Significance of Biogenic Amines in Cold-Smoked Fish and Their Relation to Microbiological Characteristics of Products Available in Portuguese Retail Markets

Studies on microbial characterization of cold-smoked salmon and salmon trout during cold storage were performed on samples available in the Portuguese market. Samples were also classified microbiologically according to guidelines for ready-to-eat (RTE) products. Further investigations on sample variability and microbial abilities to produce tyramine and histamine were also performed. The coefficient of variation for viable counts of different groups of microorganisms of samples collected at retail market point was high in the first 2 wk of storage, mainly in the Enterobacteriaceae group and aerobic plate count (APC), suggesting that microbiological characteristics of samples were different in numbers, even within the same batch from the same producer. This variation seemed to be decreased when storage and temperature were controlled under lab conditions. The numbers of Enterobacteriaceae were influenced by storage temperature, as indicated by low microbial numbers in samples from controlled refrigeration. Lactic acid bacteria (LAB) and Enterobacteriaceae were predominant in commercial products, a significant percentage of which were tyramine and less histamine producers. These results might be influenced by (1) the technological processes in the early stages of production, (2) contamination during the smoking process, and (3) conditions and temperature fluctuations during cold storage at retail market point of sale.

Cholesterolgehalte in Fischereierzeugnissen vom Deutschen Markt

Kurzfassung Zahlreiche Fischereierzeugnisse aus dem Deutschen Handel wurden auf ihren Gehalt an Cholesterol hin analysiert. Zur Analyse gelangten 38 verschiedene Dauerkonserven von acht Fischarten, 4 Produkte kalt geräucherter Atlantischer Zuchtlachs in Scheiben, 10 Garnelenarten und 25 Fischstäbchenerzeugnisse von 5 Tierarten in Verbraucherpackungen. Bei den Dauerkonserven lagen die Gehalte zwischen 24 und 40 mg/100 g. Zwei Ausnahmen bildeten Sprottenkonserven mit durchschnittlich 107 mg/100 g und Oktopuskonserven mit 196 mg/100 g. Die Garnelenarten variierten zwischen 84 und 161 mg/100g. Die kalt geräucherten Lachsscheiben wiesen nur eine kleine Bandbreite im Cholesterolgehalt zwischen 38 und 43 mg/100 g auf. Alle Fischstäbchen aus Magerfischen enthielten niedrige Gehalte an Cholesterol (Pangasius hypophthalmus 25, Seehecht 19, Seelachs 31 und Alaska Seehecht 28 mg/100 g), während die zwei Proben aus Tintenfischen über 100 mg/100 g lagen. Abstract Numerous fishery products from the German market have been analysed for their content of cholesterol. In total 38 different canned fishery products produced from 8 species, 4 products of sliced cold smoked Atlantic salmon, 10 species of crustacean shellfish and 25 different brands of consumer packages of fish fingers (produced from 5 species) were investigated. Canned fishery products contained amounts of cholesterol ranging from 24 to 40 mg/100 g. However, canned sprats exhibit cholesterol content as high as 107 mg/100g and canned octopus 196 mg/100 g. Crustacean shellfish was found to contain cholesterol content between 84 and 161 mg/100 g depending of species. Sliced cold smoked salmon in 200 g consumer packages showed only a little variation in cholesterol content (38-43 mg/100 g). In all fish fingers produced from lean fish species low cholesterol content (pangasius or sutchi catfish 25, hake 19, saithe 31, and Alaska Pollack 28 mg/100 g, respectively) was found, whereas two products produced from squid exceeded 100 mg/100 g.

Development of molecular-based techniques for the detection, identification and quantification of food-borne pathogens

La presencia de microorganismos patógenos en alimentos es uno de los problemas esenciales en salud pública, y las enfermedades producidas por los mismos es una de las causas más importantes de enfermedad. Por tanto, la aplicación de controles microbiológicos dentro de los programas de aseguramiento de la calidad es una premisa para minimizar el riesgo de infección de los consumidores. Los métodos microbiológicos clásicos requieren, en general, el uso de pre-enriquecimientos no-selectivos, enriquecimientos selectivos, aislamiento en medios selectivos y la confirmación posterior usando pruebas basadas en la morfología, bioquímica y serología propias de cada uno de los microorganismos objeto de estudio. Por lo tanto, estos métodos son laboriosos, requieren un largo proceso para obtener resultados definitivos y, además, no siempre pueden realizarse. Para solucionar estos inconvenientes se han desarrollado diversas metodologías alternativas para la detección identificación y cuantificación de microorganismos patógenos de origen alimentario, entre las que destacan los métodos inmunológicos y moleculares. En esta última categoría, la técnica basada en la reacción en cadena de la polimerasa (PCR) se ha convertido en la técnica diagnóstica más popular en microbiología, y recientemente, la introducción de una mejora de ésta, la PCR a tiempo real, ha producido una segunda revolución en la metodología diagnóstica molecular, como pude observarse por el número creciente de publicaciones científicas y la aparición continua de nuevos kits comerciales. La PCR a tiempo real es una técnica altamente sensible -detección de hasta una molécula- que permite la cuantificación exacta de secuencias de ADN específicas de microorganismos patógenos de origen alimentario. Además, otras ventajas que favorecen su implantación potencial en laboratorios de análisis de alimentos son su rapidez, sencillez y el formato en tubo cerrado que puede evitar contaminaciones post-PCR y favorece la automatización y un alto rendimiento. En este trabajo se han desarrollado técnicas moleculares (PCR y NASBA) sensibles y fiables para la detección, identificación y cuantificación de bacterias patogénicas de origen alimentario (Listeria spp., Mycobacterium avium subsp. paratuberculosis y Salmonella spp.). En concreto, se han diseñado y optimizado métodos basados en la técnica de PCR a tiempo real para cada uno de estos agentes: L. monocytogenes, L. innocua, Listeria spp. M. avium subsp. paratuberculosis, y también se ha optimizado y evaluado en diferentes centros un método previamente desarrollado para Salmonella spp. Además, se ha diseñado y optimizado un método basado en la técnica NASBA para la detección específica de M. avium subsp. paratuberculosis. También se evaluó la aplicación potencial de la técnica NASBA para la detección específica de formas viables de este microorganismo. Todos los métodos presentaron una especificidad del 100 % con una sensibilidad adecuada para su aplicación potencial a muestras reales de alimentos. Además, se han desarrollado y evaluado procedimientos de preparación de las muestras en productos cárnicos, productos pesqueros, leche y agua. De esta manera se han desarrollado métodos basados en la PCR a tiempo real totalmente específicos y altamente sensibles para la determinación cuantitativa de L. monocytogenes en productos cárnicos y en salmón y productos derivados como el salmón ahumado y de M. avium subsp. paratuberculosis en muestras de agua y leche. Además este último método ha sido también aplicado para evaluar la presencia de este microorganismo en el intestino de pacientes con la enfermedad de Crohn's, a partir de biopsias obtenidas de colonoscopia de voluntarios afectados. En conclusión, este estudio presenta ensayos moleculares selectivos y sensibles para la detección de patógenos en alimentos (Listeria spp., Mycobacterium avium subsp. paratuberculosis) y para una rápida e inambigua identificación de Salmonella spp. La exactitud relativa de los ensayos ha sido excelente, si se comparan con los métodos microbiológicos de referencia y pueden serusados para la cuantificación de tanto ADN genómico como de suspensiones celulares. Por otro lado, la combinación con tratamientos de preamplificación ha resultado ser de gran eficiencia para el análisis de las bacterias objeto de estudio. Por tanto, pueden constituir una estrategia útil para la detección rápida y sensible de patógenos en alimentos y deberían ser una herramienta adicional al rango de herramientas diagnósticas disponibles para el estudio de patógenos de origen alimentario.

Optimization of growth and bacteriocin activity of the food bioprotective Carnobacterium divergens V41 in an animal origin protein free medium

Optimization of Carnobacterium divergens V41 growth and bacteriocin activity in a culture medium deprived of animal protein, needs for food bioprotection, was performed by using a statistical approach. In a screening experiment, twelve factors (pH, temperature, carbohydrates, NaCl, yeast extract, soy peptone, sodium acetate, ammonium citrate, magnesium sulphate, manganese sulphate, ascorbic acid and thiamine) were tested for their influence on the maximal growth and bacteriocin activity using a two-level incomplete factorial design with 192 experiments performed in microtiter plate wells. Based on results, a basic medium was developed and three variables (pH, temperature and carbohydrates concentration) were selected for a scale-up study in bioreactor. A 23 complete factorial design was performed, allowing the estimation of linear effects of factors and all the first order interactions. The best conditions for the cell production were obtained with a temperature of 15°C and a carbohydrates concentration of 20 g/l whatever the pH (in the range 6.5-8), and the best conditions for bacteriocin activity were obtained at 15°C and pH 6.5 whatever the carbohydrates concentration (in the range 2-20 g/l). The predicted final count of C. divergens V41 and the bacteriocin activity under the optimized conditions (15°C, pH 6.5, 20 g/l carbohydrates) were 2.4 x 1010 CFU/ml and 819200 AU/ml respectively. C. divergens V41 cells cultivated in the optimized conditions were able to grow in cold-smoked salmon and totally inhibited the growth of Listeria monocytogenes (< 50 CFU g-1) during five weeks of vacuum storage at 4° and 8°C.

Viability of tallow inclusion in Atlantic salmon diet, as assessed by an on-farm grow out trial

The results of a recent increase in research interest directed at the inclusion of tallow in fish feed formulations are suggesting tallow is viable as a potential substitute for other alternative lipid sources such as poultry by-product oil. Although strong growth performance data has been shown, reservations still exist regarding reduced digestibility and the potential impacts this could have on performance over the duration of a grow-out period in low temperature conditions. Also little information is yet available on the potential effect of dietary tallow inclusion on final product quality. A large scale farm based study testing the inclusion of tallow at 40% inclusion, partially replacing poultry by-product oil, in commercial diets of Atlantic salmon over a winter grow-out period in southern Tasmania, Australia was conducted. Tallow inclusion had no impact on growth performance or nutrient digestibility. Tallow resulted in a slight improvement in fillet quality exhibiting a significant reduction in n - 6 PUFA and the n6:n3 ratio, and an increased n - 3LC-PUFA tissue deposition. Consumers were unable to display any preference in liking between 3 salmon products (cold smoked, hot smoked, and cooked) as a result of tallow inclusion. This study demonstrates the viability of partial inclusion of tallow in Atlantic salmon diets over a winter grow-out period. Statement of relevance: Improved knowledge of alternative dietary energy sources (oils and fats) to be used in aquafeed, (replacing the increasingly expensive, and diminishingly available, fish oil) is a key area of research towards improved environmental sustainability and economic viability of the aquaculture sector. Following a promising laboratory based, research scale, in vivo trial aimed at assessing the viability of tallow in salmon feed, a larger and longer duration farm-based trial was implemented to validate initial findings. Consumer test of final products (fresh-cooked, hot smoked and cold smoked fillets) showed no modification of sensorial attributes. Tallow is hereto shown to be a highly viable alternative oil for the salmon aquafeed industry.

Lagerversuch von frischem bzw. tief gefrorenen gelagerten kalt geräuchertem Atlantischem Lachs (Salmo salar) in 200-g-Fertigpackungen mit sensorischer Beurteilung in Abhängigkeit von der Lagerzeit

After 4 months frozen storage at –18 °C cold smoked Atlantic salmon in consumer packages can hardly be differentiated from the freshly smoked product by sensory assessment by an expert panel and cannot be differentiated by consumers.

Effects of hot and cold smoking processes on organoleptic properties, yield and composition of matrinxa fillet

Foi avaliado o efeito do processo de defumação a quente (45-90ºC/5 horas) e a frio (27-45ºC/10 horas) nas propriedades organolépticas, no rendimento e na composição dos filés de matrinxã (Brycon cephalus). Não houve diferença significativa no rendimento de filés defumados e não-defumados. As perdas no processo de defumação foram significativamente maiores para defumação a quente (19,37%) em comparação à defumação a frio (17,08%). O processo de defumação reduziu a umidade (in natura = 72,91%; defumado a quente = 58,51%; e defumado a frio = 59,68%) e aumentou os teores de proteína bruta, lipídios e cinzas. Houve diferença significativa somente nos teores de proteína no defumado a quente (28,07%) e defumado a frio (27,14%). O processo a frio resultou em melhor aparência e cor de filé, enquanto o processo a quente melhorou o sabor, o teor de sal e a aparência geral. O aroma e a textura não diferiram significativamente entre os processos. O processo de defumação a quente melhora as propriedades organolépticas e os níveis de proteína do filé de matrinxã.

Effects of hot and cold smoking processes on organoleptic properties, yield and composition of matrinxa fillet

Foi avaliado o efeito do processo de defumação a quente (45-90ºC/5 horas) e a frio (27-45ºC/10 horas) nas propriedades organolépticas, no rendimento e na composição dos filés de matrinxã (Brycon cephalus). Não houve diferença significativa no rendimento de filés defumados e não-defumados. As perdas no processo de defumação foram significativamente maiores para defumação a quente (19,37%) em comparação à defumação a frio (17,08%). O processo de defumação reduziu a umidade (in natura = 72,91%; defumado a quente = 58,51%; e defumado a frio = 59,68%) e aumentou os teores de proteína bruta, lipídios e cinzas. Houve diferença significativa somente nos teores de proteína no defumado a quente (28,07%) e defumado a frio (27,14%). O processo a frio resultou em melhor aparência e cor de filé, enquanto o processo a quente melhorou o sabor, o teor de sal e a aparência geral. O aroma e a textura não diferiram significativamente entre os processos. O processo de defumação a quente melhora as propriedades organolépticas e os níveis de proteína do filé de matrinxã.

Flavour components of some processed. fish and fishery products of Japan

A study was conducted to examine the flavour components of some processed fish and fishery products of Japan by gas chromatography-mass spectrometry (GC-MS). In brief the method was to absorb the headspace volatiles at 70°C into the fused silica fibre of needle of the solid phase micro extraction fibre. The absorbed components were injected to the GC-MS. The components were identified by computer matching with library database as well as by authentic standard components. In general the number of flavour components were higher in the processed fish and fishery products (except frozen prawn) than that of the raw fish and prawn. The concentration (quantity) of the f1avour components in processed fish and fishery products was much higher than that of the raw fish and prawn. Smoked salmon and baked salmon possessed double number of flavour components than that of the raw salmon. Smoking resulted the highest number of flavour components followed by baking (grilling) and canning, surimi products (kamaboko and chikuwa), drying and lastly salting. However, freezing and frozen storage resulted loss of flavour components in prawn.

Qualidade microbiológica e vida útil de filés defumados de tilápia-do-nilo sob refrigeração ou congelamento

The objective of this work was to evaluate the microbiological quality and shelf life of Nile tilapia fillets subjected to different smoking methods and storing conditions. Two smoking processes (cold or hot) were used in fillets with or without pigmentation. Products were stored under refrigeration or freezing, and monitored continually for 28 days for evaluation of their shelf life. Frozen fillets were monitored for 146 days for analysis of thiobarbituric acid (TBA) only. Hot- and cold-smoking reduced the coliform quantity, respectively, by 99.78% and 97.80%. Product storage under refrigeration allowed a 99.73% coliform reduction, and storage under freezing reduced them by 99.83%. Fecal coliform values were within the allowed limits. TBA values in fillets reached their maximum on the 14th storage day. TBA values were higher in treatments under refrigeration storage than in those under freezing, as well as in cold-smoked fillets in comparison to the hot-smoked ones. Hot-smoked process, followed by refrigeration storage, is the most adequate technique to ensure quality and a larger shelf life for Nile tilapia fillets, regardless of pigmentation process.

Enterococcus faecium isolated from Lombo, a Portuguese traditional meat product: characterisation of antibacterial compounds and factors affecting bacteriocin production

Strain ST211CH, identified as a strain of Enterococcus faecium, isolated from Lombo produced a bacteriocin that inhibited the growth of Enterococcus spp., Listeria spp., Klebsiella spp., Lactobacillus spp., Pseudomonas spp., Staphylococcus spp. and Streptococcus spp. The mode of action of the bacteriocin named as bacteriocin ST211Ch was bactericidal against Enterococcus faecalis ATCC19443. As determined by Tricine-SDS-PAGE, the approximate molecular mass of the bacteriocin was 8.0 kDa. Loss in antimicrobial activity was recorded after treatment with proteolytic enzymes. Maximum activity of bacteriocin ST211Ch was measured in broth cultures of E. faecium strain ST211Ch after 24 h; thereafter, the activity was reduced. Bacteriocin ST211Ch remained active after exposure to various temperatures and pHs, as well as to Triton X-100, Tween-80, Tween-20, sodium dodecyl sulfate, NaCl, urea and EDTA. Effect of media components on production of bacteriocin ST211Ch was also studied. On the basis of PCR reactions targeting different bacteriocin genes, i.e. enterocins, curvacins and sakacins, no evidences for the presence of these genes in the total DNA of E. faecium strain ST211Ch was obtained. The bacterium most probably produced a bacteriocin different from those mentioned above. Based on the antimicrobial spectrum, stability and mode of action of bacteriocin ST211CH, E. faecium strain ST211Ch might be considered as a potential candidate with beneficial properties for use in biopreservation to control food spoilage bacteria.

Tn6198, a novel transposon containing the trimethoprim resistance gene dfrG embedded into a Tn916 element in Listeria monocytogenes

OBJECTIVES: To characterize Tn6198, a novel conjugative transposon from the clinical Listeria monocytogenes strain TTH-2007, which contains the tetracycline and trimethoprim resistance genes tet(M) and dfrG, respectively, and to assess its transferability in vitro and in situ. METHODS: The complete sequence of Tn6198 was determined using a primer walking strategy. Horizontal gene transfer studies were performed by filter matings, as well as on the surface of smear-ripened cheese and smoked salmon. The presence of Tn916-like circular intermediates was determined by PCR. Antibiotic resistance was determined by the broth microdilution method and microarray hybridization. RESULTS: Sequencing of Tn6198 revealed that a 3.3 kb fragment containing dfrG was integrated between open reading frames 23 and 24 of Tn916. Furthermore, an additional copy of Tn916 was present in L. monocytogenes TTH-2007. Both elements were transferred simultaneously and separately in vitro to recipients L. monocytogenes 10403S and Enterococcus faecalis JH2-2 by conjugation, resulting in either tetracycline- and trimethoprim-resistant or solely tetracycline-resistant transconjugants. On the surface of cheese and salmon, only L. monocytogenes 10403S transconjugants were detected. CONCLUSIONS: This study reports the first Tn916-like element associated with a trimethoprim resistance gene, as well as the first fully characterized transposon conferring multidrug resistance in L. monocytogenes. This is of concern, as trimethoprim is administered to listeriosis patients with β-lactam allergy and as Tn6198 has a large potential for dissemination, indicated by both intra-species and inter-genus transfer.

Monitoring changes in nisin susceptibility of Listeria monocytogenes Scott A as an indicator of growth phase using FACS

Listeria monocytogenes has previously been shown to adapt to a wide variety of environmental niches, principally those associated with low pH, and this compromises its control in food environments. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. The present Study aimed to gain a further understanding of the physiological basis for the differential effects of one control strategy, namely the use of the lantibiotic nisin. Using propidium iodide (PI) to probe membrane integrity it was shown that L. monocytogenes Scott A was sensitive to nisin (8 ng mL(-1)) but this was growth phase dependent with stationary phase cells (OD600=1.2) being much more resistant than exponential phase cells (OD600=0.38). We demonstrate that, using a combination of techniques including fluorescence activated cell sorting (FACS), the membrane adaptations underpinning nisin resistance are triggered much earlier (OD600 < 0.5) than the onset of stationary phase. The significance of these findings in terms of mechanism and application are discussed. (c) 2005 Elsevier B.V.All rights reserved.

From the river to the open sea - a critical life phase of young Atlantic salmon migrating from the Simojoki river

Long-term monitoring data collected from wild smolts of Atlantic salmon (Salmo salar) in the Simojoki river, northern Finland, were used in studying the relationships between the smolt size and age, smolt and postsmolt migration, environmental conditions and postsmolt survival. The onset of the smolt run was significantly dependent on the rising water temperature and decreasing discharge of the river in the spring. The mean length of smolts migrating early in the season was commonly higher and the mean age always older than among smolts migrating later. Many of the smolts migrating early in the season and almost all smolts migrating later had started their new growth in spring in the river before their sea entry. Among postsmolts, the time required for emigration from the estuary was dependent on the sea surface temperature (SST) off the river, being significantly shorter in years with warm than cold sea temperatures. After leaving the estuary, the postsmolts migrated southwards along the eastern coast of the northern Gulf of Bothnia, the geographical distribution of the tag recoveries coinciding with the warm thermal zone in spring in the coastal area. After arriving in the southern Gulf of Bothnia in late summer the postsmolts mostly migrated near the western coast, reaching the Baltic Main Basin in late autumn. Until the early 1990s there was only a weak positive association between smolt length and postsmolt survival. However, following a subsequent decrease in the mean smolt size, a significant positive dependence was observed between smolt size and the reported recapture rate of tagged salmon. The differences in recapture rates between smolts tagged during the first and second half of the annual migration season were insignificant, indicating that the seasonal variation in smolt size and age seem to be too small to affect survival. Among the climatic factors examined, the summer SST in the Gulf of Bothnia was most clearly related to the survival of the wild postsmolts. Postsmolt survival appeared to be highest in years when the SST in June in the Bothnian Bay varied between 9 and 12 ºC. In addition, the survival of wild postsmolts showed a significant positive dependence on the SST in July in the Bothnian Sea, but not on the abundance of the prey fish (0+ herring, Clupea harengus and sprat, Sprattus sprattus) in the Bothnian Sea and in the Baltic Main Basin. The results suggest, that if the incidence of extreme weather conditions were to increase due to climatic changes, it would probably reduce the postsmolt survival of wild salmon populations. For improving the performance of hatchery-reared smolts, it could be useful to examine opportunities to produce smolts that are in their smolt traits and abilities more similar to the wild smolts described in this thesis.