libraries, respectively, they had only four patterns in common. This result underlined the differences in the microbial composition of these communities. In conclusion, breweries represent a habitat with high cleaning and disinfecting pressure, which might have selected for a limited number of more resistant or adopted species. Instead, the community structures of biofilms in industrial environments were found to be diverse and variable in their compositions.

Le poisson est un produit très périssable dont l’altération résulte essentiellement de la croissance bactérienne. Comparé aux régions tempérées, peu d’études portent sur le microbiote d’altération des poissons tropicaux. En Martinique, le thon jaune (Thunnus albacares) et l’ombrine ocellée (Sciaenops ocellatus) représentent des poissons d’intérêt pour les filières pêche et aquaculture. Dans le but de mieux connaître le microbiote d’altération de ces poissons, des analyses culturales et aculturales (séquençage de nouvelle génération des amplicons d’ARNr 16S, Illumina MiSeq) ont été réalisées.Une grande diversité d’espèces bactériennes a été retrouvée dans le thon et l’ombrine fraîchement pêchés (104 et 887 OTUs, respectivement) et la plupart d’entre elles sont communément isolées des poissons (Chryseobacterium, Burkholderia, Flavobacterium, Psychrobacter, Arthrobacter, Staphylococcus). Certaines, comme Ralstonia sp. et Rhodanobacter terrae, en quantité importante dans le thon frais, sont plus atypiques. Au cours de l’entreposage du thon sous-glace, Pseudomonas et Brochothrix deviennent dominants. L’emballage sous atmosphère modifiée (MAP) ou sous vide (VP) entraine clairement la sélection de Brochothrix dans un cas et d’un mélange de Brochothrix, bactéries lactiques (Lactococcus piscium, Carnobacterium maltaromaticum) et d’entérobactéries (Hafnia paralvei) dans l’autre, et ne permet pas une augmentation significative de la durée de conservation. Pour les filets d’ombrine, peu de différences sont observées entre MAP et VP dont le microbiote se compose essentiellement de bactéries lactiques (Carnobacterium spp., Vagococcus spp., Lactococcus spp., Leuconostoc spp.). La durée de conservation est étendue de 15 jours par rapport au poisson entier sous air.L’inoculation de différentes espèces bactériennes dans de la chair pauci-microbienne de thon ou d’ombrine a montré que Hafnia paralvei et Serratia spp. sont les espèces les plus altérantes. Brochothrix thermosphacta et Carnobacterium spp. produisent aussi des odeurs indésirables mais de façon plus modérée. Chez Pseudomonas, les espèces ne sont pas toutes altérantes et présentent même parfois des capacités à empêcher le développement des mauvaises odeurs induites par d’autres bactéries (Pseudomonas psychrophila/fragi) et à dégrader l’histamine (Pseudomonas cedrina, Pseudomonas plecoglossicida/monteilii). En parallèle, des tests sensoriels et des dosages physico-chimiques ont également été réalisés pour comprendre les conséquences de la croissance bactérienne et identifier des indicateurs fiables pour l’évaluation du degré d’altération des produits
Fish is a highly perishable product and spoilage is mainly due to the bacterial growth. Compared to temperate regions, few studies examined the spoilage microbiota of tropical fish. In Martinique, yellowfin tuna (Thunnus albacares) and red drum (Sciaenops ocellatus) are essential fish of fisheries and aquaculture sectors. For a better characterization of the microbial ecosystem, culture-dependent and culture-independent (next-generation sequencing of 16S rRNA amplicons, Illumina MiSeq) methods were carried out.A wide diversity of species was found in freshly caught tuna and red drum (104 and 887 OTUs, respectively) and most of them are commonly isolated from fish (Chryseobacterium, Burkholderia, Flavobacterium, Psychrobacter, Arthrobacter, Staphylococcus). Others, such as Ralstonia sp. and Rhodanobacter terrae, largely present in fresh tuna, are less familiar. During the ice-storage of tuna, Pseudomonas and Brochothrix became dominant. The modified atmosphere packaging (MAP) and vacuum packaging (VP) clearly leaded to the selection of Brochothrix in one case and to a mixture of Brochothrix, lactic acid bacteria (Lactococcus piscium, Carnobacterium maltaromaticum) and enterobacteria (Hafnia paralvei) in the other case, and not conduct to a significant increase of the shelf-life. For red drum fillets, few differences were observed between MAP and VP with a microbiota essentially composed by lactic acid bacteria (Carnobacterium spp., Vagococcus spp., Lactococcus spp., Leuconostoc spp.). The shelf-life was extended by 15 days compared to the whole fish ice-stored.The inoculation of different bacterial species into the pauci-microbial flesh of tuna or red drum showed that Hafnia paralvei and Serratia spp. were the most spoiling bacteria. Brochothrix thermosphacta and Carnobacterium spp. produced more moderate undesirable odors. Among the Pseudomonas genus, not all species induced spoiling effects and some of them are even able to prevent the development of unpleasant odors from other bacteria (Pseudomonas psychrophila/fragi) and to degrade histamine (Pseudomonas cedrina, Pseudomonas plecoglossicida/monteilii).At the same time, sensory tests and physico-chemical assays were performed to understand the consequences of the bacterial growth and to identify reliable indices for the evaluation of the spoilage degree of the products